US/Ireland Emerging Technologies,

Univ. of Massachusetts, Lowell, Oct 19-20th., 2009.

'Developing Immunoassays for

Bioprocess Analysis and Diagnostics'

Richard O’Kennedy,

Professor of Biological Sciences/Vice-President,

School of Biotechnology, Biomedical Diagnostics Institute and National Centre for Sensors Research,

Dublin City University, Dublin 9, Ireland.

SUMMARY:

'Developing Immunoassays for Bioprocess Analysis and Diagnostics'

• Background

• Antibodies: ideal reagents for assay development

• Key Characteristics: specificity, sensitivity,

structural format, stability and immobilization.

• Generation

• Genetic engineering facilitates optimisation

• Potential applications

Core Expertise

Biorecognition molecule:Antibodies, fragments, peptides, protein scaffolds and DNA

Antibody Production:Monoclonal, polyclonal and recombinant (human, chicken, mouse and rabbit)

Fermentation:Large scale protein expression

Cloning:Cloning, expression and purification of antigens and biomakers

Protein Kinetics:Determination of interaction rate constants and thermodynamic profiles

Liposomes:Antibody labelling and dye/contrast agent encapsulation

Automated Screening:Custom written software for high throughput screening

Mutagenesis:Random and site-specific mutagenesis for protein improvement

Biosensor Assays:Incorporation of biorecognition elements into biosensor platforms

Display and Selection:Phage, yeast and ribosomal display of proteins

Lateral Flow Assays:Point of care tests for environmental and clinical applications

Research Interests

Marine ToxinsProstate Cancer

Markers

Aflatoxins

Food

Contaminants:

Drugs of Abuse

Cardiac Markers

Multiple Myeloma

Sialic Acids

They didn’t mention this in my contract

VH

VL

CH1

CL

F(ab')2

VH

VL

CH1

CL

Fab

scFv

IgG

CH1

CL

CH2

VL

VH

VL

VHV

L

VH

VL

VHV

L

VH

VL

VH

Dimeric scFv

Dimeric bifunctional

scFv

Recombinant antibodies

Characteristics Polyclonal Monoclonal Recombinant

Ease of production ++++ +++ ++

Low cost ++++ +++ ++

Stability* +++ ++ ++

Availability ++++ +++ + ()

Ease of immobilization ++++ ++++ ++++++

Sensitivity* ++++ ++++ +++++

Capacity for improvement* - - +++++

Antibody Types

Avian Antibodies: IgY

• Phylogenetically distant from humans • Single primer sets• Stable and long half-life• Do not react with RF, HAMA or surface Fc receptors

Picture obtained from www.beckman.com

Development of Target-specific tests using a range of antibody labels

Kinetic and Thermodynamic Characterisation

Time

RU

Derivatization of TargetTarget-Carrier Conjugate Design and Synthesis

Target

Linker

Biosensor-basedImmunoassay

Target Conc.Res

pons

e (R

U)

Immunize Rabbit

Polyclonal antibodies

Immunize Mouse

Recombinant antibodies Monoclonal antibodies

Antibody Production, Characterication and Applications

Nature 446, 964-966 (26 April 2007)

Pimp My Antibody

Antibodies for Use – Key Issues

Issue Strategy for Optimisation

Sensitivity High-throughput screen / improve

Specificity Increase / broaden

Stability Genetic modification

Immobilisation Chemical- multiple chemistries

Orientation Insert tags e.g. biotin

Labelling Chemical/Biological

Size Smaller facilitates denser packing

Multi-use Stabilise / immobilise antigen

Non-specific binding Use blockers / adequate controls

Phage Display scFv antibodies

PRODUCTION AND CHARACTERISATION OF MURINE SINGLE CHAIN FV ANTIBODIES

Extract RNA

Reverse Transcription

Genetic source of antibody

fragments

- Non-immunised/Immunised Mouse

- Hybridomas

SfiI digest of SOE product

Ligation of amplified DNA into plasmid

Chloramphenicol

F1

Transform into E.coli

VH

VL

SOE-PCR anneals the VH and VL regions together

Vector containing VH and VL DNA sequences

Recombinant scFv antibody fragment

pIII phage coat protein

Phage displaying scFv antibody fragment

Diagram of filamentous phage expressing scFv on the surface of the

phage as a fusion with the pIII phage coat protein. Light and heavy

chain genes are present in the vector contained within the phage and

the phage is ready for infection into E.coli.

Phage selection

cycle2. Incubate with antigenon immunotube

4. Elute specific phage

8. Prepare phage particles

7. Amplify bacteria

6. Re-infect selected phages into E. coli

5. Analyse eluted phage by phage-ELISA

3. Wash and remove non-binders

1. Phage antibody library

• Screening capacity: 4000 clones per day•Automated screening (custom designed)

Robotic Screening

A100 – rapid kinetic characterisation

• High throughput hardware and software

• High quality, high content data

• Parallel analysis array format system

A100 Screening approach

Complex Stability of 95 clones

Stability early Stability late

%left =Stability late

Stability early

X 100

Listeria monocytogenes

• Gram-positive bacterium, motileGram-positive bacterium, motile

• Ubiquitous in the environmentUbiquitous in the environment

• ‘‘Listeriosis’ - manifested as Listeriosis’ - manifested as

• food poisoning (‘Influenza-like’)food poisoning (‘Influenza-like’)

• spontaneous abortion (2spontaneous abortion (2ndnd/3/3rd rd trimester)trimester)

• meningitis/encephalitismeningitis/encephalitis

• >20% mortality rate>20% mortality rate

Food recalls

• In 2002, 27.4 million pounds of turkey produce

were recalled due to suspected contamination with

L. monocytogenes

•largest meat recall in the history of the United States

•resulted in several fatalities and a subsequent

nationwide class-action lawsuit

Limits allowed: 1 micro-organism per 50 g

Listeriamonocytogenes

Listeriolysin (60 kDa),PI-PLC,PC-PLC(escape from vacuole)

p60 (60kDa),Hpt(cell growth/division)

Internalin A (InlA)80 kDa

Internalin B (InlB)~65 kDa

Actin Tail Filaments (ActA)90 kDa(Intracellular motility)

P66 (kDa)Aminopeptidase

Virulence proteins

Protein Antibodies produced

Polyclonal Monoclonal scFv

InlA/rInlA - -

InlB/rInlB

p60/rp60 -

Whole cells

Antibodies

1

3

5

7

9

11

13

15

A/A

o

mAb2b3 cross - reactivity• Direct capture ELISA ( Anti-Inl A antibody)Direct capture ELISA ( Anti-Inl A antibody)

– mAb2b3 only bound to mAb2b3 only bound to L. monocytogenesL. monocytogenes cells tested cells tested

Listeria monocytogenes determination

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.E+04 1.E+05 1.E+06 1.E+07 1.E+08 1.E+09 1.E+10

Cell Concentration (Cells/ml)

A/A

0..

Anti-InlA antibody-linked red light emitting quantum dots (605nm)

The development of rapid fluorescence-based immunoassays, using quantum dot-labelled antibodies for the detection of Listeria monocytogenes cell surface proteins - Int. J. Biol. Macromol., 39(1-3), 127-134. Tully, E., Hearty, S., Leonard, P. and O’Kennedy, R. (2006).

Detection of Listeria

AFB1

Aflatoxin B1

• Potent naturally occurring carcinogen

• Ability to induce specific mutations in specific mammalian genes

• Reported 1 million cases of liver cancer per year due to aflatoxins • Linked to human hepatocellular carcinoma

• Listed as group 1 carcinogens by the IARC

Anti-aflatoxin scFv preincubated with aflatoxin

Anti-aflatoxin scFv Aflatoxin

Biacore assay development

Antibody and free aflatoxin injected over aflatoxin-

immobilised CM5 chip surface

Conversion of scFv to Fab format

• Anti-AFB1 murine scFv was converted to a chimeric Fab format by the addition of human constant regions using a number of overlap extension polymerase chain reactions (PCRs)

• Potential benefits of converting

an scFv to Fab format in terms of

stability, sensitivity and specificity

were evaluated

• Influence of additional constant regions

and presence of the interchain disulphide

bonds in the Fab fragment was assessed

scFv

Fab

scFv

Fab

Heavy chain CDR3 Q E X X X Y S M D

NNK NNK NNK

Improving Affinity• in vitro (directed) evolution strategies

Antibody Fragment ELISA Limit of Detection

Biacore Limit of detection

Monomeric scFv 12ppb 0.390ppb

Dimeric scFv 3ppb 0.19ppb

Bifunctional dimeric scFv

3ppb n/a

D11 Fab 4.2ppb 0.29ppb

G6 Fab (mutant) 1.3ppb 0.12ppb

Comparison of Antibodies:Detection of aflatoxin B1

Aim: offer perspective on selection and use

of antibodies

• Specificity

• Sensitivity

• Sources

• Selection

• Screening

• Sensing

Six-s = success

Summary of Lecture

Acknowledgements

• Lab group

• Biacore

• FIRM

• SFI

• Fusion

• EI