DEVELOPMENT OF A LOW COST, RAPID

DETECTION METHOD FOR

ESCHERICHIA COLI

By

PHILLIP JOHN GRAMMER

JOE BROWN, COMMITTEE CHAIR

PAULINE JOHNSON

PHILLIP JOHNSON

A THESIS

Submitted in partial fulfillment of the requirements for the

degree of Master of Science in the Department of

Civil, Construction, and Environmental

Engineering in the Graduate School of

The University of Alabama

TUSCALOOSA, ALABAMA

2011

© Phillip John Grammer 2011

ALL RIGHTS RESERVED

ii

ABSTRACT

Reducing the burden of water-related illness requires better monitoring for fecal

contamination so that risks can be identified and controlled. Currently used tests do not provide

rapid feedback on drinking water risks, are often bulky and expensive, and are not well suited for

field use. We developed a novel test method based on a specific antigen/antibody reaction to

create a visual indication of the presence of E. coli in a water sample. We used a latex

agglutination technique employing E. coli specific antibody coated microparticles (MPs) as an

identification method, preceded by a recovery step and growth period to enhance concentrations

to detectable levels. E. coli in laboratory prepared waters were recovered via membrane

filtration, grown to titers sufficient for MP use, and interacted with MPs to provide a quantal

(presence/absence). The growth rate of E. coli in liquid media after concentration by membrane

filtration was investigated, as well as the role of elution of E. coli from the membrane filter. The

detection limit of antibody coated microparticles was also determined. Results showed low

correlation (-0.150) between average recovery rate and average 0 – 6 hour doubling time,

indicating low need for bacterial elution. The subsequent growth step yielded an average

replication of 660,000 the original membrane filter count at 9 hours, with an average doubling

time in log phase growth of 21.5 minutes. Results suggest a lower detection limit of

approximately 13,000,000 cfu/mL for E. coli using antibody coated microparticles. Coupled

together, the test can positively detect a single E. coli cfu in the original sample after incubation

for 11 hours (95% CI). Required minimum incubation times for detection of 10 cfu and 100 cfu

iii

were 9 hours 50 minutes, and 8 hours 40 minutes (95% CI), respectively. The novel method

provides a promising method of decreasing both the cost of field water testing and time required

to provide results. Doing so would greatly improve the ability to identify and reduce the effect

of fecal contamination in drinking water sources.

iv

DEDICATION

I dedicate this to my fiancée Nikki and our dog Cory. I love you both very much.

v

LIST OF ABBREVIATIONS AND SYMBOLS

Ab Antibody

atm Standard atmosphere

AD Agglutination Delta, a measure of positive sample agglutination

AceB Acetate Buffer

ATCC American Type Culture Collection

BBS Borate Buffered Saline

BSA Bovine Serum Albumin

ccc Critical Coagulation Concentration, NaCl concentration at which particles are

stable, yet agglutination readily.

cfu Colony Forming Units

C Celsius

CI Confidence Interval

CPB Citrate Phosphate Buffer

DI Deionized

DNA Deoxyribonucleic acid

vi

DT0 Doubling Time, with respect to the filter count

DT1 Doubling Time, with respect to post recovery tube solution count

EPA Environmental Protection Agency

FC Filter Count, total number of bacteria on a membrane filter

g Gram

GBS Glycine Buffered Saline

GES Growth-Eluent Solution, solution used to facilitate bacterial recovery and provide

essential requirements of bacterial growth

FC Filter Count, number of colony forming units of bacteria on a membrane filter

ELISA Enzyme-linked Immunosorbent Assay

ESI Electron Spray Ionization

Fab Fragment Antibody

FAB Fast-atom Bombardment

Fc Fragment Crystallizable

FISH Flourescent in situ Hybridization

h Hour

IgG Immuno-γ globulin G

JMP Joint Monitoring Program

vii

k Specific growth rate of bacteria

Kd Disassociation Constant

L Liters

LAT Latex Agglutination Test

m Meter

MALDI Matrix-assisted Laser Desorption/Ionization

MF Membrane Filter

MPa Megapascals

MPN Most Probable Number

mg milligram

mL milliliter

mm millimeter

M Molarity

MDG Millennium Development Goal

MP Microparticle

MS Mass Spectroscopic

MW Molecular Weight

viii

NRC National Research Council

NSA Non Specific Aggregation, agglutination of particles when antigen is not present

OC Overnight Culture

OCR Original Count Replication, number of replications a single colony forming unit

makes in a given time

P/A Presence/Absence

PBS Phosphate Buffered Saline

PCR Polymerase Chain Reaction

POU Point of Use

pH Pouvoir hydrogène

RA Rheumatoid Arthritis

RNA Ribonucleic Acid

RPM Revolutions per Minute

RT Room Temperature

s Second

SELDI Surface Enhanced Laser Desorption/Ionization

TSB Tryptic Soy Broth

TBS Tris-Buffered Saline

ix

TC Total Coliform

TSC Tube Solution Count, the total number of bacteria in solution in a growth tube

WBDO Waterborne Disease Outbreak

WHO World Health Organization

μg Microgram

µm Micrometer

μL Microliter

x

ACKNOWLEDGMENTS

I thank God for creating such an amazing universe to study. I am also pleased to thank

the many people who have helped me in so many ways with this project. I am forever grateful of

the patience and wisdom provided by my advisors, Dr. Pauline Johnson and Dr. Joe Brown. I

am also thankful for Dr. Philip Johnson serving on my committee and assisting me with the

project. Thank you to Dr. Julie Olson, who provided me a laboratory to finish this work when I

had nowhere else to go. None of the research I conducted would have been possible without the

undergraduate lab team who sacrificed for this project: Lissa Petri, Eric Lang, Alex Tietlebaum,

Anika Kuczynski, Camille Perret, Rebecca Midkiff, Norm Pelak, Hugh Sherer, Derek McElveen,

and Josh Thomas. Thanks also to Mrs. Sue Keel, Mrs. Patty McCaffery and April Jones for so

much help along the way. Thank you to my wonderful fiancée Nikki and to my family and

friends who motivated me and kept me focused on the goals I so often got distracted from.

Finally, I would like to thank the trillions of E. coli who quietly sacrificed their lives in

servitude for me every day, and hopefully for the good of many other people around the world.

This research was made possible by funding from The Bill and Melinda Gates

Foundation.

xi

CONTENTS

ABSTRACT .................................................................................................................................... ii

DEDICATION ............................................................................................................................... iv

LIST OF ABBREVIATIONS AND SYMBOLS ............................................................................v

ACKNOWLEDGMENTS ...............................................................................................................x

LIST OF TABLES ....................................................................................................................... xiv

LIST OF FIGURES .................................................................................................................... xvii

1. INTRODUCTION .......................................................................................................................1

1.1 Introduction ..........................................................................................................................1

1.2 Research Objectives .............................................................................................................2

1.3 Thesis Organization .............................................................................................................3

2. LITERATURE REVIEW ............................................................................................................4

2.1 Waterborne Pathogens .........................................................................................................4

2.2 Indicators for Microbial Risk ...............................................................................................7

2.3 Escherichia coli ...................................................................................................................9

2.4 Methods of E. coli and TC Detection ................................................................................10

2.5 Latex Agglutination Testing ..............................................................................................15

2.5.1 Microparticle Preparation .........................................................................................18

2.5.2 Antibody Immobilization ..........................................................................................19

2.6 E. coli Growth ....................................................................................................................22

xii

2.7 Summary ............................................................................................................................24

3. METHODOLOGY ....................................................................................................................27

3.1 E. coli Famp Growth and Recovery ....................................................................................27

3.1.1 Bacterial Stock Production .......................................................................................28

3.1.2 Spot Plate Procedure .................................................................................................29

3.1.3 Membrane Filtration Method ....................................................................................29

3.1.4 Spike Sample Preparation .........................................................................................30

3.1.5 Recovery and Growth Testing ..................................................................................31

3.1.6 Growth and Recovery Calculations ..........................................................................31

3.2 Microparticle Analysis .......................................................................................................33

3.2.1 Microparticle Production ..........................................................................................34

3.2.1.1 Buffer Preparation ............................................................................................35

3.2.1.2 Antibody Preparation .......................................................................................37

3.2.1.3 Bacterial Culture Production............................................................................37

3.2.1.4 Particle Washing ..............................................................................................37

3.2.1.5 Antibody Immobilization .................................................................................38

3.2.2 Antibody and Strain Reactivity Testing ....................................................................39

3.2.3 Function and Detection Limit of Coated Microparticles ..........................................40

4. RESULTS ..................................................................................................................................42

4.1 E. coli Recovery .................................................................................................................42

4.2 E. coli Growth ....................................................................................................................45

4.3 Antibody and Strain Reactivity Testing .............................................................................47

4.4 Microparticle Function.......................................................................................................48

xiii

4.3.1 Polystyrene Microparticles .......................................................................................49

4.3.2 Streptavidin Coated Microparticles ..........................................................................61

5. DISCUSSION ............................................................................................................................65

5.1 E. coli Recovery .................................................................................................................65

5.2 E. coli Growth ....................................................................................................................68

5.3 Microparticle Function.......................................................................................................70

5.4 Summary ............................................................................................................................76

5.5 Future Work .......................................................................................................................77

6. CONCLUSION ..........................................................................................................................80

REFERENCES ..............................................................................................................................82

APPENDIX ....................................................................................................................................87

A.1 Per Test Cost Determination of Novel Test Method .........................................................87

A.2 List of Growth and Recovery Trials .................................................................................88

A.3 Results of Individual Latex Agglutination Tests: Streptavidin Coated Particles..............96

A.4 Results of Individual Latex Agglutination Tests: PS Particles .......................................100

xiv

LIST OF TABLES

Table 2.1. Comparison of E. coli and TC Detection Methods ......................................................25

Table 3.1. Summary of Microparticles Tested..............................................................................34

Table 3.2. Summary of Antibodies Tested ...................................................................................35

Table 3.3. Citrate-Phosphate Buffer Constituents ........................................................................36

Table 3.4. MP Agglutination Rating Criteria................................................................................41

Table 4.1. Analysis of Mechanical Methods of Bacterial Recovery ............................................42

Table 4.2. Analysis of PBS and Tween20 on Bacterial Recovery................................................43

Table 4.3. Analysis of Tube Size and Fill on Bacterial Recovery ...............................................43

Table 4.4. Analysis of Media Concentration on Bacterial Recovery ...........................................44

Table 4.5. Analysis of Tween20, TSB, and Tube Size on E. coli Growth Rate ...........................45

Table 4.6. Average DT0 and DT1 of 15 and 50 mL Growth Tubes with Tween20 and TSB .......46

Table 4.7. DT0 of 9-hour Growth with and without Shaking .......................................................47

Table 4.8. DT0 of 9-hour Growth with 95% CI ............................................................................49

Table 4.9. E. coli Antibody Reactivity .........................................................................................48

Table 4.10. Agglutination Results: DI, PBS, and BBS, 2 hr RT Incubation, DI Suspension, E.

coli O1:K1:H7 ....................................................................................................................49

Table 4.11. Agglutination Results: DI, PBS, and BBS, with 0.05% Tween20, 2 hr RT

Incubation, DI Suspension, E. coli O1:K1:H7 ...................................................................50

Table 4.12. Agglutination Results: DI, PBS, and BBS, 24 hr 4°C Incubation, DI Suspension, E.

coli O1:K1:H7 ....................................................................................................................50

Table 4.13. Agglutination Results: DI, PBS, and BBS, 0.05% Tween20, 24 hr 4°C Incubation,

DI Suspension, E. coli O1:K1:H7 ......................................................................................51

xv

Table 4.14. Agglutination Results: TBS, GBS, CPB, and AceB; 24 hr 4° C Incubation ............52

Table 4.15. Agglutination Results: TBS, CPB, BBS, and AceB; with 0.05% Tween20, 24 hr 4°

C Incubation .......................................................................................................................53

Table 4.16. Agglutination Results: Acetate Buffer, 2 and 24 Hour RT Incubation, DI

Suspension, E. coli O1:K1:H7 ...........................................................................................54

Table 4.17. Agglutination Results: Acetate Buffer + 0.15M NaCl, 2 Hour RT and 24 Hour 4° C

Incubation, DI Suspension, E. coli O1:K1:H7 ...................................................................55

Table 4.18. Agglutination Results: Acetate Buffer + 0.15M NaCl, 2 Hour RT and 24 Hour 4° C

Incubation, DI Suspension, E. coli Famp .............................................................................56

Table 4.19. Agglutination Results: Acetate Buffer + 0.15M NaCl, 2 Hour RT and 24 Hour 4° C

Incubation, DI Suspension Retest, E. coli O1:K1:H7 ........................................................57

Table 4.20. Agglutination Results: Acetate Buffer + 0.15M NaCl, 2 Hour RT and 24 Hour 4° C

Incubation, DI Resuspension Retest, E. coli Famp ..............................................................57

Table 4.21. Agglutination Results: Agglutination Deltas of Acetate Buffer Preparations, E. coli

O1:K1:H7 ...........................................................................................................................58

Table 4.22. Detection Limit of Prepared MPs with E. coli O1:K1:H7, BSA Suspension ............59

Table 4.23. Effect of NaCl on Agglutination ................................................................................60

Table 4.24. Agglutination with MF Concentrated E. coli O1:K1:H7, after 9-hour Growth ........61

Table 4.25. Agglutination Results: Streptavidin Coated Particles, PBS and BBS, 2 and 24 Hour

RT Incubation, E. coli Famp ................................................................................................62

Table 4.26. Agglutination Results: Streptavidin Coated Particles, PBS and BBS, 2 and 24 Hour

RT Incubation, E. coli Famp ................................................................................................63

Table 4.27. Investigation of NSA in CM01N Particles ................................................................64

Table 5.1. Effect of BSA on NSA and Detection Limit ...............................................................73

Table 5.2. Effect of Particle Size and Antibody on NSA and Detection Limit ............................73

xvi

Table 5.3. Average Times to Detection, Log Phase DT = 21.4 Minutes ......................................77

Table 5.4. Times to Detection, 95% CI, Log Phase DT = 22.3 Minutes ......................................77

xvii

LIST OF FIGURES

Figure 2.1. Diagram illustrating function of antibody coated particles in LATs ..........................16

Figure 4.1. Comparison of recovery rates using 20 second hand shake, 1.0% and 0.5% Tween20,

15 mL and 50 mL tubes, and 15 g/L and 30 g/L TSB .......................................................44

Figure 4.2. DT0 9-hour growth with and without shaking ............................................................47

Figure 5.1. 0 – 6 hour doubling time (DT0) vs. recovery for 15 mL and 50 mL tubes ................67

Figure 5.2. 0 – 6 hour doubling time (DT1) vs. recovery for 15 mL and 50 mL tubes ................68

Figure 5.3. Comparison of AD values for ab13627, ab31499, and ab354 in acetate buffer

preparations, E. coli O1:K1:H7 .........................................................................................72

1

1. INTRODUCTION

1.1 Introduction

According to the WHO, every year an estimated 1.6 million children under the age of

five die from diarrheal diseases caused by unsafe drinking water, inadequate sanitation, and poor

hygiene (WHO & UNICEF, 2006). Worldwide, diarrheal diseases caused 17% of deaths of post-

neonatal children under 5 years old, compared to 8% and 3% of deaths caused by Malaria and

HIV/AIDS, respectively, from 2000-2003 (WHO, 2005). Access to safe drinking water is a

fundamental necessity; however, 1.1 billion, or 17 % of the global population, live without daily

access to an improved drinking water source. Improved drinking water sources include piped

water, rainwater, other sources thought to be of lower microbiological risk; pathogens originating

from fecal sources are the principle concern for safety of drinking water globally (WHO, 2006).

In 2000, the United Nations and other stakeholders agreed to a Millennium Development Goal

(MDG) of reducing by half the population of the world living without access to safe drinking

water by 2015 (WHO & UNICEF, 2006). Current methods of determining drinking water safety

rely on identifying human and animal fecal contamination, the ingestion of which creates the

greatest risk of infection (WHO, 2011). The detection of fecal contamination in drinking water

relies on indicator organisms, and current detection methods of these organisms requires trained

analysts, and supporting facilities of a microbiological lab or field analysis kit (Sobsey and

Pfaender, 2002). A need exists for a simpler, quicker, and more affordable test method that can

reliably detect fecal contamination in potential drinking-water sources.

2

A latex agglutination test (LAT) is a simple immunodetection method using antibody

coated particles to detect the presence or absence of a target antigen in a sample. The method

was first developed and used in 1956 for the detection of the rheumatoid factor in patients

(Singer & Plotz, 1956). Since then, LATs have been developed for numerous target antigens,

including human immunodeficiency virus, E. coli O157, tuberculosis, and human chorionic

gonadotropin (the pregnancy hormone) (TechNote 301, 2008). LAT are advantageous because

they are portable, rapid, efficient, and require minimum training and equipment to utilize. LAT

offer a unique method for detection of E. coli in water samples that could be used affordably in a

variety of settings. Development of a method to detect E. coli using LAT also requires

concentration of E. coli using a method such as membrane filtrations, and enhancement of the

bacterial titer to detectable levels using a short growth step.

1.2 Research Objectives

The goal of this research was to investigate the feasibility of a novel testing method for

the detection of E. coli in water samples. The test relied on the membrane filtration technique

for concentration of E. coli, a subsequent growth step to enhance titers to detectable levels, and a

LAT to signal the presence of E. coli in the samples. Specifically, this research aimed to meet

the following goals:

Determine the significance of elution of E. coli from membrane filter papers to

growth and if necessary, optimize this process.

Maximize and quantify the growth rate of E. coli recovered from membrane

filters.

3

Investigate the factors involved in developing a latex agglutination test for

detection of E. coli.

Prepare microparticles for use in a latex agglutination test for E. coli and quantify

and minimize their detection limit.

1.3 Thesis Organization

This thesis is divided into five sections: Introduction, Literature Review, Methods,

Results, Discussion, and Conclusions. The Literature Review discusses the current state of

drinking water, pathogens and indicators of water microbial safety, E. coli, an overview and

history of latex agglutination testing, analysis of latex agglutination development, and overview

of E. coli growth. The Methodology sections details experimental methods that were used in E.

coli growth and recovery, and LAT particle preparation. The Results section presents the

findings from experiments performed. The Discussion section provides an analysis of the

results. The Conclusion addresses the research objectives and summarizes the findings. This

thesis is concluded with list of references and an Appendix, which includes results of all growth,

recovery, and particle trials.

4

2. LITERATURE REVIEW

2.1 Waterborne Pathogens

The quality of drinking water is a serious concern to consumers, as well as suppliers,

regulators, and public health officials (Medema et al., 2003). According to a 2002 study, water,

sanitation, and hygiene were responsible for 5.7% of disease and 4.0% of deaths worldwide

(Prüss-Üstün et al., 2002). Most waterborne diseases are associated with enteric infections, and

are usually mild and non-life threatening. However, certain parts of the population, including

people with weak immune systems, children, and elderly people, will suffer more greatly,

particularly in areas where health care is lacking (Medema et al., 2003). Potential waterborne

pathogens include certain bacteria, viruses, protozoa, and helminthes. Most pathogens do not

grow in water, but are instead introduced from fecal contamination from human or animal feces

(WHO, 2006). In public water systems, the protozoa Giardia and bacteria Cryptosporidium are

common causes of waterborne disease outbreak (WBDO) in developing countries, accounting for

25 and 15, respectively, WBDOs in the United States from 1991-2002 (Craun et al., 2006).

Similarly, England and Wales experienced high numbers of WBDOs of Cryptosporidium and

Campylobacter; from 1991- 2000 they caused 26 and 9 outbreaks, respectively (Stanwell-Smith,

Anderson, & Levy, 2003). Other agents causing large numbers of illness in the United States

include Norovirus, Shigella, Campylobacter, Legionella, and E. coli O157:H7 (Craun et al.,

2006).

5

The likelihood and severity of illnesses caused by waterborne pathogens vary greatly.

Generally, all pathogens present a greater danger to those in “vulnerable subpopulations.” These

include infants, children, the elderly, and people who are immunocomprimised (National

Resource Council [NRC], 2004). Many waterborne pathogens cause acute diarrhea as the

principal symptom of infection. These pathogens include Campylobacter, Cryptosporidium, and

Giardia, as well as numerous others. The mortality and chronic effect of these infections are

highly dependent susceptibility of the infected individual. Other pathogens can cause more

severe symptoms of infection; Helicobacter pylori causes chronic active gastritis and ulceration,

and is classified as a Class I carcinogen. The severity of E. coli O157 infection can lead to

thrombotic thrombocytopaenic purpura and haemorrhagic colitis, the later resulting in fatality in

10% of cases (Percival et al., 2004). Some waterborne pathogens have been linked to chronic

diseases, with diabetes, myocarditis, and gastric cancer linked to Coxsackie B4 virus, echovirus,

and Helicobacter sp., respectively (Medema et al., 2003).

Due to the nature of large distribution systems, drinking water has the potential to

transmit disease to large numbers of people very rapidly, as in the case of the 1993 Milwaukee,

WI Cryptosporidium outbreak, when 400,000 people were infected over the course of a few

weeks (MacKenzie et al., 1994). This large outbreak raised the profile of microbial threats in

distribution systems in the USA (Percival et al., 2004). Smaller-scale distribution systems can

limit the scale of outbreaks, but they may introduce greater risks for supply contamination.

Historically in the United States, the majority of WBDOs are caused by deficiencies in water

treatment, including inadequate filtration or disinfection. Recently, however, the percent of

WBDOs caused by deficient water treatment has dropped, and the percentage caused by

deficiencies in the water distribution system has risen, from 15% during 1981 to 1990, to 54%

6

during 2001-2002 (Craun et al., 2006). This change in nature of WBDOs creates new challenges

in assessing drinking water safety, since localized WBDOs occurring in the distribution system

cannot be identified at the treatment location.

In lower-income countries, the burden of environmental health-related gastrointestinal

illness is high, and safe water sources may be limited. The Joint Monitoring Program (JMP) of

the World Health Organization and UNICEF tracks the distribution of and access to drinking

water sources globally. According to the JMP, water sources are divided into two categories

based on associated risk of waterborne disease: improved and unimproved. Improved water

sources are more likely to provide safe drinking water, and include piped supply systems, public

taps, water from rainwater catchments, and protected wells and springs. Unimproved sources are

less likely to provide safe water, and include carted or trucked water, surface water, and

unprotected wells and springs. According to the latest JMP report on global drinking water

supply, 884 million people lack access to improved sources of drinking water, or about 13% of

the global population. Almost all of these live in developing regions, particularly Sub-Saharan

Africa and Southeast Asia, where 334 million and 222 million lack access, respectively. This

number has been falling quickly, however, and it is predicted the MDG goal will be met for

drinking water (WHO & UNICEF, 2010). Improved water sources are not, however, guarantees

of microbiologically safe water: it is important to note that even untreated surface water may be

considered “improved” according to the global metrics, if it is delivered to a households via a

pipe.

Because of the high risks associated with consuming unsafe drinking water, is it critical

that safe and adequate access be maintained. In developed nations, developed water distribution

networks must be monitored to ensure safety. Failure in this monitoring can lead to outbreaks,

7

with cases of infection ranging from a handful to many thousand, in the case of the Milwaukee,

WI outbreak. Individuals, whom receive water from other sources, whether they are improved or

unimproved, are of greater risk of infection. Outbreaks are more likely, and can be small and

short-lived, possibly never noticed. For these reason, it is imperative to have methods of water

testing that can easily, quickly, and cheaply validate the safety or danger of drinking water.

2.2 Indicators for Microbial Risk

Public health personnel in the United States have relied primarily on indicator organisms

as the primary assessment of microbial drinking water quality. The principal indicators for

waterborne pathogens in the United States presently in use are total coliform (TC), fecal

coliform, E. coli, and enterococci (NRC, 2004). Coliform bacteria, commonly referred to as

coliform, are a category of microorganisms present in the gut flora of all warm blooded animals,

and consist of bacteria from the genera Escherichia, Enterobacter, Citobactor, and Klebsiella

(Tchobanoglous, Burton, & Stensel, 2003). Coliform is not a taxonomic classification but

instead a “working definition group of enteric gram-negative, facultative anaerobic rod-shaped

bacteria that ferments lactose to produce acid and gas within 48 hours at 35 degrees C” (Feng,

Weagant, & Grant, 2002). Detection of coliform bacteria with the total coliform (TC) test in a

water sample is evidence of fecal contamination. However, there are many organisms in the

coliform group, and the presence of coliform does not always signify fecal contamination.

Included in TC is fecal coliform, which is a group of coliform capable of surviving at 44.5

degrees C. Escherichia coli are a principal member of this group, and it is more representative

of fecal contamination than coliform genera (Tchobanoglous, Burton, & Stensel, 2003).

8

Therefore, E. coli are members of the group fecal coliform, and fecal coliform are members of

the group total coliform. Of the three, E. coli is the most specific indicator of fecal

contamination, and total coliform the least (Love, 2007).

In 1966, Bonde (as cited in NRC, 2004) specified the attributes of an ideal indicator of

water pollution. The ideal indicator should be present when pathogens are present, be present

only when pathogens pose immediate danger, occur in greater number than pathogens, be more

resistant to disinfection than pathogens, grow easily, offer means of unambiguous identification,

be random in the sample, grow independent of other organisms when inoculated in artificial

media. Advances in detection methods have created separate categories for ideal attributes of

indicators and methods. Ideally, an indicator should have a correlated health risk, similar

survivability and transport to pathogens, present in greater number than pathogens, and source

specific to animal digestive tracks. A method should be specific to the target organism, precise,

rapid, sensitive, quantifiable, broadly applicable, and logistically feasible (NRC 2004).

Problems exist related to the use of current indicator organisms in determining microbial

water safety. With E. coli and TC, there is the possibility of false positives and negatives. While

primarily an inhabitant of free living microbe, E. coli grows in soil, leading to the possibility of

false positives (Sobsey and Pfaender, 2002). It is also possible to detect other fecal bacteria,

such as enterococci, when TC and E. coli are undetectable and the water considered

bacteriologically safe (Sobsey and Pfaender, 2002). This can lead to discrepancies among the

results of tests using the indicator. In water systems, the correlation between protozoa and TC

detection is low; this can be attributed to the differing susceptibility to chlorine disinfection

(Sobsey, 1989). For these reasons, it is apparent the appropriate role of coliforms in water safety

needs to be addressed. The WHO Guidelines for Drinking Water Quality (GDWQ) support the

9

use of bacterial indicators in determining the microbial safety of drinking water (WHO, 2006).

As indicators TC, fecal coliform, E. coli, and entercocci are acceptable. They meet most

desirable attributes of an indicator, and are invaluable as indicators of fecal contamination in

certain situations.

2.3 Escherichia coli

Escherichia coli are gram negative rod shaped bacteria, members of the family

Enterobacteriaceae. It was discovered in 1885 by its namesake, Theodor Escherich, a German

Pediatrician (Feng, Weagant, & Grant, 2002). E. coli is present in the gut of all warm-blooded

animals and in some birds as well. It makes up about 1% of the biomass of feces, and divides

1.2 times per day in the gut (Janda and Abbot, 2006). There are numerous strains of E. coli, and

most strains are harmless and beneficial to humans, as they assist in digestion. Beneficial strains

of E. coli can be divided into two categories: transient and resident. Transient strains live in the

host for only a few days, while resident strains can reside in the gut for years. Most humans

harbor one or two resident strains at a time, with a variable number of transient strains (Janda &

Abbot, 2006). A few strains, such as E. coli O157:H7, are pathogenic and cause gastrointestinal

illness upon ingestion. Contamination from pathogenic E. coli can come from numerous sources

including raw or undercooked meat, vegetables, drinking water, and medical equipment such as

catheters.

All E. coli strains may be placed into one of three groups: commensal, diarrheagenic, and

extraintestinal. Commensal are normal strains found in the intestine, do not normally cause

infection, and are beneficial to the host by aiding in the process of digestion. Commensal strains,

10

however, can cause infection in immune-compromised patients. Diarrheagenic strains of E. coli

are pathogenic, and can cause infection within the GI tract. Diarrheagenic strains are divided

into six pathogenic groups: enteropathogenic (EPEC), enteroinvasive (EIEC), enterotoxigenic

(ETEC), shiga toxin producing (STEC), enteroaggregative (EAEC), and diffuse adhering

(DAEC). EPEC stains traditionally caused large many cases of disease in children in the

developed and developing world. These numbers have since declined, due to improved

sterilization procedures and pasteurization. EIEC infections are much rarer and cause the fewest

cases of infection worldwide. ETEC strains cause many cases of diarrhea each year, particularly

in children in developing countries. Between 1970 and 1999, 280 million cases of ETEC

diarrhea occurred in children younger than 5 years old. ETEC strains are also believed to be the

leading cause of traveler’s diarrhea; it is estimated to cause 20 to 75% of all cases. STEC

infections are also rare, although due to the presence of STEC strains in animals, they can be a

cause of waterborne illness to runoff from farms as well vegetable contamination from animal

fertilizers. Recent studies show EAEC strains are supplanting ETEC strains as the leading cause

of diarrheal disease in developing countries. Extraintestinal strains, designated as ExPEC, refer

to strains that cause infection outside of the GI tract. The cases of ExPEC are large; in 2000 it

was estimated ExPEC sepsis causes 40,000 deaths in the United States. ExPEC strains can also

cause numerous other illnesses, such as pneumonia, uncomplicated cystitis, and catheter-

associated UTI (Janda & Abbot, 2006, pp 23- 37).

2.4 Methods of E. coli and TC detection

Shardinger originally proposed the use of E. coli as an indicator organism in 1892, due to

its ability to ferment lactose abundance in human animal feces and absence from other niches

11

(Feng, Weagant, & Grant, 2002). E. coli was the target organism for the total coliform (TC) test;

however, it was found that the test was not specific to E. coli (Tchobanoglous, Burton, &

Stensel, 2003). Regardless, due to relative simplicity and cost associated with testing, TC

became the primary method of detecting fecal contamination in water samples. The two primary

detection methods for the quantification of TC are the membrane filtration (MF) technique and

the most probable number (MPN) technique. The membrane filtration technique involves

filtering an appropriate volume of sample water through a 0.45 µm pore size filter and placing

the filter on a plate of agar. Presence of TC or E. coli is confirmed using MI agar, which is a

specific growth medium (Feng, Weagant, & Grant, 2002). E. coli, because they contain a

specific enzyme (β-glucoronidase), are able to cleave the flourogenic substrate MUG to produce

a bright blue fluorescence (Tchobanoglous, Burton, & Stensel, 2003). Additionally, the

flourogenic substrate MUG can detect the presence of β-galactosidase and confirm the presence

of total coliform (Feng, Weagant, & Grant, 2002). After incubation at 35 degrees C for 24 hours,

the number of blue and fluorescent colonies can be counted to determine the concentration of E.

coli or total coliform in the sample. This process is utilized in the standard method for E. coli

and TC determination according to EPA Method 1604 (EPA, 2002), ISO method ISO 9308-

1:2000 (as cited in Fricker et al., 2008), and SM 9222 (APHA, AWWA, & WEF, 1999). The

method has been adapted for use in the field in the Oxfam Delagua Kit.

Another method for detection of total coliform and E. coli is the MPN method. The

MPN method is based on an application of the Poisson distribution to analyze the number of

positive and negative results, and determine a statistical estimation of the colonies present. The

test determines presence or absence of TC or E. coli by observing gas production in a growth

media containing lactose, and analyzes in multiple portions of equal volume, and of different

12

volumes in geometric series. Commercial methods utilizing MPN include the IDEXX Colilert, a

method for the detection and enumeration of fecal coliform that uses a preformed 96 well

Quanti-Trays, that has been approved by the EPA as an alternative testing method for E. coli

(Alternative Testing Methods, 2010). The 100 ml sample volume is distributed across the wells,

and once incubated 24 h with the reagent, reports TC or E. coli titer from 1 to 2,419 per 100 mL

(IDEXX, 2011). The MF technique is advantageous to the MPN method since it is a direct count

of the number of bacteria, reported as cfu/volume, while MPN is only an estimate, reported as

MPN/volume (Tchobanoglous, Burton, & Stensel, 2003). It has, however, been shown to yield

results that are consistent with MF standard methods, and in some forms (like IDEXX Colilert),

the method can result in significant time savings.

Other techniques for E. coli or TC enumeration involve immunochemical processes,

chiefly immunoassays. Immunoassays use antibodies specific to the target organism for

detection. The most widely used immunoassay is the enzyme-linked immunosorbent assay

(ELISA). In this test, antibodies specific to either E. coli or TC are obtained and attached to

surface, usually a polystyrene microtiter plate. The sample is exposed to the surface, and any

organisms with the matching antigen will bind to the antibodies. A second antibody conjugated

to a reporter enzyme with specificity for the same antigen but different binding site is then

introduced, creating a “sandwich”. A fluorogenic or chromogenic substrate is added and the

resulting amount of color formation is proportional to the amount of antigen, and a measure of

the amount of E. coli or TC in the sample. A method of detecting E. coli using fluorescent in

situ hybridization, or FISH, has also been developed.

In addition to immunoassays, nucleic acid detection methods also exist. These are based

on DNA or RNA analysis and can be performed in a variety of different ways. An important

13

DNA amplification technique called polymerase chain reaction, or PCR, is often employed to

generate rapid results. PCR expands the DNA sequence of interest exponentially while all other

sequences increase arithmetically. A reporter system can then be used to detect pathogens. A

method known as “real time PCR” allows rapid identification of organisms by adding a

fluorescent signal during the amplification process to provide rapid results (NRC, 2004). DNA

can also be detected in a process called gel electrophoresis, a process where DNA molecules are

place in a well near a gel medium (often agarose) and an electric current applied. The molecules

distribute in the gel medium, their larger molecules moving more slowly than the small. After

staining, different sized molecules form distinct bands in the gel, indicating the components of

the DNA, and enabling strain typing of the organism (Genetic Science Learning Center, 2011).

Mass spectroscopic (MS) methods can also be used which can detect proteins by

measuring the mass of the molecule after it has been converted to ions. Techniques for

ionization include fast-atom bombardment (FAB), electron spray ionization (ESI), pyrolysis

followed by MS, matrix-assisted laser desorption/ionization (MALDI), and surface enhanced

laser desorption/ionization (SELDI) (Sen, 2011). MS methods detect the most abundant and

most accessible proteins, enabling identification of particular organisms, including indicators

such as E. coli (Sen, 2011). All DNA, RNA, and MS methods require significant amounts of the

target organism, require significant training and equipment, and have high per sample costs.

In addition to these quantitative techniques, tests are available that can determine

Presence or Absence (P/A) of E. coli in a sample. While these test techniques are not as

descriptive as a quantitative technique, they still provide important information on the safety of

drinking water and presence of fecal contamination. P/A tests are performed on 100 mL water

samples, and attempt to confirm absence of E. coli in the water sample, in order to conform to

14

most drinking water standards (Sobsey and Pfaender, 2002). P/A methods can be adapted to be

quantitative by reproducing the sample volumes to be tested, and obtaining results based on

statistical likelihood, replicating the function of the MPN technique.

One simple technique for detecting the possibility of fecal contamination is the Hydrogen

Sulfide Producing Test. Manja et al. noticed that coliform presence in water often accompanied

H2S, and most bacteria that form H2S are of fecal origin (1982). The test is conducted by placing

a paper strip in a bottle containing bacterial culture and observing the presence or absence of an

iron sulfide precipitate formed by the reaction of H2S and iron. The test doesn’t necessarily test

for specifically TC, fecal coliform, or a specific species, but instead a group of bacteria that are

typically associated with the gut flora of warm- blooded animals, although false positives are

possible with the test. The test requires incubation, with most protocols incubating at 22 to 35

degrees C for 12-24 hours, although best results in detecting low levels of H2S producing

bacteria required 24-48 hours. The test’s simplicity suits it for use in a variety of situations and

prepared test kits have the advantage of being used with little or no training (Sobsey and

Pfaender, 2002).

Significant variation exists in E. coli and TC detection methods, including time to results,

sensitivity, logistical requirements (such as cost), and measure of viability and/or infectivity. A

particular need that is addressed only by the Delagua kit and Hydrogen Sulfide Producing Test is

the ability of the test to be used in the field. DNA and RNA methods, MS methods, ELISA, and

the MF technique require sometimes substantial equipment and sterilization. Microbial water

safety testing is often demanded in undeveloped areas, significant distances from laboratories.

Even in developed areas, transportation requirements for samples may not be possible. In these

situations, a reliable field test is a requirement. Often, the 24 hour time requirement of current

15

field deployable methods can be problematic. The need clearly exists for a test that is simple and

field deployable like the Delagua Kit and Hydrogen Sulfide Producing Test, but can also

generate rapid results.

2.5 Latex Agglutination Testing

The latex agglutination test (LAT) is a procedure that makes use of antibody-labeled

latex particles to detect target antigen proteins. According to Collings and Caruso (1997),

antibodies (Ab) are highly selective chemical attractor molecules made up of proteins and

produced by mammals in response to antigens. Antigens are substances that elicit Ab formation,

and can be molecules, hormones, viruses, bacteria, etc. The reaction between Ab and antigens

are highly specific; reacting Ab attract and attach to antigens (p. 1403). LAT utilizes this

interaction by coating particles with Ab specific to a target antigen. When the Ab coated

particles are in the presence of the antigen, they react by clumping together, the antigens forming

links between the particles, as in Figure 2.1. Uses of LAT vary widely, from detecting

pregnancy hormones (proteins) in urine samples, to the presence of microbe-associated proteins,

as well as detection of autoimmune disorders such as lupus erythematosus and rheumatoid

arthritis. LAT tests have been performed since the late 1950’s, and continue to be a common

clinical diagnostic tool today for numerous conditions as well as typing application such as

blood. Interpretation of a LAT test results involve “grading” agglutination, usually on a scale

with categories such as strong positive, weak positive, and negative. Criteria for a positive test

vary based on the nature of the LAT test being performed.

16

Figure 2.1

Diagram illustrating function of antibody coated particles in LATs. Adapted from Bangs (1996).

The earliest accounts of latex agglutination tests being performed were for the diagnosis

of rheumatoid arthritis (Singer & Plotz, 1956) and later lupus erythematosus by Christian,

Mendez-Bryan, and Larson (1958). Wilson, Morison, and Wright (1960) analyzed the

performance of an early latex slide test, a variant of LAT, compared to differential agglutination

testing for the diagnosis of rheumatoid arthritis (RA) and found the latex slide test positive in

85% of patients with definite or probable RA. The research also concluded the latex slide test to

be more practical for laboratory use as compared the traditional differential agglutination test

(DAT). Greenbury (1960) compared the performance of the four RA tests: the bentonite

flocculation test, the latex fixation test (a precursor to the LAT) of Singer and Plotz (1956), a

commercially available LAT method the Hyland “RA-test” (Hyland Laboratories), and the Rose-

Walker method, the established RA diagnosis tool of the time. Greenbury concluded the

performance of the RA-test was superior to the other three, and also commented on its speed,

ease, and minimal equipment requirement. In 1971, soon after the discovery of the hepatitis

associated antigen, Leach and Ruck developed a LAT for detection of Hepatitis. Severin

developed a similar technique for the detection of meningococcal meningitis using cerebrospinal

Antibodies bound to particles When antigen is present,

agglutination occurs

Where = Microparticle = antibody, and = antigen

17

fluid in 1972. In 1974 a method was developed for the detection of E. coli antigens using latex

agglutination testing. The test was performed by coating latex particles with E. coli antisera

from rabbits (Hechemy, Stevens, and Gaafar 1974). In 1989, March and Ratnam evaluate a

commercially available LAT test specific to E. coli O157:H7 (Oxoid Ltd.). The test had 100%

sensitivity and specificity on laboratory strains. Love (2007) developed a LAT method capable

of detecting F+ coliphages in water in as quickly as 120 minutes using E. coli as host bacteria.

Numerous methods have been developing involving LAT process, and many different

variations of the principal. All aspects of the method can be altered, such as microparticle size

and type, antibody binding method, preparation and reading procedures. Over time, methods

have become more complex to involve more sophisticated technologies for more specific and

niche applications. Most LAT tests follow a simple protocol. The fluid to be tested is prepared

to contain the target antigen or antibody in sufficient concentration. A volume of LAT Ab

coated MP suspension is mixed with a volume of the fluid of interest on a card, glass microscope

slide, or well. The solution is then gently agitated for a short period of time (1-3 minutes), and

the degree of agglutination read by turbidity, nephelometry, quasi-elastic light scattering, angular

dissymmetry, or visually or microscopic particle observation. (Collings & Caruso, 1997).

Agglutination observed visually will appear as a gradual clearing of the milky MP suspension

with formation of small clumps (Bangs, 1996). This can be recorded as positive or negative or

more commonly rated on a scale, such as 1-4. This is beneficial due to the possibility of non-

specific aggregation (NSA), or clumping of LAT particles without the presence of the target

antigen. NSA is a significant drawback to LAT methods, caused by the exposed hydrophobic

domain of latex particles (Yoon, Kim, Choi, Kim, & Kim, 2001). As such, colloidal stability is

18

the most important parameter affecting the performance of the LAT (Ortega-Vinuesa & Bastos-

González, 2001).

Due to the large variety of LAT methods created; there are significant variations among

LAT methods. In an early LAT test for E. coli, Hechemy et al. (1974) hand tilts the LAT for 2

minutes on a glass slide. Other methods describe a similar process, tilting slides for 2 to 5

minutes before reading the results. In each case, agglutination is recorded as 4+, 3+, 2+, or 1+,

in descending agglutination quality (Hechemy 1974). Severin (1974) shakes the agglutination

test for three minutes as opposed to plate tilting, and agglutination read immediately afterward.

Improvements to slide test LAT include Roche’s OnTrak device, which removes most

manipulation of the slide by the operator. Dried reagents are also produced, where the Ab coated

MPs are supplied dried on the LAT card, and are rehydrated with the sample (Bangs, 1996).

According to Price and Newman (1991) reading of LAT results by turbidimetric or

nephelometric procedures as opposed to visually can increase the sensitivity of the assays by two

to three orders of magnitude (as cited in Ortega-Vinuesa & Bastos-González, 2001). Particle

counters can improve the sensitivity 10 - 15 times better than turbidimetric or nephelometric

procedures by counting MPs before and after agglutination (Bangs, 1996).

2.5.1 Microparticle Preparation

There exists a wide variety of microparticles (MPs) available commercially from various

sources. MPs are available in diameters as small as 0.01 µm. An abundance of bead

compositions are also available, including polystyrene (PS), PS with the crosslinker

divinylbenzene (DVB), Silica, COOH- modified polymers, polymethacrylate (PMMA), as well

19

as fluorescent and magnetic MPs (TechNote 100, 2010). The MP material used depends on the

Ab used, the method of Ab immobilization, and the method of LAT evaluation. Different beads

are well suited for various applications, with PS MPs diameter 0.4 – 1 µm the most widely used

for LAT with visual inspection. Other MP polymers have been used to attempt to mitigate NSA,

including poly(hydroxyethyl methacrylate) and poly(glycidyl methacrylate) (Kawagachi, 2000).

Use of polymers other than PS often requires more complex methods of Ab immobilization.

As a first step in producing microparticles, it is generally recommended to clean any

microparticles to be used to remove any surfactants before attempting to bind proteins.

Suggested cleaning techniques include: washing, dialysis, cross flow filtration, mixed ion

exchange resin, and column methods. These techniques vary in complexity and time, with the

simplest technique and most common being washing. Washing is performed by suspending MPs

in a wash buffer and agitating for a period of time, then centrifuging and decanting the

supernatant. The process is then repeated usually 3 times and finally the MPs are suspended in a

buffer selected for protein adsorption (TechNote 201, 2008). Most studies clean beads by

washing multiple times before attempting to attach antibodies.

2.5.2 Antibody Immobilization

Most LAT procedures are developed using polyclonal immuno-γ globulin G (IgG) Ab

from various mammalian species. Polyclonal Ab differ from monoclonal Ab in their specificity.

Monoclonal Ab have advantages in LAT in that they limit NSA, but they can fail to aggregate

when the antigen is present if there is only reactive epitope on the antigen surface (Ortega-

Vinuesa & Bastos-González, 2001). IgG is popular because of its ease of production and

20

purification. IgG Ab are composed proteins arranged into three fragments, two identical Fab

(fragment antibody) and one Fc (fragment crystallizable), arranged in a Y-shape. At the ends of

the Fab are variable regions where amino acids are arranged as binding sites for the antigen. At

the end of the Fc portion are carboxyl amino acids which permit attachment to solids structures

(such as MPs) (Collins & Caruso, 1997).

Generally, Ab are prepared using live animal injections, with rabbits often being used,

followed by purification. One early method involved immunizing rabbit and extracting antisera,

then precipitating the antisera through a column of ammonium sulfate and diethylaminoethyl-

cellulose. These were then assayed for purity and bound to latex particles (Hechemy, Stevens, &

Gaafar, 1974). In a LAT method developed by Huang, cell surface antigens, extracted with urea,

were injected into New Zealand White rabbits, a common method of production (2001). The

rabbits were injected 4 times in 4 week intervals. Ten days after the final injection, ear arterial

blood was drawn and the antisera were purified using diethylaminoethyl ion-exchange

chromatography. The Ab were then bound to latex particles through passive adsorption with

mixing and room temperature incubation (Huang, Chang, & Chang 2001). Other studies have

utilized commercially available antiserum for specific applications (Baldrich & Munoz, 2008;

Pyle, Broadaway, & McFeters, 1999). Most commercially available antiserum is monoclonal

strain specific, often for E. coli O157, although there is a wide variety of antiserum available.

Pyle et al. obtained O157 specific antiserum from Difco (1999). Baldrich used a polyclonal

biotinylated antiserum produced from rabbits available from AbCam (Cambridge, UK) described

as being reactive to “most” O and K serotypes (AbCam website; Baldrich, personal

communication 2010).

21

The three primary methods of Ab-MP immobilization are affinity binding, covalent

ligand attachment, and passive protein adsorption. Affinity binding is a method to aid in binding

protein to MPs utilizing the high affinity of streptavidin for Biotin (Vitamin B7). In this

procedure, the antibodies to be bound are biotinylated, meaning a biotin molecule has been

attached to the antibody. The biotinylated antibodies are attached to MPs coated with

streptavidin. The streptavidin-biotin complex is known as one of the strongest non-covalent

bonds in nature; the dissociation constant Kd for the biotin-streptavidin relationship is

approximately 10-14

mol/L (Green, 1975). Standard antibodies can be biotinylated by incubating

antiserum with a biotin reagent for several hours in darkness (Yu & Bruno, 1996). Additionally,

biotinylated antibodies are available commercially. Covalent ligand attachment is a method of

protein that is often employed “for the immobilization of biomolecules when a very active and

stable microsphere reagent is required” (TechNote 205, 2008). Covalent ligand attachment is

considerably more complex than either passive protein adsorption or affinity binding and used

only for specific applications.

Passive protein adsorption involves incubating Ab in solution with latex MPs and

allowing hydrophobic Van Der Waals forces between antibodies in suspension and the MP

surface to facilitate attachment. Early methods of latex agglutination testing relied on methods

similar to passive protein adsorption for immobilization of antibodies to MPs (Fessel, 1959).

Passive protein adsorption is known as the simplest technique, but highly dependent on a number

of factors, most notably pH and ionic strength of the immobilization buffer (Serra et al., 1992).

Improper attachment can cause non-functioning microspheres, where antibodies are immobilized

in a non-functioning position, or where little or no antibodies are bound (TechNote 204, 2008).

MPs produced with passive protein adsorption are also at high risk of NSA. After Ab

22

immobilization, the MPs are washed to remove excess Ab. After washing, the MPs are

suspended in the reaction buffer. To avoid NSA, the pH and ionic strength of the reaction buffer

is critical, and highly dependent on the immobilization conditions (Serra et al., 1992).

2.6 E. Coli growth

Various E. coli detection techniques, including LAT test methods, only function properly

when E. coli titer is sufficiently high, at least higher than the test detection limit that varies based

on the test. For most cases, this does not create an issue, because of the high titers which will be

present in the case of food contamination, or in clinical samples. The situation for detecting E.

coli is much different in environmental samples, however, considering titers in environmental

samples are usually very low (often < 10 cfu/mL), and the test ideally would be able to detect the

presence of individual organisms. Thus, E. coli titer must be enhanced to detectable levels

before a LAT can be performed. Two primary methods of titer enhancement are concentration

and growth. The former refers usually to membrane or other filtration, a process where a large

volume of water is filtered in order to concentration E. coli on a membrane filter. Additionally,

small quantities of E. coli can be grown to detectable concentrations in an enrichment culture. In

laboratory investigations of LAT protocols, E. coli is typically is grown to sufficient

concentrations using overnight incubation with growth media (Bettleheim, 2001; March, 1989;

Huang, 2001). In the interest of creating a rapid test for E. coli in water, methods of rapid E. coli

growth are investigated to create titers sufficient for LAT testing.

23

E. coli reproduce by binary fission, a process by which individual cells divide repeatedly

during active growth. A measure of this growth is doubling time (DT), or the amount of time it

takes the population to double (Equation 2.1).

Equation 2.1

The microbial growth life cycle of a population is divided into four different phases: the lag

phase, the log phase, the stationary phase, and the death phase. The lag phase is characterized by

slow microbial growth as cells adapt to their environments. The log phase is the most active

growth period, as cells double most rapidly. As resources available to the organism become

limited, the bacteria reach the stationary phase, where growth equals death and the population

remains constant. During the death phase, the death rate outpaces the growth rate as resources

are exhausted, until the population dies out entirely (Csuros & Csuros, 1999). E. coli follow the

same growth pattern, and when used in culture methods the log phase is most beneficial to titer

enhancement.

Bacteria are capable or sustained growth in a wide range of pH (1 – 11) due to the large

range of naturally occurring environments found on earth. E. coli is a neutrophilic bacterium,

meaning growth occurs fastest between pH 6 to 8, with generally falling growth rates outside that

range. Air pressure also has an effect on bacterial growth rate. E. coli, while fairly pressure

tolerant and capable of growing at pressures of 56 MPa, grows most rapidly at or around 1 atm

(Ingraham & Marr, 1996). The specific growth rate, k, is a measure of the rate of growth of

bacteria. It is a function of temperature, and over a certain range for all bacteria (the normal

24

range), log k has a linear relationship with 1/temperature. Outside of the normal range, k

decreases with increasing or decreasing temperature. E. coli can grow between about 7.5 and 49

degrees C, and its normal range is between 21 to 37 degrees C. E. coli grow most rapidly

between about 37 to 42 degrees C, at the top of the normal range (Ingraham & Marr, 1996). For

this reason, incubation requirements for culture method often specify 35 – 37 degrees C.

Considering the wide range of temperature E. coli can grow, other incubation temperatures are

possible, and even ambient temperature incubation has been demonstrated to be effective,

particularly in tropical climates (Brown, et al. 2011). Optimal E. coli growth conditions lead to a

maximum k value of around 2 hr-1

(Herendeen,VanBogelen, & Neidhardt, 1979), and a

subsequent minimum doubling time of around 20 minutes during the log phase, with a lag phase

dependent on specific conditions.

2.7 Summary

Sophisticated lab based E. coli and TC detection techniques perform satisfactorily for a

wide variety of applications. A significant gap in available methods, however, are those which

are well suited for use in field conditions with limited training, yet are rapid and affordable.

Conditions in rural areas, developing countries, and disaster areas are often very lacking of the

resources required for the more complex methods such as PCR. MF is a method best suited for

use in a lab, although the Delagua kit creates a field deployable system. The H2S method

provides a valid option for P/A detection that is cheap and suited for field use, but while it is a

suitable measure of microbial water quality, it cannot test for E. coli or TC, and still requires a

25

lengthy incubation period. Table 2.1 summarizes the options and estimated costs for water

quality test methods.

Table 2.1

Comparison of E. coli and TC Detection Methods

Technique

Incubation

Time

Requireda

Simple and

Field

Deployable Quantitative

Estimated

Unit Costb Target

MF 18 - 24 h No Yes $0.62 TC and E. coli

Delagua MF 18 - 24 h Yes Yes $0.85 TC and E. coli

MPN: IDEXX 18 – 24 h No Yes $6.18 TC

H2S: Hach 24 – 48h Yes No $1.00 H2S producing

bacteria

Realtime PCR None No Yes $20.00 TC and E. coli

Latex Agglutination 6-18 h Yes No $0.81c E. coli

Notes: a incubation time required to detect bacterial concentration of 1 cfu/100mL at 35° C;

b

based on average cost of one disposable test; c See appendix A.1 for cost calculation.

The Latex Agglutination Test presents a very favorable test method in its rapidity and

minimum logistical requirements. However, the lower detection limit of LAT tests is far too

high to be used reliably to detect E. coli in water samples. This research is concerned with

creating a proof of concept of a detection method coupling a LAT with the MF technique and a

short incubation period for the detection of E. coli. The test will consist of coating MPs with

polyclonal Ab reactive with E. coli, and using these to validate the presence of E. coli in samples

that have been filtered using the MF technique and enriched by a short incubation step. This

method would be advantageous in it rapidity, which would be 6 - 12 hours as opposed to 18 – 24.

The test would improve on the logistical requirements of current rapid methods; it could be

performed with limited training at per unit costs similar to the MF technique. Additionally, the

adaptability of the LAT test would enable this method to be modified for other current indicators

such as enterococci, for emerging indicators, or for the direct detection of pathogens.

26

Improvements on this proof of concept could lead to variations, including dipstick

immunoassays and immunochromatography.

27

3. METHODOLOGY

Generally, laboratory procedures were divided into two sections, which are summarized

as follows:

1. E. coli Famp growth and recovery. Recovery and growth methods were analyzed in

conjunction with the membrane filtration technique to determine the appropriate method for

enhancing bacterial titers, with doubling time of bacteria being the primary outcome of interest.

2. Microparticle production. Methods of bead production were investigated with the goal of

obtaining agglutination with E. coli Famp or E. coli O1:K1:H7 in low titers, with the detection

limit being the primary outcome of interest.

3.1 E. coli Famp Growth and Recovery

It was determined early in the project that concentrations of E. coli found in

environmental samples would not be sufficient to achieve latex agglutination. Therefore,

experiments were performed to determine the most effective ways of increasing bacteria titers to

detectable levels. All tests were performed in conjunction with the membrane filtration

technique described in EPA Method 1604 (2002) as a standard method for E. coli recovery and

concentration from environmental samples.

28

3.1.1 Bacteria Stock Preparation

Non-pathogenic strains of E. coli Famp (a laboratory strain resistant to the antibiotics

streptomycin and ampicillin) were used in all growth and recovery tests due to its safety, ease of

growth, ease of isolating and removing interfering bacteria with antibiotics, and interaction with

antibodies used in bead production procedures. Using an sterilized inoculating loop, small

amounts of culture from the slants were streaked onto 100 x 15 mm sterile plastic petri dish

containing RAPID’E.coli 2 agar (Bio-Rad). These plates were incubated overnight at 37 °C and

bacterial growth verified for violet color indicating presence of E. coli. Streptomycin ampicillin

solution was prepared according to EPA Method 1601 7.2.2. 0.15 g of ampicillin sodium salt

and 0.15 g streptomycin sulfate were dissolved into 100 mL of deionized (DI) water. This

solution was then filtered through a 0.22 µm pore size membrane filter assembly (Pall Corp.) to

sterilize. The solution was stored in 10 mL vials by freezing at -20 °C until ready for use.

Dehydrated tryptic soy broth (TSB) growth medium (Becton, Dickenson, and Company)

consisted of 17 g tryptone, 3 g peptic digest of soybean meal, 2.5 g glucose, 5.0 g sodium

chloride, and 2.5 g dipotassium hydrogen phosphate. It was prepared by dissolving 15g of

dehydrated TSB in 500 mL DI water. The solution was boiled, autoclaved at 121° C for 30

minutes, cooled to RT, and stored at 4° C until ready for use. Overnight culture (OC) bacterial

stocks were prepared by transferring a small amount of bacterial culture from select violet

colonies on the streak plate to a 125 mL sterile culture flask containing 30 mL TSB medium and

1 mL of stock streptomycin ampicillin solution. This was incubated for 20-24 hours at 35 ° C

and refrigerated up to 7 days or until ready for use.

29

3.1.2 Spot Plate Procedure

The spot plate method was the primary method of bacterial enumeration, although the

membrane filtration technique was used in situations where the estimate of the titer of the fluid

was below +/- 50 cfu/mL. To perform the spot plate method, three replicate 10 µL spots of the

fluid (serial 1:10 dilutions, vortexed) were placed on a spot plate (65 mm x 15 mm petri dish)

containing E. coli selective media (Rapid’E. coli 2 Agar, Bio-Rad) using a pipettor (VWR

Signature, VWR) equipped with a sterile 200 µL tip (VWR). Each plate was replicated along

with a negative control, for a total assay volume of 60 µL per sample. Plates were air dried for

20 minutes then incubated inverted at 37 °C for 18-24 hours. After incubation, violet colonies

(indicating E. coli by cleavage of a flourogenic β-glucuronide substrate) were counted; each

violet colony indicating one colony forming unit (cfu).

3.1.3 Membrane Filtration Method

The membrane filtration method was performed as described in EPA method 1604 (2002)

and was used as both an assay method and the initial concentration step of the novel method. A

47-mm diameter, 0.45 µm pore size cellulose ester filter (Pall Corp.) was placed in a sterile

magnetic vacuum filter funnel apparatus (Pall Corp.) using sterile tweezers. 1 mL, 10 mL, or

100 mL of the fluid was transferred to the filter funnel using a pipettor equipped with a sterile 1

mL pipette tip. Vacuum was applied until all the fluid had been filtered, then the filter funnel

was washed three times with sterile DI water. For E. coli assays, the filter was then removed

using sterile tweezers and placed face up on a 60 x 15 mm sterile petri dish containing

30

RAPID’E.coli 2 agar (Bio-Rad), an alternative to MI agar. Plates were incubated inverted at 37

°C for 18-24 hours, and each violet colony indicated one cfu. When used in the novel method,

after sample filtration, the membrane filter was placed in a tube containing growth eluent

solution (GES) with sterile tweezers.

3.1.4 Spike Sample Preparation

Samples of water containing E. coli, or spiked samples, were needed to evaluate the

various components of the detection method. The preparation of spiked samples began by

washing bacteria culture; a 5 mL aliquot of E. coli OC was transferred to a sterile centrifuge tube

and centrifuged (VWR Clinical 50, VWR) at 2000 g for 20 minutes. The supernatant was

carefully pipetted off with care not to disturb the pellet. 5 mL of sterile DI water was placed

back into the tube, and agitated using a vortex mixer (VWR) for 10 seconds. This wash

procedure was repeated twice more, for a total of three washes. After the final wash, the bacteria

were suspended in 5 mL of sterile DI water and the titer enumerated using the spot plate

procedure (section 3.1.2). The washed bacteria suspension was used within four days of

preparation. The spiked samples were prepared by transferring an amount of washed bacteria

stock to a flask containing 250-400 mL of sterile deionized water. The amount of stock used was

based on the desired spike titer (5 - 200 cfu/mL), and the titer of the washed bacterial culture,

estimated by prior enumerations(s). The final titer of the spiked water samples was determined

using the membrane filtration method (see section 3.1.3).

31

3.1.5 Recovery and Growth Testing

Spiked water samples were used to place E. coli cells on membrane filter papers to

simulate membrane filters prepared by filtering contaminated environmental sample water.

This was performed following the filtration technique described in EPA Method 1604 (2002) and

section 3.1.3. 1, 10, or 100 mL volumes of spiked sample water were filtered through 47-mm

diameter, 0.45 um pore size cellulose ester filters in sterile, magnetic vacuum filter funnel

apparatuses. The filters with bacterial populations of approximately 10 – 5000 cfu were then

placed in a 15 or 50 mL dilution tube containing and 10 – 40 mL GES using sterile tweezers.

The tube was capped, and agitated (by either hand-shaking or vortexing) to stimulate bacterial

recovery from the membrane filter. To estimate bacterial recovery, the tube was sampled for

bacterial enumeration using the spot plate procedure (section 3.1.2). If the growth rate of the

tube was to be determined, the tube was incubated after assaying at 35° C. Growth rate of E. coli

was determined by sampling the tubes for bacterial enumeration by spot plating at 1.5 or 3 hour

intervals.

3.1.6 Growth and Recovery Calculations

The concentration of bacteria in a liquid was determined by dividing the number bacteria

(colony forming units, or cfu) counted in either the spot plate method (section 3.1.2) or the

membrane filtration technique (section 3.1.3) by the volume of the sample. The concentration

was calculated using Equation 3.1.

Equation 3.1

32

Recovery of bacteria in liquid eluent was evaluated by determining the approximate total number

of bacteria originally on the membrane filter and the total number of bacteria eluted. The total

number of bacteria on the membrane filter placed in the tube was known as filter count (FC), and

the total number of bacteria in the GES solution was known as tube solution count (TSC). These

values were determined using the volumes and calculated titers of both the spiked sample and the

GES as in Equations 3.2 and 3.3.

Equation 3.2

Equation 3.3

The recovery was calculated by comparing the total number of colonies in solution in the tube to

the total number of colonies on the membrane filter, as in Equation 3.4, and expressed as a

percent.

Equation 3.4

Growth was analyzed using two parameters: doubling time (DT) and original count replication

(OCR). Doubling time was calculated using Equation 3.5. Because of the nature of the analysis,

when calculating DT over the entire growth period, two different values could be used for the

initial count: the FC or the TSC. The results of this research use both, and denote them as DT0

when using the FC, and DT1 when using the TSC.

Equation 3.5

33

The ratio OCR refers to the number of times the bacterial population has replicated, and

represents the bacterial population would be if a single E. coli cell had been introduced to the

sample. It was calculated by comparing the TSC of the GES at a given time to the original FC as

shown in Equation 3.6. Immediately after recovery, OCR will be equivalent to recovery

expressed as a ratio.

Equation 3.6

3.2 Microparticle Analysis

Microparticles (MPs) used in this research were prepared and analyzed with the objective

of reliable function and minimum detection limit. MPs were produced with variation in particle

type; antibody type; incubation time, temperature, and agitation; immobilization buffer; and

reaction buffer. MPs were then tested for function using E. coli Famp and O1:K1:H7 (NCTC

9001) [American Type Culture Collection (ATCC) no. 11775]. E. coli O1:K1:H7 was used to do

its serological properties, which matched several antibodies used in this project indicating likely

antibody-antigen interactions.

34

3.2.1 Microparticle Production

Six different particle types were evaluated for use in the LAT. Particles used varied in

coating, size, and magnetism, but all fell within the general size guidelines for LAT of 0.4 to 1.0

µm. The MPs tested can be found in Table 3.1.

Table 3.1

Summary of Microparticles Tested

Name Type Size Coating Supplier

C1 MyOne Magnetic 1.0 µm Streptavidin Invitrogen Life Sciences

PMS1N ProMag 1 Series Magnetic 1.0 µm Streptavidin Bangs Laboratories

CM01N ProActive Magnetic 0.83 µm Streptavidin Bangs Laboratories

CP01F ProActive PS 0.97 µm Streptavidin Bangs Laboratories

PS03N PS 0.95 µm None Bangs Laboratories

PS02N PS 0.40 µm None Bangs Laboratories

Antibodies were tested for reactivity with the three E. coli strains used in the project.

Antibodies selected for use in the project fit the description of having high sensitivity and

specificity toward all strains of E. coli, being easy to bind to polystyrene MPs or streptavidin

coated MPs, and being polyclonal, as polyclonal antibodies are generally superior for LAT.

Antibodies of different levels of purification were also used to try to find the optimal purity for

LAT testing.

Antibodies evaluated for use in the test can be found in Table 3.2 with properties as

described by the supplier. The antibody ab20640 was biotin conjugated, meaning a biotin

molecule had been attached to the protein. It was used exclusively with streptavidin coated MPs,

attempting to employ the high affinity between biotin and streptavidin in MP production.

Antibodies ab354 and ab986 were used in an attempt to find a reaction between E. coli and the

two reactive compounds (Alkaline Phosphatase and β-galactosidase) present in the cells.

35

Table 3.2

Summary of Antibodies Tested

Antibody Supplier Reactivity Conjugation Purity Speciesa

ab986 Millipore β-galactosidase None Whole antiserum Rabbit

ab13627 abcam E. coli (most O + K serotypes) None IgG fraction Goat

ab31499 abcam E. coli (all O + K serotypes) None Protein A Rabbit

ab354 abcam Alkaline Phosphatase None Whole antiserum Rabbit

ab78993 abcam E. coli H-7 antigen None Whole antiserum Rabbit

ab20640 abcam E. coli (many O + K serotypes) Biotin Protein A Rabbit

Note.a Species refers to the species of animal antibody was raised in.

In conjunction to E. coli Famp strain used in growth and recovery testing (see section 3.1),

two additional E. coli strains were used to confirm positive cell-antibody reactions. E. coli B

was provided by Dr. Joe Brown, and E. coli O1:K1:H7 was obtained from ATCC (Manassas,

VA). E. coli O1:K1:H was selected due its serotype matching the descriptions of the reactivity

of four of the selected antibodies(ab13627, ab31499, ab78993, and ab20640), its biosafety level

1, and its use as a control culture in numerous publications, including the quality control

procedure for IDEXX Colilert-18 (IDEXX, 2010).

3.2.1.1 Buffer Preparation

Immobilization buffers were varied to find the ideal buffer to facilitate MP coating and

function. Buffers evaluated included deionized water (DI), phosphate buffered saline (PBS)

(Okubo et al., 1987), borate buffered saline (BBS), tris buffered saline (TBS), acetate buffer

(AceB) (Yoon et al., 2003; TechNote 204, 2008), glycine buffered saline (GBS) (Shekarchia,

Fuccillo, Sever, & Madden, 1988; Okubo et al., 1987), and citrate-phosphate buffer (CPB)

(TechNote 204, 2008) of varying pHs. Buffers were tested with and without the addition of

Tween20 (polysorbate 20) non-ionic surfactant. PBS (pH 7.2) was prepared by combining 100

36

mL 10x OmniPur sterile PBS concentrate (EMD Millipore) and 900 mL DI water in a flask.

BBS (pH 8.0) was prepared by combining equal parts BBS 2x solution (G-Biosciences) and DI

water in a flask. TBS (pH 7.6) was prepared by dissolving 1 TBS concentrate tablet (Amresco)

in 100 mL DI water. AceB (Ricca Chemical)(pH 4.6) was supplied already prepared. GBS (pH

6.9) was prepared by dissolving 3.75 g glycine and 8.78 g sodium chloride in 1 L DI water as

described in Okubo et al. (1987). CPB was prepared in 4 different concentrations for four target

pHs, as described in Bangs Laboratories TechNote 204 (2008). 0.1 M citric acid solution was

first prepared by dissolving 3.84 g of anhydrous citric acid (MW: 192.13) in 200 mL DI water.

0.2 M sodium phosphate solutions was prepared by dissolving 10.72 g sodium phosphate dibasic

heptahydrate (MW: 268.07 g/mol) in 200 mL DI water. The solutions were mixed in different

volumes dependent on desired buffer pH (see Table 3.3), and the volume adjusted to 100 mL by

adding DI water. The pH of each buffer was measured after preparation (Accumet Basic, Fisher

Scientific). For buffers to be supplemented with surfactants, 100 mL of the buffer was

transferred to a separate flask. Using a pipettor, 10 or 50 uL of Tween20 (Amresco) was added,

creating concentrations of 0.01% and 0.05%, respectively. The solutions were stored until ready

for use, up to a month.

Table 3.3

Citrate-Phosphate Buffer Constituents

mL for pH =

Solution 6.6 5.8 5.0 4.2

0.1 M Citric Acid 13.6 19.7 24.3 29.4

0.2 M Na Phosphate 36.4 30.3 25.7 20.6

DI water 100.0 100.0 100.0 100.0

Measured pH 6.8 6.2 5.3 4.6

Note. Adapted from Bangs Laboratories TechNote 204.

37

3.2.1.2 Antibody preparation

Antibodies were prepared by first removing an aliquot of antibody, and transferring the

remaining volume to a sterile 1.5 mL screw cap dilution tube (VWR, Inc.), and placing it in

storage at -20° C for long term usage. Depending on the desired concentration of antibody in

solution during immobilization, antibodies were diluted before use. Antibody concentration was

based on the saturation surface value of IgG, which is approximately 2.5 mg/m2

(TechNote 204,

2008). Antibodies concentrations used were calculated based on the surface area of the particles,

and usually above and below the saturation surface value.

3.2.1.3 Bacterial culture preparation

E. coli Famp, E. coli B, and ATCC 11775 cultures were prepared by following the

procedure for E. coli Famp described in section 3.1.2, excepting that streptomycin-ampicillin (SA)

solution was not added to overnight cultures of E. coli B and ATCC 11775. The bacteria were

subsequently washed as described in section 3.1.4 prior to use.

3.2.1.4 Particle washing

For the three magnetic particle types (C1, PMS1N, CM01N), a method of particle

preparation adapted from Bangs Laboratories Product Data Sheet 721 (2010) was used. This

preparation method was used for the three magnetic bead types. The 1% solids particle

suspension supplied was removed from refrigeration and vortexed for 30 seconds to suspend the

MPs. A 500 μL aliquot of particle suspension was transferred into a sterile 1.5 mL screw-cap

38

centrifuge tube (VWR). The MPs were washed by adding 1 mL of sterile DI water to each tube,

and vortexing for 30 seconds. The tube was placed on a rack with a magnet attached for 10

minutes until all MPs had gathered in a tight cluster on the side. The supernatant fluid was then

removed while keeping the magnet in place to prevent bead loss. The MPs were washed two

more times using the same process. After the final wash, the beads were suspended in 1 mL of

the desired immobilization buffer to bring the final particle concentration to 5 mg/mL (0.5%

solids).

The preparation method of non-magnetic streptavidin coated latex particles (CP01F) and

latex particles (PS02N, PS03N) differed from preparation of magnetic particles in that a

centrifuge was used to separate the particles and remove supernatant when washed and protein

coated. The tube containing the washed particles was placed in a centrifuge for 15 minutes at

2000g (PS02N) or 1000g (PSO3N, CP01F). Afterward, the supernatant was carefully removed

using a pipettor (VWR) as to not disturb the MPs. Since PS02N and PS03N particles were

supplied at a higher particle concentration, the procedure was adjusted accordingly. The 500 μL

aliquot of particle suspension was transferred to a 15 mL screw-cap centrifuge tube (VWR), and

10 mL of DI water was added for washing. After the final wash, PS02N or PS03N particles were

suspended with 10 mL of the desired immobilization buffer to bring the final particle

concentration to 5 mg/mL (0.5% solids).

3.2.1.5 Antibody Immobilization

Particle coating began by first centrifuging washed particles for 15 minutes at 1000 g for

non-magnetic particle types, or magnetic separation for magnetic particle types. After

39

separation, the supernatant wash buffer was removed carefully via pipette. The desired

immobilization buffer was added at a volume to achieve a particle concentration of 5 mg/mL

(0.5% solids), and vortexed for 10 seconds. 1.5 mL screw cap centrifuge tubes (VWR) were

prepared by adding 100 μL of the desired antibody dilution, 800 μL of the immobilization buffer,

and 100 μL of the particle suspension, creating a final particle concentration of 500 μg/mL

(0.05% solids). Antibody concentration was based on surface area of the particles. The highest

antibody concentration tested was 3.5 mg/m2, just above the saturation concentration of IgG.

The tubes were then placed in an orbital mixer (HulaMixer, Invitrogen Life Sciences,

Carlsbad, CA) set to either 10 or 30 RPM, and moved to a refrigerator for 4° C incubations, an

incubator for 37° C incubations, or left on the bench top RT incubation. The mixer was allowed

to run for the required time duration. After incubation, the particles were separated from the

immobilization buffer and antibodies. Magnetic particle preparations were placed on a rack with

a magnet attached for 10 minutes at which point all MPs gathered into a small cluster in the tube.

Non-magnetic particles were separated by centrifugation at 1000g for 15 minutes. Afterward, the

supernatant fluid was carefully removed mitigating MP loss. Coated MPs were washed 2 more

times by adding 1 mL of sterile DI water to each tube and agitation gently by hand. After the

final wash, the particles were suspended in 1 mL of reaction buffer for evaluation

3.2.2 Antibody and Strain Reactivity Testing

Antibodies were tested for reactions with E. coli strains used in the project. On a glass

microscope slide, 75 x 25 mm (VWR), four circles of diameter 1 cm were drawn with a wax

pencil. Equal 5 uL volumes of washed bacteria culture and antibody dilution (1:10, vortexed)

40

were transferred to each circle with a pipettor (VWR) equipped with a sterile 100 uL pipette tip

(VWR). Negative controls were also prepared, for each antibody/bacteria combination. The

slide was tilted for 60 seconds to facilitate bacterial agglutination. The slide was then inspected

at 40x magnification. Positive (+) reactions were those where the majority of bacterial cells

appeared to clump to one another in the presence of antibodies, with no clumping present in the

negative control. Negative (-) reactions were those where the antibody appeared to have to

agglutinative effect on the bacteria. Reactions where limited clumping occurred to a minority of

cells were rated as slight (s).

3.2.3 Function and Detection Limit of Coated MPs

After MPs were coated, washed, and suspended, they were tested for function by

observing interaction and assess agglutination with particles and E. coli cells. E. coli OC was

washed as described in section 3.1.4. Four to eight circles of diameter 10 mm were drawn on

glass microscope slides, 75 x 25 mm (VWR), with a wax pencil. Equal 5 uL volumes of washed

overnight bacteria culture and coated MPs were transferred to each circle with a pipettor (VWR)

equipped with a sterile 100 uL pipette tip (VWR). A negative control of DI also prepared, for

each antibody/MP combination. The slide was placed on a nutating mixer (Clay Adams) for 120

seconds to facilitate agglutination, and the slide was then inspected. The agglutination observed

both visually and microscopically and recorded as 0 - 4. The approximate criteria applied to rate

the level agglutination present on the microscope slide can be found in Table 3.4. The

agglutination delta (AD) served as a measure of proper particle function, and was determined by

41

subtracting the grade of NSA in the negative sample from the grade of positive agglutination

with E. coli present (Equation 3.7)

Equation 3.7

In certain particle preparations that performed well, the detection limit was determined.

A dilution series of E. coli O1:K1:H7 (1:4, vortexed) was prepared. The particle suspension was

interacted with each dilution in the method described above, and the agglutination rated. The

detection limit was determined by observing the interacting MPs, and noting the bacterial

suspension where agglutination becomes visibly indistinguishable from the negative control.

This usually corresponded to an agglutination delta of 1. To the naked eye, particles with an AD

greater than 1 appeared to agglutinate; particles with an AD less than 1 appeared to not be

agglutinating. The titers of the bacterial samples were determined using the spot plate procedure,

described in section 3.1.2.

Table 3.4

MP Agglutination Rating Criteria

Size of majority of

largest clumps

Rating

Recorded

Agglutination

Type

No clumps present 0 None

< 0.05 mm 1 Slight

0.05 – 0.5 mm 2 Partial

0.5 mm – 1 mm 3 Good

> 1 mm 4 Total

42

4. RESULTS

4.1 E. coli Recovery

Initial tests were performed under the assumption high levels of bacterial recovery would

be necessary for the novel test method. Therefore, preliminary bacterial testing evaluated

methods of bacterial recovery for the optimal mechanical action and eluent. Initially, recovery

rates were measured using 40 mL DI in 50 mL tubes as a simple eluent to recover from MF

papers treated with DI water spiked with E. coli Famp. Averages of tested mechanical methods

are shown in Table 4.1. Increased energy and time associated with recovery yielded higher

bacterial concentrations in solution.

Table 4.1

Analysis of Mechanical Methods of Bacterial Recovery

Action Time (s) Recovery (%)

None 0 3.66

Shake 20 25.34

Shake 60 30.93

Vortex 20 31.68

Vortex 60 34.66

A 20 second hand shake was selected as the baseline mechanical action. The effect of DI, PBS,

and Tween20 on recovery was tested using 40 mL GES in a 50 mL tube. The average recovery

rates for these configurations are shown in Table 4.2. GES containing PBS with the addition of

Tween20 improved recovery greatly, to 65.4% with 0.5% Tween20.

43

Table 4.2

Analysis of PBS and Tween20 on Bacterial Recovery

Tween20

(%)

Recovery (%) with Buffer

DI PBS

1.0 34.46 61.57

0.5 32.26 65.37

None 17.63 17.61

PBS with 0.5% and 1.0% Tween20 were chosen as the ideal GES in testing of the effect

on tube size on recovery. 15 mL tubes with 10 mL of GES, along with 50 mL tubes with 10 mL

and 40 mL of GES were evaluated using a 20 second hand shake as the baseline mechanical

action. The results of this analysis can be found in Table 4.3. The concentration of Tween20

and fill of the 50 mL tubes had limited effect on recovery. The 15 mL tube size limited recovery

(46.72% compared to 60.55% for tubes containing 0.5% Tween20).

Table 4.3

Analysis of Tube Size and Fill on Bacterial Recovery

Tween20 (%) Tube Size (mL) GES Volume (mL) Recovery (%)

1.0 50 40 61.57

1.0 50 10 61.51

1.0 15 10 47.81

0.5 50 40 65.37

0.5 50 10 60.55

0.5 15 10 46.72

Because growth of recovered bacteria was required, Tryptic Soy Broth (TSB) was added

to the GES and recovery was analyzed. Tubes of 50 mL were filled only to 10 mL to limit the

volume of GES required, and the use of 15 mL tubes filled with 10 mL was continued since

because of their potential to increase growth rate. Average rates of recovery with TSB can be

found in Table 4.4 and Figure 4.1. Increasing TSB concentration in the eluent had a positive

effect on recovery, regardless of tube size or Tween20 concentration. The combination of 30 g/L

44

TSB, 0.5 % Tween20 in a 50 mL tube exhibited the highest average recovery (83.35%) of all

configurations thus far. This was positive, considering growth media would be required in the

tubes in order to facilitate growth. The 15 mL tubes continued to limit recovery. The

concentration of Tween20 showed no consistent effect when using the larger 50 ml tubes, but

higher Tween20 concentration benefitted recovery in the 15 mL tubes.

Table 4.4

Analysis of Media Concentration on Bacterial Recovery

TSB (g/L) Tween20 (%)

Recovery (%) with GES

Volume (mL)=

15 50

30 1.0 70.04 61.58

30 0.5 51.61 83.35

15 1.0 68.35 67.67

15 0.5 51.49 66.52

0 1.0 47.81 61.51

0 0.5 46.72 60.55

Figure 4.1

Comparison of recovery rates using 20 second hand shake, 1.0% and 0.5% Tween20, 15 mL and

50 mL tubes, and 15 g/L and 30 g/L TSB

0

10

20

30

40

50

60

70

80

90

100

30 15 0

Ave

rage

Bac

teri

al R

eco

very

(%

)

TSB (g/L)

1.0% Tween 20, 50 mL Tube

0.5% Tween20, 50 mL Tube

1.0% Tween20, 15 mL Tube

0.5% Tween20, 15 mL Tube

45

4.2 E. coli Growth

Growth of E. coli Famp was measured after recovery from MF, with the goal of

determining the optimal conditions for rapid E. coli multiplication. Additionally, the effect of

recovery was analyzed to determine whether elution of E. coli from the MF paper was necessary

or beneficial to growth. First, the effects of Tween20, TSB concentration, and tube size on

growth rate were tested. In all configurations, a 20 second hand shake was used for mechanical

action to facilitate recovery, and 10 mL of GES with PBS was placed in the tube. The titer of the

GES was assayed immediately after shaking and at 90 minute intervals. The results of this

analysis can be found in Table 4.5. While the 50 mL tubes continued to benefit recovery, the

effect was negated by hour 6, when increased growth rate in the small tubes created higher titers.

The final titers were higher when using 15 mL tubes, regardless of recovery. This is evident in

the average DTs of the configurations, in Table 4.6.

Table 4.5

Analysis of Tween20, TSB, and Tube Size on E. coli Growth Rate

Tube Size

Tween20

(%)

TSB

(g/L)

Average OCR at time (h) =

0a 1.5 3 4.5 6

15 mL

1.0 30 0.76 0.91 6.60 66.7 1254

1.0 15 0.63 0.93 4.54 55.9 1691

0.5 30 0.60 0.87 5.50 53.7 1415

0.5 15 0.54 0.73 5.18 50.1 1337

50 mL

1.0 30 0.71 0.96 4.94 31.7 1005

1.0 15 0.93 0.96 5.42 55.5 1417

0.5 30 0.97 1.14 7.47 51.9 1206

0.5 15 0.88 1.28 6.06 56.0 1209

Note: a Values at time zero represent fraction recovered.

46

Table 4.6

Average DT0 and DT1 of 15 and 50 mL Growth Tubes with Tween20 and TSB

Tube

Size

Tween20

(%)

TSB

(g/L)

Average DT (min) between hours:

0 - 3 0 - 6 3 - 6

DT0 DT1 DT0 DT1 DT

15 mL

1.0 30 66.1 57.6 35.0 33.7 23.8

1.0 15 82.4 63.1 33.6 31.6 21.1

0.5 30 73.2 56.1 34.4 32.1 22.5

0.5 15 75.8 55.1 34.7 31.9 22.5

50 mL

1.0 30 78.1 64.4 36.1 34.4 23.5

1.0 15 73.8 70.7 34.4 34.0 22.4

0.5 30 62.1 61.0 35.2 35.0 24.5

0.5 15 69.3 64.8 35.2 34.5 23.6

The data in Tables 4.5 and 4.6 indicate the concentrations of Tween20 and TSB seem to

be independent of growth rate. It is likely, if growth was allowed to continue for a longer period

of time, that the reduction of TSB concentration would reduce the growth rate as bacterial

competition for resources become high. The analysis was repeated for longer time periods to

determine the titers that would be achieved in the desired 9-hour test time period. The analyses

were performed with 15 mL tubes filled with 10 mL of GES, consisting of 0.5% Tween20, 30

g/L TSB, and PBS. The effect of shaking on growth was evaluated once again. The results of

the experiment can be found in Table 4.7. The overall average DT0 with 95% CI of all 9- hour

growth tubes are shown in Table 4.8 and Figure 4.2.

Table 4.7

DT0 of 9-hour Growth with and without Shaking

Shaking

Average DT0(min) between hours:

0-3 0-6 0-9 3-9 6-9

20-sec 61.7 33.2 27.8 22.0 21.6

None 85.9 34.9 28.2 20.7 21.3

All 68.7 34.0 27.9 21.5 21.4

47

Figure 4.2

DT0 9-hour growth with and without shaking. Error bars represent 95% CI.

Table 4.8

DT0 of 9-hour Growth with 95% CI

Average DT0(min) between hours:

0-3 0-6 0-9 3-9 6-9

Upper 95% CI 62.9 33.1 27.6 21.0 20.9

Average 68.7 34.0 27.9 21.5 21.4

Lower 95% CI 78.0 35.3 28.4 22.0 22.3

4.3 Antibody and Strain Reactivity Testing

Antibodies were reacted with three strains of E. coli used in the project. Table 4.9

displays the results of the interactions. The broad reactivity E. coli O1:K1:H7 exhibited with the

various test antibodies was evident. Conversely, E. coli Famp and E. coli B exhibited limited

20

21

22

23

24

25

26

27

28

29

30

0 - 9 3 - 6 3 - 9 6 - 9

DT 0

(min

)

Time Intervals (hours)

All

20 s Shake

No Shake

48

reactivity, not as broad as O1:K1:H7. The strongest observed interactions occurred between E.

coli O1:K1:H7 and ab13627 and ab31499. Based on the results of this test, it was determined to

proceed with antibodies ab13627, ab31499, and ab354 primarily. E coli O1:K1:H7 would be

used as the primary test strain, although E. coli Famp would be used in parallel.

Table 4.9

E. coli Antibody Reactivity

Antibody E. coli Famp E. coli B

E. coli

O1:K1:H7

Negative

Control

ab986 s + + -

ab13627 + s ++ -

ab31499 + - ++ -

ab354 s s + -

ab78993 - - + -

ab20640 - - + -

4.4 Microparticle Function

Microparticle function was assessed by evaluating the prepared MPs for microscopic

agglutination in the presence of the aforementioned three lab E. coli strains. Protein assays were

attempted but found to be ineffective at measuring the binding efficiencies of protein due to the

protein concentrations being much lower than the detection limit for spectrophotometric protein

assays (1 μg/mL < 5 μg/mL), and the low amount of binding capacity of the particles. As such,

it was impossible to measure directly the amount of protein bound to the particles, however, due

to confirmation of the interaction between antibody and cell (see section 4.3), the coated MP’s

performance served as a suitable metric for the assessment of the binding process, while

simultaneously delivered direct results as they apply to the test method.

49

4.3.1 Polystyrene Particles

PS particles were the primarily particle type tested due to their widespread use in

immunoassays. Passive protein absorption was used to bind Ab to the particle surface, in

methods similar to those described by Molina-Bolivar & Galisteo-Gonzalez (2004), Yoon et al.

(2000), Okubo et al. (1987), and Bangs Laboratories TechNote 204 (2008). All methods were

first tested using 0.97 um PS particles (PS03N), orbital agitation at 10 RPM, with two incubation

conditions: 1 hour at RT, and 24 hours at 4° C. Three immobilization buffers, PBS, BBS, and

DI, all with and without the addition of 0.05% Tween20, were tested. Five antibodies: ab986,

ab136327, ab31499, ab354, and ab78993, at three concentrations 3.50, 0.88, and 0.22 mg/m2

were also used. DI water was used as a reaction buffer in all trials. Agglutination results with

prepared MPs and E. coli O1:K1:H7 can be found in Tables 4.10 through 4.13.

Table 4.10

Agglutination Results: DI, PBS, and BBS, 2 hr RT Incubation, DI suspension, E. coli O1:K1:H7

Buffer

Ab

(mg/m2)

Antibody Type, Result (arbitrary units) with O1:H1:K7

ab986 ab13627 ab31499 ab354 ab78993

Neg Pos Neg Pos Neg Pos Neg Pos Neg Pos

DI

3.50 1 1 1 1 1 1 1 1 1 1

0.88 1 1 1 1 1 1 1 1 1 1

0.22 1 1 1 1 1 1 1 1 1 1

PBS

3.50 1 1 1 1 1 1 1 1 1 1

0.88 1 1 1 1 1 1 1 1 1 1

0.22 1 1 1 1 1 1 1 1 1 1

BBS

3.50 1 1 1 1 1 1 1 1 1 1

0.88 1 1 1 1 1 1 1 1 1 1

0.22 1 1 1 1 1 1 1 1 1 1

50

Table 4.11

Agglutination Results: DI, PBS, and BBS, with 0.05% Tween20, 2 hr RT Incubation, DI

suspension, E. coli O1:K1:H7

Buffer

(+ 0.05%

Tween20)

Ab

(mg/m2)

Antibody Type, Result (arbitrary units) with O1:H1:K7

ab986 ab13627 ab31499 ab354 ab78993

Neg Pos Neg Pos Neg Pos Neg Pos Neg Pos

DI

3.50 0 0 0 0 0 0 0 0 0 0

0.88 0 0 0 0 0 0 0 0 0 0

0.22 0 0 0 0 0 0 0 0 0 0

PBS

3.50 0 0 0 0 0 0 0 0 0 0

0.88 0 0 0 0 0 0 0 0 0 0

0.22 0 0 0 0 0 0 0 0 0 0

BBS

3.50 0 0 0 0 0 0 0 0 0 0

0.88 0 0 0 0 0 0 0 0 0 0

0.22 0.5 0.5 0 0 0 0 0 0 0 0

Table 4.12

Agglutination Results: DI, PBS, and BBS, 24 hr 4°C Incubation, DI suspension, E. coli

O1:K1:H7

Buffer

Ab

(mg/m2)

Antibody Type, Result (arbitrary units) with O1:H1:K7

ab986 ab13627 ab31499 ab354 ab78993

Neg Pos Neg Pos Neg Pos Neg Pos Neg Pos

DI

3.50 1 1 1 1 1 1 1 1 1 1

0.88 1 1 1 1 1 1 1 1 1 1

0.22 1 1 1 1 1 1 0.5 0.5 1 1

PBS

3.50 1 1 1 1 1 1 1 1 1 1

0.88 1 1 1 1 1 1 1 1 1 1

0.22 1 1 1 1 1 1 1 1 1 1

BBS

3.50 1 1 1 1 1 1 1 1 1 1

0.88 1 1 1 1 1 1 1 1 1 1

0.22 1 1 1 1 1 1 1 1 1 1

51

Table 4.13

Agglutination Results: DI, PBS, and BBS, 0.05% Tween20, 24 hr 4°C Incubation, DI suspension,

E. coli O1:K1:H7

Buffer (+

0.05% T20)

Ab

(mg/m3)

Antibody Type, Result (arbitrary units) with O1:H1:K7

ab986 ab13627 ab31499 ab354 ab78993

Neg Pos Neg Pos Neg Pos Neg Pos Neg Pos

DI

3.50 0 0 0 0 0 0 0 0 0 0

0.88 0 0 0 0 0 0 0 0 0 0

0.22 0 0 0 0 0 0 0 0 0 0

PBS

3.50 0 0 0.5 0.5 0 0 0 0 0 0

0.88 0 0 0 0 0 0 0 0 0 0

0.22 0 0 0 0 0 0 0 0 0 0

BBS

3.50 0.5 0.5 0 0 0 0 0 0 0 0

0.88 0.5 0.5 0 0 0 0 0 0 0 0

0.22 0.5 0.5 0 0 0 0 0 0 0 0

The six initial immobilization buffers did not create any functioning particles. In general,

all the particles prepared with immobilization buffers that did not have Tween20 showed slight

NSA independent of E. coli presence. The experiments were expanded to include seven

additional immobilization buffers (TBS, BBS, CPB [pH = 6.8, 6.2, 5.3, 4.6], and AceB),

incubated for 24 hours at 4° C with and without the addition of Tween20. Due to their limited

reactivity (see section 4.2), antibodies ab986 and ab78993 were no longer used to limit the

number of trials required. The other three antibodies (ab13627, ab31499, and ab354) were tested

at five concentrations: 3.50, 0.88, 0.22, 0.055, and 0.014 mg/m2.The results of prepared MP

interactions with E. coli O1:K1:H7of can be found in Tables 4.14 and 4.15.

52

Table 4.14

Agglutination Results: TBS, GBS, CPB, and AceB; 24 hr 4° C Incubation

Buffer Ab (mg/m2)

Antibody Type, Result with E. coli O1:K1:H7 (arbitrary units)

ab13627 ab31499 ab354 BSA Control

Neg Pos Neg Pos Neg Pos Neg Pos

TBS

3.50 1 1 1 1 1 1 1 1

0.88 1 1 1 1 1 1

0.22 1 1 1 1 1 1

0.055 1 1 1 1 1 1

0.014 1 1 1 1 1 1

GBS

3.50 1 1 1 1 1 1 1 1

0.88 1 1 1 1 1 1

0.22 1 1 1 1 1 1

0.055 1 1 1 1 1 1

0.014 1 1 1 1 1 1

CPB pH =

6.8

3.50 1 1 1 1 1 1 1 1

0.88 1 1 1 1 1 1

0.22 1 1 1 1 1 1

0.055 1 1 1 1 1 1

0.014 1 1 1 1 1 1

CPB pH =

6.2

3.50 1 1 1 1 1 1 1 1

0.88 1 1 0.5 0.5 1 1

0.22 1 1 1 1 1 1

0.055 0.5 0.5 1 1 1 1

0.014 1 1 1 1 1 1

CPB pH =

5.3

3.50 1 1 1 1 1 1 0.5 0.5

0.88 0.5 1 0 0 1 1

0.22 1 1 0 0 1 1

0.055 1 1 1 1 1 1

0.014 1 1 0.5 0.5 0 0

CPB pH =

4.6

3.50 1 1 1 1 1 1 1 1

0.88 1 1 1 1 1 1

0.22 1 1 1 1 1 1

0.055 1 1 1 1 1 1

0.014 1 1 1 1 1 1

AceB

3.50 2 2 2 2 1 1.5 1 1

0.88 1 2 1 2 1 3

0.22 1 2 1 3 1 2

0.055 1 1.5 1 2.5 1 2

0.014 1 1 1 1 1 2

53

Table 4.15

Agglutination Results: TBS, CPB, BBS, and AceB; with 0.05% Tween20, 24 hr 4° C Incubation

Buffer Ab (mg/m2)

Antibody Type, Result with E. coli O1:K1:H7 (arbitrary units)

ab13627 ab31499 ab354 BSA Control

Neg Pos Neg Pos Neg Pos Neg Pos

TBS

3.50 0 0 1 1 0 0 0 0

0.88 0 0 0 0 0 0

0.22 0 0 0 0 0 0

0.055 0 0 0 0 0 0

0.014 0 0 0 0 0 0

GBS

3.50 0 0 0 0 0 0 0 0

0.88 0 0 0 0 0 0

0.22 0 0 0 0 0 0

0.055 0 0 0 0 0 0

0.014 0 0 0 0 0 0

CPB pH =

6.8

3.50 0 0 0 0 0 0 0 0

0.88 0.5 0.5 0 0 0 0

0.22 0 0 0 0 0 0

0.055 0 0 0 0 0 0

0.014 0 0 0 0 0 0

CPB pH =

6.2

3.50 0 0 0 0 0 0 0 0

0.88 0 0 0 0 0 0

0.22 0 0 0 0 0 0

0.055 0 0 0 0 0 0

0.014 0 0 0 0 0 0

CPB pH =

5.3

3.50 0 0 0 0 0 0 0 0

0.88 0 0 0 0 0 0

0.22 0 0 0 0 0 0

0.055 0 0 0 0 0 0

0.014 0 0 0 0 0 0

CPB pH =

4.6

3.50 0 0 0 0 0.5 0.5 0 0

0.88 0 0 0 0 0.5 0.5

0.22 0 0 0 0 0 0

0.055 0 0 0.5 0.5 0 0

0.014 0 0 0 0 0 0

AceB

3.50 0 0 0 0 0.5 0.5 0 0

0.88 0 0 0 0 0.5 0.5

0.22 0 0 0 0 0 0

0.055 0 0 0 0 0 0

0.014 0 0 0 0 0 0

54

The results showed continued negative agglutination for particles using all buffers except

acetate buffer. All particles prepared with Tween20 present in the buffer showed little to no

agglutination. Other buffers continued to create slight agglutination independent of the presence

of E. coli. MPs prepared with acetate buffer showed slight agglutination until interacted with E.

coli O1:K1:H7. Agglutination increased in a few batches after about a minute of tilting. A few

batches showed increased NSA without E. coli present in the sample. Several of the batches

exhibited positive AD values in around 2-3. Afterward, it was decided to continue testing

protein immobilization using acetate buffer only.

Two additional methods of incubation were then evaluated: 2 hour RT incubation, and 24

hour RT incubation. Acetate buffer without Tween20 was used in all preparations, and the same

antibody suite was used as in the previous results. The results of this analysis can be seen in

Table 4.16.

Table 4.16

Agglutination Results: Acetate Buffer, 2 and 24 Hour RT Incubation, DI Suspension, E. coli

O1:K1:H7

Incubation

Time Ab (mg/m2)

Antibody Type, Result (arbitrary units)

ab13627 ab31499 ab354 BSA Control

Neg Pos Neg Pos Neg Pos Neg Pos

2 Hours

3.50 1 3 1 3 1 2 1 1

0.88 1 2.5 1 1.5 1 3

0.22 1 2 1 2.5 1 3

0.055 1 2 1 2 1 2

0.014 1 2.5 1 3 1 3

24 Hours

3.50 2 2.5 2 2 2 2.5 1 1

0.88 1 2 1 1.5 1 2.5

0.22 1 1.5 1 1 1 2

0.055 1 1 1 2 1 1

0.014 1 1 1 1 1 2

55

Generally, the MPs prepared using two hour RT incubation presented better

agglutination. Almost all preparations showed some degree of agglutination, and all exhibited at

least slight NSA. A few preparations, particularly those with 3.50 mg/m3

antibody concentration

incubated for 24 hours, exhibited higher degrees of NSA. The experiment was repeated, but with

0.15 M Sodium Chloride added to the immobilization buffer as described by Bangs Laboratories

TechNote 204 (2008). Due to the low performance of 24 hour RT incubation, 24 hour 4° C

incubation was used instead. The results of this analysis can be found in Table 4.17.

Agglutination results with E. coli Famp were weaker, although somewhat similar (Table 4.18).

Table 4.17

Agglutination Results: Acetate Buffer + 0.15M NaCl, 2 Hour RT and 24 Hour 4° C Incubation,

DI Suspension, E. coli O1:K1:H7

Incubation

Time/ Temp Ab (mg/m2)

Antibody Type, Result (arbitrary units)

ab13627 ab31499 ab354 BSA Control

Neg Pos Neg Pos Neg Pos Neg Pos

2 Hours/ RT

3.50 1 1.5 2 3 2 2.5 1 1

0.88 1 1.5 1 3.5 1 3

0.22 1 1 1 3 1 3

0.055 0.5 1 1 3 1 3

0.014 1 3.5 1 3 1 3

24 Hours/

4° C

3.50 1 1.5 2 3.5 2 2.5 1 1

0.88 1 2 1 3 1 1

0.22 1 3 1 3 1 2

0.055 1 1 1 2 1 2

0.014 1 2 2 3 1 3

56

Table 4.18

Agglutination Results: Acetate Buffer + 0.15M NaCl, 2 Hour RT and 24 Hour 4° C Incubation,

DI Suspension, E. coli Famp

Incubation

Time/ Temp Ab (mg/m2)

Antibody Type, Result (arbitrary units)

ab13627 ab31499 ab354 BSA Control

Neg Pos Neg Pos Neg Pos Neg Pos

2 Hours/ RT

3.50 1 1.5 2 2 2 2 1 1

0.88 1 1.5 1 1.5 1 2

0.22 1 1 1 2.5 1 2

0.055 0.5 1 1 2 1 3

0.014 1 2.5 1 2 1 2

24 Hours/

4° C

3.50 1 1.5 2 3 2 2.5 1 1

0.88 1 2 1 3 1 1

0.22 1 3 1 2 1 2

0.055 1 1 1 2 1 2

0.014 1 2 2 3 1 2

The addition of sodium chloride to the immobilization buffer did seem to improve

agglutination, with many preparations showing good agglutination. NSA continued to be present

slightly in all samples, and more extensively in a few. The degree of agglutination with antibody

ab13627 in the 2 hour RT incubation was very limited, except for the 0.014 mg/m3 preparation,

which was unexpected. Because of this, the experiment was repeated to verify the results (Table

4.19). Agglutination results were similar but slightly weaker when using E. coli Famp, as shown

in Table 4.20.

57

Table 4.19

Agglutination Results: Acetate Buffer + 0.15M NaCl, 2 Hour RT and 24 Hour 4° C Incubation,

DI Suspension Retest, E. coli O1:K1:H7

Incubation

Time/ Temp Ab (mg/m2)

Antibody Type, Result (arbitrary units)

ab13627 ab31499 ab354 BSA Control

Neg Pos Neg Pos Neg Pos Neg Pos

2 Hours/ RT

3.50 1 2.5 1.5 2 1 1 1 1

0.88 1 2 1 1 1 1

0.22 1.5 4 1 1 1 1

0.055 1 2 1 1 1 2

0.014 1 1 1 1.5 1 1

24 Hours/

4° C

3.50 2 3 1 1 1 1 1 1

0.88 1 3 1 1 1 1

0.22 1 1 1 1 1 1

0.055 1 1 1 1 1 2

0.014 1 1 1 1 1 1

Table 4.20

Agglutination Results: Acetate Buffer + 0.15M NaCl, 2 Hour RT and 24 Hour 4° C Incubation,

DI Suspension Retest, E. coli Famp

Incubation

Time/ Temp Ab (mg/m2)

Antibody Type, Result (arbitrary units)

ab13627 ab31499 ab354 BSA Control

Neg Pos Neg Pos Neg Pos Neg Pos

2 Hours/ RT

3.50 1 2 1.5 2 1 1 1 1

0.88 1 2 1 1 1 1

0.22 1.5 3 1 1 1 1

0.055 1 1 1 1 1 1

0.014 1 1 1 1 1 1

24 Hours/

4° C

3.50 2 3 1 1 1 1 1 1

0.88 1 3 1 1 1 1

0.22 1 1 1 1 1 1

0.055 1 1 1 1 1 2

0.014 1 1 1 1 1 1

The retest yielded unexpected results; almost all the preparations exhibited zero AD.

NSA continued to be present in similar degrees as previous experiments in negatives, however

58

limited positive agglutination was observed. Only ab13627 exhibited good agglutination, but the

degree varied. It was desired that the past experiments would show a trend in the performance of

a particular antibody or concentration. The results, unfortunately, fail to indicate an antibody or

antibody concentration performing superior to the others. A summary of all agglutination deltas

of each antibody/concentration from all acetate buffer preparations can be seen in Table 4.21.

Table 4.21

Agglutination Results: Agglutination Deltas of Acetate Buffer Preparations, E. coli O1:K1:H7

Incubation

Time/ Temp

Ab

(mg/m2)

Incubation Temp, Time (h), AD (arbitrary units)

Average

AD

RT 4° C

2 2 2 24 24 24 24

ab13627

3.50 2 0.5 1.5 0.5 0 0.5 1 0.9

0.88 1.5 0.5 1 1 1 1 2 1.1

0.22 1 0 2.5 0.5 1 2 0 1.0

0.055 1 0.5 1 0 0.5 0 0 0.4

0.014 1.5 2.5 0 0 0 1 0 0.7

ab31499

3.50 2 1 0.5 0 0 1.5 0 0.7

0.88 0.5 2.5 0 0.5 1 2 0 0.9

0.22 1.5 2 0 0 2 2 0 1.1

0.055 1 2 0 1 1.5 1 0 0.9

0.014 2 2 0.5 0 0 1 0 0.8

ab354

3.50 0 0.5 0 0.5 0.5 0.5 0 0.3

0.88 0.5 2 0 1.5 2 0 0 0.9

0.22 0 2 0 1 1 1 0 0.7

0.055 1 2 1 0 1 1 1 1.0

0.014 0 2 0 1 1 2 0 0.9

BSA Control 3.50 0 0 0 0 0 0 0 0.0

Experiments were conducted that used BSA as a blocker after washing particles to try to

minimize NSA. Antibodies ab13627 and ab31499 were immobilized at concentration 3.5

mg/m2. 0.97 μm PS03N and 0.4 μm PS02N particles were used. After immobilization, particles

were washed twice in DI and then suspended in DI with BSA in varying concentrations. After

59

incubation for 1 hour, MPs were tested for function and detection limit. The results of this

analysis are shown in Table 4.22.

Table 4.22

Detection Limit of Prepared MPs with E. coli O1:K1:H7, BSA Suspension

Particle

Size

(μm) Ab

BSA

(mg/m2) Neg

O1:K1:H7 Titer (106cfu/mL), Result

(arbitrary units) Detection

Limit

(106cfu/mL) 833 208 52.1 13.0 3.26 0.81

0.97

ab13627

0.0 2 3 3 2.5 2 2 2 52

0.5 2 3 2 2 2 2 2 833

1.0 1.5 4 3.5 2.5 1.5 1.5 1.5 52

5.0 1 3 3 2 1.5 1 1 52

20.0 2 4 3 2 2 2 2 208

ab31499

0.0 1.5 3 3 2.5 1.5 1.5 1.5 52

0.5 1.5 3 3 2 1.5 1.5 1.5 208

1.0 1.5 3 3 2 1.5 1.5 1.5 208

5.0 1 2.5 2.5 1.5 1 1 1 208

20.0 1 2.5 2 1.5 1 1 1 208

0.40

ab13627

0.0 1.5 3 3 2.5 2 1.5 1.5 52

0.5 2 3 2.5 2 2 2 2 208

1.0 2 3 2 2 2 2 2 208

5.0 1 3 2.5 2.5 2 1.5 1 13

20.0 2 4 3 2 2 2 2 208

ab31499

0.0 2 3 3 3 2 2 2 52

0.5 1 3 3 2 1 1 1 52

1.0 1.5 3 3 2 2 2 1.5 13

5.0 1.5 3.5 3 2.5 2 2 1.5 52

20.0 2 3 2.5 2 2 2 2 208

Note: Lowest titer at which agglutination is evident in bold.

60

Particles with immobilized antibodies were also evaluated with varying NaCl

concentrations in the reaction buffers. Particles were prepared using acetate buffer and antibody

ab13627, washed twice, and then suspended in DI with varying concentrations NaCl to

investigate its effect on NSA and the possible existence of a critical coagulation concentration.

Agglutination results with E. coli O1:K1:H7 are shown in Table 4.23.

Table 4.23

Effect of NaCl on Agglutination

Ab

(mg/m2)

NaCl concentration (M), Result (arbitrary units)

0 0.05 0.15 0.5 1.5 5.0

3.50

Pos 3 4 4 4 4 4

Neg 2 2 2 4 4 4

AD 1 2 2 0 0 0

1.75

Pos 3 3 4 4 4 4

Neg 1 1 3 4 4 4

AD 2 2 1 0 0 0

Antibody immobilized particles were evaluated in detection of E. coli O1:K1:H7 grown

using the methods described in section 3.1. The key difference between this analysis and other

performed is the presence of the GES, which is required for bacteria growth. DI water with E.

coli O1:K1:H7 added was filtered in three different volumes (1, 10, and 100 mL), recovered,

grown for 9 hours, and evaluated using 0.40 μm PS02N labeled with 1.75 μg/m2 ab13627 for 1

hour at RT in acetate buffer. 10 mL PBS with 30 g/L TSB in a 15 mL tube was used with and

without 0.5% Tween20 because of the previously observed affect Tween20 can have on

agglutination. This analysis also evaluates NaCl present in the reaction buffer to help facilitate

agglutination. The results of this analysis are shown in Table 4.24.

61

Table 4.24

Agglutination with MF Concentrated E. coli O1:K1:H7, after 9-hour Growth

NaCl concentration (M), Result (arbitrary units)

GES

Volume

Filtered

(mL)

Hour 9 Titer

(cfu/mL) OCRa 0 0.05 0.15 0.5 1.5 5.0

TSB

1 1,818,182 356,506 1 1 1 1 2 2

10 20,303,030 398,099 1 2 2 3 3 2

100 345,454,545 677,362 1 2.5 2.5 3 4 2

Neg 0 0 1 1 1 1 2 2

TSB +

Tween20

1 2,242,424 439,691 1 1 1 1 1 1

10 13,636,364 267,380 1 1 1 1 1 1

100 287,878,788 564,468 1 1 1 1 1 1

Neg 0 0 1 1 1 1 1 1

Note: a Original sample titer = 51 cfu/mL.

4.3.2 Streptavidin Coated Microparticles

Streptavidin-coated MPs were used initially to take advantage of the high affinity for

streptavidin to biotin. Several magnetic particle types were tested due to the potential

advantages they offered over PS particles both in immobilization and their potential role in the

novel test method. The biotin conjugated antibody ab20640 was used in concentrations of 3.5,

0.7, and 0.14 mg/m2, with four streptavidin coated MP types: C1, CM01N, PMS1N, CP01F. Of

the four, all but CP01F were magnetic, which helped streamline the production process, and

presented favorable options for usage if prepared successfully. Particles were prepared using DI

and PBS as immobilization buffers, 30 RPM orbital mixing, and 2 and 24 hour incubation

periods at RT. The agglutination results with E. coli Famp are shown in Table 4.25.

62

Table 4.25

Agglutination Results: Streptavidin Coated Particles, PBS and BBS, 2 and 24 Hour RT

Incubation, E. coli Famp

Buffer

Incubation

Time (hr)

Antibody

(mg/m2)

Particle Type, Result (arbitrary units)

C1 CM01N PMS1N CP01F

Neg Pos Neg Pos Neg Pos Neg Pos

PBS

1

3.50 1 1 1 3 0 0 0 0

0.70 1 1 1 3 0 0 0 0

0.14 1 1 1 3 0 0 0 0

24

3.50 0 0 1 3 0 0 0 0

0.70 0 0 1 3 0 0 0 0

0.14 0 0 1 2.5 0 0 0 0

DI

1

3.50 0 0 1 2 0 0 0 0

0.70 0 0 1 2 0 0 0 0

0.14 0 0 1 2 0 0 0 0

24

3.50 0 0 1 2 0 0 0 0

0.70 0 0 1 2 0 0 0 0

0.14 0 0 1 1.5 0 0 0 0

The results show good agglutination for the CM01N particles, with minimal positive

agglutination with the others. The agglutination of CMO1N particles was investigated further

with continued use of PBS and DI as immobilization buffers, and RT incubation for 30 and 60

minutes. The results of this analysis are found in Table 4.26.

63

Table 4.26

Agglutination Results: Streptavidin Coated Particles, PBS and BBS, 2 and 24 Hour RT

Incubation, E. coli Famp

Buffer

Incubation

Time (min)

Antibody

(mg/m2)

E. coli Famp Titer (106

cfu/mL), Result (Arbitrary Units)

Neg

PBS

30

3.50 1 2 2 2 2 2 1 1

1.25 1 2 2 2 2 2 2 1

0.63 1 2 2 2 2 2 2 1

0.10 1 1 1 1 1 1 1 1

60

3.50 1 3 3 2 2 2 2 1

1.25 1 3 3 2 2 2 2 1

0.63 1 2 2 2 2 2 2 1

0.10 1 2 2 1 1 1 1 1

DI

30

3.50 1 3 3 3 2 2 1 1

1.25 1 2 2 2 2 2 1 1

0.63 1 2 2 2 2 2 1 1

0.10 1 3 3 2 2 1 1 1

60

3.50 1 3 3 2 2 2 1 1

1.25 1 3 3 2 2 2 1 1

0.63 1 2 2 2 2 2 2 1

0.10 1 2 1 1 1 1 1 1

The results of the analysis showed good results. The agglutination appears to be

occurring at titers too low for agglutination than possible. It was suspected that NSA was

somehow affecting the results. This was investigated by manipulating the particles in different

ways using different buffers, then evaluating any NSA. The results of this analysis are shown in

Table 4.27. The particles showed significant NSA after washing and mixing, and incubation

with ab20640 only increased this effect. In some cases, NSA after incubation and mixing was

total. For this reason, the agglutination present in previous preparations of CM01N particles was

considered non-specific.

64

Table 4.27

Investigation of NSA in CM01N Particles

Process

Buffer, NSA After Process (arbitrary units)

Buffer Alone Buffer

+ 0.05% Tween20

DI PBS BBS DI PBS BBS

Negative Control (As Is) 0 0 0 0 0 0

Pre-Wash 1 0 1 0 0 1 0

Pre- Wash 2 1 1 1 1 0 0

60 s Mixa 2 1 2 2 0 1

Post Incubationb 2 3 3 2 3 3

Post-Wash 3 1 2 2 1 2 2

Post-Wash 4 1 2 2 1 2 2

60 s Mixa 1 4 3 3 4 3

Suspension in BSAc 1 2 2 2 2 1

60 s Mixa 2 4 3 3 4 3

Notes: a Using platform shaker @ 120 RPM (New Brunswick Innova)

b Incubation with ab20640

for 30 minutes at RT with 30 rpm rotation. c BSA at 1 mg/mL.

65

5. DISCUSSION

5.1 E. coli Recovery

The initial process of the proposed novel detection method involved concentrating E. coli

using the MF technique, subsequently placing the filter in growth media, and growing the E. coli

to higher concentrations. The role that E. coli elution, separation from the filter to the broth,

played on growth was unknown. It was discovered that simple methods could be used to

generate high levels of bacterial recovery (see Table 4.4). Elution required to primary

components: mechanical action and a surfactant (Tween20 in this case). Research also revealed

evidence supporting the idea that high levels of recovery were not necessary to maximize growth

rate.

The effect of various eluents, methods of mechanical action, and tube sizes were

investigated. Simple hand shaking for 20 seconds was the mechanical method selected that

increased recovery while still being simple and easy to perform, although increased levels of

mechanical input generally increased recovery (see Table 4.1). No significant role of TSB in the

eluent was determined. However, the presence of Tween20 was important to increasing

recovery, but the ideal concentration was not determined. Also, when larger tubes were used,

recovery tended to be higher, probably because they permitted increased agitation of the

membrane filter. Placing the 45 mm membrane filter in a 50 mL tube containing 10 mL of GES

consisting of DI, 30 g/L TSB, and 0.5% Tween20 and shaking for 20 seconds generated average

recovery of around 80% (see Table 4.4).

66

Research also revealed that high recovery was not necessary or beneficial to growth. The

plot of 0 – 6 hour DT0 times and recovery values (Figure 5.1) of tubes used in Table 4.6 shows

low correlation between the two. Indeed, there exists little correlation (-0.150) between 0 – 6

hour DT0 values and recovery. The plot of 0 – 6 hour DT1 times and recovery values (Figure

5.2) of the same tubes does show moderate correlation (0.568). This is due to an apparent

increase in growth rate caused by non-eluted bacteria on the MF contributing to bacteria growth.

DT1 times are based on TSC bacterial count, so in cases where recovery was low the growth rate

would appear higher than it actually is due to non eluted bacteria not represented in the DT

calculation contributing to growth. DT0 values are based on the FC bacterial count, so the

growth rate is based on all bacteria, regardless whether or not they are eluted. Since DT0 and not

DT1 is independent of recovery, it is likely all bacteria contribute to growth, regardless of

whether or not they are eluted. This greatly reduces the significance of on high recovery rates in

the function of the novel method.

67

Figure 5.1

0 – 6 hour doubling time (DT0) vs. recovery for 15 mL and 50 mL tubes. This data includes all

tests from Tables 4.6. Note the lack of correlation between DT0 and Recovery.

28

30

32

34

36

38

40

0.00 0.20 0.40 0.60 0.80 1.00 1.20

DT 0

(m

in)

Recovery

15 mL Tube Size

50 mL Tube Size

68

Figure 5.2

0 – 6 hour doubling time (DT1) vs. recovery for 15 mL and 50 mL tubes. This data includes all

tests from Tables 4.6. An apparent correlation exists when using DT1, but this is misleading

since DT1 bases growth on TSC and would therefore overestimate growth rate when non-eluted

bacteria replicate.

5.2 E. coli Growth

The growth rate of E. coli is well understood, and a significant increase in growth rate

above the accepted maximums was the not anticipated. The rate of growth following the

aforementioned recovery of bacteria following membrane filtration was of interest. Research

showed similar growth rates for E. coli regardless of TSB and Tween20 concentration (Tables

4.5 and 4.6). Lower concentrations of TSB did seem to raise growth rates slightly. Average DT

between hours 3 and 6 in a 50 mL tube with 1.0% Tween20 was 22.4 minutes with 15 g/L TSB,

but only 23.5 minutes with 30 g/L TSB. This increase in growth rate would likely be neutralized

over a longer growing period, as competition for resources increases.

28

30

32

34

36

38

40

0.00 0.20 0.40 0.60 0.80 1.00 1.20

DT 1

(min

) Recovery

15 mL Tube

50 mL Tube

Linear (15 mL Tube)

Linear (50 mL Tube)

69

The effect of tube size on growth was more pronounced. Table 4.6 shows an increase in

growth rate using 15 mL tubes, as indicated by the 0 – 6 hour average DT0s. This is probably

likely to the improved proportions of the 15 mL tubes, which have a higher specific surface area

(surface area/volume) than the 50 mL tubes, allowing them to increase temperature faster upon

incubation. The bacteria will then more quickly reach log phase growth, which is suggested by

the decreased DT0 times for hours 3 – 6. GES consisting of 1.0% Tween20 and 15 g/L of TSB

had a DT of 21.1 minutes between hours 3 and 6 in a 15 mL tube. The same GES in a 50 mL

tube had a DT of 22.4 minutes between hours 3 and 6. Higher DT0 values for hours 0 - 3 for 15

mL tubes are a result of decreased recovery. In the novel test method, an ideal growth container

for the membrane filter would be one that allowed easy placement of the filter in the GES, but

had as a high specific surface area as possible.

Growth trials were extended to 9 hours to better quantify the log phase growth rates of E.

coli since recovery plays a marginal role in growth. The effect of shaking the tubes on growth

was evaluated. As apparent in Figure 4.2, shaken tubes have a slightly higher growth rate over

the 9 hour period, although the non-shaken tubes grew faster from hours 3 to 9. This could be

due to some effect the bacteria growing in place on the filter have on the titer, or more likely due

to experimental variation, which is high for the non-shaken tubes. Not shaking the tubes creates

more opportunity for variability to be introduced, since it is possible for the filter to not be

completely submerged in the GES without shaking. Although not necessary to increase recovery,

in practice, a short shake is still necessary to ensure the filter is properly submerged in the

growth media and any E. coli captured is able to replicate.

Overall, the growth rate of E. coli behaved predictably, with about a three hour lag phase

of slow growth, followed by an acceleration to log phase. The average DT in log phase of 21.4

70

minutes is similar to the generally accepted minimum DT values for E. coli. At the average rate

measured, a single E. coli cell will replicate to 660,000 cells in 9 hours and 220 million in 12

hours. Using the lower confidence limit (α = 0.05), a single E. coli cell will replicate to 540,000

cells in 9 hours and 140 million in 12 hours. The growth rate results follow closely to growth of

E. coli measured in other studies. While this is not surprising, it does show that using MF as a

method of concentration, followed by suspension of the filter in TSB and incubation will grow

bacteria as efficiently as other methods. This process could be modified for convenient use in a

field deployable apparatus, such as the Oxfam Delagua kit, that uses a liquid based

immunoassay. Based on the results of the growth analysis and other research, it seems there is

very little room to increase growth rate of E. coli. If the process was optimized and the average

DT was decreased 60 seconds to 20.5 minutes from hour 3 onward, a single cell would replicate

to 522 million at hour 12. While this increase of 237% seems significant, E. coli replicating at

the average original rate (DT = 21.5 minutes) would reach the same count in 27 additional

minutes of incubation. For application in the novel test method, much more significant gains in

required incubation time can be made by lowering the detection limit of the immunoassay.

Using the measured DT of 21.5 minutes, each order of magnitude reduction in test sensitivity

would reduce the required incubation time by 72 minutes.

5.3 Microparticle Function

Microparticles (MPs) can be prepared that agglutinate in the presence of E. coli. The

detection limit of the best functioning particles prepared in this research is about 13,000,000

cfu/mL (see Table 4.22). Research supports the conclusion that based on the selection of

71

antibodies available on the market, MPs can be prepared that are highly sensitive and specific to

most environmental E. coli strains. The extent of this was not evaluated in this research,

although it is critical to the development of the novel test method. The use of streptavidin coated

particles in this research was problematic; many prepared particles showed significant and

unpredictable NSA. NSA continued to be a problem with PS particles, but to a lesser extent.

Despite the use of various buffers in other studies, of all the buffers tested for

immobilization, only acetate buffer generated positive results. This is contrary to the claim that

absorption most readily occurs when buffer pH is at or around the isoelectric point of the protein,

which for IgG is approximately 6.5 to 8.0. The pH of acetate buffer was measured to be 4.7,

well below the range. Furthermore, PBS, GBS, TBS, and BBS all had pH within the range,

measured to be 7.2, 6.9, 7.6, and 8.0, respectively. The reason for this could be due to low

isoelectric points of the antibodies used. Also, more likely, was the salt concentration of the

acetate buffer was optimal for agglutination with the reaction conditions tested. pH is important

to antibody binding, but low pH probably still permited binding, just to a lesser extent. Had the

effect of salt been analyzed more thouroughly from the onset, properly functioning

microparticles might have been prepared with other buffers as well.

The results also showed little evidence of an ideal antibody or concentration. The highest

overall AD averages occur with ab13627 at 0.88 mg/m2 and ab31499 at 0.22 mg/m

2. The

variation in the average AD values is fairly low. They are plotted in Figure 5.3, and although

weak, it seems there is a general trend for optimal performance at 0.88 mg/m2, with decreasing

performance at higher and lower concentrations. Decreasing performance at 3.5 mg/m2

is mainly

due to increased NSA at that concentration. If NSA could be decreased, 3.5 mg/m2 might

perform better than other concentrations.

72

Figure 5.3

Comparison of AD values for ab13627, ab31499, and ab354 in acetate buffer preparations, E.

coli O1:K1:H7

These results underscore the complexity of the protein adsorption process. Proper

function of coated microparticle requires careful control of the surface charge density of the

coated particles to balance particle stability. If the charge density is too high, the particles will

be too stable and similarly charged particles will repulse each other and not agglutinate. If the

surface charge density is too low, the particles will be too unstable and attract to one another,

causing NSA (Ortega-Vinuesa & Bastos-González, 2000). Blocking proteins, such as bovine

serum albumin (BSA), can also be employed to help reduce NSA. Adding NaCl or MgCl is also

used to help control particle stability. Reducing NSA is one of the significant challenges facing

the design of a latex agglutination test.

BSA was tested to control NSA, with mixed results (Table 5.1). Batches not treated with

BSA had an average NSA of 1.75, while those treated 5 mg/m3 BSA had average NSA of 1.13.

0

0.2

0.4

0.6

0.8

1

1.2

0.01 0.1 1 10

ab13627

ab31499

ab354

73

However, the particles treated with 20 mg/m2 had average NSA of 1.75. The lowest average

detection limit was for MPs not treated with BSA. Conversely, the two preparations with the

lowest detection limit were treated with 1 mg/m2 and 5 mg/m

2 BSA respectively. It is unclear

the role BSA played in the development of these particles. The better particle size, however,

seemed much more clearly to be 0.40 μm. The average detection limit was about half of that of

the 0.97 μm particles, and the two preparations with the lowest detection limit were both 0.40 μm

MPs (Table 5.2). The difference between the two antibodies was less pronounced, although the

average detection limit of particles coated with ab13627 was lower. Neither particle size nor

antibody seemed to greatly affect NSA. The role of BSA in limiting NSA could be dependent on

the particular microparticle preparation, and requires more investigation.

Table 5.1

Effect of BSA on NSA and Detection Limit

BSA

(mg/m3)

Average

NSA

Average DL

(106cfu/mL)

0.0 1.75 52.1

0.5 1.63 325.3

1.0 1.63 120.3

5.0 1.13 81.3

20.0 1.75 208.0

Table 5.2

Effect of Particle Size and Antibody on NSA and Detection Limit

Particle Size

(μm), Antibody

Average

NSA

Average DL

(106cfu/mL)

0.97 1.50 208.1

0.40 1.65 106.6

ab13627 1.70 188.6

ab31499 1.45 126.1

74

Additionally, the stability of particles can be controlled using salt (NaCl) in both the

immobilization buffer and reaction buffer. Increasing concentration of salt in the reaction buffer

decreases particle stability leading to specific aggregation and eventually NSA. The ideal salt

concentration is known as the critical coagulation concentration (ccc), at which NSA is limited

but agglutination occurs readily. Increasing levels of bound antibodies decreases stability,

leading to increased NSA and a lower ccc (Serra, et al., 1992). The effect of NaCl was analyzed,

and the results shown in Table 4.23. Increasing NaCl concentrations did increase agglutination.

The benefits were limited, however, since in most cases agglutination increased in both the

positive and negative samples. MPs immobilized with 3.50 μm/m2 antibody were positively

affected by low (0.05 and 0.15 M) concentrations of NaCl. NaCl showed no positive effect on

MPs immobilized with 1.75 μm/m2 antibody, 0.05 M caused similar performance to the control,

with increased concentrations causing significant NSA. For all samples, NaCl concentrations at

and above 0.5 M caused total NSA in the control. Greater analysis would have to be performed

on the antibody immobilized particles to determine the ccc to use in the novel method.

Complicating the process is the effect of the immobilization buffer, the pH and NaCl

concentration of which will cause different levels of particle stability during reaction. For every

attempt at antibody immobilization, a different ccc will result.

Joining the two processes (concentration and growth, LAT detection) presented a new

problem. The presence of Tween20 and TSB in the GES had the potential to greatly affect

agglutination, based on the erratic performance of the microparticles. This was evaluated by

testing agglutination with bacteria concentrated and grown using the novel test method. Table

4.24 shows the result of this analysis, and it is clear that Tween20 present in the GES completely

negated all agglutination. This was somewhat expected, considering the results of agglutination

75

using Tween20 in the reaction buffer (Table 4.15). TSB also inhibited agglutination. Adding

NaCl to the TSB did facilitate agglutination, with the ideal concentration, or ccc, at about 0.5 M.

For use in the novel test method NaCl could be used to help cause agglutination, although the

NaCl concentration would have to be optimized in conjunction with changes to the GES. Any

modification to the GES would causes effect the agglutination process, thereby changing the ccc.

It is possible the required NaCl could be added to the GES, although if it caused problems to

bacterial growth it could also be added to the particle suspension or added as another reagent in

the test.

Uncertainty could arise from use of strains other than O1:K1:H7 in agglutination testing.

While the interaction of antibody ab78993 and the E. coli H-7 flagellar antigen is not typical or

likely to occur in environmental samples, it does provide a strong confirmation for organism

interaction, and it benefited the troubleshooting process in creating functioning MPs. Reactions

with other E. coli strains might be better or worse. Antibodies ab13627 and ab31499 represent

good candidates for use in a novel test method due to their higher specificity for E. coli, although

the exact sensitivities of these antibodies are unknown and would have to be evaluated.

Although the specifics of interactions between antibodies and different E. coli strains are

beyond the scope of this project, it is a critical element in the design of the functioning rapid

detection method. As demonstrated in Table 4.9, reactivity between E. coli antibodies and cells

is irregular and unpredictable. This aspect is critical to the function of a detection method, as the

method would function most suitably with high sensitivity and specificity. Regardless of the

level, the sensitivity and specificity would have to be absolutely known, in order to determine the

redundancy requirements to give accurate results. While other known and unknown factors are

likely to interfere with the sensitivity of the test, a central determinant would be antibody

76

reactivity. The ideal antibody for use in the method would be absolutely specific and sensitive to

all strains of E. coli. An antibody of this nature is not likely to be found, partially due to the high

likelihood of cross-over interactions of antibodies reactive to E. coli to other members of the

family Escherichia.

5.4 Summary

The results of this research show promise for the novel detection method. Coupled

together, the growth, recovery, and detection methods investigated in this research would be able

to detect a single E. coli cfu after a minimum required incubation time of 10 hours and 55

minutes (95% CI). 10 cfu and 100 cfu would be detectable with required minimum incubation

times of 9 hours 41 minutes, and 8 hours, 27 minutes, respectively (Tables 5.3 and 5.4).

Improvements in the LAT method that lower detection limit could significantly reduce these

times. To bring the test into the desired six hour window, the detection limit of the LAT would

need to be around 1,000 cfu. This is not impossible, but more advanced detection technologies

would have to be employed, such as nephelometry or particle counters. This would add

unwanted complexity to the detection method, so the ideal configuration for the method would

need to balance simplicity with required incubation time. Even if the required incubation time

stayed as high as 9 hours, it would be significant improvement over current methods.

77

Table 5.3

Average Times to Detection, Log Phase DT = 21.4 Minutes

LAT Detection

Limit

Time to detect initial bacteria population of (cfu):

1 10 100

13,000,000 10:29 9:18 8:07

1,000,000 9:10 7:59 6:48

100,000 7:59 6:48 5:37

10,000 6:48 5:37 4:26

1,000 5:37 4:26 3:15

Table 5.4

Times to Detection, CI = 95%, Log Phase DT = 22.3 Minutes

LAT Detection

Limit

Time to detect initial bacteria population of (cfu):

1 10 100

13,000,000 10:55 9:41 8:27

1,000,000 9:33 8:18 7:04

100,000 8:18 7:04 5:50

10,000 7:04 5:50 4:36

1,000 5:50 4:36 3:22

5.5 Future Work

There are significant areas for continuation of this research. Specific areas that could be

researched further are:

Optimization of the MP production process

Select the ideal antibody for the best possible sensitivity and specificity

Understand the effect of competing bacteria in the sample

Application of new innovation in immunoassays to improve the test

Of these four, the two most critical are probably the optimization of the MP production process

and understanding the effects of competing bacteria in the sample. Further evaluation of the

78

antibodies that were used in this research is necessary, but the broad selectivity of the antibody

used might suit it for continued use.

It is clear through the research that the conclusions of Molina-Bolivar and Galisteo-

Gonzalez (2004) are very true: protein absorption on latex particles is a complex and difficult to

optimize process, due to a lack of experimental reproducibility. The results of this research show

promise for the method, however the detection limit found with Ab coated particles requires a

growth period of 9-12 hours for the E. coli to reach detectable titers. If the production of the

MPs could be optimized to drive down the detection limit, less growth time would be required

for the E. coli, and the method would be improved. The research also demonstrated significant

variation in the performance of the MPs. This would absolutely have to be amended for use in

the novel test method. It is clear that MP coating is the most complex aspect of this research and

leaves significant room for improvement.

An important consideration of the development of the novel method is the effect of

competing bacteria in the sample. The scope of this research was limited to understand the role

of E. coli in the novel method. However, in actual environmental samples, there will always be a

host of other bacteria captured through MF. These bacteria will likely grow and compete for

nutrients, possible slowing the growth rate of E. coli. It is important to understand the degree to

which this will occur, and if there are any techniques for limiting it.

There are a wide selection of new technologies for use in biosensors and immunoassays.

The application of these techniques to this novel method will vary. Techniques that increase the

sensitivity of the method are of most interest. Based on the lab measured doubling time of E.

coli during log phase growth (21.4 min), each order of magnitude increase in test sensitivity

reduces the required growth time of the E. coli sample by about 70 minutes. A 3-order of

79

magnitude increase in test sensitivity as described by Ortega-Vinuesa and Bastos-González

(2001) would reduce the time required for growth to near the 6 hour window desired. With

successful optimization of the immunodetection method, there exist possibilities of modifying

this method into a quantitative immunoassay. It might also be possible to develop a strip test for

the detection of E. coli that could simply be dipped in the growth solution. While this research

experienced problems with the operation of magnetic particles, they do present numerous

possibilities in immunoassay for E. coli in environmental water samples. Magnetic particles

could be used to concentrate E. coli in a sample, by allowing the magnetic particles to bind to the

E. coli, then magnetically separating them.

80

6. CONCLUSION

The research conducted demonstrated three central findings relating to the research

objectives concerning the development of a novel test method using LAT:

E. coli recovered via membrane filtration can be reliably grown in tryptic soy

broth regardless of measures taken to enhance elution.

E. coli grown from MF suspended in TSB will have an average doubling time of

about 28 minutes through 9 hours, although in log phase (which is achieved at

around 3 hours, titers will double every 21.4 minutes.

Polystyrene microparticles can be coated with polyclonal antibodies that are

broadly specific to strains of E. coli, and be used as a detection method with a

sensitivity of about 13 million cfu/mL.

The development of a latex agglutination test is a complex process, particularly

the production of antibody labeled microparticles, but it shows promise as a

method of E. coli detection.

The research partially succeeds in investigating the concept of a rapid detection method for E.

coli in environmental samples using latex agglutination. Further research is required, but latex

agglutination, coupled with a prior concentration and growth step, is a promising alternative to

current E. coli detection methods.

81

MF and subsequent growth in liquid media does seem to be a reliable method of

enhancing titers of E. coli, and with improvements LAT offers a very elegant method of E. coli

detection. If successful, such a test method would be an appealing alternative to other E. coli

detection methods, particularly in cases where speed and cost are of concern. Depending on the

requirements of the LAT detection method, this test method would also present less logistical

requirements than other methods; the test could be conducted with minimal operator training

without access to a lab.

The most significant problem encountered was considerable difficulty in the production

of antibody immobilized MPs, which is a very complex process. The function of the novel test

method very much depends on the capabilities of antibody coated latex particles or some

variation thereof. This research does show they can function properly and detect E. coli at

concentrations of 13,000,000 cfu/mL. The concentration and growth steps demonstrated

acceptable performance, and the average replication time at log phase is 21.4 minutes. At that

detection limit and growth rate, a single E. coli captured on a membrane filter would be

detectable in 10 hours, 29 minutes. Using the determined lower 95% CI, it would be detectable

in 10 hours 55 minutes. Increases in E. coli in the sample or reduction in detection limit would

only reduce the incubation time required. Additionally, improvements to the detection limit of

the LAT would reduce required incubation times as well.

82

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Wilson, J. V., Morison, R. A. H., & Wright., V. (1960). The Latex Slide Test in Rheumatic

Disorders. Journal of Clinical Pathology, 13, 453-455.

World Health Organization (2006). Guidelines for Drinking-water Quality (Vol 1). (3rd

ed.).

Geneva: World Health Organization.

86

Worlth Health Organization & UNICEF (2010). Progress on Sanitation and Drinking-Water,

2010 Update. Geneva, World Health Organization.

World Health Organization (2011). Guidelines for Drinking-water Quality (4th ed.). Geneva:

World Health Organization.

World Health Organization & UNICEF (2006). Meeting the MDG drinking water and sanitation

target: the urban and rural challenge of the decade. Geneva: World Health

Organization.

World Health Organization (2005). The World Health Report 2005 – Make Every Mother and

Child Count. Geneva: World Health Organization.

Yoon, J. Y., Kim, K. H., Choi, S. W., Kim, J. H., Kim, W. S. (2001). Effects of Surface

Characteristics on Non-Specific Agglutination in Latex Immunoagglutination Antibody

Assay. Colloids and Surfaces B: Biointerfaces, 27, 3-9.

Yu, H., & Bruno, J. G. (1995). Immunomagnetic-electrochemiluminescent Detection of

Escherichia coli O157 and Salmonella typhimurium in Foods and Environmental Water

Samples. Applied and Environmental Microbiology, 62, 587-592.

87

APPENDIX

A.1 Per Test Cost Determination of Novel Test Method

Material Supplier Bulk

Cost ($)

Bulk

Quantity

Amount per

Test

Test/Bulk

Quantity

Cost per Test ($)a

Media (TSB) EMD Millipore 1828.35 25 kg 0.36 g 27778 0.062

15 mL Growth Tube VWR 173.24 500 1 500 0.346

Antibody abcam 325.00 2 mg 0.3 μg 6667 0.048

Membrane Filter Membrane Solutions 211.16 600 1 600 0.352

Microparticles Bangs Labs 596.00 5 g 7.5 μg 666667 0.001

Tween20 EMD Millipore 59.00 1 L 0.0075 μL 130 mil 0.000

Total per test 0.809

Note: a Each test consists of one membrane filtration growth tube and three replicate agglutination tests.

88

A.2 Results of Growth and Recovery Trials

GES

Buffer

GES

Volume

(mL)

Tube

Size

(mL)

Tween20

(%)

TSB

(g/L) Action FC

TSC (cfu) at time (hr):

0 1.5 3 4.5 6 9

PBS 10 15 0.5 30 20 s Shake 51

2242424

PBS 10 15 0.5 30 20 s Shake 51

1575758

PBS 10 15 0.0 30 20 s Shake 51

1818182

PBS 10 15 0.0 30 20 s Shake 51

818182

PBS 10 15 0.5 30 20 s Shake 510

16666667

PBS 10 15 0.5 30 20 s Shake 510

9393939

PBS 10 15 0.0 30 20 s Shake 510

20303030

PBS 10 15 0.0 30 20 s Shake 510

13636364

PBS 10 15 0.5 30 20 s Shake 5100

342424242

PBS 10 15 0.5 30 20 s Shake 5100

345454545

PBS 10 15 0.0 30 20 s Shake 5100

330303030

PBS 10 15 0.0 30 20 s Shake 5100

287878788

PBS 10 15 0.5 30 20 s Shake 4563 2125

21125

4625000 1950000000

PBS 10 15 0.5 30 None 4563 625

15125

9125000 2562500000

PBS 10 15 0.5 30 20 s Shake 4563 2250

30500

6625000 2975000000

PBS 10 15 0.5 30 None 4563 125

22250

11375000 2475000000

PBS 10 15 0.5 30 20 s Shake 4563 2500

33500

11125000 2912500000

PBS 10 15 0.5 30 None 4563 375

11375

4375000 2250000000

PBS 10 15 0.5 30 20 s Shake 4563 2125

36375

14625000 3062500000

PBS 10 15 0.5 30 None 4563 250

15500

7875000 3175000000

PBS 10 15 0.5 30 20 s Shake 99 80

1125

272727 87837838

PBS 10 15 0.5 15 20 s Shake 99 78

875

90909 65315315

PBS 10 15 0.0 30 20 s Shake 99 100

1750

363636 101351351

PBS 10 15 0.0 15 20 s Shake 99 57

750

136364 9009009

PBS 10 15 0.0 15 20 s Shake 99 68

375

68182 20270270

PBS 10 15 0.0 30 20 s Shake 99 61

750

227273 92342342

89

GES

Buffer

GES

Volume

(mL)

Tube

Size

(mL)

Tween20

(%)

TSB

(g/L) Action FC

TSC (cfu) at time (hr):

0 1.5 3 4.5 6 9

PBS 10 15 0.5 15 20 s Shake 99 73

625

113636 20270270

PBS 10 15 0.5 30 20 s Shake 99 87

1500

295455 112612613

PBS 10 15 0.5 30 20 s Shake 99 68

875

250000 54054054

PBS 10 15 0.5 15 20 s Shake 99 76

750

90909 54054054

PBS 10 15 0.0 30 20 s Shake 99 65

625

227273 92342342

PBS 10 15 0.0 15 20 s Shake 99 59

125

90909 11261261

PBS 5 15 0.5 30 20 s Shake 99 45

250

45455 3378378

PBS 5 15 0.5 15 20 s Shake 99 50

688

79545 15765766

PBS 5 15 0.0 30 20 s Shake 99 50

250

68182 15765766

PBS 5 15 0.0 15 20 s Shake 99 50

438

56818 27027027

PBS 5 15 0.0 15 20 s Shake 99 54

563

68182 15765766

PBS 5 15 0.0 30 20 s Shake 99 65

125

90909 29279279

PBS 5 15 0.5 15 20 s Shake 99 58

125

56818 9009009

PBS 5 15 0.5 30 20 s Shake 99 63

188

79545 7882883

PBS 5 15 0.5 30 20 s Shake 99 60

750

147727 22522523

PBS 5 15 0.5 15 20 s Shake 99 71

750

79545 22522523

PBS 5 15 0.0 30 20 s Shake 99 34

125

102273 18018018

PBS 5 15 0.0 15 20 s Shake 99 62

313

56818 14639640

PBS 10 50 0.5 30 20 s Shake 119 75

766

42793

PBS 10 50 0.5 30 20 s Shake 119 113

586

51802

PBS 10 15 0.5 30 20 s Shake 119 63

653

94595

PBS 10 15 0.5 30 20 s Shake 119 150

676

81081

PBS 10 50 0.5 30 None 119 13

405

51802

PBS 10 50 0.5 30 None 119 13

270

51802

PBS 10 15 0.5 30 None 119 38

563

103604

PBS 10 15 0.5 30 None 119 13

541

63063

PBS 10 50 0.5 30 20 s Shake 119 113

811

96847

PBS 10 50 0.5 30 20 s Shake 119 75

901

105856

90

GES

Buffer

GES

Volume

(mL)

Tube

Size

(mL)

Tween20

(%)

TSB

(g/L) Action FC

TSC (cfu) at time (hr):

0 1.5 3 4.5 6 9

PBS 10 15 0.5 30 20 s Shake 119 63

698

132883

PBS 10 15 0.5 30 20 s Shake 119 50

473

123874

PBS 10 50 0.5 30 None 119 13

428

87838

PBS 10 50 0.5 30 None 119 13

743

94595

PBS 10 15 0.5 30 None 119 63

586

105856

PBS 10 15 0.5 30 None 119 25

698

92342

PBS 10 50 0.5 30 20 s Shake 325 313

2500

850000

PBS 10 50 0.5 30 20 s Shake 325 425

1750

475000

PBS 10 50 0.5 30 20 s Shake 325 275

2500

500000

PBS 10 50 0.5 15 20 s Shake 325 350

3500

450000

PBS 10 50 0.5 15 20 s Shake 325 325

1750

225000

PBS 10 50 0.5 15 20 s Shake 325 225

2250

500000

PBS 10 50 1.0 15 20 s Shake 325 325

4250

425000

PBS 10 50 1.0 15 20 s Shake 325 425

4250

550000

PBS 10 50 1.0 15 20 s Shake 325 275

2500

250000

PBS 10 50 0.5 30 20 s Shake 900 1017

CPB pH=6 10 50 0.5 30 20 s Shake 900 1067

CPB pH=5 10 50 0.5 30 20 s Shake 900 667

CPB pH=5 10 50 0.5 30 20 s Shake 900 767

CPB pH=6 10 50 0.5 30 20 s Shake 900 900

PBS 10 50 0.5 30 20 s Shake 900 733

PBS 10 50 0.5 30 20 s Shake 900 833

CPB pH=6 10 50 0.5 30 20 s Shake 900 800

CPB pH=5 10 50 0.5 30 20 s Shake 900 767

CPB pH=5 10 50 0.5 30 20 s Shake 900 833

CPB pH=6 10 50 0.5 30 20 s Shake 900 800

PBS 10 50 0.5 30 20 s Shake 900 883

DI 40 50 0.0 0 60 s Vortex 2439394 884848

91

GES

Buffer

GES

Volume

(mL)

Tube

Size

(mL)

Tween20

(%)

TSB

(g/L) Action FC

TSC (cfu) at time (hr):

0 1.5 3 4.5 6 9

DI 40 50 0.0 0 60 s Shake 2439394 878788

DI 40 50 0.0 0 20 s Vortex 2439394 812121

DI 40 50 0.0 0 60 s Shake 2439394 806061

DI 40 50 0.0 0 20 s Vortex 2439394 733333

DI 40 50 0.0 0 20 s Shake 2439394 690909

DI 40 50 0.0 0 60 s Shake 2439394 630303

DI 40 50 0.0 0 20 s Shake 2439394 545455

DI 40 50 0.0 0 None 2439394 139394

DI 40 50 0.0 0 None 2439394 39039

DI 40 50 0.0 0 20 s Shake 1878788 400000

DI 40 50 0.0 0 20 s Shake 1878788 351515

DI 40 50 0.0 0 20 s Shake 1878788 339394

DI 40 50 0.0 0 20 s Shake 1878788 309091

DI 40 50 0.0 0 20 s Shake 1878788 303030

DI 40 50 0.0 0 20 s Shake 1878788 303030

DI 40 50 0.0 0 20 s Shake 1878788 290909

DI 40 50 0.0 0 20 s Shake 1878788 272727

DI 40 50 0.0 0 20 s Shake 1878788 224242

DI 40 50 0.0 0 20 s Shake 1878788 175758

DI 40 50 1.0 0 20 s Shake 5416667 2066667

DI 40 50 1.0 0 20 s Shake 5416667 1666667

DI 40 50 0.0 0 20 s Shake 5416667 812121

PBS 40 50 1.0 0 20 s Shake 5416667 4000000

PBS 40 50 1.0 0 20 s Shake 5416667 1933333

DI 40 50 0.0 0 20 s Shake 5416667 648485

PBS 40 50 0.0 0 20 s Shake 5416667 800000

PBS 40 50 0.0 0 20 s Shake 5416667 703030

PBS 40 50 1.0 0 20 s Shake 863636 624242

92

GES

Buffer

GES

Volume

(mL)

Tube

Size

(mL)

Tween20

(%)

TSB

(g/L) Action FC

TSC (cfu) at time (hr):

0 1.5 3 4.5 6 9

PBS 40 50 1.0 0 20 s Shake 863636 551515

DI 40 50 0.0 0 20 s Shake 863636 218182

DI 40 50 0.0 0 20 s Shake 863636 181818

PBS 40 50 0.5 0 20 s Shake 863636 618182

PBS 40 50 0.5 0 20 s Shake 863636 496970

PBS 40 50 1.0 0 20 s Shake 1416667 1090909

PBS 40 50 1.0 0 20 s Shake 1416667 933333

PBS 40 50 1.0 0 20 s Shake 1416667 781818

PBS 40 50 1.0 0 20 s Shake 1416667 690909

PBS 40 50 0.5 0 20 s Shake 1416667 1139394

PBS 40 50 0.5 0 20 s Shake 1416667 1054545

PBS 40 50 0.5 0 20 s Shake 1416667 830303

PBS 40 50 0.5 0 20 s Shake 1416667 703030

PBS 40 50 0.0 0 20 s Shake 1416667 406061

PBS 40 50 0.0 0 20 s Shake 1416667 169697

PBS 10 15 2.0 0 20 s Shake 1007576 483333

PBS 10 15 2.0 0 20 s Shake 1007576 383333

PBS 10 15 1.0 0 20 s Shake 1007576 666667

PBS 10 15 1.0 0 20 s Shake 1007576 466667

PBS 10 15 0.5 0 20 s Shake 1007576 500000

PBS 10 15 0.5 0 20 s Shake 1007576 416667

PBS 10 15 0.1 0 20 s Shake 1007576 600000

PBS 10 15 0.1 0 20 s Shake 1007576 516667

PBS 40 50 0.0 0 20 s Shake 1007576 212121

PBS 40 50 0.0 0 20 s Shake 1007576 163636

PBS 10 15 0.0 0 20 s Shake 1007576 93939

PBS 10 15 0.0 0 20 s Shake 1007576 59091

PBS 10 50 1.0 0 20 s Shake 878788 683333

93

GES

Buffer

GES

Volume

(mL)

Tube

Size

(mL)

Tween20

(%)

TSB

(g/L) Action FC

TSC (cfu) at time (hr):

0 1.5 3 4.5 6 9

PBS 10 15 1.0 0 20 s Shake 878788 433333

PBS 10 15 0.5 0 20 s Shake 878788 650000

PBS 10 50 0.5 0 20 s Shake 878788 383333

PBS 10 15 0.1 0 20 s Shake 878788 500000

PBS 10 50 0.1 0 20 s Shake 878788 433333

PBS 10 50 0.0 0 20 s Shake 878788 266667

PBS 10 15 0.0 0 20 s Shake 878788 216667

PBS 10 50 1.0 0 20 s Shake 1141667 516667

PBS 10 50 1.0 15 20 s Shake 1141667 850000

PBS 10 50 1.0 30 20 s Shake 1141667 750000

PBS 10 50 1.0 15 20 s Shake 1141667 566667

PBS 10 50 1.0 15 20 s Shake 1141667 516667

PBS 10 50 0.0 15 20 s Shake 1141667 183333

PBS 10 50 0.0 0 20 s Shake 1141667 166667

PBS 10 50 0.0 30 20 s Shake 1141667 133333

PBS 10 50 0.0 15 20 s Shake 1141667 133333

PBS 10 50 0.0 15 20 s Shake 1141667 100000

PBS 10 15 1.0 15 20 s Shake 9250 8667

PBS 10 50 0.5 15 20 s Shake 9250 7333

PBS 10 50 1.0 30 20 s Shake 9250 6833

PBS 10 50 1.0 15 20 s Shake 9250 6833

PBS 10 50 0.5 30 20 s Shake 9250 6667

PBS 10 15 0.5 30 20 s Shake 9250 6167

PBS 10 15 0.5 15 20 s Shake 9250 5333

PBS 10 15 1.0 30 20 s Shake 9250 5000

PBS 10 50 0.5 0 20 s Shake 3167 2500

PBS 10 15 0.5 0 20 s Shake 3167 1167

PBS 10 50 0.1 0 20 s Shake 3167 1833

94

GES

Buffer

GES

Volume

(mL)

Tube

Size

(mL)

Tween20

(%)

TSB

(g/L) Action FC

TSC (cfu) at time (hr):

0 1.5 3 4.5 6 9

PBS 10 15 0.1 0 20 s Shake 3167 833

PBS 10 50 0.5 30 20 s Shake 7750 7333

PBS 10 15 1.0 30 20 s Shake 7750 6667

PBS 10 50 1.0 15 20 s Shake 7750 6500

PBS 10 50 0.5 0 20 s Shake 3167 2500

PBS 10 15 0.5 0 20 s Shake 3167 1833

PBS 10 15 0.5 0 20 s Shake 3167 1833

PBS 10 50 0.5 15 20 s Shake 7750 4167

PBS 10 50 0.1 0 20 s Shake 3167 1500

PBS 10 15 0.5 15 20 s Shake 7750 3667

PBS 10 50 1.0 30 20 s Shake 7750 3500

PBS 10 15 1.0 15 20 s Shake 7750 3333

PBS 10 50 0.1 0 20 s Shake 3167 1167

PBS 10 15 0.5 30 20 s Shake 7750 2833

PBS 10 50 0.5 0 20 s Shake 3167 833

PBS 10 15 0.1 0 20 s Shake 3167 667

PBS 10 15 0.1 0 20 s Shake 3167 167

PBS 10 50 0.5 0 20 s Shake 367 217

PBS 10 15 0.5 0 20 s Shake 367 117

PBS 10 50 0.1 0 20 s Shake 367 133

PBS 10 15 0.1 0 20 s Shake 367 133

PBS 10 50 0.5 0 20 s Shake 367 233

PBS 10 50 0.5 0 20 s Shake 367 183

PBS 10 15 0.5 0 20 s Shake 367 167

PBS 10 15 0.1 0 20 s Shake 367 150

PBS 10 50 0.1 0 20 s Shake 367 150

PBS 10 15 0.5 0 20 s Shake 367 133

PBS 10 50 0.1 0 20 s Shake 367 83

95

GES

Buffer

GES

Volume

(mL)

Tube

Size

(mL)

Tween20

(%)

TSB

(g/L) Action FC

TSC (cfu) at time (hr):

0 1.5 3 4.5 6 9

PBS 10 15 0.1 0 20 s Shake 367 50

PBS 10 50 1.0 15 20 s Shake 7750 6500 3939 36364 181818 10909091

PBS 10 15 1.0 30 20 s Shake 7750 6667 5758 66667 424242 12727273

PBS 10 15 0.5 30 20 s Shake 7750 2833 5455 45455 272727 9393939

PBS 10 50 0.5 15 20 s Shake 7750 4167 10000 39394 303030 9393939

PBS 10 50 0.5 30 20 s Shake 7750 7333 6970 21212 121212 6666667

PBS 10 15 0.5 15 20 s Shake 7750 3667 6364 24242 272727 9090909

PBS 10 15 1.0 15 20 s Shake 7750 3333 3939 42424 242424 11515152

PBS 10 50 1.0 30 20 s Shake 7750 3500 7879 33333 242424 7272727

PBS 10 50 1.0 30 20 s Shake 9250 6833 10152 48485 242424 9696970

PBS 10 15 1.0 15 20 s Shake 9250 8667 9091 30303 696970 16969697

PBS 10 15 0.5 15 20 s Shake 9250 5333 6212 54545 575758 8484848

PBS 10 50 0.5 30 20 s Shake 9250 6667 9394 42424 333333 5757576

PBS 10 50 0.5 15 20 s Shake 9250 7000 11061 27273 606061 6060606

PBS 10 15 0.5 30 20 s Shake 9250 6167 8636 42424 606061 10909091

96

A.3 Results of Individual Latex Agglutination Tests: Streptavidin Coated Particles

Particle

Type Batch

Time

(h) RPM

Immobilization

Buffer a

Ab

(mg/m2)

Particle

Mass (mg)

Temp

(° C)

Post

Wash

Reaction

Buffer

Reaction With:

DI Famp

C1 1 1 30 PBS 3.5 1 RT DI DI 1 1

C1 2 1 30 PBS 0.7 1 RT DI DI 1 1

C1 3 1 30 PBS 0.14 1 RT DI DI 1 1

CM01N 4 1 30 PBS 3.5 1 RT DI DI 1 3

CM01N 5 1 30 PBS 0.7 1 RT DI DI 1 3

CM01N 6 1 30 PBS 0.14 1 RT DI DI 1 3

PMS1N 7 1 30 PBS 3.5 1 RT DI DI 0 0

PMS1N 8 1 30 PBS 0.7 1 RT DI DI 0 0

PMS1N 9 1 30 PBS 0.14 1 RT DI DI 0 0

CP01F 10 1 30 PBS 3.5 1 RT DI DI 0 0

CP01F 11 1 30 PBS 0.7 1 RT DI DI 0 0

CP01F 12 1 30 PBS 0.14 1 RT DI DI 0 0

C1 13 24 30 PBS 3.5 1 RT DI DI 0 0

C1 14 24 30 PBS 0.7 1 RT DI DI 0 0

C1 15 24 30 PBS 0.14 1 RT DI DI 0 0

CM01N 16 24 30 PBS 3.5 1 RT DI DI 1 3

CM01N 17 24 30 PBS 0.7 1 RT DI DI 1 3

CM01N 18 24 30 PBS 0.14 1 RT DI DI 1 2.5

PMS1N 19 24 30 PBS 3.5 1 RT DI DI 0 0

PMS1N 20 24 30 PBS 0.7 1 RT DI DI 0 0

PMS1N 21 24 30 PBS 0.14 1 RT DI DI 0 0

CP01F 22 24 30 PBS 3.5 1 RT DI DI 0 0

CP01F 23 24 30 PBS 0.7 1 RT DI DI 0 0

CP01F 24 24 30 PBS 0.14 1 RT DI DI 0 0

C1 25 1 30 DI 3.5 1 RT DI DI 0 0

C1 26 1 30 DI 0.7 1 RT DI DI 0 0

97

Particle

Type Batch

Time

(h) RPM

Immobilization

Buffer a

Ab

(mg/m2)

Particle

Mass (mg)

Temp

(° C)

Post

Wash

Reaction

Buffer

Reaction With:

DI Famp

C1 27 1 30 DI 0.14 1 RT DI DI 0 0

CM01N 28 1 30 DI 3.5 1 RT DI DI 1 2

CM01N 29 1 30 DI 0.7 1 RT DI DI 1 2

CM01N 30 1 30 DI 0.14 1 RT DI DI 1 2

PMS1N 31 1 30 DI 3.5 1 RT DI DI 0 0

PMS1N 32 1 30 DI 0.7 1 RT DI DI 0 0

PMS1N 33 1 30 DI 0.14 1 RT DI DI 0 0

CP01F 34 1 30 DI 3.5 1 RT DI DI 0 0

CP01F 35 1 30 DI 0.7 1 RT DI DI 0 0

CP01F 36 1 30 DI 0.14 1 RT DI DI 0 0

C1 37 24 30 DI 3.5 1 RT DI DI 0 0

C1 38 24 30 DI 0.7 1 RT DI DI 0 0

C1 39 24 30 DI 0.14 1 RT DI DI 0 0

CM01N 40 24 30 DI 3.5 1 RT DI DI 1 2

CM01N 41 24 30 DI 0.7 1 RT DI DI 1 2

CM01N 42 24 30 DI 0.14 1 RT DI DI 1 1.5

PMS1N 43 24 30 DI 3.5 1 RT DI DI 0 0

PMS1N 44 24 30 DI 0.7 1 RT DI DI 0 0

PMS1N 45 24 30 DI 0.14 1 RT DI DI 0 0

CP01F 46 24 30 DI 3.5 1 RT DI DI 0 0

CP01F 47 24 30 DI 0.7 1 RT DI DI 0 0

CP01F 48 24 30 DI 0.14 1 RT DI DI 0 0

C1 25 1 30 DI 3.5 1 37 DI DI 0 0

C1 26 1 30 DI 0.7 1 37 DI DI 0 0

C1 27 1 30 DI 0.14 1 37 DI DI 0 0

CM01N 28 1 30 DI 3.5 1 37 DI DI 1 1

CM01N 29 1 30 DI 0.7 1 37 DI DI 1 1

CM01N 30 1 30 DI 0.14 1 37 DI DI 1 1

98

Particle

Type Batch

Time

(h) RPM

Immobilization

Buffer a

Ab

(mg/m2)

Particle

Mass (mg)

Temp

(° C)

Post

Wash

Reaction

Buffer

Reaction With:

DI Famp

PMS1N 31 1 30 DI 3.5 1 37 DI DI 0 0

PMS1N 32 1 30 DI 0.7 1 37 DI DI 0 0

PMS1N 33 1 30 DI 0.14 1 37 DI DI 0 0

CP01F 34 1 30 DI 3.5 1 37 DI DI 0 0

CP01F 35 1 30 DI 0.7 1 37 DI DI 0 0

CP01F 36 1 30 DI 0.14 1 37 DI DI 0 0

C1 37 24 30 DI 3.5 1 37 DI DI 0 0

C1 38 24 30 DI 0.7 1 37 DI DI 0 0

C1 39 24 30 DI 0.14 1 37 DI DI 0 0

CM01N 40 24 30 DI 3.5 1 37 DI DI 1 1

CM01N 41 24 30 DI 0.7 1 37 DI DI 1 1

CM01N 42 24 30 DI 0.14 1 37 DI DI 1 1

PMS1N 43 24 30 DI 3.5 1 37 DI DI 0 0

PMS1N 44 24 30 DI 0.7 1 37 DI DI 0 0

PMS1N 45 24 30 DI 0.14 1 37 DI DI 0 0

CP01F 46 24 30 DI 3.5 1 37 DI DI 0 0

CP01F 47 24 30 DI 0.7 1 37 DI DI 0 0

CP01F 48 24 30 DI 0.14 1 37 DI DI 0 0

CM01N 49 0.5 30 PBS 3.5 1 RT DI DI 1 2

CM01N 50 0.5 30 PBS 1.25 1 RT DI DI 1 2

CM01N 51 0.5 30 PBS 0.63 1 RT DI DI 1 2

CM01N 52 0.5 30 PBS 0.1 1 RT DI DI 1 1

CM01N 53 1 30 PBS 3.5 1 RT DI DI 1 3

CM01N 54 1 30 PBS 1.25 1 RT DI DI 1 3

CM01N 55 1 30 PBS 0.63 1 RT DI DI 1 2

CM01N 56 1 30 PBS 0.1 1 RT DI DI 1 2

CM01N 57 0.5 30 BBS 3.5 1 RT DI DI 1 3

CM01N 58 0.5 30 BBS 1.25 1 RT DI DI 1 2

99

Particle

Type Batch

Time

(h) RPM

Immobilization

Buffer a

Ab

(mg/m2)

Particle

Mass (mg)

Temp

(° C)

Post

Wash

Reaction

Buffer

Reaction With:

DI Famp

CM01N 59 0.5 30 BBS 0.63 1 RT DI DI 1 2

CM01N 60 0.5 30 BBS 0.1 1 RT DI DI 1 3

CM01N 61 1 30 BBS 3.5 1 RT DI DI 1 3

CM01N 62 1 30 BBS 1.25 1 RT DI DI 1 3

CM01N 63 1 30 BBS 0.63 1 RT DI DI 1 2

CM01N 64 1 30 BBS 0.1 1 RT DI DI 1 2

100

A.4 Results of Individual Latex Agglutination Tests: PS Particles

Particle

Type Batch

Time

(h) RPM

Immobilization

Buffer a Ab

Ab

(mg/m2)

Temp

(° C) Post Wash Reaction Buffer

Reaction With:

DI O1:K1:H7 Famp

PS03N 1 2 10 DI A 3.5 RT 2x DI DI 1 1 1

PS03N 2 2 10 DI A 0.88 RT 2x DI DI 1 1 1

PS03N 3 2 10 DI A 0.22 RT 2x DI DI 1 1 1

PS03N 4 2 10 DI B 3.5 RT 2x DI DI 1 1 1

PS03N 5 2 10 DI B 0.88 RT 2x DI DI 1 1 1

PS03N 6 2 10 DI B 0.22 RT 2x DI DI 1 1 1

PS03N 7 2 10 DI C 3.5 RT 2x DI DI 1 1 1

PS03N 8 2 10 DI C 0.88 RT 2x DI DI 1 1 1

PS03N 9 2 10 DI C 0.22 RT 2x DI DI 1 1 1

PS03N 10 2 10 DI D 3.5 RT 2x DI DI 1 1 1

PS03N 11 2 10 DI D 0.88 RT 2x DI DI 1 1 1

PS03N 12 2 10 DI D 0.22 RT 2x DI DI 1 1 1

PS03N 13 24 10 DI A 3.5 4 2x DI DI 1 1 1

PS03N 14 24 10 DI A 0.88 4 2x DI DI 1 1 1

PS03N 15 24 10 DI A 0.22 4 2x DI DI 1 1 1

PS03N 16 24 10 DI B 3.5 4 2x DI DI 1 1 1

PS03N 17 24 10 DI B 0.88 4 2x DI DI 1 1 1

PS03N 18 24 10 DI B 0.22 4 2x DI DI 1 1 1

PS03N 19 24 10 DI C 3.5 4 2x DI DI 1 1 1

PS03N 20 24 10 DI C 0.88 4 2x DI DI 1 1 1

PS03N 21 24 10 DI C 0.22 4 2x DI DI 1 1 1

PS03N 22 24 10 DI D 3.5 4 2x DI DI 1 1 1

PS03N 23 24 10 DI D 0.88 4 2x DI DI 1 1 1

PS03N 24 24 10 DI D 0.22 4 2x DI DI 1 1 1

PS03N 25 2 10 PBS A 3.5 RT 2x DI DI 1 1 1

101

Particle

Type Batch

Time

(h) RPM

Immobilization

Buffer a Ab

Ab

(mg/m2)

Temp

(° C) Post Wash Reaction Buffer

Reaction With:

DI O1:K1:H7 Famp

PS03N 26 2 10 PBS A 0.88 RT 2x DI DI 1 1 1

PS03N 27 2 10 PBS A 0.22 RT 2x DI DI 1 1 1

PS03N 28 2 10 PBS B 3.5 RT 2x DI DI 1 1 1

PS03N 29 2 10 PBS B 0.88 RT 2x DI DI 1 1 1

PS03N 30 2 10 PBS B 0.22 RT 2x DI DI 1 1 1

PS03N 31 2 10 PBS C 3.5 RT 2x DI DI 1 1 1

PS03N 32 2 10 PBS C 0.88 RT 2x DI DI 1 1 1

PS03N 33 2 10 PBS C 0.22 RT 2x DI DI 1 1 1

PS03N 34 2 10 PBS D 3.5 RT 2x DI DI 1 1 1

PS03N 35 2 10 PBS D 0.88 RT 2x DI DI 1 1 1

PS03N 36 2 10 PBS D 0.22 RT 2x DI DI 1 1 1

PS03N 37 2 10 BBS A 3.5 RT 2x DI DI 1 1 1

PS03N 38 2 10 BBS A 0.88 RT 2x DI DI 1 1 1

PS03N 39 2 10 BBS A 0.22 RT 2x DI DI 1 1 1

PS03N 40 2 10 BBS B 3.5 RT 2x DI DI 1 1 1

PS03N 41 2 10 BBS B 0.88 RT 2x DI DI 1 1 1

PS03N 42 2 10 BBS B 0.22 RT 2x DI DI 1 1 1

PS03N 43 2 10 BBS C 3.5 RT 2x DI DI 1 1 1

PS03N 44 2 10 BBS C 0.88 RT 2x DI DI 1 1 1

PS03N 45 2 10 BBS C 0.22 RT 2x DI DI 1 1 1

PS03N 46 2 10 BBS D 3.5 RT 2x DI DI 1 1 1

PS03N 47 2 10 BBS D 0.88 RT 2x DI DI 1 1 1

PS03N 48 2 10 BBS D 0.22 RT 2x DI DI 1 1 1

PS03N 49 24 60 DI A 3.5 RT 2x DI DI 1 1 1

PS03N 50 24 60 DI A 0.88 RT 2x DI DI 1 1 1

PS03N 51 24 60 DI A 0.22 RT 2x DI DI 1 1 1

PS03N 52 24 60 DI B 3.5 RT 2x DI DI 1 1 1

PS03N 53 24 60 DI B 0.88 RT 2x DI DI 1 1 1

102

Particle

Type Batch

Time

(h) RPM

Immobilization

Buffer a Ab

Ab

(mg/m2)

Temp

(° C) Post Wash Reaction Buffer

Reaction With:

DI O1:K1:H7 Famp

PS03N 54 24 60 DI B 0.22 RT 2x DI DI 1 1 1

PS03N 55 24 60 DI C 3.5 RT 2x DI DI 1 1 1

PS03N 56 24 60 DI C 0.88 RT 2x DI DI 1 1 1

PS03N 57 24 60 DI C 0.22 RT 2x DI DI 1 1 1

PS03N 58 24 60 DI D 3.5 RT 2x DI DI 1 1 1

PS03N 59 24 60 DI D 0.88 RT 2x DI DI 1 1 1

PS03N 60 24 60 DI D 0.22 RT 2x DI DI 1 1 1

PS03N 61 24 60 PBS A 3.5 RT 2x DI DI 1 1 1

PS03N 62 24 60 PBS A 0.88 RT 2x DI DI 1 1 1

PS03N 63 24 60 PBS A 0.22 RT 2x DI DI 1 1 1

PS03N 64 24 60 PBS B 3.5 RT 2x DI DI 1 1 1

PS03N 65 24 60 PBS B 0.88 RT 2x DI DI 1 1 1

PS03N 66 24 60 PBS B 0.22 RT 2x DI DI 1 1 1

PS03N 67 24 60 PBS C 3.5 RT 2x DI DI 1 1 1

PS03N 68 24 60 PBS C 0.88 RT 2x DI DI 1 1 1

PS03N 69 24 60 PBS C 0.22 RT 2x DI DI 1 1 1

PS03N 70 24 60 PBS D 3.5 RT 2x DI DI 1 1 1

PS03N 71 24 60 PBS D 0.88 RT 2x DI DI 1 1 1

PS03N 72 24 60 PBS D 0.22 RT 2x DI DI 1 1 1

PS03N 73 2 60 DI A 3.5 RT None DI 1 1 1

PS03N 74 2 60 DI A 0.88 RT None DI 1 1 1

PS03N 75 2 60 DI A 0.22 RT None DI 1 1 1

PS03N 76 2 60 DI B 3.5 RT None DI 1 1 1

PS03N 77 2 60 DI B 0.88 RT None DI 1 1 1

PS03N 78 2 60 DI B 0.22 RT None DI 1 1 1

PS03N 79 2 60 DI C 3.5 RT None DI 1 1 1

PS03N 80 2 60 DI C 0.88 RT None DI 1 1 1

PS03N 81 2 60 DI C 0.22 RT None DI 1 1 1

103

Particle

Type Batch

Time

(h) RPM

Immobilization

Buffer a Ab

Ab

(mg/m2)

Temp

(° C) Post Wash Reaction Buffer

Reaction With:

DI O1:K1:H7 Famp

PS03N 82 2 60 DI D 3.5 RT None DI 1 1 1

PS03N 83 2 60 DI D 0.88 RT None DI 1 1 1

PS03N 84 2 60 DI D 0.22 RT None DI 1 1 1

PS03N 85 2 60 DI + T20 A 3.5 RT 2x DI DI 1 1 1

PS03N 86 2 60 DI + T20 A 0.88 RT 2x DI DI 1 1 1

PS03N 87 2 60 DI + T20 A 0.22 RT 2x DI DI 1 1 1

PS03N 88 2 60 DI + T20 B 3.5 RT 2x DI DI 1 1 1

PS03N 89 2 60 DI + T20 B 0.88 RT 2x DI DI 1 1 1

PS03N 90 2 60 DI + T20 B 0.22 RT 2x DI DI 1 1 1

PS03N 91 2 60 DI + T20 C 3.5 RT 2x DI DI 1 1 1

PS03N 92 2 60 DI + T20 C 0.88 RT 2x DI DI 1 1 1

PS03N 93 2 60 DI + T20 C 0.22 RT 2x DI DI 1 1 1

PS03N 94 2 60 DI + T20 D 3.5 RT 2x DI DI 1 1 1

PS03N 95 2 60 DI + T20 D 0.88 RT 2x DI DI 1 1 1

PS03N 96 2 60 DI + T20 D 0.22 RT 2x DI DI 1 1 1

PS03N 97 2 60 DI E 3.5 RT 2x DI DI 1 1 1

PS03N 98 2 60 DI E 0.88 RT 2x DI DI 1 1 1

PS03N 99 2 60 DI E 0.22 RT 2x DI DI 1 1 1

PS03N 100 2 60 PBS E 3.5 RT 2x DI DI 1 1 1

PS03N 101 2 60 PBS E 0.88 RT 2x DI DI 1 1 1

PS03N 102 2 60 PBS E 0.22 RT 2x DI DI 1 1 1

PS03N 103 24 45 DI A 3.5 35 SW DI 1 1 1

PS03N 104 24 45 DI B 3.5 35 SW DI 1 1 1

PS03N 105 24 45 DI C 3.5 35 SW DI 1 1 1

PS03N 106 24 45 DI D 3.5 35 SW DI 1 1 1

PS03N 107 24 45 DI E 3.5 35 SW DI 1 1 1

PS03N 108 24 45 DI + T20 A 3.5 35 SW + T20 DI 0 0 0

PS03N 109 24 45 DI + T20 B 3.5 35 SW + T20 DI 0 0 0

104

Particle

Type Batch

Time

(h) RPM

Immobilization

Buffer a Ab

Ab

(mg/m2)

Temp

(° C) Post Wash Reaction Buffer

Reaction With:

DI O1:K1:H7 Famp

PS03N 110 24 45 DI + T20 C 3.5 35 SW + T20 DI 0 0 0

PS03N 111 24 45 DI + T20 D 3.5 35 SW + T20 DI 0 0 0

PS03N 112 24 45 DI + T20 E 3.5 35 SW + T20 DI 0 0 0

PS03N 113 24 45 PBS A 3.5 35 PBS DI 1 1 1

PS03N 114 24 45 PBS B 3.5 35 PBS DI 1 1 1

PS03N 115 24 45 PBS C 3.5 35 PBS DI 1 1 1

PS03N 116 24 45 PBS D 3.5 35 PBS DI 1 1 1

PS03N 117 24 45 PBS E 3.5 35 PBS DI 1 1 1

PS03N 118 24 45 PBS + T20 A 3.5 35 PBS + T20 DI 0 0 0

PS03N 119 24 45 PBS + T20 B 3.5 35 PBS + T20 DI 0 0 0

PS03N 120 24 45 PBS + T20 C 3.5 35 PBS + T20 DI 0 0 0

PS03N 121 24 45 PBS + T20 D 3.5 35 PBS + T20 DI 0 0 0

PS03N 122 24 45 PBS + T20 E 3.5 35 PBS + T20 DI 0 0 0

PS03N 123 24 45 DI A 0.88 35 SW DI 1 1 1

PS03N 124 24 45 DI B 0.88 35 SW DI 1 1 1

PS03N 125 24 45 DI C 0.88 35 SW DI 1 1 1

PS03N 126 24 45 DI D 0.88 35 SW DI 1 1 1

PS03N 127 24 45 DI E 0.88 35 SW DI 1 1 1

PS03N 128 24 45 DI + T20 A 0.88 35 SW + T20 DI 0 0 0

PS03N 129 24 45 DI + T20 B 0.88 35 SW + T20 DI 0 0 0

PS03N 130 24 45 DI + T20 C 0.88 35 SW + T20 DI 0 0 0

PS03N 131 24 45 DI + T20 D 0.88 35 SW + T20 DI 0 0 0

PS03N 132 24 45 DI + T20 E 0.88 35 SW + T20 DI 0 0 0

PS03N 133 24 45 PBS A 0.88 35 PBS DI 1.5 1.5 1

PS03N 134 24 45 PBS B 0.88 35 PBS DI 1 1 1

PS03N 135 24 45 PBS C 0.88 35 PBS DI 1 1 1

PS03N 136 24 45 PBS D 0.88 35 PBS DI 1 1 1

PS03N 137 24 45 PBS E 0.88 35 PBS DI 1 1 1

105

Particle

Type Batch

Time

(h) RPM

Immobilization

Buffer a Ab

Ab

(mg/m2)

Temp

(° C) Post Wash Reaction Buffer

Reaction With:

DI O1:K1:H7 Famp

PS03N 138 24 45 PBS + T20 A 0.88 35 PBS + T20 DI 0 0 1

PS03N 139 24 45 PBS + T20 B 0.88 35 PBS + T20 DI 0 0 1

PS03N 140 24 45 PBS + T20 C 0.88 35 PBS + T20 DI 0 0 1

PS03N 141 24 45 PBS + T20 D 0.88 35 PBS + T20 DI 0 0 1

PS03N 142 24 45 PBS + T20 E 0.88 35 PBS + T20 DI 0 0 1

PS03N 143 24 45 PBS A 3.5 4 2x DI DI 1 1

PS03N 144 24 45 PBS A 0.88 4 2x DI DI 1 1

PS03N 145 24 45 PBS A 0.22 4 2x DI DI 1 1

PS03N 146 24 45 PBS B 3.5 4 2x DI DI 1 1

PS03N 147 24 45 PBS B 0.88 4 2x DI DI 1 1

PS03N 148 24 45 PBS B 0.22 4 2x DI DI 1 1

PS03N 149 24 45 PBS C 3.5 4 2x DI DI 1 1

PS03N 150 24 45 PBS C 0.88 4 2x DI DI 1 1

PS03N 151 24 45 PBS C 0.22 4 2x DI DI 1 1

PS03N 152 24 45 PBS D 3.5 4 2x DI DI 1 1

PS03N 153 24 45 PBS D 0.88 4 2x DI DI 1 1

PS03N 154 24 45 PBS D 0.22 4 2x DI DI 1 1

PS03N 155 24 45 PBS E 3.5 4 2x DI DI 1 1

PS03N 156 24 45 PBS E 0.88 4 2x DI DI 1 1

PS03N 157 24 45 PBS E 0.22 4 2x DI DI 1 1

PS03N 158 24 45 BBS A 3.5 4 2x DI DI 1 1

PS03N 159 24 45 BBS A 0.88 4 2x DI DI 1 1

PS03N 160 24 45 BBS A 0.22 4 2x DI DI 1 1

PS03N 161 24 45 BBS B 3.5 4 2x DI DI 1 1

PS03N 162 24 45 BBS B 0.88 4 2x DI DI 1 1

PS03N 163 24 45 BBS B 0.22 4 2x DI DI 1 1

PS03N 164 24 45 BBS C 3.5 4 2x DI DI 1 1

PS03N 165 24 45 BBS C 0.88 4 2x DI DI 1 1

106

Particle

Type Batch

Time

(h) RPM

Immobilization

Buffer a Ab

Ab

(mg/m2)

Temp

(° C) Post Wash Reaction Buffer

Reaction With:

DI O1:K1:H7 Famp

PS03N 166 24 45 BBS C 0.22 4 2x DI DI 1 1

PS03N 167 24 45 BBS D 3.5 4 2x DI DI 1 1

PS03N 168 24 45 BBS D 0.88 4 2x DI DI 1 1

PS03N 169 24 45 BBS D 0.22 4 2x DI DI 1 1

PS03N 170 24 45 BBS E 3.5 4 2x DI DI 1 1

PS03N 171 24 45 BBS E 0.88 4 2x DI DI 1 1

PS03N 172 24 45 BBS E 0.22 4 2x DI DI 1 1

PS03N 173 24 45 PBS + T20 A 3.5 4 2x DI DI 0 0

PS03N 174 24 45 PBS + T20 A 0.88 4 2x DI DI 0 0

PS03N 175 24 45 PBS + T20 A 0.22 4 2x DI DI 0 0

PS03N 176 24 45 PBS + T20 B 3.5 4 2x DI DI 0.5 0.5

PS03N 177 24 45 PBS + T20 B 0.88 4 2x DI DI 0 0

PS03N 178 24 45 PBS + T20 B 0.22 4 2x DI DI 0 0

PS03N 179 24 45 PBS + T20 C 3.5 4 2x DI DI 0 0

PS03N 180 24 45 PBS + T20 C 0.88 4 2x DI DI 0 0

PS03N 181 24 45 PBS + T20 C 0.22 4 2x DI DI 0 0

PS03N 182 24 45 PBS + T20 D 3.5 4 2x DI DI 0 0

PS03N 183 24 45 PBS + T20 D 0.88 4 2x DI DI 0 0

PS03N 184 24 45 PBS + T20 D 0.22 4 2x DI DI 0 0

PS03N 185 24 45 PBS + T20 E 3.5 4 2x DI DI 0 0

PS03N 186 24 45 PBS + T20 E 0.88 4 2x DI DI 0 0

PS03N 187 24 45 PBS + T20 E 0.22 4 2x DI DI 0 0

PS03N 188 24 45 BBS + T20 A 3.5 4 2x DI DI 0.5 0.5

PS03N 189 24 45 BBS + T20 A 0.88 4 2x DI DI 0.5 0.5

PS03N 190 24 45 BBS + T20 A 0.22 4 2x DI DI 0.5 0.5

PS03N 191 24 45 BBS + T20 B 3.5 4 2x DI DI 0 0

PS03N 192 24 45 BBS + T20 B 0.88 4 2x DI DI 0 0

PS03N 193 24 45 BBS + T20 B 0.22 4 2x DI DI 0 0

107

Particle

Type Batch

Time

(h) RPM

Immobilization

Buffer a Ab

Ab

(mg/m2)

Temp

(° C) Post Wash Reaction Buffer

Reaction With:

DI O1:K1:H7 Famp

PS03N 194 24 45 BBS + T20 C 3.5 4 2x DI DI 0 0

PS03N 195 24 45 BBS + T20 C 0.88 4 2x DI DI 0 0

PS03N 196 24 45 BBS + T20 C 0.22 4 2x DI DI 0 0

PS03N 197 24 45 BBS + T20 D 3.5 4 2x DI DI 0 0

PS03N 198 24 45 BBS + T20 D 0.88 4 2x DI DI 0 0

PS03N 199 24 45 BBS + T20 D 0.22 4 2x DI DI 0 0

PS03N 200 24 45 BBS + T20 E 3.5 4 2x DI DI 0 0

PS03N 201 24 45 BBS + T20 E 0.88 4 2x DI DI 0 0

PS03N 202 24 45 BBS + T20 E 0.22 4 2x DI DI 0 0

PS03N 203 24 10 DI A 3.5 4 2x DI DI 1 1

PS03N 204 24 10 DI A 0.88 4 2x DI DI 1 1

PS03N 205 24 10 DI A 0.22 4 2x DI DI 1 1

PS03N 206 24 10 DI B 3.5 4 2x DI DI 1 1

PS03N 207 24 10 DI B 0.88 4 2x DI DI 1 1

PS03N 208 24 10 DI B 0.22 4 2x DI DI 1 1

PS03N 209 24 10 DI C 3.5 4 2x DI DI 1 1

PS03N 210 24 10 DI C 0.88 4 2x DI DI 1 1

PS03N 211 24 10 DI C 0.22 4 2x DI DI 1 1

PS03N 212 24 10 DI D 3.5 4 2x DI DI 1 1

PS03N 213 24 10 DI D 0.88 4 2x DI DI 1 1

PS03N 214 24 10 DI D 0.22 4 2x DI DI 0.5 0.5

PS03N 215 24 10 DI E 3.5 4 2x DI DI 1 1

PS03N 216 24 10 DI E 0.88 4 2x DI DI 1 1

PS03N 217 24 10 DI E 0.22 4 2x DI DI 1 1

PS03N 218 2 10 PBS + T20 E 3.5 RT 2x DI DI 1 1

PS03N 219 2 10 PBS + T20 E 0.88 RT 2x DI DI 1 1

PS03N 220 2 10 PBS + T20 E 0.22 RT 2x DI DI 1 1

PS03N 221 2 10 PBS + T20 E 0.055 RT 2x DI DI 1 1

108

Particle

Type Batch

Time

(h) RPM

Immobilization

Buffer a Ab

Ab

(mg/m2)

Temp

(° C) Post Wash Reaction Buffer

Reaction With:

DI O1:K1:H7 Famp

PS03N 222 2 10 PBS + T20 E 0.027 RT 2x DI DI 1 1

PS03N 223 2 10 PBS + T20 E 6.84E-03 RT 2x DI DI 1 1

PS03N 224 2 10 PBS + T20 E 1.71E-03 RT 2x DI DI 1 1

PS03N 225 2 10 PBS + T20 E 4.27E-04 RT 2x DI DI 1 1

PS03N 226 2 10 PBS + T20 E 1.07E-04 RT 2x DI DI 1 1

PS03N 227 24 10 PBS + T20 E 3.5 RT 2x DI DI 1.5 1.5

PS03N 228 24 10 PBS + T20 E 0.88 RT 2x DI DI 1 1

PS03N 229 24 10 PBS + T20 E 0.22 RT 2x DI DI 1 1

PS03N 230 24 10 PBS + T20 E 0.055 RT 2x DI DI 1 1

PS03N 231 24 10 PBS + T20 E 0.027 RT 2x DI DI 1 1

PS03N 232 24 10 PBS + T20 E 6.84E-03 RT 2x DI DI 1 1

PS03N 233 24 10 PBS + T20 E 1.71E-03 RT 2x DI DI 1 1

PS03N 234 24 10 PBS + T20 E 4.27E-04 RT 2x DI DI 1 1

PS03N 235 24 10 PBS + T20 E 1.07E-04 RT 2x DI DI 1 1

PS03N 236 24 10 PBS + T20 B 3.5 4 2x DI DI 0 0

PS03N 237 24 10 PBS + T20 B 0.88 4 2x DI DI 0 0

PS03N 238 24 10 PBS + T20 B 0.22 4 2x DI DI 0 0

PS03N 239 24 10 PBS + T20 B 0.055 4 2x DI DI 0 0

PS03N 240 24 10 PBS + T20 C 3.5 4 2x DI DI 0 0

PS03N 241 24 10 PBS + T20 C 0.88 4 2x DI DI 0 0

PS03N 242 24 10 PBS + T20 C 0.22 4 2x DI DI 0 0

PS03N 243 24 10 PBS + T20 C 0.055 4 2x DI DI 0 0

PS03N 244 24 10 PBS + T20 E 3.5 4 2x DI DI 0 0

PS03N 245 24 10 PBS + T20 E 0.88 4 2x DI DI 0 0

PS03N 246 24 10 PBS + T20 E 0.22 4 2x DI DI 0 0

PS03N 247 24 10 PBS + T20 E 0.055 4 2x DI DI 0 0

PS03N 248 24 10 BBS + T20 B 3.5 4 2x DI DI 0 0

PS03N 249 24 10 BBS + T20 B 0.88 4 2x DI DI 0 0

109

Particle

Type Batch

Time

(h) RPM

Immobilization

Buffer a Ab

Ab

(mg/m2)

Temp

(° C) Post Wash Reaction Buffer

Reaction With:

DI O1:K1:H7 Famp

PS03N 250 24 10 BBS + T20 B 0.22 4 2x DI DI 0 0

PS03N 251 24 10 BBS + T20 B 0.055 4 2x DI DI 0 0

PS03N 252 24 10 BBS + T20 C 3.5 4 2x DI DI 0 0

PS03N 253 24 10 BBS + T20 C 0.88 4 2x DI DI 0 0

PS03N 254 24 10 BBS + T20 C 0.22 4 2x DI DI 0 0

PS03N 255 24 10 BBS + T20 C 0.055 4 2x DI DI 0 0

PS03N 256 24 10 BBS + T20 E 3.5 4 2x DI DI 0 0

PS03N 257 24 10 BBS + T20 E 0.88 4 2x DI DI 0 0

PS03N 258 24 10 BBS + T20 E 0.22 4 2x DI DI 0 0

PS03N 259 24 10 BBS + T20 E 0.055 4 2x DI DI 0 0

PS03N 260 24 10 PBS + T20 B 3.5 4 2x DI PBS + T20 0 0

PS03N 261 24 10 PBS + T20 B 0.88 4 2x DI PBS + T20 0 0

PS03N 262 24 10 PBS + T20 B 0.22 4 2x DI PBS + T20 0 0

PS03N 263 24 10 PBS + T20 B 0.055 4 2x DI PBS + T20 0 0

PS03N 264 24 10 PBS + T20 C 3.5 4 2x DI PBS + T20 0 0

PS03N 265 24 10 PBS + T20 C 0.88 4 2x DI PBS + T20 0 0

PS03N 266 24 10 PBS + T20 C 0.22 4 2x DI PBS + T20 0 0

PS03N 267 24 10 PBS + T20 C 0.055 4 2x DI PBS + T20 0 0

PS03N 268 24 10 PBS + T20 E 3.5 4 2x DI PBS + T20 0 0

PS03N 269 24 10 PBS + T20 E 0.88 4 2x DI PBS + T20 0 0

PS03N 270 24 10 PBS + T20 E 0.22 4 2x DI PBS + T20 0 0

PS03N 271 24 10 PBS + T20 E 0.055 4 2x DI PBS + T20 0 0

PS03N 272 24 10 BBS + T20 B 3.5 4 2x DI PBS + T20 0 0

PS03N 273 24 10 BBS + T20 B 0.88 4 2x DI PBS + T20 0 0

PS03N 274 24 10 BBS + T20 B 0.22 4 2x DI PBS + T20 0 0

PS03N 275 24 10 BBS + T20 B 0.055 4 2x DI PBS + T20 0 0

PS03N 276 24 10 BBS + T20 C 3.5 4 2x DI PBS + T20 0 0

PS03N 277 24 10 BBS + T20 C 0.88 4 2x DI PBS + T20 0 0

110

Particle

Type Batch

Time

(h) RPM

Immobilization

Buffer a Ab

Ab

(mg/m2)

Temp

(° C) Post Wash Reaction Buffer

Reaction With:

DI O1:K1:H7 Famp

PS03N 278 24 10 BBS + T20 C 0.22 4 2x DI PBS + T20 0 0

PS03N 279 24 10 BBS + T20 C 0.055 4 2x DI PBS + T20 0 0

PS03N 280 24 10 BBS + T20 E 3.5 4 2x DI PBS + T20 0 0

PS03N 281 24 10 BBS + T20 E 0.88 4 2x DI PBS + T20 0 0

PS03N 282 24 10 BBS + T20 E 0.22 4 2x DI PBS + T20 0 0

PS03N 283 24 10 BBS + T20 E 0.055 4 2x DI PBS + T20 0 0

PS03N 284 24 10 PBS + T20 B 3.5 4 2x DI PBS + T20 0 0

PS03N 285 24 10 PBS + T20 B 0.88 4 2x DI BBS + T20 0 0

PS03N 286 24 10 PBS + T20 B 0.22 4 2x DI BBS + T20 0 0

PS03N 287 24 10 PBS + T20 B 0.055 4 2x DI BBS + T20 0 0

PS03N 288 24 10 PBS + T20 C 3.5 4 2x DI BBS + T20 0 0

PS03N 289 24 10 PBS + T20 C 0.88 4 2x DI BBS + T20 0 0

PS03N 290 24 10 PBS + T20 C 0.22 4 2x DI BBS + T20 0 0

PS03N 291 24 10 PBS + T20 C 0.055 4 2x DI BBS + T20 0 0

PS03N 292 24 10 PBS + T20 E 3.5 4 2x DI BBS + T20 0 0

PS03N 293 24 10 PBS + T20 E 0.88 4 2x DI BBS + T20 0 0

PS03N 294 24 10 PBS + T20 E 0.22 4 2x DI BBS + T20 0 0

PS03N 295 24 10 PBS + T20 E 0.055 4 2x DI BBS + T20 0 0

PS03N 296 24 10 BBS + T20 B 3.5 4 2x DI BBS + T20 0 0

PS03N 297 24 10 BBS + T20 B 0.88 4 2x DI BBS + T20 0 0

PS03N 298 24 10 BBS + T20 B 0.22 4 2x DI BBS + T20 0 0

PS03N 299 24 10 BBS + T20 B 0.055 4 2x DI BBS + T20 0 0

PS03N 300 24 10 BBS + T20 C 3.5 4 2x DI BBS + T20 0 0

PS03N 301 24 10 BBS + T20 C 0.88 4 2x DI BBS + T20 0 0

PS03N 302 24 10 BBS + T20 C 0.22 4 2x DI BBS + T20 0 0

PS03N 303 24 10 BBS + T20 C 0.055 4 2x DI BBS + T20 0 0

PS03N 304 24 10 BBS + T20 E 3.5 4 2x DI BBS + T20 0 0

PS03N 305 24 10 BBS + T20 E 0.88 4 2x DI BBS + T20 0 0

111

Particle

Type Batch

Time

(h) RPM

Immobilization

Buffer a Ab

Ab

(mg/m2)

Temp

(° C) Post Wash Reaction Buffer

Reaction With:

DI O1:K1:H7 Famp

PS03N 306 24 10 BBS + T20 E 0.22 4 2x DI BBS + T20 0 0

PS03N 307 24 10 BBS + T20 E 0.055 4 2x DI BBS + T20 0 0

PS03N 308 24 10 TBS B 3.5 4 2x DI DI 1 1

PS03N 309 24 10 TBS B 0.88 4 2x DI DI 1 1

PS03N 310 24 10 TBS B 0.22 4 2x DI DI 1 1

PS03N 311 24 10 TBS B 0.055 4 2x DI DI 1 1

PS03N 312 24 10 TBS B 0.014 4 2x DI DI 1 1

PS03N 313 24 10 TBS C 3.5 4 2x DI DI 1 1

PS03N 314 24 10 TBS C 0.88 4 2x DI DI 1 1

PS03N 315 24 10 TBS C 0.22 4 2x DI DI 1 1

PS03N 316 24 10 TBS C 0.055 4 2x DI DI 1 1

PS03N 317 24 10 TBS C 0.014 4 2x DI DI 1 1

PS03N 318 24 10 TBS D 3.5 4 2x DI DI 1 1

PS03N 319 24 10 TBS D 0.88 4 2x DI DI 1 1

PS03N 320 24 10 TBS D 0.22 4 2x DI DI 1 1

PS03N 321 24 10 TBS D 0.055 4 2x DI DI 1 1

PS03N 322 24 10 TBS D 0.014 4 2x DI DI 1 1

PS03N 323 24 10 TBS+ T20 B 3.5 4 2x DI DI 0 0

PS03N 324 24 10 TBS+ T20 B 0.88 4 2x DI DI 0 0

PS03N 325 24 10 TBS+ T20 B 0.22 4 2x DI DI 0 0

PS03N 326 24 10 TBS+ T20 B 0.055 4 2x DI DI 0 0

PS03N 327 24 10 TBS+ T20 B 0.014 4 2x DI DI 0 0

PS03N 328 24 10 TBS+ T20 C 3.5 4 2x DI DI 1 1

PS03N 329 24 10 TBS+ T20 C 0.88 4 2x DI DI 0 0

PS03N 330 24 10 TBS+ T20 C 0.22 4 2x DI DI 0 0

PS03N 331 24 10 TBS+ T20 C 0.055 4 2x DI DI 0 0

PS03N 332 24 10 TBS+ T20 C 0.014 4 2x DI DI 0 0

PS03N 333 24 10 TBS+ T20 D 3.5 4 2x DI DI 0 0

112

Particle

Type Batch

Time

(h) RPM

Immobilization

Buffer a Ab

Ab

(mg/m2)

Temp

(° C) Post Wash Reaction Buffer

Reaction With:

DI O1:K1:H7 Famp

PS03N 334 24 10 TBS+ T20 D 0.88 4 2x DI DI 0 0

PS03N 335 24 10 TBS+ T20 D 0.22 4 2x DI DI 0 0

PS03N 336 24 10 TBS+ T20 D 0.055 4 2x DI DI 0 0

PS03N 337 24 10 TBS+ T20 D 0.014 4 2x DI DI 0 0

PS03N 338 24 10 TBS BSA 3.5 4 2x DI DI 1 1

PS03N 339 24 10 TBS+ T20 BSA 3.5 4 2x DI DI 0 0

PS03N 340 24 10 AceB B 3.5 4 2x DI DI 2 2

PS03N 341 24 10 AceB B 0.88 4 2x DI DI 1 2

PS03N 342 24 10 AceB B 0.22 4 2x DI DI 1 2

PS03N 343 24 10 AceB B 0.055 4 2x DI DI 1 1.5

PS03N 344 24 10 AceB B 0.014 4 2x DI DI 1 1

PS03N 345 24 10 AceB C 3.5 4 2x DI DI 2 2

PS03N 346 24 10 AceB C 0.88 4 2x DI DI 1 2

PS03N 347 24 10 AceB C 0.22 4 2x DI DI 1 3

PS03N 348 24 10 AceB C 0.055 4 2x DI DI 1 2.5

PS03N 349 24 10 AceB C 0.014 4 2x DI DI 1 1

PS03N 350 24 10 AceB D 3.5 4 2x DI DI 1 1.5

PS03N 351 24 10 AceB D 0.88 4 2x DI DI 1 3

PS03N 352 24 10 AceB D 0.22 4 2x DI DI 1 2

PS03N 353 24 10 AceB D 0.055 4 2x DI DI 1 2

PS03N 354 24 10 AceB D 0.014 4 2x DI DI 1 2

PS03N 355 24 10 AceB BSA 3.5 4 2x DI DI 1 1

PS03N 356 24 10 AceB + T20 B 3.5 4 2x DI DI 0 0

PS03N 357 24 10 AceB + T20 B 0.88 4 2x DI DI 0 0

PS03N 358 24 10 AceB + T20 B 0.22 4 2x DI DI 0 0

PS03N 359 24 10 AceB + T20 B 0.055 4 2x DI DI 0 0

PS03N 360 24 10 AceB + T20 B 0.014 4 2x DI DI 0 0

PS03N 361 24 10 AceB + T20 C 3.5 4 2x DI DI 0 0

113

Particle

Type Batch

Time

(h) RPM

Immobilization

Buffer a Ab

Ab

(mg/m2)

Temp

(° C) Post Wash Reaction Buffer

Reaction With:

DI O1:K1:H7 Famp

PS03N 362 24 10 AceB + T20 C 0.88 4 2x DI DI 0 0

PS03N 363 24 10 AceB + T20 C 0.22 4 2x DI DI 0 0

PS03N 364 24 10 AceB + T20 C 0.055 4 2x DI DI 0 0

PS03N 365 24 10 AceB + T20 C 0.014 4 2x DI DI 0 0

PS03N 366 24 10 AceB + T20 D 3.5 4 2x DI DI 0.5 0.5

PS03N 367 24 10 AceB + T20 D 0.88 4 2x DI DI 0.5 0.5

PS03N 368 24 10 AceB + T20 D 0.22 4 2x DI DI 0 0

PS03N 369 24 10 AceB + T20 D 0.055 4 2x DI DI 0 0

PS03N 370 24 10 AceB + T20 D 0.014 4 2x DI DI 0 0

PS03N 371 24 10 AceB + T20 BSA 3.5 4 2x DI DI 0 0

PS03N 372 24 10 GBS B 3.5 4 2x DI DI 1 1

PS03N 373 24 10 GBS B 0.88 4 2x DI DI 1 1

PS03N 374 24 10 GBS B 0.22 4 2x DI DI 1 1

PS03N 375 24 10 GBS B 0.055 4 2x DI DI 1 1

PS03N 376 24 10 GBS B 0.014 4 2x DI DI 1 1

PS03N 377 24 10 GBS C 3.5 4 2x DI DI 1 1

PS03N 378 24 10 GBS C 0.88 4 2x DI DI 1 1

PS03N 379 24 10 GBS C 0.22 4 2x DI DI 1 1

PS03N 380 24 10 GBS C 0.055 4 2x DI DI 1 1

PS03N 381 24 10 GBS C 0.014 4 2x DI DI 1 1

PS03N 382 24 10 GBS D 3.5 4 2x DI DI 1 1

PS03N 383 24 10 GBS D 0.88 4 2x DI DI 1 1

PS03N 384 24 10 GBS D 0.22 4 2x DI DI 1 1

PS03N 385 24 10 GBS D 0.055 4 2x DI DI 1 1

PS03N 386 24 10 GBS D 0.014 4 2x DI DI 1 1

PS03N 387 24 10 GBS BSA 3.5 4 2x DI DI 1 1

PS03N 388 24 10 GBS+ T20 B 3.5 4 2x DI DI 0 0

PS03N 389 24 10 GBS+ T20 B 0.88 4 2x DI DI 0 0

114

Particle

Type Batch

Time

(h) RPM

Immobilization

Buffer a Ab

Ab

(mg/m2)

Temp

(° C) Post Wash Reaction Buffer

Reaction With:

DI O1:K1:H7 Famp

PS03N 390 24 10 GBS+ T20 B 0.22 4 2x DI DI 0 0

PS03N 391 24 10 GBS+ T20 B 0.055 4 2x DI DI 0 0

PS03N 392 24 10 GBS+ T20 B 0.014 4 2x DI DI 0 0

PS03N 393 24 10 GBS+ T20 C 3.5 4 2x DI DI 0 0

PS03N 394 24 10 GBS+ T20 C 0.88 4 2x DI DI 0 0

PS03N 395 24 10 GBS+ T20 C 0.22 4 2x DI DI 0 0

PS03N 396 24 10 GBS+ T20 C 0.055 4 2x DI DI 0 0

PS03N 397 24 10 GBS+ T20 C 0.014 4 2x DI DI 0 0

PS03N 398 24 10 GBS+ T20 D 3.5 4 2x DI DI 0 0

PS03N 399 24 10 GBS+ T20 D 0.88 4 2x DI DI 0 0

PS03N 400 24 10 GBS+ T20 D 0.22 4 2x DI DI 0 0

PS03N 401 24 10 GBS+ T20 D 0.055 4 2x DI DI 0 0

PS03N 402 24 10 GBS+ T20 D 0.014 4 2x DI DI 0 0

PS03N 403 24 10 GBS+ T20 BSA 3.5 4 2x DI DI 0 0

PS03N 404 24 10 CPB 5.3 B 3.5 4 2x DI DI 1 1

PS03N 405 24 10 CPB 5.3 B 0.88 4 2x DI DI 0.5 1

PS03N 406 24 10 CPB 5.3 B 0.22 4 2x DI DI 1 1

PS03N 407 24 10 CPB 5.3 B 0.055 4 2x DI DI 1 1

PS03N 408 24 10 CPB 5.3 B 0.014 4 2x DI DI 1 1

PS03N 409 24 10 CPB 5.3 C 3.5 4 2x DI DI 1 1

PS03N 410 24 10 CPB 5.3 C 0.88 4 2x DI DI 0 0

PS03N 411 24 10 CPB 5.3 C 0.22 4 2x DI DI 0 0

PS03N 412 24 10 CPB 5.3 C 0.055 4 2x DI DI 1 1

PS03N 413 24 10 CPB 5.3 C 0.014 4 2x DI DI 0.5 0.5

PS03N 414 24 10 CPB 5.3 D 3.5 4 2x DI DI 1 1

PS03N 415 24 10 CPB 5.3 D 0.88 4 2x DI DI 1 1

PS03N 416 24 10 CPB 5.3 D 0.22 4 2x DI DI 1 1

PS03N 417 24 10 CPB 5.3 D 0.055 4 2x DI DI 1 1

115

Particle

Type Batch

Time

(h) RPM

Immobilization

Buffer a Ab

Ab

(mg/m2)

Temp

(° C) Post Wash Reaction Buffer

Reaction With:

DI O1:K1:H7 Famp

PS03N 418 24 10 CPB 5.3 D 0.014 4 2x DI DI 0 0

PS03N 419 24 10 CPB 5.3 BSA 3.5 4 2x DI DI 0.5 0.5

PS03N 420 24 10 CPB 5.3 + T20 B 3.5 4 2x DI DI 0 0

PS03N 421 24 10 CPB 5.3 + T20 B 0.88 4 2x DI DI 0 0

PS03N 422 24 10 CPB 5.3 + T20 B 0.22 4 2x DI DI 0 0

PS03N 423 24 10 CPB 5.3 + T20 B 0.055 4 2x DI DI 0 0

PS03N 424 24 10 CPB 5.3 + T20 B 0.014 4 2x DI DI 0 0

PS03N 425 24 10 CPB 5.3 + T20 C 3.5 4 2x DI DI 0 0

PS03N 426 24 10 CPB 5.3 + T20 C 0.88 4 2x DI DI 0 0

PS03N 427 24 10 CPB 5.3 + T20 C 0.22 4 2x DI DI 0 0

PS03N 428 24 10 CPB 5.3 + T20 C 0.055 4 2x DI DI 0 0

PS03N 429 24 10 CPB 5.3 + T20 C 0.014 4 2x DI DI 0 0

PS03N 430 24 10 CPB 5.3 + T20 D 3.5 4 2x DI DI 0 0

PS03N 431 24 10 CPB 5.3 + T20 D 0.88 4 2x DI DI 0 0

PS03N 432 24 10 CPB 5.3 + T20 D 0.22 4 2x DI DI 0 0

PS03N 433 24 10 CPB 5.3 + T20 D 0.055 4 2x DI DI 0 0

PS03N 434 24 10 CPB 5.3 + T20 D 0.014 4 2x DI DI 0 0

PS03N 435 24 10 CPB 5.3 + T20 BSA 3.5 4 2x DI DI 0 0

PS03N 436 24 10 CPB 4.6 B 3.5 4 2x DI DI 1 1

PS03N 437 24 10 CPB 4.6 B 0.88 4 2x DI DI 1 1

PS03N 438 24 10 CPB 4.6 B 0.22 4 2x DI DI 1 1

PS03N 439 24 10 CPB 4.6 B 0.055 4 2x DI DI 1 1

PS03N 440 24 10 CPB 4.6 B 0.014 4 2x DI DI 1 1

PS03N 441 24 10 CPB 4.6 C 3.5 4 2x DI DI 1 1

PS03N 442 24 10 CPB 4.6 C 0.88 4 2x DI DI 1 1

PS03N 443 24 10 CPB 4.6 C 0.22 4 2x DI DI 1 1

PS03N 444 24 10 CPB 4.6 C 0.055 4 2x DI DI 1 1

PS03N 445 24 10 CPB 4.6 C 0.014 4 2x DI DI 1 1

116

Particle

Type Batch

Time

(h) RPM

Immobilization

Buffer a Ab

Ab

(mg/m2)

Temp

(° C) Post Wash Reaction Buffer

Reaction With:

DI O1:K1:H7 Famp

PS03N 446 24 10 CPB 4.6 D 3.5 4 2x DI DI 1 1

PS03N 447 24 10 CPB 4.6 D 0.88 4 2x DI DI 1 1

PS03N 448 24 10 CPB 4.6 D 0.22 4 2x DI DI 1 1

PS03N 449 24 10 CPB 4.6 D 0.055 4 2x DI DI 1 1

PS03N 450 24 10 CPB 4.6 D 0.014 4 2x DI DI 1 1

PS03N 451 24 10 CPB 4.6 BSA 3.5 4 2x DI DI 1 1

PS03N 452 24 10 CPB 4.6 + T20 B 3.5 4 2x DI DI 0 0

PS03N 453 24 10 CPB 4.6 + T20 B 0.88 4 2x DI DI 0 0

PS03N 454 24 10 CPB 4.6 + T20 B 0.22 4 2x DI DI 0 0

PS03N 455 24 10 CPB 4.6 + T20 B 0.055 4 2x DI DI 0 0

PS03N 456 24 10 CPB 4.6 + T20 B 0.014 4 2x DI DI 0 0

PS03N 457 24 10 CPB 4.6 + T20 C 3.5 4 2x DI DI 0 0

PS03N 458 24 10 CPB 4.6 + T20 C 0.88 4 2x DI DI 0 0

PS03N 459 24 10 CPB 4.6 + T20 C 0.22 4 2x DI DI 0 0

PS03N 460 24 10 CPB 4.6 + T20 C 0.055 4 2x DI DI 0.5 0.5

PS03N 461 24 10 CPB 4.6 + T20 C 0.014 4 2x DI DI 0 0

PS03N 462 24 10 CPB 4.6 + T20 D 3.5 4 2x DI DI 0.5 0.5

PS03N 463 24 10 CPB 4.6 + T20 D 0.88 4 2x DI DI 0.5 0.5

PS03N 464 24 10 CPB 4.6 + T20 D 0.22 4 2x DI DI 0 0

PS03N 465 24 10 CPB 4.6 + T20 D 0.055 4 2x DI DI 0 0

PS03N 466 24 10 CPB 4.6 + T20 D 0.014 4 2x DI DI 0 0

PS03N 467 24 10 CPB 4.6 + T20 BSA 3.5 4 2x DI DI 0 0

PS03N 468 24 10 CPB 6.2 B 3.5 4 2x DI DI 1 1

PS03N 469 24 10 CPB 6.2 B 0.88 4 2x DI DI 1 1

PS03N 470 24 10 CPB 6.2 B 0.22 4 2x DI DI 1 1

PS03N 471 24 10 CPB 6.2 B 0.055 4 2x DI DI 0.5 0.5

PS03N 472 24 10 CPB 6.2 B 0.014 4 2x DI DI 1 1

PS03N 473 24 10 CPB 6.2 C 3.5 4 2x DI DI 1 1

117

Particle

Type Batch

Time

(h) RPM

Immobilization

Buffer a Ab

Ab

(mg/m2)

Temp

(° C) Post Wash Reaction Buffer

Reaction With:

DI O1:K1:H7 Famp

PS03N 474 24 10 CPB 6.2 C 0.88 4 2x DI DI 0.5 0.5

PS03N 475 24 10 CPB 6.2 C 0.22 4 2x DI DI 1 1

PS03N 476 24 10 CPB 6.2 C 0.055 4 2x DI DI 1 1

PS03N 477 24 10 CPB 6.2 C 0.014 4 2x DI DI 1 1

PS03N 478 24 10 CPB 6.2 D 3.5 4 2x DI DI 1 1

PS03N 479 24 10 CPB 6.2 D 0.88 4 2x DI DI 1 1

PS03N 480 24 10 CPB 6.2 D 0.22 4 2x DI DI 1 1

PS03N 481 24 10 CPB 6.2 D 0.055 4 2x DI DI 1 1

PS03N 482 24 10 CPB 6.2 D 0.014 4 2x DI DI 1 1

PS03N 483 24 10 CPB 6.2 BSA 3.5 4 2x DI DI 1 1

PS03N 484 24 10 CPB 6.2 + T20 B 3.5 4 2x DI DI 0 0

PS03N 485 24 10 CPB 6.2 + T20 B 0.88 4 2x DI DI 0 0

PS03N 486 24 10 CPB 6.2 + T20 B 0.22 4 2x DI DI 0 0

PS03N 487 24 10 CPB 6.2 + T20 B 0.055 4 2x DI DI 0 0

PS03N 488 24 10 CPB 6.2 + T20 B 0.014 4 2x DI DI 0 0

PS03N 489 24 10 CPB 6.2 + T20 C 3.5 4 2x DI DI 0 0

PS03N 490 24 10 CPB 6.2 + T20 C 0.88 4 2x DI DI 0 0

PS03N 491 24 10 CPB 6.2 + T20 C 0.22 4 2x DI DI 0 0

PS03N 492 24 10 CPB 6.2 + T20 C 0.055 4 2x DI DI 0 0

PS03N 493 24 10 CPB 6.2 + T20 C 0.014 4 2x DI DI 0 0

PS03N 494 24 10 CPB 6.2 + T20 D 3.5 4 2x DI DI 0 0

PS03N 495 24 10 CPB 6.2 + T20 D 0.88 4 2x DI DI 0 0

PS03N 496 24 10 CPB 6.2 + T20 D 0.22 4 2x DI DI 0 0

PS03N 497 24 10 CPB 6.2 + T20 D 0.055 4 2x DI DI 0 0

PS03N 498 24 10 CPB 6.2 + T20 D 0.014 4 2x DI DI 0 0

PS03N 499 24 10 CPB 6.2 + T20 BSA 3.5 4 2x DI DI 0 0

PS03N 500 24 10 CPB 6.8 B 3.5 4 2x DI DI 1 1

PS03N 501 24 10 CPB 6.8 B 0.88 4 2x DI DI 1 1

118

Particle

Type Batch

Time

(h) RPM

Immobilization

Buffer a Ab

Ab

(mg/m2)

Temp

(° C) Post Wash Reaction Buffer

Reaction With:

DI O1:K1:H7 Famp

PS03N 502 24 10 CPB 6.8 B 0.22 4 2x DI DI 1 1

PS03N 503 24 10 CPB 6.8 B 0.055 4 2x DI DI 1 1

PS03N 504 24 10 CPB 6.8 B 0.014 4 2x DI DI 1 1

PS03N 505 24 10 CPB 6.8 C 3.5 4 2x DI DI 1 1

PS03N 506 24 10 CPB 6.8 C 0.88 4 2x DI DI 1 1

PS03N 507 24 10 CPB 6.8 C 0.22 4 2x DI DI 1 1

PS03N 508 24 10 CPB 6.8 C 0.055 4 2x DI DI 1 1

PS03N 509 24 10 CPB 6.8 C 0.014 4 2x DI DI 1 1

PS03N 510 24 10 CPB 6.8 D 3.5 4 2x DI DI 1 1

PS03N 511 24 10 CPB 6.8 D 0.88 4 2x DI DI 1 1

PS03N 512 24 10 CPB 6.8 D 0.22 4 2x DI DI 1 1

PS03N 513 24 10 CPB 6.8 D 0.055 4 2x DI DI 1 1

PS03N 514 24 10 CPB 6.8 D 0.014 4 2x DI DI 1 1

PS03N 515 24 10 CPB 6.8 BSA 3.5 4 2x DI DI 1 1

PS03N 516 24 10 CPB 6.8 + T20 B 3.5 4 2x DI DI 0 0

PS03N 517 24 10 CPB 6.8 + T20 B 0.88 4 2x DI DI 0.5 0.5

PS03N 518 24 10 CPB 6.8 + T20 B 0.22 4 2x DI DI 0 0

PS03N 519 24 10 CPB 6.8 + T20 B 0.055 4 2x DI DI 0 0

PS03N 520 24 10 CPB 6.8 + T20 B 0.014 4 2x DI DI 0 0

PS03N 521 24 10 CPB 6.8 + T20 C 3.5 4 2x DI DI 0 0

PS03N 522 24 10 CPB 6.8 + T20 C 0.88 4 2x DI DI 0 0

PS03N 523 24 10 CPB 6.8 + T20 C 0.22 4 2x DI DI 0 0

PS03N 524 24 10 CPB 6.8 + T20 C 0.055 4 2x DI DI 0 0

PS03N 525 24 10 CPB 6.8 + T20 C 0.014 4 2x DI DI 0 0

PS03N 526 24 10 CPB 6.8 + T20 D 3.5 4 2x DI DI 0 0

PS03N 527 24 10 CPB 6.8 + T20 D 0.88 4 2x DI DI 0 0

PS03N 528 24 10 CPB 6.8 + T20 D 0.22 4 2x DI DI 0 0

PS03N 529 24 10 CPB 6.8 + T20 D 0.055 4 2x DI DI 0 0

119

Particle

Type Batch

Time

(h) RPM

Immobilization

Buffer a Ab

Ab

(mg/m2)

Temp

(° C) Post Wash Reaction Buffer

Reaction With:

DI O1:K1:H7 Famp

PS03N 530 24 10 CPB 6.8 + T20 D 0.014 4 2x DI DI 0 0

PS03N 531 24 10 CPB 6.8 + T20 BSA 3.5 4 2x DI DI 0 0

PS03N 532 2 10 AceB B 3.5 RT 2x DI DI 1 3

PS03N 533 2 10 AceB B 0.88 RT 2x DI DI 1 2.5

PS03N 534 2 10 AceB B 0.22 RT 2x DI DI 1 2

PS03N 535 2 10 AceB B 0.055 RT 2x DI DI 1 2

PS03N 536 2 10 AceB B 0.014 RT 2x DI DI 1 2.5

PS03N 537 2 10 AceB C 3.5 RT 2x DI DI 1 3

PS03N 538 2 10 AceB C 0.88 RT 2x DI DI 1 1.5

PS03N 539 2 10 AceB C 0.22 RT 2x DI DI 1 2.5

PS03N 540 2 10 AceB C 0.055 RT 2x DI DI 1 2

PS03N 541 2 10 AceB C 0.014 RT 2x DI DI 1 3

PS03N 542 2 10 AceB D 3.5 RT 2x DI DI 1 2

PS03N 543 2 10 AceB D 0.88 RT 2x DI DI 1 3

PS03N 544 2 10 AceB D 0.22 RT 2x DI DI 1 3

PS03N 545 2 10 AceB D 0.055 RT 2x DI DI 1 2

PS03N 546 2 10 AceB D 0.014 RT 2x DI DI 1 3

PS03N 547 2 10 AceB BSA 3.5 RT 2x DI DI 1 1

PS03N 548 24 10 AceB B 3.5 RT 2x DI DI 2 2.5

PS03N 549 24 10 AceB B 0.88 RT 2x DI DI 1 2

PS03N 550 24 10 AceB B 0.22 RT 2x DI DI 1 1.5

PS03N 551 24 10 AceB B 0.055 RT 2x DI DI 1 1

PS03N 552 24 10 AceB B 0.014 RT 2x DI DI 1 1

PS03N 553 24 10 AceB C 3.5 RT 2x DI DI 2 2

PS03N 554 24 10 AceB C 0.88 RT 2x DI DI 1 1.5

PS03N 555 24 10 AceB C 0.22 RT 2x DI DI 1 1

PS03N 556 24 10 AceB C 0.055 RT 2x DI DI 1 2

PS03N 557 24 10 AceB C 0.014 RT 2x DI DI 1 1

120

Particle

Type Batch

Time

(h) RPM

Immobilization

Buffer a Ab

Ab

(mg/m2)

Temp

(° C) Post Wash Reaction Buffer

Reaction With:

DI O1:K1:H7 Famp

PS03N 558 24 10 AceB D 3.5 RT 2x DI DI 2 2.5

PS03N 559 24 10 AceB D 0.88 RT 2x DI DI 1 2.5

PS03N 560 24 10 AceB D 0.22 RT 2x DI DI 1 2

PS03N 561 24 10 AceB D 0.055 RT 2x DI DI 1 1

PS03N 562 24 10 AceB D 0.014 RT 2x DI DI 1 2

PS03N 563 24 10 AceB BSA 3.5 RT 2x DI DI 1 1

PS03N 564 2 10 AceB + NaCl B 3.5 RT 2x DI DI 1 1.5 1.5

PS03N 565 2 10 AceB + NaCl B 0.88 RT 2x DI DI 1 1.5 1.5

PS03N 566 2 10 AceB + NaCl B 0.22 RT 2x DI DI 1 1 1

PS03N 567 2 10 AceB + NaCl B 0.055 RT 2x DI DI 0.5 1 1

PS03N 568 2 10 AceB + NaCl B 0.014 RT 2x DI DI 1 3.5 2.5

PS03N 569 2 10 AceB + NaCl C 3.5 RT 2x DI DI 2 3 2

PS03N 570 2 10 AceB + NaCl C 0.88 RT 2x DI DI 1 3.5 1.5

PS03N 571 2 10 AceB + NaCl C 0.22 RT 2x DI DI 1 3 2.5

PS03N 572 2 10 AceB + NaCl C 0.055 RT 2x DI DI 1 3 2

PS03N 573 2 10 AceB + NaCl C 0.014 RT 2x DI DI 1 3 2

PS03N 574 2 10 AceB + NaCl D 3.5 RT 2x DI DI 2 2.5 2

PS03N 575 2 10 AceB + NaCl D 0.88 RT 2x DI DI 1 3 2

PS03N 576 2 10 AceB + NaCl D 0.22 RT 2x DI DI 1 3 2

PS03N 577 2 10 AceB + NaCl D 0.055 RT 2x DI DI 1 3 3

PS03N 578 2 10 AceB + NaCl D 0.014 RT 2x DI DI 1 3 2

PS03N 579 2 10 AceB + NaCl BSA 3.5 RT 2x DI DI 1 1 1

PS03N 580 24 10 AceB + NaCl B 3.5 4 2x DI DI 1 1.5 1.5

PS03N 581 24 10 AceB + NaCl B 0.88 4 2x DI DI 1 2 2

PS03N 582 24 10 AceB + NaCl B 0.22 4 2x DI DI 1 3 3

PS03N 583 24 10 AceB + NaCl B 0.055 4 2x DI DI 1 1 1

PS03N 584 24 10 AceB + NaCl B 0.014 4 2x DI DI 1 2 2

PS03N 585 24 10 AceB + NaCl C 3.5 4 2x DI DI 2 3.5 3

121

Particle

Type Batch

Time

(h) RPM

Immobilization

Buffer a Ab

Ab

(mg/m2)

Temp

(° C) Post Wash Reaction Buffer

Reaction With:

DI O1:K1:H7 Famp

PS03N 586 24 10 AceB + NaCl C 0.88 4 2x DI DI 1 3 3

PS03N 587 24 10 AceB + NaCl C 0.22 4 2x DI DI 1 3 2

PS03N 588 24 10 AceB + NaCl C 0.055 4 2x DI DI 1 2 2

PS03N 589 24 10 AceB + NaCl C 0.014 4 2x DI DI 2 3 3

PS03N 590 24 10 AceB + NaCl D 3.5 4 2x DI DI 2 2.5 2.5

PS03N 591 24 10 AceB + NaCl D 0.88 4 2x DI DI 1 1 1

PS03N 592 24 10 AceB + NaCl D 0.22 4 2x DI DI 1 2 2

PS03N 593 24 10 AceB + NaCl D 0.055 4 2x DI DI 1 2 2

PS03N 594 24 10 AceB + NaCl D 0.014 4 2x DI DI 1 3 2

PS03N 595 24 10 AceB + NaCl BSA 3.5 4 2x DI DI 1 1 1

PS03N 596 1 8 AceB + NaCl B 0.014 RT DI + T20 DI 0 0 0

PS03N 597 1 8 AceB + NaCl C 0.88 RT DI + T20 DI 0 0 0

PS03N 598 1 8 AceB + NaCl D 3.5 RT DI + T20 DI 0 0 0

PS03N 599 1 8 AceB + NaCl BSA 3.5 RT DI + T20 DI 0.5 0.5 0.5

PS03N 600 1 8 AceB + NaCl B 0.014 RT DI + T20 DI + T20 0 0 0

PS03N 601 1 8 AceB + NaCl C 0.88 RT DI + T20 DI + T20 0.5 0.5 0.5

PS03N 602 1 8 AceB + NaCl D 3.5 RT DI + T20 DI + T20 0 0 0

PS03N 603 1 8 AceB + NaCl BSA 3.5 RT DI + T20 DI + T20 0.5 0.5 0.5

PS03N 604 1 8 AceB + NaCl B 0.014 RT PBS + T20 PBS + T20 0 0 0

PS03N 605 1 8 AceB + NaCl C 0.88 RT PBS + T20 PBS + T20 0 0 0

PS03N 606 1 8 AceB + NaCl D 3.5 RT PBS + T20 PBS + T20 0 0 0

PS03N 607 1 8 AceB + NaCl BSA 3.5 RT PBS + T20 PBS + T20 0 0 0

PS03N 608 1 10 AceB + NaCl B 3.5 RT 2x DI DI 1 2.5 2

PS03N 609 1 10 AceB + NaCl B 0.88 RT 2x DI DI 1 2 2

PS03N 610 1 10 AceB + NaCl B 0.22 RT 2x DI DI 1.5 4 3

PS03N 611 1 10 AceB + NaCl B 0.055 RT 2x DI DI 1 2 1

PS03N 612 1 10 AceB + NaCl B 0.014 RT 2x DI DI 1 1 1

PS03N 613 1 10 AceB + NaCl C 3.5 RT 2x DI DI 1.5 2 2

122

Particle

Type Batch

Time

(h) RPM

Immobilization

Buffer a Ab

Ab

(mg/m2)

Temp

(° C) Post Wash Reaction Buffer

Reaction With:

DI O1:K1:H7 Famp

PS03N 614 1 10 AceB + NaCl C 0.88 RT 2x DI DI 1 1 1

PS03N 615 1 10 AceB + NaCl C 0.22 RT 2x DI DI 1 1 1

PS03N 616 1 10 AceB + NaCl C 0.055 RT 2x DI DI 1 1 1

PS03N 617 1 10 AceB + NaCl C 0.014 RT 2x DI DI 1 1.5 1

PS03N 618 1 10 AceB + NaCl D 3.5 RT 2x DI DI 1 1 1

PS03N 619 1 10 AceB + NaCl D 0.88 RT 2x DI DI 1 1 1

PS03N 620 1 10 AceB + NaCl D 0.22 RT 2x DI DI 1 1 1

PS03N 621 1 10 AceB + NaCl D 0.055 RT 2x DI DI 1 2 1

PS03N 622 1 10 AceB + NaCl D 0.014 RT 2x DI DI 1 1 1

PS03N 623 1 10 AceB + NaCl BSA 3.5 RT 2x DI DI 1 1 1

PS03N 624 24 10 AceB + NaCl B 3.5 4 2x DI DI 2 3 3

PS03N 625 24 10 AceB + NaCl B 0.88 4 2x DI DI 1 3 3

PS03N 626 24 10 AceB + NaCl B 0.22 4 2x DI DI 1 1 1

PS03N 627 24 10 AceB + NaCl B 0.055 4 2x DI DI 1 1 1

PS03N 628 24 10 AceB + NaCl B 0.014 4 2x DI DI 1 1 1

PS03N 629 24 10 AceB + NaCl C 3.5 4 2x DI DI 1 1 1

PS03N 630 24 10 AceB + NaCl C 0.88 4 2x DI DI 1 1 1

PS03N 631 24 10 AceB + NaCl C 0.22 4 2x DI DI 1 1 1

PS03N 632 24 10 AceB + NaCl C 0.055 4 2x DI DI 1 1 1

PS03N 633 24 10 AceB + NaCl C 0.014 4 2x DI DI 1 1 1

PS03N 634 24 10 AceB + NaCl D 3.5 4 2x DI DI 1 1 1

PS03N 635 24 10 AceB + NaCl D 0.88 4 2x DI DI 1 1 1

PS03N 636 24 10 AceB + NaCl D 0.22 4 2x DI DI 1 1 1

PS03N 637 24 10 AceB + NaCl D 0.055 4 2x DI DI 1 2 2

PS03N 638 24 10 AceB + NaCl D 0.014 4 2x DI DI 1 1 1

PS03N 639 24 10 AceB + NaCl BSA 3.5 4 2x DI DI 1 1 1

PS03N 640 24 10 AceB + T20 B 3.5 4 2x DI DI 0 0

PS03N 641 24 10 AceB + T20 B 0.88 4 2x DI DI 0 0

123

Particle

Type Batch

Time

(h) RPM

Immobilization

Buffer a Ab

Ab

(mg/m2)

Temp

(° C) Post Wash Reaction Buffer

Reaction With:

DI O1:K1:H7 Famp

PS03N 642 24 10 AceB + T20 B 0.22 4 2x DI DI 0 0

PS03N 643 24 10 AceB + T20 B 0.055 4 2x DI DI 0 0

PS03N 644 24 10 AceB + T20 B 0.014 4 2x DI DI 0 0

PS03N 645 24 10 AceB + T20 C 3.5 4 2x DI DI 0 0

PS03N 646 24 10 AceB + T20 C 0.88 4 2x DI DI 0 0

PS03N 647 24 10 AceB + T20 C 0.22 4 2x DI DI 0 0

PS03N 648 24 10 AceB + T20 C 0.055 4 2x DI DI 0 0

PS03N 649 24 10 AceB + T20 C 0.014 4 2x DI DI 0 0

PS03N 650 24 10 AceB + T20 D 3.5 4 2x DI DI 0.5 0.5

PS03N 651 24 10 AceB + T20 D 0.88 4 2x DI DI 0.5 0.5

PS03N 652 24 10 AceB + T20 D 0.22 4 2x DI DI 0 0

PS03N 653 24 10 AceB + T20 D 0.055 4 2x DI DI 0 0

PS03N 654 24 10 AceB + T20 D 0.014 4 2x DI DI 0 0

PS03N 655 24 10 AceB + T20 BSA 3.5 4 2x DI DI 0 0

PS02N 656 2 10 AceB B 3.5 RT 2x DI DI 3 4

PS02N 657 2 10 AceB C 3.5 RT 2x DI DI 2 3

PS02N 658 2 10 CP 4.6 B 3.5 RT 2x DI DI 1 1

PS02N 659 2 10 CP 4.6 C 3.5 RT 2x DI DI 1 1

PS02N 660 2 10 PBS B 3.5 RT 2x DI DI 1 1

PS02N 661 2 10 PBS C 3.5 RT 2x DI DI 1 1

PS03N 662 2 10 AceB B 3.5 RT 2x DI DI 2 3

PS03N 663 2 10 AceB C 3.5 RT 2x DI DI 3 4

PS03N 664 2 10 CP 4.6 B 3.5 RT 2x DI DI 1 1

PS03N 665 2 10 CP 4.6 C 3.5 RT 2x DI DI 2 2

PS03N 666 2 10 PBS B 3.5 RT 2x DI DI 2 2

PS03N 667 2 10 PBS C 3.5 RT 2x DI DI 1 1

PS03N 668 2 10 AceB B 3.5 RT 2x DI DI 2 3

PS03N 669 2 10 AceB C 3.5 RT 2x DI DI 1.5 3

124

Particle

Type Batch

Time

(h) RPM

Immobilization

Buffer a Ab

Ab

(mg/m2)

Temp

(° C) Post Wash Reaction Buffer

Reaction With:

DI O1:K1:H7 Famp

PS02N 670 2 10 AceB B 3.5 RT 2x DI DI 1.5 3

PS02N 671 2 10 AceB C 3.5 RT 2x DI DI 2 3

PS03N 672 2 10 AceB B 3.5 RT 2x DI 0.5 mg/mL BSA 2 3

PS03N 673 2 10 AceB C 3.5 RT 2x DI 0.5 mg/mL BSA 1.5 3

PS02N 674 2 10 AceB B 3.5 RT 2x DI 0.5 mg/mL BSA 2 3

PS02N 675 2 10 AceB C 3.5 RT 2x DI 0.5 mg/mL BSA 1 3

PS03N 676 2 10 AceB B 3.5 RT 2x DI 1.0 mg/mL BSA 1.5 4

PS03N 677 2 10 AceB C 3.5 RT 2x DI 1.0 mg/mL BSA 1.5 3

PS02N 678 2 10 AceB B 3.5 RT 2x DI 1.0 mg/mL BSA 2 3

PS02N 679 2 10 AceB C 3.5 RT 2x DI 1.0 mg/mL BSA 1.5 3

PS03N 680 2 10 AceB B 3.5 RT 2x DI 5.0 mg/mL BSA 1 3

PS03N 681 2 10 AceB C 3.5 RT 2x DI 5.0 mg/mL BSA 1 2.5

PS02N 682 2 10 AceB B 3.5 RT 2x DI 5.0 mg/mL BSA 1 3

PS02N 683 2 10 AceB C 3.5 RT 2x DI 5.0 mg/mL BSA 1.5 3.5

PS03N 684 2 10 AceB B 3.5 RT 2x DI 20.0 mg/mL BSA 2 4

PS03N 685 2 10 AceB C 3.5 RT 2x DI 20.0 mg/mL BSA 1 2.5

PS02N 686 2 10 AceB B 3.5 RT 2x DI 20.0 mg/mL BSA 2 4

PS02N 687 2 10 AceB C 3.5 RT 2x DI 20.0 mg/mL BSA 2 3

PS02N 688 2 10 AceB B 3.5 RT 2x DI DI 2 3

PS02N 689 2 10 AceB B 3.5 RT 2x DI 0.05 M NaCl 2 4

PS02N 690 2 10 AceB B 3.5 RT 2x DI 0.15 M NaCl 2 4

PS02N 691 2 10 AceB B 3.5 RT 2x DI 0.50 M NaCl 4 4

PS02N 692 2 10 AceB B 3.5 RT 2x DI 1.50 M NaCl 4 4

PS02N 693 2 10 AceB B 3.5 RT 2x DI 5.0 M NaCl 4 4

PS02N 694 2 10 AceB B 3.5 RT 2x DI DI 1 3

PS02N 695 2 10 AceB B 3.5 RT 2x DI 0.05 M NaCl 1 3

PS02N 696 2 10 AceB B 3.5 RT 2x DI 0.15 M NaCl 3 4

PS02N 697 2 10 AceB B 3.5 RT 2x DI 0.50 M NaCl 4 4

125

Particle

Type Batch

Time

(h) RPM

Immobilization

Buffer a Ab

Ab

(mg/m2)

Temp

(° C) Post Wash Reaction Buffer

Reaction With:

DI O1:K1:H7 Famp

PS02N 698 2 10 AceB B 3.5 RT 2x DI 1.50 M NaCl 4 4

PS02N 699 2 10 AceB B 3.5 RT 2x DI 5.0 M NaCl 4 4