development of monoclonal antibody and enzyme linked

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LSU Historical Dissertations and Theses Graduate School

1999

Development of Monoclonal Antibody andEnzyme Linked Immunosorbent for Detection ofOff -Flavor Compound 2-Methylisoborneol.Eun Sung ParkLouisiana State University and Agricultural & Mechanical College

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DEVELOPMENT OF MONOCLONAL ANTIBODY AND ENZYME LINKED IMMUNOSORBENT ASSAY FOR DETECTION OF OFF-FLAVOR

COMPOUND 2-METHYLISOBORNEOL

A Dissertation

Submitted to the Graduate Faculty o f the Louisiana State University and

Agricultural and Mechanical College in partial fulfillment of the

requirements for the degree o f Doctor o f Philosophy

in

The Department of Food Science

byEun Sung Park

B.S., Korea University, 1991 M.S., Korea University, 1993

December 1999


UMI Number 9951614

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UMIUMI Microform9951614

Copyright 2000 by Bell & Howell Information and Learning Company. All rights reserved. This microform edition is protected against

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P.O. Box 1346 Ann Arbor, Ml 48106-1346


DEDICATION

To my lovely wife Young Ju Lee, whose love and patience gave me the

power to overcome whenever there were troubles and difficulties. I am glad that I

have a chance to show my love and my appreciation to her.

To my lovely boy Han Bin Park, his presence in this world brings the most

joy and happiness to me. My boy, I will try hard to be a good father.

To my mother and father, I wouldn’t have been able to complete this study

without their love and support. I have always loved you and I am proud of you.

a


ACKNOWLEDGEMENT

I would like to thank to Dr. Leslie Plhak for her help, support and

wonderful advice as my major advisor. I also thank Dr. Douglas L. Park, Dr. J.

Samuel Godber and Dr. Ronald J. Siebeling for their kind advice, suggestions,

encouragement and support on this thesis.

I would like to thank to Dr. Witoon Prinyawiwatukul and Dr. Joan M.

King for their encouragement and advice whenever I had a frustration and

difficulties. It was really big help for me to overcome the difficulties while I was

in this Ph. D. program.

I feel that 1 had enjoyed studying in this food science department and I

really appreciate the help and support from all o f the students and faculty in this

department.

iii


TABLE OF CONTENTS

DEDICATION........................................................................................................... ii

ACKNOWLEDGEMENT....................................................................................... iii

LIST OF TABLES...................................................................................................-vi

LIST OF FIGURES.................................................................................................vii

ABSTRACT............................................................................................................... x

CHAPTER 1. INTRODUCTION............................................................................ 1

CHAPTER 2. REVIEW OF LITERATURE......................................................... 32.1 Off-flavor problem in water and aquaculture industries............................ 3

2.1.1 Off-flavor problems in water and aquaculture industries............... 32.1.2 Occurrence of off-flavor compounds in natural waters...................52.1.3 Earthy-musty or muddy flavor in fish..............................................72.1.4 Off-flavor problems in the catfish industry......................................92.1.5 Economic burden............................................................................. 112.1.6 Detection thresholds o f off-flavor compounds...............................13

2.2 Production of off-flavor compounds by microorganism........................ 152.2.1 Production of off flavor compounds by actinomycetes.................152.2.2 Production of off-flavor compounds by blue-green algae.............16

2.3 Control o f off-flavor compounds in the aquacuture industry................ 232.3.1 Uptake of odorous compounds in fish............................................242.3.2 Depuration of off-flavor compounds from fish .............................272.3.3 Pond management and off-flavor....................................................30

2.4 Analysis o f off-flavor compounds............................................................332.4.1 Analysis of off-flavor compounds by gas chromatography mass

spectrometry (GC-MS)..................................................................... 342.4.2 Analysis o f off-flavor compounds by sensory analysis................ 392.4.3 Analysis o f off-flavor compounds by enzyme linked immuno

sorbent assay (ELISA)..................................................................... 392.5 Enzyme immunoassay and production o f monnoclonal antibody 40

2.5.1 Enzyme immunoassay..................................................................... 402.5.2 Development o f monoclonal antibody........................................... 41

2.6 Current research objectives....................................................................... 44

CHAPTER 3. MATERIALS AND METHODS.................................................453.1 Materials...................................................................................................... 453.2 Procedures................................................................................................... 46

3.2.1 Preparation of immunogen and solid phase protein conjugates... 463.2.1.1. Preparations o f bomeol-hemisuccinate, isobomeol-

hemisuccinate and MIB-hemisuccinate................................. 46

iv


3.2.12 . Preparation of immunogens.................................................. 483.2.1.3 Preparation of solid phase protein conjugates.....................49

3.2.2 Preparation o f monoclonal antibody............................................. 513.2.2.1 Immunization..........................................................................513.2.2.2 Cell fusion and selection........................................................ 513.2.2.3 Cell cloning.............................................................................533.2.2.4 Ammonium sulfate precipitation o f monoclonal antibody ...54

3.2.3 Enzyme immunoassay (E l).............................................................563.2.3.1 Testing mice sera for determination of antibody tite r.........563.2.3.2 Competitive enzyme immunoassay......................................57

CHAPTER 4. RESULTS AND DISCUSSION...................................................614.1 Production o f immunogen and solid phase protein conjugate................ 6 14.2 Production o f monoclonal antibody......................................................... 68

4.2.1 Test o f mouse polyclonal antibody................................................684.2.2 Fusion and screening of mouse 1 and 2 immunized with

MIB-LPH......................................................................................... 744.2.3 Fusion and screening o f mouse 3 that had been immunized with

bomeol-LPH.................................................................................... 784.2.4 Cloning of anti bomeol monoclonal antibody..............................78

4.3 Effects o f Ab and solid phase conjugate concentrations on the sensitivity of ELISA................................................................................ 804.3.1 Effect of Ab concentration on the sensitivity o f ELISA............ 804.3.2 Effect of solid phase conjugate concentrations on the sensitivity

o f ELISA........................................................................................ 924.4 Effect o f solid phase conjugate protein structure on the sensitivity o f

ELISA.......................................................................................................... 984.5 Specificity o f antibody............................................................................. 1054.6 Standard curve..........................................................................................110

CHAPTER 5. SUMMARY AND CONCLUSION...........................................114

REFERENCES...................................................................................................... 117

APPENDIX I. EPITOPE DENSITY DETERMINATION............................... 130APPENDIX H. SCREENING (1)........................................................................ 131APPENDIX Iff. SCREENING (2)....................................................................... 132APPENDIX IV. RESULT OF 1st CLONING..................................................... 133APPENDIX V. RESULT OF 2nd CLONING..................................................... 134APPENDIX VI. RESULT OF 3rd CLONING.................................................... 137

VITA.......................................................................................................................138

V


LIST OF TABLES

Table

Table

Table

Table

Table

Table

Table

Table

Table

Table

Table

Table

. Threshold odor concentrations o f geosmin and MIB reported in the literature.......................................................................................... 14

L Blue green algae previously reported to produce geosmin in the literature........................................................................... 19

. Blue green algae previously reported to produce MIB in the Literature.................................................................................................. 21

k Gas chromatography Mass spectrometry method reported in the literature for the detection o f off-flavor compounds, geosmin and M IB............................................................................................................. 37

k R f values o f hemisuccination reaction product...................................... 67

Molecular weight and hapten numbers o f solid phase protein conjugates. Each molecular weight was determined by M ALDI............................. 71

r. Effects o f Ab concentrations on the sensitivity o f ELISA when MIB- BSA was used as coating protein..............................................................86

!. Effects o f Ab concentrations on the sensitivity o f ELISA when bomeol-BSA was used as coating protein.............................................. 91

k Effects o f solid phase conjugate (MIB-BSA) concentrations on the sensitivity o f ELISA................................................................................. 97

0. Effects o f solid phase conjugate (bomeol-BSA) concentrationson the sensitivity o f ELISA................................................................... 102

1. Test of solid phase structure effect...................................................... 109

2. Cross reactivity o f compounds that have similar structurewith M IB ................................................................................................I l l

vi


LIST OF FIGURES

Figure 1. The structure o f off-flavor compounds, 2-methyIisobomeoland geosmin................................................................................................ 4

Figure 2. Preparation o f immunogen (bomeol-LPH)...........................................47

Figure 3. Production of monoclonal antibody by fusion o f splenocytes o fmouse with myeloma cells.......................................................................55

Figure 4. Procedure of competitive ELISA...........................................................59

Figure 5. Structures o f compounds that are used for the conjugation o fimmunogen and solid phase conjugate, 2-methylisobomeol, bomeol and isobomeol........................................................................................... 64

Figure 6. TLC result of MIB-hemisuccination..................................................... 65

Figure 7. TLC result of bomeol-hemisuccination........................................ 66

Figure 8. Matrix assisted laser desorption ionization (MALDI) spectrums for bovine serum albumin (BSA) and 2-methylisobomeol (MIB)-BSA conjugate....................................................................................................69

Figure 9. Matrix assisted laser desorption ionization (MALDI) spectrums for isobomeol-bovine serum albumin (BSA) and bomeol-BSA conjugate....................................................................................................70

Figure 10. Checker board ELISA o f mice (mouse land 2) sera afterimmunization of MIB-LPH two times................................................. 72

Figure 11. Checker board ELISA o f mice (mouse 3and 4) sera afterimmunization of bomeol-LPH two times............................................ 73

Figure 12. Test of mouse sera for the competitive inhibition o f binding to solid phase protein conjugate..........................................................................75

Figure 13. Cloning of cells for the production of Ab that specifically bind to free MIB after fusion o f splenocytes of mouse 2 (immunized with MIB-LPH) with myeloma cells............................................................77

Figure 14. Screening o f cells for the production of antibody after 5 days and 9 days fsuion o f splenocyets (mouse 3 immunized with bomeol-LPH) with myeloma cells...................................................... 79

vii


Figure 15. Standard curves constructed using cell culture supernatants fromthree times cloned cell line f6b4g7b4.................................................. 81

Figure 16. Test o f primary Ab concentrations effect when high concentration(1 pg/ml) o f MIB-BSA was used as coating protein........................... 83

Figure 17. Test o f primary Ab concentrations effect when medium concentration (0.5pg/ml) of MIB-BSA was used as coating protein.............84

Figure 18. Test o f primary Ab concentrations effect when low concentration(0.25pg/ml) o f MIB-BSA was used as coating protein...................... 85

Figure 19. Test of primary Ab concentrations effect when high concentration(lpg/m l) o f bomeol-BSA was used as coating protein....................... 88

Figure 20. Test o f primary Ab concentrations effect when medium concentration (0.5pg/mi) of bomeol-BSA was used as coating protein....... 89

Figure 21. Test of primary Ab concentrations effect when low concentration(0.25pg/ml) o f bomeol-BSA was used as coating protein..................90

Figure 22. Test o f solid phase protein conjugate (MIB-BSA) concentrations effect when high concentration o f Ab (F6b4g7b4, 1/125 times) was used as a 1st A b ...................................................................................... 94

Figure 23. Test of solid phase protein conjugate (MIB-BSA) concentrations effect when medium concentration o f Ab (F6b4g7b4, 1/250 times) was used as a 1st A b............................................................................ 95

Figure 24. Test of solid phase protein conjugate (MIB-BSA) concentrations effect when low concentration o f Ab (F6b4g7b4,1/500 times) was used as a 1st A b............................................................................... 96

Figure 25. Test o f solid phase protein conjugate (bomeol-BSA) concentrations effect when high concentrations o f Ab (F6b4g7b4, 1/125 times) was used as a 1st A b...............................................................................99

Figure 26. Test o f solid phase protein conjugate (bomeol-BSA) concentrations effect when medium concentration o f Ab (F6b4g7b4,1/250 times) was used as a Ist A b............................................................................ 100

Figure 27. Test o f solid phase protein conjugate (bomeol-BSA) concentrations effect when low concentration o f Ab (F6b4g7b4, 1/500 times) was used as a 1st A b .................................................................................... 101

viii


Figure 28. Comparison o f Ab titer using three different solid phase proteinconjugates ............................................................................................ 106

Figure 29. Comparison o f Solid phase conjugate affinity to anti bomeolmonoclonal antibody (F6b4g7b4)....................................................... 106

Figure 30. Test o f solid phase conjugate structure effect using 1/250 times o f 1st Ab (Cell line f6b4g7b4) and 0.5 pg/ml o f solid phase conjugates.............................................................................................. 107

Figure 31. Test o f solid phase conjugate structure effect using 1/125 times of 1st Ab (Cell line f6b4g7b4) and 1 pg/ml o f solid phase conjugates ............................................................................................ 108

Figure 32. Standard curve constructed using Ab concentration o f 1/250(f6b4g7b4) and 0.5 fig/ml o f MIB-BSA as a solid phase protein conjugate ............................................................................................ 113

ix


A B S T R A C T

2-Methylisobomeol (MIB) is a secondary metabolite o f cyanobacteria and

fungi that causes earthy and musty taste and odor. This is a significant problem in

the aquaculture industry and large scale water supplies. Water and aquaculture

products exposed to this compound may become unacceptable to consumers.

Especially the accumulation of this compound in the fish flesh is a major problem

for the channel catfish industry. To monitor the levels o f this compound for

quality control and abatement, rapid, sensitive and inexpensive methods are

needed.

This research reports the development o f an indirect enzyme linked

immunosorbent assay (ELISA) for MIB using monoclonal antibodies. For the

preparation o f monoclonal antibodies against MEB, MLB-Limitius polyphemus

hemocyanin (LPH) and bomeol-LPH were synthesized as an immunogen. In

order to compare and optimize the effect of solid phase conjugate structure,

bomeol-bovine serum albumin (BSA), isobomeol-BSA, MIB-BSA were

synthesized as a solid phase protein conjugate. To produce monoclonal

antibodies, two mice (3-4 weeks, female, BALB/c) were immunized with

bomeol-LPH protein conjugate and another two mice were immunized with MIB-

LPH. Hybridoma cells were made by the fusion o f myeloma cells and spleen cells

o f the mice that showed high antibody titer and specificity. Hybridoma cells that

secreted high affinity monoclonal antibodies were cloned by the limiting dilution

method three times to ensure clonality. For the optimization o f ELISA, different

x


Ab concentrations and different solid phase conjugate concentrations were tested.

At lower concentrations of antibody and solid phase conjugate, the sensitivity was

greatest, however the signal also began to decrease. The effect o f solid phase

conjugate structure was studied by comparing the sensitivity o f ELISA using

bomeol-BSA, isobomeoI-BSA, and MIB-BSA as a solid phase conjugate. When

MIB-BSA was used as a solid phase conjugate, the affinity for the free MIB was

improved against the affinity to MIB-BSA and the sensitivity o f ELISA was

greatest. By using anti-bomeol monoclonal antibody (1/250) and MIB-BSA (0.5

pg/ml) as a solid phase conjugate, standard curve was constructed from 100 mg/L

to 0.1 ng/L range. The linear range o f this standard curve was found to occur

between lng/L and I mg/L MIB. The detection limit, defined as the MIB

concentration giving an A/Ao value of 0.8, was found to be approximately lng/L.

xi


CHAPTER 1. INTRODUCTION

Geosmin and 2-methylisobomeol are produced by blue green algae,

filamentous bacteria and fungi (Gerber, 1967; Dionigi et al., 1992; Safferman et

al., 1967; Lovell and Sackey, 1973). These compounds can impart musty and

earthy off-flavors to potable water, aquaculture raised fishes and food, causing

economic losses in these and related industries (Lorio et al., 1992). As low

amounts of these compounds are required for “off-flavor” and microorganisms

that produce these compounds are ubiquitous, the musty flavor compounds

geosmin and 2-methylisobomeol are a problem in the potable water and

aquaculture industries.

These two main compounds responsible for earthy and musty flavors are

synthesized by blue green algae and actinomycetes. They are taken up through the

gills and accumulate in the fatty tissues (Tucker and Martin, 1991).

Farm raised catfish (Ictalurus punctatus) has gained wide acceptance

among consumers as a high quality product with a mild flavor. Fish producers are

most affected by the off-flavor problem because o f their inability to market their

fish crop when desired. The first step of quality control in the processing o f farm

raised catfish is a flavor evaluation to check for the presence of off-flavors. Off-

flavor fish can not be harvested and brought to the market. If harvested fish arrive

at a processing plant and are determined to be off-flavor, they are returned to the

pond. The most often used practice for off-flavor improvement is depurating the

1


undesirable odor or flavor by changing the environment and holding for an

indefinite period o f time (Arganosa and Flick, 1992).

Producers are doing a thorough job o f screening and taste testing fish and

water for flavor quality to maintain standards (Chung et al., 1991). Sensory

evaluation is the most sensitive method o f analysis currently available (Johnsen

and Kelly, 1990). However, as it is a subjective method, it may show large

variation between tasters and between replicate samples.

Gas chromatographic (GC) methods for the determination o f geosmin

have been developed and are non-subjective. However the lack o f sensitivity,

expense, technical training, extensive sample preparation and variability limit the

application of these methods by industry.

Immunochemical methods can be accurate, simple and sensitive. There is

a great demand for a rapid, sensitive and non-subjective method for monitoring

off-flavor problems in the catfish production and processing industries as well as

for research.

Polyclonal antibody produced by Chung et al. (1990) was not acceptable

because of poor sensitivity. In this study a ELISA system was developed specific

for MIB and having a detection limit low enough to detect MIB from fish or water

without sample concentration.


CHAPTER 2. REVIEW OF LITERATURE

2.1 Off-flavor problem In water and aquaculture industries

2.1.1 Off-flavor problems in water and in aquaculture industries

"Off-flavors" are objectionable tastes or odors in water or foods. Off-

flavors in fish can be caused by feed ingredients, natural foods, post-mortem

oxidative rancidity, or odorous compounds absorbed from the environment.

Sources o f environment related off-flavors include chemical pollution and extra

cellular metabolites of aquatic bacteria or algae. Organic compounds responsible

for off-flavors are rapidly absorbed by fish from water and stored in lipid rich

tissues; elimination is relatively slow (Tucker and Martin, 1991).

Two metabolites, which are the primary compounds responsible for earthy

or musty "off-flavor" in fresh catfish, are geosmin (la , 10b-dimethyl-9a-decalol)

(Figure 1) and 2-methylisobomeol (1-R-exo-l,2,7,7,-tetramethyl-bicycIo-[2.2.1]-

heptan-2-ol) (MIB) (Figure 1). These two compounds are metabolites synthesized

by some cyanobacteria, actinomycetes and fungi, and cause problems for the

potable water and aquaculture industries because o f the ubiquitousness of the

organisms that produce these metabolites (Hansen, 1964; Gerber, 1967;

Safferman et al., 1967; Lovell and Sackey, 1973; Izaguire et al., 1982; Persson,

1982; Yagi, 1983; Yagi et al., 1983; Aoyama, 1990; Izaguire and Taylor,1995).

A causal relationship between certain odors in surface waters and aquatic

microorganisms was suspected before the turn of the century. This relationship

was suggested by the similarity o f odors in water and odors produced in cultures

3


of certain bacteria. The most common odor produced by these cultures has been

variously described as "muddy", "musty" or "earthy". Berthelot and Andre (1891,

as cited by Gerber 1979) considered the odors produced by actinomycete cultures

to be similar to those o f freshly plowed soil. They noted that the substance

was chemically neutral. Thaysen (1936) described the compound that caused the

“earthy” odor as an organic compound which is slightly soluble in water, volatile

in steam, soluble in ether and partly soluble in alcohol. Similar flavors have been

noticed in fish for centuries.

Figure 1. The structure o f off-flavor compounds, 2-methylisobomeol andgeosmin.

Gerber and Lechevalier (1965) analyzed an odor concentrate from an

actinomycetes culture by gas chromatography and found a single sharp peak

corresponding to the earthy odor detected at the exit port. They named the

compound geosmin from the greek "ge", for earth, and "osme", for odor. It was

described as a colorless neutral oil which darkens very slightly on long storage

and is unstable in an acidic environment (Gerber 1979). Chemical synthesis

responsible for the typical odor of soil could be extracted from soil by steam and

2-methylisobomeol geosmin

4


revealed that geosmin had four isomers, but only one gave the earthy odor

(Marshall and Hochestettler, 1968).

Soon after the identity o f geosmin was known, another earthy-smelling

metabolite of actinomycetes was described (Gerber, 1969; Medsker et al., 1969).

This compound, 2-methyisobomeoI, was previously known as a synthetic product

prepared by methylation o f camphor. The odor of 2-methyisobomeol is musty or

earthy in dilute solution, but camphorous in concentrated solutions. This

compound has been isolated from soils and fresh waters worldwide (Tucker and

Martin, 1991). MIB is a small terpenoid compound with a boiling point o f 210 °C

(Rosen etal., 1970).

Geosmin and 2-methyIsiobomeol are synthesized via the isoprenoid

biosynthetic pathway (Bentley and Meganathan, 1981). Geosmin is probably

derived from a C-15 sesquiterpenoid by loss o f an isopropyl side group. 2-

methylisobomeol appears to be derived from methyl addition to a monoterpenoid.

Intermediate products and mechanisms controlling bisosynthesis are unknown.

The biosynthetic pathways for geosmin and 2-methylisobomeol are presumably

common to all organisms found to produce these compounds (Tucker and Martin,

1991).

2.1.2. Occurrence o f off-flavor compounds in natural waters

Offensive tastes and odors in potable water may arise in the raw water

supply, during treatment procedures at water works (chlorination), or by microbial

growth in the distribution systems. Off-flavors in fish are generally due to the

same compounds that cause a bad taste and odor in the water (Persson, 1983). In

5


U.S. water supplies, offensive odors have been noticed since the 1850s, and in

European and Australian fisheries have been recorded since the beginning o f the

century. Off-flavors in natural waters have been known for a long time and they

are a world wide problem (Persson, 1983).

Various water supply systems have experienced elusive taste and odor

episodes not attributable to planktonic algae or actinomycetes. One such system is

that o f The Metropolitan Water District o f Southern California (U.S j V). It is a

major water wholesaler, serving about 12 million people in six counties, that

receives its water from the Colorado river and from northern California via the

massive State Water Project. In late 1974 and early 1975, there was an off-flavor

problem affecting the Colorado river portion o f the system, but no cause was

conclusively pinpointed. Again in September and October o f 1979, numerous

complaints o f musty taste were received, but despite extensive sampling

throughout the system, nothing was found that could explain the problem. In

September 1980, there was a recurrence o f earthy-musty odor in the water, but by

then the analytical methods for detecting odorous compounds in water had been

developed, and the problem was traced to MIB from a large source water reservoir

(McGuire et al., 1981; Izaguire et al., 1983).

The practical consequences o f off-flavors are obvious and include

consumer dissatisfaction, high treatment costs for water works, economic losses

for fisheries and reduced aesthetic value o f recreational areas (Persson, 1983). In

Finland, 75 water areas have been affected by off-flavors (Persson, 1978).

6


Lake Biwa is the largest lake in Japan. A musty odor was first found in

1969, and since then musty odor problems commonly occurred from May to early

June due to the metabolites produced by Phormidium tenue in 1970s. The

problem in the summer of 1981 was found to be caused by an algal bloom o f

Anabaena macrospora. MIB was found to be produced by Phormidium tenue,

whereas geosmin by Anabaena macrospora (Yagi et al., 1983).

2.1.3. Earthy-musty or muddy flavor in fish

The first complete description o f the origin and nature of earthy off-flavors

in fish was provided by Thayson (1936) and Thayson and Pentelow (1936). They

associated the cause o f off-flavor in Atlantic salmon (Salmo salar) from certain

rivers in Scotland with actinomycetes present in decaying beds o f submerged

reeds along the river bank. Thayson (1936) speculated that the earthy compound

produced by these actinomycetes is absorbed across the gills and transported to

various tissues via the bloodstream. The earthy flavor was also experimentally

produced in fish exposed to concentrated distillates from cultures of

actinomycetes (Thayson and Pentelow, 1936). A casual relationship between

certain odors in surface waters and aquatic microorganisms was suspected before

the turn o f the century and production o f earthy odors by cultures of

actinomycetes has been recognized since before 1900 (Thaysen, 1936).

In experimental aquaculture areas in Manitoba studied by Tabachek and

Yurokowski (1976), 30% of the lakes were affected by muddy odor. In the

autumn o f 1969, a severe problem o f muddy odor in fish in the Oulu sea area in

Finland caused hundreds o f fisherman to lose income (Persson, 1974). A muddy-

7


earthy flavor in rainbow trout (Salmo gairdnert), channel catfish (Ictalurus

punctatus) and carp (Cyprinrts carpio) has been associated with the presence of

species o f actinomycetes and blue green algae in aquatic environments (Arganosa

and Flick, 1992). The culturing o f rainbow trout in prairie pot-hole lakes in central

Canada has been impaired by the occurrence of this muddy-earthy flavor. Off-

flavor also has been reported as a comm on occurrence in carp ponds in China,

Japan, and Europe (Arganosa and Flick, 1992). Considerable research has been

conducted on earthy flavors in rainbow trout (Oncorhyncfms mykiss) cultured in

prairie pot-hole lakes in central Canada. Two highly odorous compounds,

geosmin and MIB, were confirmed as causes of the flavors, and cyanobacteria

capable o f producing these compounds were isolated and identified (Yurokowski

and Tabachek, 1974; Tabachek and Yurokowski, 1976). These two compounds

also caused off-flavor in walleye (Stizostedium vitreum), cisco (Coregonus

artedii), lake whitefish (Coregonus clupeaformis) and northern pike (Esox lucius)

from Cedar lake, an important commercial fishery in Manitoba, Canada

(Yurokowski and Tabachek, 1980).

Kuusi and Suihko (1983) surveyed off-flavors in fish from Finland from

1969 to 1981. Most o f the off-flavors were related to man-made pollution, but

earthy off-flavors were common, particularly in carp. Ashner et al. (1967)

reported from Israel that carp cultured in a sandy soil pond which was practically

mud-free and was supplied by water rich in plant nutrients were off-flavor. They

suspected the blue-green alga Oscillatora tenuis that was present in the pond to be

the cause o f earthy flavor.

8


Bream, Abramis brama, from a shallow eutropic brackish water bay in the

Gulf o f Finland had a muddy flavor. This flavor was significantly correlated with

the amount o f Oscillatoria agardhii in the area (Persson, 1978, 1979, 1981).

Oscillatoria agardii and O. princeps were also suspected o f being the cause o f an

off-flavor which affected a lake in Germany (Comellius and Bandt, 1933 as cited

by Aschner et al., 1967).

Lovell (1971) reported that a characteristic, objectionable earthy-musty

flavor is frequently found in intensively cultured catfish in south-central and

southeastern United States. Heavy concentrations o f odor-producing

actinomycetes and blue-green algae have been identified in ponds with “earthy-

musty” flavored catfish and were suspected of being the organisms responsible for

the problem (Lovell and Sackey, 1973). It is clear from this evidence that an

earthy-musty or muddy flavor in fish is a common problem throughout the world

(Persson, 1983).

2.1.4. Off-flavor problems in the catfish industry

Off-flavors, associated with algal blooms in aquaculture are a major

problem for the channel catfish industry. These undesirable tastes in catfish result

from accumulation o f geosmin and 2-methylisobomeol in fish flesh. Warm water

temperature and high feeding rates are associated with the incidence of off-flavor

in catfish ponds ( Johnsen, 1989 and Johnsen and Dupree, 1991).

Earthy off-flavors in pond raised channel catfish were initially described

by Lovell and Sackey (1973) and Malgalig et al. (1973). The incidence o f off-

9


flavor in channel catfish has increased dramatically as culture practices have

changed to increase fish yields (Brown and Boyd, 1982).

When ‘musty’ fish cannot be harvested and brought to the market, the fish

are held and fed until deemed “on-flavor’ by an experienced human taster

employed by the processing plant. Earthy/musty flavors constitute a significant

restriction to the growth o f the catfish industry. Fish that do not meet the

processor’s quality standards are called off-flavor and are not harvested until

flavor quality has improved enough for fish to be considered acceptable to

consumers. If the flavor of the fish sampled is unacceptable, the fish are rejected

for processing; fish are either not harvested or returned to the pond from the

transport truck (Tucker and Martin, 1991). The Research Committee o f the

Catfish Fanners o f America identified off-flavor as the most serious problem in

the industry. Reasons for the seriousness o f the problem are the high rate of

occurrence o f off-flavor, the damage that can be done to product image if off-

flavor fish get into market channels, and the lack of control measures for off-

flavor (Lelana, 1987).

Sensory scores for channel catfish sampled from four experimental ponds

at Stoneville, Mississippi (Tucker and Martin, 1991) illustrate the dynamic nature

of the incidence and severity o f off-flavor. On the initial sampling in July, all four

ponds were judged to contain acceptably flavored fish; in early October, three of

the four samples were judged off-flavor to varying degrees. Off-flavor episodes

varied from two weeks to over three months. The highest incidence o f off-flavor

occurred in September and October with samples from 10 of the 14 ponds judged

10


off-flavor. The most intense off-flavors also occurred at this time; the flavor

described as "blue-green" (confirmed as being caused by MIB) was most common

and the most severe (Tucker and Martin, 1991). Studies of catfish culture ponds in

Alabama showed fish from 50-75 % o f the ponds sampled in late summer or early

fall were off-flavor and unacceptable for harvesting (Brown and Boyd, 1982;

Armstrong et al., 1986; Lovell et al., 1986). These two off-flavor compounds,

MIB and geosmin, afflicted 50-70% of the ponds involved in channel catfish

culture in Western Mississippi (Martin et al., 1987). The predominant off-flavor

was "earthy-musty". Geosmin was confirmed in 80% o f the fish with earthy-

musty off-flavors; MIB was not detected in any fish sampled (Lovell et al., 1986).

The reasons for the different chemical etiologies o f earthy-musty off-flavors in

Alabama and Mississippi are not known.

Total incidence of ponds with off-flavor fish, and the incidence o f earthy-

musty flavors increased during the summer growing season as phytoplankton

biomass increased in response to higher water temperatures and amounts o f feed

added to ponds. During the summer and autumn months approximately half o f the

commercially cultured channel catfish presented for processing at a given time,

are rejected because of objectionable flavor and odor (Martin et al., 1988a and

1990; Brown, 1996).

2.1.5. Economic burden

Producers o f channel catfish consistently identify environment related off-

flavors as their major production problem (Tucker and Martin, 1991). Pond

culture o f channel catfish is the largest aquaculture industry in the United States.

11


In 1988, over 150 million kg o f fish were produced. About 80% o f the total was

produced in a limited geographical area o f west central Mississippi. In 1990,

about 62,000 ha of water were used to produce about 164 million kg o f catfish in

the United States. Alabama and Mississippi had about 12% and 60% of the total

area and catfish production (Hariyadi et al., 1994).

Keenum and Waldrop (1988) attempted to estimate the effects o f off-

flavor on production costs of Mississippi pond-raised channel catfish. When off-

flavor occurred during the winter, the only additional cost charged was an

opportunity cost for delayed income. When off-flavor occurred during the

summer growing season, interest was charged on the quantity o f fish that couldn't

be sold. Other potential costs accruing from off-flavor are difficult to estimate.

Fish may be lost to infectious diseases or poor water quality while the fish are in

inventory waiting for off-flavor to abate. Market constraints (not being able to sell

fish when they reach market size, also called quotas) also impact annual

production cost.

After production o f geosmin or MIB ceases, the compound is purged from

the fish. However the costs associated with delayed harvest o f off-flavor fish can

be substantial. For example, it has been estimated that environment-derived off-

flavor may add 10-20% to the cost o f producing channel catfish in the

southeastern United States (Paerl and Tucker, 1995). Holding market size fish in

inventory because o f off-flavor, restricts sales and cash-flow and increase the cost

of production by $0.02-0.20/lb fish because of feeding, aeration, and lost

opportunities to sell fish (Weirich, 1995).

12


2.1.6. Detection thresholds o f off-flavor compounds

The human gustatory thresholds o f geosmin or MIB are reported to be

0.004 pg/L to 0.2 pg/L (Safferman et al., 1967; Buttery et al., 1976; Persson,

1980; Johnsen and Kuan, 1987; Mallevialle and Suffet, 1987; Arganosa and Flick,

1992). Table 1 depicts the threshold odor concentrations o f geosmin and MIB that

have been previously reported in the literature. The results of the threshold tests

for geosmin and MIB are variable depending on the panelist. As the definition o f

the threshold concentration is different between the groups, the threshold

concentrations are variable in each previous report. Persson (1979b) defined the

threshold odor concentration o f MIB as the concentration that 75% o f the judges

considered muddy and found that threshold concentration of MIB in water was

from 0.018 to 0.042 pg/L.

The sensory threshold concentration o f geosmin in fish ranges from 0.6 to

6.5 pg/kg (Yurokowski and Tabachek, 1974; Persson, 1980). Sensory threshold

flavor concentrations vary with the species tested; geosmin is most easily detected

in mild-flavored fish than in fish that have a fairly strong flavor, such as rainbow

trout (Tucker and Martin, 1991).

The threshold concentration o f geosmin in rainbow trout was found to be

6.5 pg/kg (Yurokowski and Tabachek, 1974), in bream it was found to be 0.9

pg/kg (Persson, 1980) and in channel catfish it was found to be 8.4 pg/kg (Lelana,

1983). The threshold odor concentration of 2-methylisobomeol is about 0.04 pg/L

(Persson, 1979b). This is higher than the threshold odor concentration for geosmin

13


Table 1. Threshold odor concentrations of geosmin and MIB reported inliterature.

Sample Off flavor compound

Odor Threshold Reference

Bream geosmin 0.9 pg/kg Persson, 1980Catfish geosmin 8.4 pg/kg Lelana, 1983Pike geosmin 0.59 pg/kg Persson, 1980Rain bow trout

geosmin 6.5 pg/kg Persson, 1980

Rainbowtrout

geosmin 6.5 pg/kg Yurokowski and Tabachek, 1974

Water geosmin 0.004 pg/L Mallevialle and Suffet, 1987Water geosmin O.OIpg/L Cees et al., 1974Water geosmin 0.015 pg/L Persson, 1979Water geosmin 0.02 pg/L Zoetman, 1980Water geosmin 0.02 pg/L Sano, 1988Water geosmin 0.021 pg/L Buttery et al., 1976Water geosmin 0.022 pg/L Ito et al, 1988Water geosmin 0.03 pg/L Johnsen and Kuan, 1987Water geosmin 0.05 pg/L Arganosa et al., 1992Water geosmin 0.05 pg/L Medsker et al., 1968Water geosmin 0.2 pg/L Safferman et al., 1967Pike MIB 0.085 pg/kg Persson, 1980Rainbowtrout

MIB 0.55 pg/kg Persson, 1980

Bream MIB 0.095 pg/kg Persson, 1980Cat fish MIB 100-200 pg/kg Martin etal., 1988bWater MIB 0.1 pg/L Wood and Snoeyink, 1977Water MIB 0.1 pg/L Medsker et al., 1969Water MIB 10 pg/L Martin etal., 1988bWater MIB 0.042 pg/L Persson, 1979

14


in water. However, the flavor imparted to fish by 2-methyisobomeol appears to be

detected by taste panels at concentrations about an order of magnitude lower than

geosmin. The sensory threshold concentration in pike is about 0.09 |ig/kg, and in

rainbow trout about 0.6 pg/kg (Persson 1980).

2.2 Production of off-flavor compounds by microorganism

2.2.1 Production of off flavor compounds by actinomycetes

Actinomycetes are unicelluar, filamentous microorganisms that frequently

occupy a position between the fungi and the bacteria as a separate group.

Actinomycetes are widely distributed in nature and account for a large part o f the

normal microbial populations o f soils, and lake and river muds (Romano and

Safferman, 1963).

Actinomycetes are most abundant in eutrophic lakes or ponds than in

nutrient poor waters. Abundance o f actinomycetes increase dramatically

following development of phytoplankton blooms. This suggests that

actinomycetes are important in the decomposition o f organic matter produced by

phytoplankton (Silvey and Wyatt, 1970).

Thaysen (1936) investigated a salmon rich stream in the British isles for

the origin o f earthy taints in fish, and he found that those odors varied with the

degree o f actinomycete growth at the place o f sampling. Since the isolation of

geosmin from cultures of Streptomyces griseus (Gerber and Lechevalier, 1965),

production o f geosmin has been confirmed in numerous actinomycetes, most

notably species o f Streptomyces and Norcardia. MIB was initially isolated from

15


cultures of Streptomyces antibioticus, S. praecox and S1 griseus (Medsker et al.,

1969) and has also been found in cultures o f several other actinomycetes.

Production o f geosmin and 2-methylisobomeol by actinomycetes in

culture varies with carbon source, temperature, and pH. Geosmin production by a

strain of Streptomyces was greatest at 25 °C and a culture pH o f 10 (Weete et al.,

1977). Geosmin was not produced at a temperature below 15 °C and geosmin

production was markedly decreased at pH values less than 7. Growth o f

Streptomyces and subsequent production o f geosmin is greatest in environments

with high dissolved oxygen concentration (Blevins, 1980; Wood et al., 1983).

2.2.2. Production o f off-flavor compounds by blue-green algae

Cyanobacteria occur in a wide variety o f environments (Carr and Whitten

1982; Reynolds, 1984), and represent at least 22 genera, including over 90 species

that have been identified from freshwater habitats.

Development o f cyanobacterial blooms is favored under conditions o f high

nutrient loading rates, low rates of vertical mixing and warm water temperature.

Unique physiological attributes of cyanobacteria such as N2 fixation and

buoyancy regulation allow cyanobacteria to compete effectively with other

phytoplankton and make cyanobacteria the dominant phytoplankton communities

(Paerl and Tucker, 1995).

Symploca muscorum is the first blue green algae that was identified to

produce gesomin in culture medium and this discovery lead to the idea that

organisms other then actinomycetes could be possible sources o f earthy and odor

problems in water supplies (Safferman et al., 1967).

16


Ashner et al. (1967) provided the first complete account o f earthy off-

flavors in fish produced by cyanobacteria. They reported that Oscillatoria tenuis

growing in fish culture ponds in Israel was responsible for earthy off-flavors in

carp, Cyprinus carpio, and suggested that the fish acquired the off-flavor either by

absorbing the compound from the water or by ingesting masses o f cyanobacteria.

During the spring o f 1969, a bloom o f Anabaena circinalis occurred in

Garza-little Elm Reservoir, which serves as the water supply for Denton, Texas.

Coincident with this bloom occurrence, a taste and odor problem o f the earthy to

musty variety developed in the Denton municipal water supply. The isolation of

Anabaena circinalis into unialgal culture and analysis o f noxious odorous

metabolites was conducted by North Texas State University and geosmin was

identified to be responsible for the odorous nature of the water supply during the

algal bloom incident (Henley, 1970).

Eleven axenic or unialgal cultures o f blue green algae, 10 producing

geosmin and 1 producing MIB were isolated from saline lakes in southwestern

Manitoba- Algae producing geosmin were Oscillatoria cf. prolifica (Greville)

Gomont, O. tenuis Agardh, O. cf. cortiana Meneghini, O. cf. variabilis Rao, O.

agardhii Gomont, O. cf. splendida Greville, O. sp., Symploca cf. muscorum

(Agardh) Gomont, Lyngbya cf. aestuarii (Mertens) Liebman.

O f the geosmin producing blue green algae isolated from the saline lakes

in Manitoba, it has been reported that Oscillatoria agardhii, O. prolifica, O.

tenuis and Symploca muscorum produced muddy odors. (Cornelius and Bandt,

1933; Ashner et al., 1967; Safferman et al., 1967; Medsker et al., 1968) and the

17

. . . I '•


latter two algae have been shown to produce geosmin (Safferman et al., 1967;

Medsker et al., 1968). It was also reported that O.princeps Vaucher, O. limosa

Agardh and O. chalybea Mertens produced a muddy odor (Cornelius and Bandt,

1933; Ashner et al., 1967; Leventer and Eren, 1970). Tabachek and Yurokowski

(1976) also found that O. splendida, O. prolificca, O. cortiana and Lyngbya

aestuarii produced the most geosmin, approximately 4 times more than O. tenuis

and Symploca muscorum, 20 times more than O. agardhii, and 50 times more than

O.variabilis. Lyngbya cryptovaginata isolated from a lake in Manitoba, Canada,

used to culture rainbow trout, is the first cyanobacteria reported to produce MIB

(Tabachek and Yurokowski, 1976).

Two Oscillatoria strains have been isolated from three different water

supply systems in California that have experienced taste and odor problems in

their drinking water. The organisms, Oscillatoria curviceps and O. tenuis variant

levis Gardner, yielded 2-methylisobomeol (MIB) at 60-150 pg/L in culture

(Izaguire et al., 1983).

Oscillatoria chalybea is a single organism that is responsible for the

production o f MIB in Mississippi catfish ponds. Growth o f algae and MIB

production by Oscillatoria chalybea are limited to the months May through

November and most likely to occur from early June through late September

(Weirich, 1995).

As with the actinomyetes, the production o f odorous metabolites by

cyanobacteria appears to be dependent on environmental conditions, but little

18


Table 2. Blue green algae reported to produce geosmin in the literature.

Blue green algae off -flavor compound

reference

Anabaena circinalis geosmin Henley, 1970Anabaena laxa geosmin Rashash et al., 1995Anabaena macrospora geosmin Yagi, 1983Anabaena macrospora geosmin Yagi et al., 1983Anabaena macrospora klebahn

geosmin Negoro et al., 1988

Anabaenascheremetievi

geosmin Izaguire, 1982

Anabaena viguieri geomin Wu et al., 1991Aphanizomenon flos- aquae

geosmin Matsumoto and Tsuchiya, 1988

Lyngbya cf. aestarii (Mertens) Liebman

geosmin Tabachek and Yurokowski, 1976

Nostoc sp geosmin, MIB HuandChiang, 1996Oscillatoria agardhii geosmin Persson, 1979aOscillatoria agardhii Gomont


Oscillatoria amoena geosmin Matsumoto and Tsuchiya, 1988Oscillatoria animalis geosmin Kaji, 1974 (cited in Yagi et al.,

1983)Oscillatoria bometii f. tenuis

geosmin Berglind et al., 1983

Oscillatoria brevis geosmin Naes et al., 1989Oscillatoria cf. Prolifica (Greville) Gomont

geosmin Tabachek and Yurogowski, 1976

Oscillatoria cf. Splendida Greville


Oscillatoria cf. Variabilis Rao


Oscillatoria cf.cortiana Meneghini


Oscillatoriasimplicissima

geosmin Izaguire et al., 1982

Oscillatoria splendida geosmin Matsumoto and Tsuchiya, 1988Oscillatoria tenuis Agardh


Oscillatoria tenuis geosmin, MIB Wu and Juttner, 1988

19


Table 2. continued.

Phormidium calcicola geosmin, MIB R ash ash et al., 1995Schizothrix mulleri geosmin Hariyama et al., 1972 (cited in

Yagi e ta l., 1983)Symploca muscorum geosmin Safferman et al., 1967Symploca muscorum (Agardh) Gomont


20


Table 3. Blue green, algae reported to produce MIB in the literature.

Blue green algae Off-flavorcompound

Reference

Lyngbya cf. Cryptovaginata

MIB Tabachek and Yurokowski, 1976

Oscillatoria curviceps MIB Izaguire et al., 1982Oscillatoria curviceps MIB Izaguire et al., 1983Oscillatoria germinata MIB Matsumoto and Tsuchiya, 1988Oscillatoria limnetica MIB Matsumoto and Tsuchiya, 1988Oscillatoria tenuis MIB Izaguire et al., 1982Oscillatoria tenuis Agardh

MIB Negoro et al., 1988

Oscillatoria tenuis var. levis gardner

MIB Izaguire et al., 1983

Oscillatoria sp MIB Martin et al., 1991Phormidium tenue MIB Yagi etal., 1983Phormidium tenue (Meneghini) Gomont

MIB Negoro eta l., 1988

21


information is available describing these interactions (Tucker and Martin, 1991).

The majority of odor and taste episodes in eutropic lakes and ponds (such as

aquaculture ponds) are believed to be caused by proliferation o f cyanobacteria

rather than actinomycetes (Persson 1981, 1982,1983).

The continuous addition o f large amounts o f nutrients in the form o f fish

waste makes channel catfish ponds ideal habitats for aquatic plants. Although

filamentous algae and vascular plants at times become established and grow

luxuriantly, the most common plant form is phytoplankton.

Phytoplankton density and taxonomic composition differ among ponds at

any given time, but in general, phytoplankton growths are dominated by

cyanobacteria during the summer months and diatoms during the winter months.

The most common cyanobacteria found during the warmer months are species of

Microcystis, Oscillatoria, Rhaphidopsis and Anabaena (Tucker and Martin,

1991).

Primary production by phytoplankton is the base o f the food chain in pond

cultures that depend upon natural foods to support fish or crustacean production.

They are part o f the pond microbial community that acts to maintain adequate

environmental conditions for culture. They are net producers o f dissolved oxygen,

they assimilate ammonia as a nitrogen source for growth, thereby reducing the

accumulation o f un-ionized ammonia, which can be toxic to aquatic animals at

relatively low concentrations (Paerl and Tucker, 1995).

The sudden death o f dense, nearly monospecific cyanobacterial

communities can have disastrous consequences in aquaculture ponds. As dead

22


algal cells decompose, photosynthetic oxygen production nearly ceases and large

amounts o f dissolved oxygen are consumed. The odorous metabolites produced

by certain species are released when cells decompose. The occurrence o f earthy,

musty off-flavors in pond-raised fish is episodic, coinciding with the appearance

and eventual disappearance o f the cyanobacterial species responsible for synthesis

of odorous compounds.

2.3 Control o f off-flavor compounds In the aquacutnre industry

Research related to quality control o f catfish flavor has been focused at the

pond level by studying factors that influence the growth o f algae or fungi

populations such as water quality, pH, oxygen levels, soil quality, fish density,

treatment of algicide, feed quality and application rates, and water temperature

(Tucker and Boyd, 1978a, 1978b; Lovell, 1983; Tucker et al., 1983; Persson,

1984; Tucker and Llyod, 1987; Johnsen, 1989; Johnsen and Dupree, 1991;

Tucker and Martin, 1991; Whitmore and Denny, 1992; Dionigi, 1994; Dionigi

and Ingram, 1994; Dionigi, 1995; Dionigi and Champagne, 1995; Petersson et

al., 1995; Velzeboer et al., 1995).

Because o f the large numbers o f factors that affect algae/fungal growth,

controlling the “off-flavor” by pond management has proven difficult. Three

approaches have been used to deal with off-flavor in pond-raised fish; manage

around off-flavor episodes, prevent off-flavor episodes, or remove the flavor from

the fish once they have developed it (Tucker and Martin, 1991).


2.3.1. Uptake o f odorous compounds in fish

It is generally believed that odorous compounds are absorbed across the

gills and deposited in the flesh (Weirich, 1995). MIB is also taken up by fish

through their gills and stored in fatty tissue (Martin et al., 1988c and 1990;

Persson, 1984). Fish with higher fat reserves are more prone to “musty” flavor

(Johnsen et al., 1996). In freshwater fish, due to osmoregulation, water is

absorbed into the body through the gills and the excess water in the body excreted

through the kidney (Smith, 1983). Therefore, many compounds or ions that

dissolve in water will enter the body through the gills and be distributed in the

body through the circulatory system. Thaysen (1936) demonstrated that live fish

absorb flavor compounds from their environment but dead fish do not.

Channel catfish, which were put into water containing dimethyl-sulfide or

2-pentanone at concentrations o f 25 to 125 mg/L, absorbed sensorily detectable

levels of those chemicals within 10 to 15 minutes (Maligalig et al., 1975b). Lovell

and Sackey (1973) produced earthy-musty flavored channel catfish by placing the

fish in water containing either a culture of geosmin producing blue green algae or

a filtrate of the culture. Fish held in tanks containing algae free filtrate from algae

culture tanks acquired the off-flavor but at a slower rate than the fish in the culture

tanks.

Johnsen (1989) transferred catfish to temperature controlled experimental

tanks when they were exposed to geosmin and MIB. A t 20 °C, fish exposed to 1

pg/L of geosmin showed a rapid uptake of material. The concentration o f geosmin

24


in the tissue increased to 3.5 pg/L within two hours. Studies with MIB indicated

that concentrations o f 0.5 pg/L can flavor fish in two hours when evaluated by

sensory panels (Arganosa and Flick, 1992).

The absorption o f organic compounds from water can occur at the gills,

through the skin, or across the intestinal epithelium from water swallowed while

drinking or incidentally while feeding. From and Horlyck (1984) placed geosmin

producing cyanobacteria Symploca muscorum at isolated locations on live

rainbow trout and recorded the development of earthy off-flavor in fillets obtained

after various exposure periods. The most rapid absorption occurred through the

gills, with only 6 min o f exposure required to obtain an earthy flavor. Absorption

was slower through the skin (1.5 h), small intestine (4 h) and stomach (7 h).

Lovell and Sackey (1973) and Maligalig et al. (1975) also suggested that

gills are the major routes o f uptake o f odorous compounds from water. However

Persson and York (1978) noted that total uptake o f 2-methylisobomeol from water

was higher in fish that were fed compared to those not fed. They speculated that

the alimentary tract may be a significant route o f uptake o f odorous compounds

and that these compounds are absorbed from water incidentally swallowed during

feeding.

The uptake o f earthy flavor in fish increases during feeding (Persson,

1980), but the earthy-odor compounds in the water is alone sufficient to taint fish.

Ingestion o f organisms is o f minor importance in the development o f off-flavor in

intensively cultured fish because virtually all nutrients are derived from feeds. The

25


major route o f uptake is absorption o f the compounds from water across the gills

(Tucker and Martin, 1991). Uptake o f odorous compounds by fish is rapid and

elimination is relatively slow.

Martin et al. (1988b) studied the uptake o f 2-methylisobomeol at two

concentrations in water by small (5-10 g) channel catfish. At steady state, the

concentration o f 2-methylisobomeol in fish muscle was about 10 times greater

than in water. Assuming a sensory threshold concentration o f about 0.1 mg/kg,

these fish would have developed a detectable off-flavor in less than 2 hr exposure

to either concentration o f 2-methylisobomeol. Highest concentrations of 2-

methylisobomeol were found in lipid rich tissues such as skin and visceral fat;

concentrations o f 2-methylisobomeol in visceral fat were almost 100 fold greater

than in water.

According to Lelana (1987), if the concentration o f geosmin in water were

1 mg/L, only about 2 hr would be required for the fish to absorb enough geosmin

to reach the sensory threshold concentration (estimated to be 8 mg/kg). I f the

concentration of geosmin in the water were 3 mg/L, the time to reach the sensory

threshold is reduced to only about 2 min (Tucker and Martin, 1991).

Factors affecting the rate o f uptake are the concentration of odorous

compounds in the fresh water and exposure time, the species of fish, the

physiological state of fish, water temperature and other environmental conditions

(Persson, 1984).

26


2.3.2. Depuration o f off-flavor compounds from fish

Nonpolar, lipophilic compounds are eliminated from fish by passive

diffusion across the gills or skin, or by metabolism to more polar compounds that

are excreted from the kidney or secreted in gallbladder bile. Elimination through

the gills is probably the major route, but this may not be true for strongly

lipophilic substances that are not readily partitioned from lipid stores into the

blood (Tucker and Martin, 1991).

The purging rate is affected by quality o f holding water and holding

condition (static vs continuous flow), water temperature and the amounts o f

odorous compounds absorbed (Iredale and York, 1979; Lovell, 1974; Maligalig et

al., 1975).

Iredale and York (1979) determined the length o f time required to purge

undesirable flavor taints from pond cultured rainbow trout transferred to two

different clear water environments. Sensory data from trained judges showed that

this required 5 days for fish transferred to a rapidly changing, purified, artificial

water environment and 16 days for fish transferred to a relatively static natural

water environment to reduce this taint to or below threshold levels o f recognition.

Lovell and Sackey (1973) held channel catfish in a 25 °C tank having a

distinct earthy-musty flavor for 14 days. The fish were then placed in a clean

water aquarium; and after 3, 6, 10 and 15 days, fish were removed and evaluated

for flavor by four experienced judges. After 3 days in clean water at 25 °C, the

flavor o f fish had significantly (P<0.05) improved. After 10 days, the flavor was

27


not significantly (P<0.05) different from that o f control fish that had never been

exposed to algae.

Water temperature must be 16 °C to allow rapid purging. MIB off-flavor is

expected to disappear within a week to 2 weeks when water temperatures are

above 20 °C. Fish transfer may cause fish to be stressed and become more

susceptible to disease. Fish should not be transferred during hot weather or when

nitrite is present in the purging pond. Fish lose weight during the purging process

unless they are feed (Weirich, 1995).

As with the uptake o f MIB, the depuration o f MIB was affected by time

and temperature. Removing MIB related off-flavors at temperatures below 15 °C

is not practical for fish with fat contents that exceed 7%. Fish with a high fat

content held at low water temperature (15% fat, 6.5 °C) did not regain acceptable

flavor until after 72 hrs o f depuration. Under most favorable conditions (5% fat,

34 °C), it required 61 hrs for fish containing the greatest MIB burden (11.4 pg

MIB/kg fish) to regain an acceptable flavor. When water temperature exceeds 30

°C, recovery o f flavor quality may occur in less than 80 hrs (Johnsen et al., 1996).

At 25 °C, 50 to 100 % o f the geosmin in sterile solutions was lost within

24 hrs. On the other hand, data presented by Lalezary et al. (1984) indicate that

geosmin and 2-methyisobomeol were not readily volatilized from aqueous

solutions. It is generally known that aeration or air stripping is not a practical

method o f removal o f geosmin or 2-methylisobomeol from drinking water

supplies (Mallevialle and Suffet, 1987; Tucker and Martin, 1991).

28


The time required to purge fish o f earthy flavors varied from 6 to 18 days

for channel catfish (Lovell and Sackey, 1973; Maligalig et al., 1973) and 5 to 16

days for rainbow trout (Iredale and York, 1976). The chemical causes o f the

earthy off-flavors were not identified in these studies. Lovell (1983) also used

sensory analysis to evaluate the effect o f temperature on rate o f loss of an "earthy-

musty" off-flavor from channel catfish. Disappearance o f the off-flavor was most

rapid at the highest water temperature.

Geosmin appears to be eliminated somewhat more slowly from rainbow

trout than from channel catfish, probably because o f the effect of cooler water

temperatures on the elimination. Yurokowski and Tabachek (1974) found that 5 to

7 days was required to remove a muddy flavor caused by geosmin in rainbow

trout. Although this is similar to the time Lelana (1987) used to purge channel

catfish of geosmin related flavor, the initial geosmin concentration in rainbow

trout (11 mg/kg) was much lower than in channel catfish (90mg/kg).

Elimination of 2-methylisobomeol from fish appears to be more rapid than

for geosmin. Martin et al. (1988) exposed small (5-10 g) channel catfish to 2-

methylisobomeol at concentrations o f 5 and 50 pg/L. Fish were exposed for 7

days and steady-state conditions were achieved. Concentrations o f 2-

methylisobomeol in both groups o f fish declined to less than 2 pg/kg over a 48 hr

period after the fish were removed to water free o f the compound (Tucker and

Martin, 1991).


2.3.3. Pond management and off-flavor

Feeding flsh causes similar effects as fertilizing the pond in enhancing

algae blooms (Boyd, 1979). This nutrient input will cause phytoplankton blooms

that are possibly associated with the incidence o f off-flavor in fish. Brown and

Boyd (1982) found a significant correlation between daily feeding rate and off-

flavor in the fish; as daily feeding rate increased, incidence o f off-flavor

increased.

Several methods have been proposed to control off-flavor in channel

catfish ponds. Application of copper sulfate to thin phytoplankton blooms is

claimed to be effective in Alabama catfish ponds. Unfortunately, this practice can

cause oxygen depletion; also phytoplankton develop resistance to copper sulfate

after continuous exposure, requiring increased application rate (Boyd, 1979).

Application o f hydrated lime in many production ponds in west Alabama in 1981-

1982 to control off-flavor was discontinued because no effect was noticed, and

also because hydrated lime can increase the pH very suddenly which might kill

the fish (Armstrong, 1984).

Because cyanobacteria are frequently implicated as the major source of

these compounds in eutropic waters, most research in off-flavor has concerned the

use of algicide to reduce cyanobacterial abundance. Algicide causes a temporary

deterioration o f water quality because they reduce oxygen production and

ammonia removal by algae. The use o f algicide is not without risk to fish health

and fish survival and must be restricted to ponds with healthy fish and facilities

for emergency aeration (Weirich, 1995).

30


Copper sulfate is very toxic to algae but it can also be toxic to fish and

must be applied with great caution (Weirich, 1995). The copper sulfate

application eliminates most but not all MIB-producing algae and the remaining

algae cause a new bloom because ponds remain favorable for the growth o f algae

and off-flavor compounds production (Weirich, 1995). Simazine is a widely used

algicide for aquatic macrophyte control in fish ponds. Application o f simazine to

channel catfish ponds for the control of phytoplankton resulted in extended

periods o f low dissolved oxygen and decreased fish yields (Tucker et al., 1983).

Tucker and Boyd (1978a) investigated the effects o f periodic application

of copper sulfate and simazine for phytoplankton control in catfish ponds.

Treatment o f channel catfish (Ictalurus punctatus) production ponds with

biweekly application o f 0.84 kg o f copper sulfate/ha did not reduce phytoplankton

density. Three periodic applications o f simazine totaling 1.3 mg/L drastically

reduced phytoplankton density. However it also resulted in decreased fish yields

and poor feed conversion ratios compared to control ponds. This is at least partly

the result o f exposure to chronically low dissolved oxygen concentration.

The effects o f simazine treatment on channel catfish and blue gill

production ponds were studied by Tucker and Boyd (1978b). They found a 19%

reduction in channel catfish yields and poorer feed conversion by fish when

compared to control ponds. The prolonged persistence o f simazine (0.2 mg/L for

more than 4 months) resulted in a lower average chlorophyll "a" concentration in

treated ponds (p<0.1) and the magnitude of chlorophyll "a" values were lower in

treated ponds than control ponds throughout the growing season. Use o f sim azine

31


in catfish ponds resulted in extended periods o f decreased concentrations o f

dissolved oxygen when compared to those in control ponds and exposure to

prolonged periods o f lowered oxygen concentration was possibly responsible for

the poorer growth observed.

When fed at libitum, fish exposed constantly to dissolved oxygen levels

60% o f saturation gained 21% less weight than those o f 100% o f saturation. The

rapid destruction and decomposition o f algal and macrophyte biomass can

contribute to increased biological oxygen demand and oxygen depletion. Oxygen

depletion can result in extensive fish mortality (Johnsen and D ionigi, 1994).

Dionigi and Champagne (1995) investigated the effects o f copper sulfate

on geosmin and biomass biosynthesis by heterotrophic cultures of the bacterium

Streptomycetes tendae and Fungus Penicillium expansum. Cultures o f S. tendae

with 12.7 mg Cu/L (copper sulfate) accumulated 44.6% more biomass and the

mycelium contained five-fold greater concentrations o f geosmin than controls.

Additionally, P. expansum cultures exposed to 12.7 mg Cu/L accumulated 9.2%

more biomass and 18-fold greater concentrations o f geosmin than controls. It is

probably because copper is an essential nutrient that contributes to biomass and

metabolic biosynthesis by heterotrophs.

Treatment of a fish culture pond with an algicide to kill cyanobacteria may

be ineffective if actinomycets are responsible for the off-flavor episode. In fact,

such a treatment may aggravate the problem by supplying actinomycetes with

nutrients (dead cyanobacteria and algae) for further growth.

32


Velzeboer et al. (1995) investigated release o f geosmin by Anabaena

circinalis following treatment with alum inum sulfate. Flocculation o f Anabaena

circinalis by alum inum sulfate was tested under laboratory condition to simulate

operating water treatment plants and resulted in removal o f cells in a healthy

condition with no additional release o f gosmin. Removal o f intact cyanobacterial

cells significantly reduced the concentrations o f taste and odor metabolites present

in water. Removal o f cells by flocculation under laboratory conditions varied 57-

81% when all cells were removed in a healthy state, with no additional release of

geosmin as a result o f alum flocculation.

Copper algicide application did not consistently improve the flavor of

farm raised channel catfish (Dionigi and Champagne, 1995; Tucker and Boyd,

1978). Several reports indicated that over 90% o f the total geosmin present was

contained within the cells rather than excreted into the media (Wu and Juttner,

1988; van der Ploeg and Boyd, 1991; Dionigi et al., 1992). As the greatest portion

of geosmin in algal cultures is contained in the cells, algicide induced lysis of

algal cells could release endogenous pools o f geosmin and increase metabolite

absorption by fish. Algicide induced destruction and decomposition o f biomass

could contribute nutrients to subsequent algal blooms and release copper-resistant

strains for competition (Dionigi and Champagne, 1995).

2.4. Analysis o f ofif-flavor compounds

The problem hindering the development o f solutions to the ‘musty flavor’

in aquaculture products is the difficulty in measuring the metabolites responsible

at the extremely low concentrations at which they cause the “musty” flavor.

33


Human tasters and gas chromatography (GC) are the two main methods that have

been used to quantitate both geosmin and MIB. Fish producers and water

suppliers are most affected by the off-flavor problem. Producers are used to doing

a thorough job o f screening and taste testing fish and water for flavor quality to

maintain standards (Chung et al., 1991).

2.4.1 Analysis o f off-flavor compounds by gas chromatography mass spectrometry (GC-MS)

Gas chromatography (GC) determination o f geosmin and MIB has been

the primary non-subjective method used to measure geosmin and MIB

(Yurokowski and Tabachek, 1974; Izaguire et al., 1982; Krasner, 1988;

Tanchotikul and Hsieh, 1990; Dionigi and Ingram, 1994; Stahl and Parkin, 1994).

Table 3 illustrates GC-MS methods that have been reported to date for the

detection o f off-flavor compounds from water samples and fish samples.

Closed loop stripping analysis, purge trap analysis and solvent extraction

are the three most popular methods for the extraction and concentration o f trace

organic compounds in water.

Closed loop stripping analysis was first developed by Grob (1973) and

used widely for the analysis o f nonpolar volatile compounds having intermediate

molecular weight. The organic compounds are stripped from the water by a

recirculating stream o f air in a closed circuit system. The organic compounds are

removed from the gas phase by a filter containing activated carbon and then the

organic compounds are extracted form the filter with carbon disulfide or

methylene chloride. This analysis is based on the extraction o f water sample by

34


evaporation followed by trapping o f the organic substances on charcoal (Grob,

and Grob, 1975). As it uses very small amounts o f solvent (lOpl) for the

extraction, it does not need a solvent concentration step and shows high sensitivity

and recovery rate. It usually showed ng/L levels o f sensitivity. Krasner et al.

(1981) used closed loop stripping analysis for the determination o f geosmin and

MIB in water, sediments and culture. Hwang et al. (1984) reported an increase in

sensitivity and stripping rate in the analysis of earthy-musty odorants in potable

water supply systems by raising the ionic strength o f the water samples with

sodium sulfate before stripping. By combining 80 g o f Na^SC^ with 1 L water

sample, stripping time was shortened to 1.5 hr and the sensitivity o f the analysis

increased to 0.8 ng/L.

The purge and trap technique is a commonly used method for the

determination o f low concentrations o f volatile organic compounds in air and

water. It is most applicable to nonpolar, low molecular weight volatile compounds

with low water solubility (Krasner et al., 1988). As the sensitivity o f this method

is low compared to the closed loop stripping analysis, usually selective ion

monitoring (SIM) is used to achieve low ng/L levels o f detection.

Johnsen et al. (1992) developed a purge trapping method for the analysis

o f MIB from the cattish. In this method, the sample is sparged with nitrogen and

trapped under vacuum (74.5kpa). Carbopack and Carbosieve HI were used as

trapping materials. It showed 38.9% recovery efficiency for MIB, 59% for

geosmin and 83% for internal standard undecanone. Its detection limit was

35


0.1 pg/kg in a 50g sample with valid quantification above 0.5 pg/kg. Stahl and

Parkin (1994) reported determination o f gesomin and MIB from soil samples by a

purge and trap extraction method. It showed 15% recovery for geosmin and 24%

recovery for MIB. The detection limit o f MIB and geosmin in soil sample was

1 pg/kg.

Solvent extraction is simple and has a wide range o f applications to

semivolatile compounds having a range o f polarities and molecular weights. Even

though it is a simple, rapid method, accumulation o f solvent impurities and severe

loss o f extracted substances during the concentration step makes this method

impracticable for the detection o f extreme trace concentrations o f organic

compounds in water (Grob and Grob, 1975). Yasuhara and Fuwa (1979) reported

determination o f geosmin in water by GC-MS after direct extraction o f water

sample (1L) with 50 ml dichloromethane. It showed a detection limit 10 pg/L

geosmin and recovery rate of 69%.

Vacuum distillation /solvent extraction has been used to determine the

levels o f geosmin in fish (Yurokowski and Tabachek, 1974). Tanchotikul and

Hsieh (1990) used an internal standard coupled with multiple-level addition to

determine amounts o f geosmin in clam samples and to determine the efficiency of

a relaying procedure for geosmin reduction.

A microwave cooking method was developed for the isolation o f MIB

from channel catfish (Ictalulus punctatus) that had been declared off-flavor

organoleptically (Martin et al., 1987). This method involves microwave cooking

36


Table 4. Gas chromatography mass spectrometry methods reported in the literature for the detection o f off-flavor compounds, geosmin and MIB.

Sample Method Recoveryrate

Detectionlimit

Reference

Geosmin in fish

High vacuum lowtemperaturedistillation

Yurokowski and Tabachek, 1974

Geosmin in water

Solvent extraction (Dichloromethane)

69% lOng/ml Yasuhara and Fuwa, 1979

Geosmin and MIB in water

Closed loop stripping analysis

Krasner et al., 1981


Closed loop stripping analysis

2ng/L Izaguire et al., 1982


Salted closed loop stripping analysis

0.8ng/L Hwang et al., 1984

Geosmin in water

Vegetable oil extraction

Ing/ml Dupuy et al., 1986

MIB in fish

Microwavecooking

60% 5ng/g fish Martin et al., 1987


Solvent extraction (Dichloromethane)

65% MIB:19.23ng/LGeosmin:9.61ng/L

Johnsen and Kuan, 1987

Geosmin in rangia clam

Vacuum distillation and hexane extraction

97.9% 2ng/g Tanchotikul and Hsieh, 1990

MIB in catfish

Purge and trap extraction

Geosmin:83.1%MIB:38.9%

0.1 pig/kg Johnson and Lloyd, 1992

Geosmin and MIB in soil

Purge and trap extraction

Geosmin:15%MIB:24%

lpg/kg Stahl and Parkin, 1994

Geosmin and MIB in catfish

Microwaveassisteddistillation-solid phase adsorbent trapping

73.5% MIB:1.7pg/kgGeosmin:l.lpg /kg

Conte et al., 1996

37


fish under a nitrogen stream and trapping the condensate at —80 °C. The fish

condensate was extracted with hexane and was reduced to 100 p i and analyzed by

GC. Concentration as low as 5 ng o f MIB/g fish was detected.

Conte et al. (1996) modified a microwave distillation-cold trapping

procedure (Martin et al., 1987), and used a thermostated condenser containing a

solid phase adsorbent instead o f using cryogen (acetone cooled with dry ice to

make -80 °C). Solid phase adsorbent analysis has the advantages o f selectivity,

collection efficiency and ease o f sample recovery compared to solvent extraction.

It gave 80% recovery o f geosmin and 85% recovery o f MIB. Detection limits of

geosmin and MIB were 1.1 pg/L for geosmin and 1.7 pg/L for MIB using

selective ion monitoring.

Quantifications of MIB and geosmin by GC are very difficult because the

concentrations o f MIB and geosmin in water are extremely low in comparison

with other organic compounds. This method requires extensive sample clean up,

specialized equipment and procedures and is time consuming. Fish samples must

be extracted and prepared using complicated procedures that suffer from low

recovery rates and reproducibility. These procedures are not acceptable for

screening a large number o f samples. More importantly, the GC methods require a

great deal of skill and training and are not adaptable to mass industry screening

needs.


2.4.2 Analysis o f off-flavor compounds by sensory analysis

Individual panelists perceive flavor intensity differently, because o f

variations in detection thresholds, adaptation, fatigue and enhancement or

suppression. Panelists experienced difficulty in determining intensity differences

o f MTR between sessions, but could determine differences in intensity o f MIB

within a session (Bett and Johnsen, 1996). Sensory evaluation is the most

sensitive method o f detection currently available, but suffers from being a

subjective method with a large degree o f variation between tasters and between

tests of the same taster (Persson, 1979; Persson, 1980; Bett and Johnsen, 1996).

Despite this problem, necessity has dictated the use o f a staff flavor-taster at

catfish processing plants with the authority to accept or reject catfish prior to

harvest or delivery-

2.4.3 Analysis o f Off-flavor compounds by enzyme linked immunosorbent assay (ELISA).

Recently, enzyme linked immunosorbent assay (ELISA) for MIB was

developed by Chung et al. (1990) and they produced polyclonal antibody (PAb)

by binding a compound similar in structure to MIB (camphor) to the carrier

protein. This PAb was found to be unacceptable because of high non-specific

binding resulting in poor sensitivity. The sensitivity achieved using this PAb was

only 1 mg/L. Thus this PAb was not useful for measuring the very small amounts

o f MIB that cause musty smells in either potable waters or in food (Chung et al.,

1990). In this immunoassay system, the antibodies were raised against a camphor

conjugate and its affinity for this compound is greater than that for MIB. Chung et

39


al. (1990) used BSA for the protein conjugate o f immunogen and ovalbumin for

the protein conjugate o f solid phase. However bovine serum albumin and

ovalbumin may be structurally related and polyclonal antibodies o f camphor-BSA

can recognize ovalbumin. This may be another cause o f high background and low

sensitivity.

2.5 Enzyme immunoassay and production of monnoclonal antibody

2.5.1 Enzyme immunoassay

Immunoassays are analytical methods that take advantage o f the

specificity o f antibody (Ab)-antigen (Ag) interaction. There are many types o f

immunoassays including radioimmunoassay, enzyme linked immunosorbent

assay (ELISA), immunofluorescence assay and luminoimmunoassay, depending

on the label used; or competitive, noncompetitive or sandwich, depending on the

set-up or whether Ab or Ag are limiting. Interaction between Ab and Ag can occur

in solution, at a solid-liquid interphase, can give rise to a soluble product or

precipitate. The method that will be used in this research is an Ab capture

competitive indirect ELISA. The binding between Ab and Ag is noncovalent and

reversible. Attractive forces include hydrophobic, Van der Waals, electrostatic

and hydrogen bonding.

Immunoassays are sensitive, simple and have advantages over other more

expensive methods. Many samples can be assayed simultaneously and

immunoassays due to their sensitivity and specificity, can eliminate the problem

of extraction and purification which has been a drawback o f previous methods

40


(Brimfield et al., 1985; Li et al., 1991; Schneider and Hammock 1992;

Meulenberg et al., 1995).

An immunoassay is accurate, simple, sensitive, and many immunoasssays

are developed for the detection o f small organic compound in the food and

environmental sciences. Plhak and Spoms (1992 and 1994) report the

development of PAb and monoclonal antibody (MAb) for glycoalkaloids. Several

types o f Ab have been developed for the detection o f pesticides, toxins, and drugs.

Thomas (1993) produced MAb for atrazine. With microtiter plate ELISA, atrazine

could be determined in the range from 0.03 to I pg/L. In dipstick format, it

showed quick visual detection of atrazine in concentrations above 0.5pg/L. An

ELISA for the herbicide bentazon was developed by Seiber et al. (1991) and its

detection limit was O.lpg/L. Stanker et al. (1993) also reported development o f a

monoclonal-based ELISA for the coccidiostat, salinomycin.

Vallejo et al. (1982) investigated effects o f hapten structure and bringing

groups on antisera parathion immunoassay developments. They improved the

sensitivity of the assay by enhancing the specificity o f the antibody response.

2.5.2. Development o f monoclonal antibody

Monoclonal antibodies produced by a single clone o f B cells offer

particular advantages such as uniform affinity and specificity o f binding,

homogeneity, and the ability to be produced in unlimited quantities (Deshpande,

1996). Monoclonal antibody production, usually using mouse cells, involves the

generation of a cloned cell line, derived from a B lymphocyte, that secretes a

41


homogeneous supply o f Ab. Plasma cells grow poorly or not at all in vitro but can

proliferate when fused with myeloma cells. The resulting hybrid cells are called

hybridoma.

Monoclonal antibody is useful because o f its specificity o f binding, its

homogeneity, and its ability to be produced in unlimited quantities. Monoclonal

antibodies give uniform affinity constants, greater reproducibility and continuity

of supply, larger linear ranges in immunoassays, more highly defined specificity

and the ability to combine different monoclonal antibodies and analyze several

analytes simultaneously. Once hybridoma is produced, production of monoclonal

antibody in vitro has many advantages. Cell culture can be done using relatively

well defined media, allowing for scale-up and continuous culture to produce a

virtually endless supply o f antibody (Plhak and Spoms, 1994).

Kohler and Milstein (1975) developed a technique that allowed the clonal

growth o f cell populations secreting antibodies with a defined specificity. By

fusing two cells, each having properties necessary for a successful hybrid cell line,

hybridoma cells can be made. The two cells that are commonly used in these

fusions are antibody secreting cells isolated from immunized animals and

myeloma cells. The myeloma cells provide the correct genes for continued cell

division in tissue culture and the antibody-secreting cells provide the functional

immunoglobulin genes.

Myelomas are neoplasms o f antibody-producing cells, each tumor

representing the proliferation of a single clone o f antibody-forming cells (Liddell

and Cryer, 1991). Myeloma can be induced in a few strains o f mice by injecting

42


mineral oil into the peritonium. Potter et al. (1972) isolated myelomas from

BALB/ C mice and these are the most commonly used partners for fusion.

The fusion between the myeloma cell and antibody secreting cell can be

affected by any fiisogen. Pontecorvo et al. (1975) first demonstrated polyethylene

glycol (PEG) as a fusing agent for mammalian cells and it is routinely used by

somatic cell genetists for hybridoma fusion. PEG fuses the plasma membranes o f

adjacent myeloma and antibody secreting cells, forming a single cell with two or

more nuclei. This heterokaryon retains these nuclei until the nuclear membranes

dissolves prior to mitosis. The cells from the immunized animal do not continue

to grow in tissue culture, however the myeloma cells are well adapted to tissue

culture.

In order to remove unfiised myeloma cells, drug selection is used.

Usually, myeloma has a mutation in one of the enzymes o f the salvage pathways

o f purine nucleotide biosynthesis (Littlefield, 1964). Selection with 8-azaguanine

yields a cell line harboring a mutated hypoxanthine-guanine phosphoribosyl

transferase gene (HGPRT). The addition of any compound that blocks the de novo

nucleotide biosynthesis pathway will force cells to use the salvage pathway.

Cells containing a nonfunctional HGPRT protein will die in these

conditions. Hybrids between myelomas with a nonfunctional HGPRT and cells

with a functional HPGRT will be able to grow. After fusion, parental myelomas

can be inhibited using a selective medium, with the remaining hybridoma cloned

and the desired MAb-producing cells selected.

43


2.6. Current research objectives

There is a need for methods that are more reliable and facile than sensory

or gas chromatographic analyses currently available for the detection o f MIB from

water samples and fish samples. Immunoassay methods have the potential to meet

these requirements. As the ELISA currently developed by Chung et al. (1990) did

not provide great enough sensitivity for the analysis o f MIB from samples without

dilution, another method will be used to prepare immunogens and solid phase

protein conjugates in an attempt to improve enzyme immunoassay o f MIB.

The objective o f this study was to obtain a sensitive and reliable

immunoassay method to measure MIB. MAb specific to MIB was produced and

used in indirect ELISA. Three solid phase protein conjugates MIB-BSA,

isobomeol-BSA and bomeol-BSA were prepared and used as coating proteins in

ELISA. Bridge heterology effect was investigated by comparing these three solid

phase protein conjugates and the protein conjugate that showed most positive

results was used for the analysis o f MIB.

44


CHAPTER 3. MATERIALS AND METHODS

3.1. Materials

Bomeol and isobomeol were purchased from Aldrich Chemical Co.,

Milwaukee, WL Succinic anhydride, 4-(Dimethyl amino) pyridine, anisaldehyde,

N-hydroxy succinimide, 1,3-dicyclohexylcarbodiimide, 2,2’-Azino-bis 3-

ethyIbenz-thiazoline-6-sulfonic acid (ABTS), ammonium carbonate, urea, bovine

serum albumin (BSA), Limulus polyphemus hemocyanin (LPH), and sterile

dimethyl sulfoxide (DMSO) were obtained from Sigma Chemical Co (St. Louis,

MO). Sterile polyethylene glycol (PEG 1500) in 75 |xM HEPES buffer was

purchased from Boehringer Mannheim (Indianapolis, IN). Ribi Adjuvant system

(for mice) was purchased from Ribi Immunochem Research, Inc. (Hamilton,

MA). Goat anti-mouse Ig G-peroxidase conjugate that was used as a secondary

antibody and was purchased from Sigma Chemical Co.

Phosphate-buffered saline (PBS) solution was made as 0.9% sodium

chloride in 0.01 M phosphate buffer, pH 7.3. To prepare PBST, tween 20 (0.05%,

v/v) was added before the pH was adjusted.

Serum-free RPMI consisted o f Rosewell Park Memorial Institute 1640

(Sigma) with the addition o f L-glutamine (2 mM), Na pyruvate (1 mM), penicillin

(100 units/mL) and streptomycin (100 pg/mL) (all purchased from Sigma).

Complete RPMI was serum free RPMI plus 20% (v/v) heat inactivated fetal

bovine serum (from Sigma). Heat inactivation was achieved at 56°C for 30 min.

HT medium consisted o f complete RPMI with 20% fetal bovine serum, sodium

45


hypoxanthine(l 00 jiM) and thymidine (16 |jM). Hypoxanthine and thymidine

were purchased sterile as 100 x supplements from Gibco BRL (Gaithersburg,

MD). HAT medium consisted o f HT medium plus aminopterin (0.4 pM) (sterile

as 100 x from Gibco BRL).

3.2. Procedures

3.2.1. Preparation of immunogen and solid phase protein conjugates

Bomeol-LPH and MIB-LPH were prepared as immunogens. MIB-BSA,

isobomeol-BSA and bomeol-BSA were prepared as solid phase protein

conjugates. Figure 2 illustrates a scheme for the production o f bomeol-LPH

immunogen. Synthesis o f MIB-LPH, MIB-BSA, isobomeol-BSA and bomeol-

BSA followed this same procedure.

3.2.1.1. Preparation o f bomeol-hemisuccinate, isobomeol-hemisuccinate and MIB-hemisuccinate

For the preparation o f bomeol-hemisucinate, 4-dimethyl amino pyridine

(31.1 mg) was first added to 9.1 mL of pyridine prior to the addition o f succinic

anhydride (487.7 mg). Bomeol (168.4 mg) was added and the reaction mixture

was heated under reflux, with stirring for 4 days, 5 hours at 58°C. The reaction

was monitored by thin layer chromatography (TLC). The TLC assay used silica

gel 60 F264 (0.2 mm thick) aluminum sheets form E. Merck, (Darmstadt,

Germany) with a mobile phase o f ethyl acetate and methanol, and 1% aqueous

ammonia in a volume ratio o f 80:20:1.

Visualization was accomplished with a mixture o f anisaldehyde, glacial

acetic acid and concentrated sulfuric acid in a volume ratio o f 0.5:50:1. The

reaction was stopped with 20 mL of H2O. The resultant product was extracted

46


bomeolSuccinic Anhydride

D imethy lam inopyridine

Pyridine

borneol-hemisuccinate

DimethylformamideN-hydroxysuccinimideN-N'-dicyclohexylcarbodiimide

bomeol-LPH

Figure 2. Preparation of immunogen (bomeol-LPH).

47


three times, each time with 25 mL methylene chloride. The organic layer (lower

layer) was collected and rotaevaporated to dryness and then dried under vacuum

in the presence o f phosphorous pentoxide (P2O5).

Preparation o f MIB-hemisuccinate and isobomeol-hemisuccinate followed

the same procedure as for bomeol-hemisuccination. To prepare MIB

hemisuccinate, first 4-dimethylamino pyridine (38.4 mg) was added to 8.9 mL of

pyridine. Into this mixture, succinic anhydride (445.7 mg) was dissolved and then

2-methylisobomeol (82 mg) was added. The mixture was heated under reflux at

74°C and monitored with TLC as described above. After 3 days reaction, most of

the MIB was esterified with succinic anhydride to yield MIB-hemisuccinate. The

reaction product was extracted with methylene chloride and then rotaevaporated

to dryness following same procedure as for bomeol-hemisuccination. The final

weight of the MIB hemisuccinate was 154 mg.

In order to prepare isobomeol-hemisuccinate, 228.9 mg o f isobomeol,

58.6 mg o f dimethylaminopyridine and 786.2 mg o f succinic anhydride were

added to 9.5 mLof dried pyridine. The reaction was conducted at 58°C for 3 days

and extracted and rotaevaporated as described previously. The final weight o f the

reaction product was 387.3 mg.

3.2.1.2. Preparation o f immunogens

In order to prepare bomeol-Limulus polyphemus hemocyanin (LPH)

conjugate, all o f the bomeol hemisuccinate produced, as described above, was

dissolved in 4 mL anhydrous dimethyl formamide (DMF). N,N'-dicyclohexyl

carbodiimide (87.9 mg) and N-hydroxy succinimide (107 mg) were added and the

48


mixture was stirred 12 hr at 4°C to form an active ester. LPH (25.1 mg) was

dissolved in 4 mL o f phosphate buffered saline (PBS), pH 7.4. Half of the active

ester reaction mixture was filtered through glass wool into the LPH/PBS solution.

The mixture was stirred 22 hr at 4°C and then dialyzed for 2 hr against 8 M urea

(0.9 L) then 1 hr against 50 mM ammonium carbonate (2 L) and finally overnight

against 25 mM ammonium carbonate(4 L). The final dialyzed product was

lyophilized to 23.1 mg.

For the preparation o f MIB-LPH conjugate, 92 mg o f MIB-hemisuccinate,

52.6 mg ofN-hydroxysuccinimide and 70.2 mg o f dicyclohexylcarbodimide were

dissolved in 3 ml o f dimethylformamide. After 1 day o f reaction, the active ester

reaction mixture was filtered through glass wool into LPH solution (50.3 mg of

LPH in 6 ml o f PBS buffer (pH 7.4). The mixture was stirred 48 hr at 4°C and

followed the same dialysis step described above. The final product after

lyophilization was 62.3 mg.

3.2.1.3. Preparation o f solid phase protein conjugates

Three solid phase protein conjugates were prepared in this research. The

solid-phase conjugate used in most ELISA assays was MIB-bovine serum

albumin conjugate. To make this conjugate, MIB hemisuccinate was prepared and

then reacted with bovine serum albumin (BSA). The MIB-hemisuccinate product

(62 mg) was dissolved in 2 ml anhydrous dimethylformamide; and then N-

hydroxysuccinimide (35 mg) and N,N'-dicyclohexylcarbodiimide (51 mg) were

added. The reaction mixture was stirred 24 hr at 4°C to form the active ester of

MIB-hemisuccinate. BSA (127.3 mg) was dissolved in 3 ml PBS. The active ester

49


reaction mixture (2 mL) was filtered through glass wool into the BSA solution.

The mixture was stirred 2 days at 4°C and then dialyzed against three successive

solutions-first against 8 M urea (0.9 L) for 2 hr, then against 50 mM ammonium

carbonate (2 L) for 4 hr, and finally, against 25 mM ammonium carbonate (4 L) 5

hr. The dialyzed sample was lyophilized and resultant product weighed (102.5

mg). To obtain only soluble conjugate, the dialysis product (80.2 mg) was

dissolved in !5mL ammonium carbonate and centrifuged at 800 x g for 15min.

The supernatant was lyophilized again. The weight o f the final purified MIB-BSA

conjugate was 27.6 mg.

For the comparison o f solid phase protein conjugate structure effects on

the sensitivity o f ELISA, isobomeol-BSA and bomeol-BSA were prepared. In

order to prepare isobomeol-BSA, 51.6 mg o f isobomeol-hemisuccinate, 161.2 mg

of N-hydroxysuccinimde and 174.2 mg o f dicyclohexylcarbodimide were

dissolved in 2 ml o f dimethylformamide. After 2 days reaction at 4°C, the active

ester reaction product was added to BSA solution dissolved in PBS buffer (403

mg of BSA in 2 mL PBS pH7.4). The dialysis method described above was

followed after 1 day reaction. The weight o f the final purified isobomeol-BSA

conjugate was 305.8 mg.

For the preparation o f bomeol-BSA, 109.4 mg o f bomeol-hemisuccinate,

117.3 mg o f N-hydroxysuccinimide and 133.9 mg o f dicyclohexylcarbodimide

were dissolved in 4 ml of dimethylformamide. After 20 hr reaction at 4°C, the

active ester reaction product was combined into BSA solution (199.6 mg of BSA

in 10 mL PBS at pH 7.4). After 2 days reaction at 4°C, the dialysis method

50


described above was followed and then the product was freeze dried. The final

weight after lyophilization was 196.8 mg.

3.2.2. Preparation o f monoclonal antibody

3.2.2.1. Immunization

BALB/c female mice at 4 months age were handled by the Life Science

Animal Service Facility at the Louisiana State University. Prior to immunization,

blood samples (200 pi) were obtained from four mice by ocular. Two mice,

mouse 1 and 2, were then immunized with 0.3 mg of MIB-LPH conjugate in 0.6

ml sterile PBS/Ribi adjuvant system and another two mice, mouse 3 and 4, were

immunized with 0.3 mg o f bomeol-LPH conjugate in 0.6 ml sterile PBS/Ribi

adjuvant system. Two 0.1 ml injections were made in each mouse, one

subcutaneously and the other intraperitoneally. Subsequent injection boosts were

made in the same manner on day 21, 46, and 67 after the initial injections for all

four mice. Mouse 2 was injected one more boost on day 154 and mouse I was

injected two more boosts on day 154 and 317. Blood samples were collected 1

wk after each immunization. Each blood sample was allowed to clot at 37°C for

30 min and then stored at 4°C overnight. The samples were then centrifuged at

15,000 x g to separate the serum from the blood cells. The serums were used to

assay for the presence o f antibodies to MIB.

3.2.2.2. Cell fusion and selection

Myeloma cells (NS-1, ATCC # TIB 18) were grown in sterile tissue

culture flasks using complete RPMI with 20 % fetal bovine serum at general

incubation conditions (37 °C, 5 % CO2 and 90-100 % humidity). Cells were

51


subcultured every 2-4 days using dilutions in the range o f 1/10 to 1/20 to maintain

cell density from about 10s to 106 cells /mL. Viable cells were counted as a 1/10

or 1/5 dilution o f cell suspension in 0.25 % (w/v) trypan blue in PBS. On the day

of fusion, actively dividing myeloma cells were centrifuged at 250 x g for 10 min.

The cells were then combined and then washed twice with 15 mL serum free

RPMI. After the final washing, the cells were resuspended in 15 mL serum free

RPMI and the viable cells counted. Three days after final injection, the selected

mouse was asphyxiated using CO2 and its spleen was removed under sterile

conditions. The spleen was washed with 10 mL serum free RPMI and moved to a

petri dish containing another 10 mL serum free RPMI. Splenocytes were gently

teased and flushed from the spleen using two sterile needles (21 gauge) and

syringes. The splenocytes were transferred to a 15 mL-conical centrifuge tube and

centrifuged at 250 x g for 5 min. The cells were washed twice with 10 mL serum

free RPMI. After the last centrifugation, red blood cells were lysed by suspending

the cells in 4 mL o f 0.8 % NH4CI for 1.5 min. The remaining intact cells

(splenocytes) were then centrifuged at 250 x g for 10 min, resuspended in 10 mL

serum free RPMI, counted and immediately used for fusion.

Splenocytes and myeloma cells were combined in a ratio of 13:1. The

combined cells were gently mixed and centrifuged at 800 x g for 5 min. One (1)

mL of 1 % polyethylene glycol ("PEG") 1500 in HEPES buffer was added to the

cells with gentle mixing and stirring for an additional 2 min at room temperature.

Serum free RPMI (1 mL) was slowly added over the next 1 min and with an

additional 9 mL added over the next 3 min. The cell suspension was then

52


centrifuged in 8 mL HAT medium and plated out by adding 100 p i cell

suspension to each well o f a 96 well sterile plate. Control myeloma cells were

added to 8 wells. The cells were cultured in HAT medium for the first 10 days.

For the next 10 days, cells were subcultured in HT medium. After 5 days,

supernatant from each well was screened for antibodies by incubating 20 pi

aliquots on microtiter plates. The plate had been previously coated with 10 pg/mL

MIB-BSA conjugate, blocked with 200 pi o f 1% BSA in PBS for I hr and washed

three times with 200 pi PBST for 5 min at room temperature. After incubating for

2 hr at room temperature wells were emptied and washed three times, as before,

with 200 pi PBST. Goat anti-mouse IgG-peroxidase conjugate was diluted 1/2000

and 200 pi of the diluted solution was added to each well. The plate was again

incubated 2 hr at room temperature and washed as before. A solution (200 pi) of

peroxidase substrate (2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid)

(ABTS), 0.5 mg/mL) with 0.01% hydrogen peroxide in 0.1 M citrate buffer (pH

3.8) was added to each well. The relative antibody (Ab) activity was expressed as

the absorbance (A 405nm) measured after 30 min peroxidase reaction at room

temperature. Cells that showed relative Ab activities o f greater than 1.0 were

subcultured in 24 well plates and cloned.

3.2.2.3. Cell cloning

Selected cells from wells producing an Ab specific for MIB were cloned

using the following method based on limiting dilution as described by Coding

(1986), Harlow and Lane (1988) and Barrett (1994). Conditioned medium was

prepared by growing myeloma cells in complete RPMI containing 20 % fetal

53


bovine semm for 2 to 4 days, centrifuging the cell suspension at 250 x g for 10

min, and passing the supernatant through a 0.2 pm filter. This filtered medium

was fortified with 20% fetal bovine serum and 1% 200 pM glutamine.

Cells were plated at concentrations o f 10, 1 and 0.1 cells per 200 pi o f

conditioned medium in 96 well plates (30 wells of each concentration).

Macroscopic colonies, as seen by looking at the under surface o f the plate,

became visible 1 week after cloning commenced. Supernatants were assayed for

the presence o f Ab following the same procedure described previously after 10

days. Positive clones were transferred to 24 well plates to test cell viability and

activity. The cloning procedure was repeated three times to ensure stability o f cell

lines.

3.2.2.4. Ammonium sulfate precipitation o f monoclonal antibody

From 200 ml o f cell (cell line F6b4G7B4, cloned three times) suspensions

cultured in RPMI media with 10 % fetal bovine serum, supernatants were

collected by centrifuging at 200 x g for 5 min. The supernatants were centrifuged

at 16,300 x g for lOmin to remove any remaining cell debris and insoluble

proteins. Ammonium sulfate (62 g/200 mL) was slowly added as a powder to

obtain 50% saturation and stirred at 4°C overnight. The mixture was centrifuged

at 16,300 x g for lOmin to obtain precipitated proteins. Precipitated pellets of

proteins were dissolved in PBS and dialyzed against 2 L PBS, 3 times, at 4°C for

3 days. Dialyzed samples were made up to 15 mLwith PBS.


splenocytes myeloma

Fusion with PEG 1500

HAT media

ScreeningCloning

Figure 3. Production o f monoclonal antibody by fusion o f splenocytes o f mouse with myeloma cells.

Y

55


3.2.3. Enzyme immunoassay (El)

3.2.3.1. Testing mice sera for determination o f antibody titer

Ab titers o f mouse sera were tested using a checker board enzyme

immunoassay. A stock solution (100 pg/mL) o f the solid phase MIB-BSA

conjugate was prepared in PBS. This solution was serially diluted three times with

PBS to obtain four different concentrations: 100 pg/mL, 10 pg/mL, 1 pg/mL, 0.1

jig/mT. and 0 pg/mL. Each row o f a microtiter plate was filled with 100 pi o f one

o f the above concentrations. The plates were then stored overnight at 4°C. The

next day the solution was removed from the plate with a sharp shake o f wrist, and

each well was blocked with 200 pi of 1 % gelatin in PBS for 30 min at 37°C. The

wells were then washed three times with 200 pi PBST for 5 min at room

temperature.

Five different dilutions o f mouse serum (1/500, 1/1000, 1/5000, 1/10000,

1/100000) were prepared by adding 1 % BSA in PBS. Fifty (50) pi o f H2O was

added to each well o f the microtiter plate, immediately followed by 50 pi diluted

serum in a manner such that each column o f the microtiter plate contained a

different serum concentration. After incubating for 30 min at 37 °C, wells were

emptied and washed three times as before with 200 p i PBST. Goat anti-mouse

IgG-peroxidase conjugate was diluted 1/1000, and 100 pi o f the diluted solution

was added to each well. The plates were again incubated for 30 min at 37 °C and

washed as before. A solution (100 pi) o f peroxidase substrate (2,2'-azino-bis(3-

ethylbenz-thiazoline-6-sulfonic acid) (ABTS, 0.5 mg/mL) with 0.01 % hydrogen

56


peroxide in 0.1 M citrate buffer (pH 3.8) was added to each well. After 30 min at

room temperature, the absorbances at 405 nm were measured.

To test the ability o f the antibody to bind to free MIB, an indirect

competitive ELISA was performed. Microplates were coated with MIB-BSA

conjugate (1 pg/mL) in PBS and then stored overnight at 4°C. The next day the

solution was removed from the plate with a sharp shake o f wrist, and each well

were blocked with 200 pi o f 1% gelatin in PBS for 30 min at 37°C. The wells

were then washed three times with 200 pi PBST for 5 min at room temperature.

Fifty (50) p i o f 5% methanol containing MIB was added to each well o f the

microtiter plate immediately followed by 50 pi diluted serum (1/5000). The MIB

solution had been serially diluted to generate different concentrations as described

above to obtain the standard curve. After incubating for 30 min at 37 °C, wells

were emptied and washed three times as before with 200 pi PBST. Goat anti

mouse IgG-peroxidase conjugate was diluted 1/1000, and 100 pi o f the diluted

solution was added to each well. The plates were again incubated for 30 min at

37°C and washed as before. A solution (100 pi) of peroxidase substrate (2,2-

azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS, 0.5 mg/mL) with 0.01%

hydrogen peroxide in 0.1 M citrate buffer (pH 3.8) was added to each well. After

30 min at room temperature, the absorbance at 405 nm was measured.

3.2.3.2. Competitive enzyme immunoassay

Figure 4 shows the principle o f competitive ELISA. For competitive

assays, inhibition curves were constructed using 0.1 ng/L to 100 mg/L MIB in 10

% methanol. Optimum concentrations o f antibody and solid phase protein

57


conjugate were predetermined by a checker board ELISA as described in the

previous section. Three different solid phase conjugates were used: MIB-BSA,

isobomeol-BSA and bomeol-BSA. Microplates were coated with different

concentrations o f solid phase protein conjugate (200 pl/well) and stored overnight

at 4°C. The next day the solution was removed from the plate with a sharp shake

of wrist, and each well blocked with 200 pi o f I % BSA in PBS 1 hr at room

temperature. The wells were then washed three times with 200 pi PBST for 5 min

at room temperature. Serially diluted MIB solution (from 10 ng/L to 100 mg/L) in

10 % methanol was added for the competition and then prediluted anti-bomeol

MAb (100 pi) was added as a primary antibody. After incubating for 2 hr at room

temperature in a shaker, the wells were washed three times with 200 pi PBST.

Prediluted goat anti-mouse IgG-peroxidase conjugate (1/2000) was added to the

wells for use as a secondary antibody and the wells were further incubated at

room temperature for 2 hr. The wells were then washed as before. A solution (100

pi) of peroxidase substrate (2,2'-azino-bis-3-ethylbenz-thiazoline-6-sulfonic acid

(ABTS, 0.5 mg/ml) with 0.01 % hydrogen peroxide in 0.1 M citrate buffer (pH

3.8)) was added to each well. After incubating for 30 min at room temperature,

the absorbance at 415nm was measured. Graphs were constructed with Ausnm

(absorbance at 415nm) as the y-axis and MIB concentration as the x-axis. The

results, expressed as A/Ao, were calculated as follows:

A/Ao= (A4i5nm sample)/( Ansnm blank)

The equations for inhibition curves were determined using SOFT max Pro

2.2 (Molecular Devices Corp. Sunnyvale, CA). The data collected for standard

58


I 2nd Ab Isubstrate

Y : anti bomeol MAb

• : MIB

•<rMIB-BSA

: Blocking solution 1% BSA

Figure 4. Procedure o f competitive ELISA.

59


solutions were used to determine values for the following parameters by the

equation:

y=(a-d)/[(l+(x/c)b] +d

In this equation, y is the response measurement (absorbance), x is MIB

concentration, a is the y-intercept, b is curvature parameter (i.e slope at the

inflection point), c is concentration o f MIB giving 50 % reduction in y ( I 5 0 value)

and d is the value o f y at infinite (saturating) x (i.e. background). Graphs are also

constructed with A/Ao as the y-axis and MIB concentration as the x-axis.

Cross reactivity was expressed as (I50 value o f MIB/ Is0 value o f the

compound tested) x 100 (%).

60


CHAPTER 4. RESULTS AND DISCUSSION

4.1 Production of Immunogen and solid phase protein conjugate

Two protein conjugates, MIB-LPH and bomeol-LPH were prepared as

immunogens and three protein conjugates, MIB-BSA, isobomeol-BSA and

bomeol-BSA were prepared as solid phase protein conjugates. Because MIB and

bomeol are small molecular weight compounds, they must be bound to a large

molecule to elicit immune response and subsequently cause the production of

antibodies. By the esterification with succinic anhydride, carboxyl groups were

introduced to hydroxyl groups o f MIB, bomeol and isobomeol.

Molecular size o f the compound and degree o f foreignness are the two

most important factors determining their immunogenicity (Abraham and Grover,

1971). Molecules with a molecular weight in excess o f 10,000 tend to be good

antigens, whereas molecules with a molecular weight in the 1,000 to 5,000 ranges

are usually poor antigens (Abraham and Grover, 1971). Peptides with a molecular

weight o f less than 1000 have been immunogenic provided they are given in

complete adjuvant (Abraham and Grover, 1971). Non-protein, small organic

molecules are generally non-immunogenic but can produce an immune response

when they are bound to protein. These molecules are termed haptens.

When a hapten contains a hydroxyl group, carboxylic acid may be

introduced by the esterification with dicarboxylic acid anhyrides (e. g. succinic

anhydride) yielding hemisuccinates, which are unstable above pH 9. Another way

to introduce carboxylic acid to a hydroxyl-containing molecules is by

61


carboxymethylation o f the hydroxyl group with bromo- or iodo-acetic acid and

reaction with pyogene. This results in the formation o f chlorocarbonates. For the

synthesis o f MIB-LPH and bomeol-LPH conjugates, a carboxylic acid group was

introduced to the MIB and bomeol. Esterification with succinic anhyride to yield

hemisuccinate derivatives has been reported in Abraham and Grover (1971) and

Plhak and Spoms (1992).

To monitor the reaction o f hemisuccination, thin layer chromatography

(TLC) was used. MIB (or bomeol) and succinic anhydride differ in their polarity,

so they can be easily separated depending on the mobile phase. As the succination

reaction proceeded, the amount o f MIB decreased and the amount o f MIB

hemisuccinate increased. When the reaction was complete, the MIB spot

disappeared.

To form covalent bonds between low molecular weight chemical

compounds, peptide bond formation is most commonly used and desirable

because it is a strong bond. For hapten-protein bond formation, carbodiimide

condensation is a common approach. Another method o f forming nonpeptide

bonds is the Schiffs base reaction using glutaraldehyde (Abraham and Grover,

1971). The coupling o f carboxyl-containing compounds to amino groups of the

polypeptide carrier is usually performed with an excess o f carboxyl groups and an

equivalent amount o f the water soluble carbodiimide around pH 5 at room

temperature. In some cases where the hapten is not water soluble, it is usually

dissolved in DMF or dioxane. In this experiment dicychlohexylcarbodiimde was


used. Dicychlohexylurea, which was one product o f the reaction, was very

insoluble and precipitated in most solvents.

The percentage o f the MIB incorporated into the protein was determined

using Matrix Assisted Laser Desorption Ionization Mass Spectroscopy (MALDI-

MS) and the trinitrobenzene sulfonate method (TNBS). To form a covalent bond

between hemisuccination reaction product and protein (LPH and BSA), a peptide

bond was introduced by the carbodiimide condensation method and in this

experiment dichlohexylcarbodiimde (DCC) was used. MIB-hemisuccinate and

bomeol-hemisuccinate were conjugated to LPH by this active ester method and

used as an immunogen. Using the same method, MIB-BSA, isobomeol-BSA and

bomeol-BSA were synthesized and used as a solid phase protein conjugate in the

enzyme immunoassay.

The reactivity o f hydroxyl groups for the esterification .is different

depending on the structure o f the carbon bearing its hydroxyl groups. For this

SN2 reaction, the phenolic hydroxyl group is most reactive and 1°> 2°> 3°

alcohols (Abraham and Grover, 1971). For the synthesis o f MIB-hemisuccinate,

the reaction was accelerated with the addition o f the catalyst 4-dimethyIamino

pyridine and by increasing the temperature and reaction time. An alternative

method for the conjugation o f MIB was to use a hydroxylated derivative o f 2°

hydroxyl group, which is similar in structure to MIB. Bomeol and isobomeol

have similar structure compared with MIB except they lack one methyl group

(Figure 5). These two compounds have 2° hydroxyl groups. There would be less


steric hindrance at these 2° hydroxyl groups in the esterification with succinic

anhydride.

CHCHCH

CHCHCH OHOH

OHCH

2-methylisobomeol bomeol isobomeol

Figure 5. Structures o f compounds that are used for the conjugation of immunogen and solid phase conjugate, 2-methylisobomeol, bomeol and isobomeol.

The reactions were monitored by TLC and visualized by anisaldehyde.

Figure 6 and Figure 7 are results o f MIB-hemisuccination and bomeol-

hemisuccination. Table 5 summarized the Rf values o f reaction products of

hemisuccination. R f values o f MIB, isobomeol and bomeol are 0.89, 0.88 and

0.88 and all three o f these compounds showed similar patterns in the TLC plate.

Rf value o f MIB-hemisuccinate was 0.63 and isobomeol-hemisuccinate and

bomeol-hemisuccinate were 0.76 and 0.78. These hemisuccination reaction

products were bound to the lysine group of LPH by the carbodiimide

condensation method. MIB-hemisuccinate and bomeol-hemisuccinate were bound

to LPH and used as immunogens. Binding o f MIB hemisuccinate to LPH was

confirmed using the TNBS method (Appendix I).

Using the same method, MIB-BSA, isobomeol-BSA, and bomeol-BSA

were prepared as a solid phase protein conjugates and effects on ELISA

64


A B

Figure 6 . TLC result o f MEB-hemisuccination. Lane A is MIB and lane B is MIB- hemisuccinate after 3 days of reaction. Mixtures o f ethylacetate, methanol and 1% ammonium hydroxide with the ratio o f 80:20:1 was used as a mobile phase. Anisaldehyde solution dissolved in acetic acid and sulfuric acid (0.5:50:1) was used to visualize the compounds.


A B

Figure 7. TLC result o f bomeol-hemisuccination. Lane A is bomeol and lane B is bomeol-hemisuccinate after 3 days o f reaction. Mixtures o f ethylacetate, methanol and 1% ammonium hydroxide with the ratio o f 80:20:1 was used as a mobile phase. Anisaldehyde solution dissolved in acetic acid and sulfuric acid (0.5:50:1) was used to visualize the compounds.

66


Table 5. R f values o f hemisuccination reaction product. Table a) is R f values o f MIB and MIB-hemisuccination product after 3 days reaction at 74°C. Table b) is R f values o f isobomeol and isobomeol-hemisuccination product after 3 days reaction at 56 °C. Table c) is R f values o f bomeol and bomeol-hemisuccination product after 4 days and 5 hr at 58 °C. Ethyl acetate and methanol and 1% aqueous ammonia mixture in a volume ratio o f 80:20:1 was used as mobile phase. Visualization was accomplished by spraying with a mixture o f anisaldehyde, glacial acetic acid and concentrated sulfuric acid volume in a ratio o f 0.5:50:1.

a)compound R f value

MIB 0.89MIB-hemisuccinate 0.63

b)compound R f valueisobomeol 0.88

isobomeol-hemisuccinate 0.76

c)compound R f value

bomeol 0.89bomeol-hemisuccinate 0.78

67


sensitivity (measured in this case as I50 values) were compared. Molecular

weights o f these conjugate proteins were measured by MALDI (Figure 8 and

Figure 9) and bound numbers of hemisuccinates (MIB-hemisuccinate, isoboraeol-

hemisuccinate and bomeol-hemisuccinate) were calculated (Table 6).

The production o f solid phase protein conjugate through hemisuccination

yielded MIB-BSA containing 10 molecules o f MIB hemisuccinate per BSA,

isobomeol-BSA containing 15 molecules o f isobomeol-hemsuccinate per BSA

and bomeol-BSA containing 13 molecules o f bomeol-hemisuccinate per BSA.

Since LPH is an aggregated protein and has a broad range of relatively high

molecular weights, MALDI is not appropriate to determine molecular weight o f

LPH conjugates.

4.2. Production o f Monoclonal antibody

4.2.1. Test o f mouse polyclonal antibody

Four female BALB/c mice were used for immunization in an attempt to

produce monoclonal antibodies of MIB. Two mice (mouse 1 and mouse 2) were

immunized with MIB-LPH protein conjugate and another two mice (mouse 3 and

mouse 4) were immunized with bomeol-LPH protein conjugate.

Figure 10 and 11 show checker board ELISA of mice sera immunized

with MIB-LPH and bomeol-LPH two times. Five different concentrations o f mice

sera (1/500, 1/1,000, 1/5,000, 1/10,000 and 1/100,000) and preimmune sera were

used for the test. MIB-BSA was serially diluted from 100 gg/mL to 0.1 fig/mL by

1/10 times and used as a coating protein. After two immunizations, all four mice

showed positive response with a slight difference in titer depending on the mouse.

68


30000 40000 50000 60000 70000 80CMass (m/2)

MIB*BSA conjugate

30000 40000 80000(mfc)

ooooo 70000

Figure 8 . Matrix assisted laser desorption ionization (MALDI) spectrums for bovine serum albumin (BSA) and 2-methylisobomeol (MIB)-BSA conjugate.

69


4666-tao-bomaol-BSA

30000

n

800006000020000 30000 7000040000

Figure 9. Matrix assisted laser desorption ionization (MALDI) spectrums for isobomeol-bovine serum albumin (BSA) conjugate and bomeol - BSA conjugate.

70


Table 6 . Molecular weight and hapten numbers o f solid phase protein conjugates. Each molecular weight was determined by MALDI.

Molecularweight

Haptennumber

MIB-BSA 68990.2 10IsobomeoI-BSA 70084.2 15

Bomeol-BSA 69792.3 13BSA 66431 0

71


Ecino•<r

a)2.5

2

1.5

1

0.5

0100 10 1 0.1

solid phase concentration (ug/ml)

Eeino

b)2

1.5

1

0.5

0100 10 1 0.1

solid phase concentration (ug/ml)

Figure 10. Checker board ELISA of mice (mouse 1 and 2) sera after immunization o f MIB-LPH two times. Five different concentrations of Ab (1/500, 1/1,000, 1/5,000, 1/10,000 and 1/100,000) were used as a primary Ab source and four different concentrations o f MIB-BSA (100pg/ml, 10|xg/ml, lpg/ml and O.lpg/ml) were used as a solid phase protein conjugates. Graph a) is checker board ELISA result of mouse #1 and graph b) is checker board ELISA result o f mouse #2 .

72


a)

2.5

0.5

00.1100 10 1solid phase concentration (ug/ml)

b)2.5

1.5Ei=ino■»r

0.5

0.1 0100 10 1solid phase concentration (ug/ml)

Figure 11. Checker board ELISA o f mice (mouse 3 and 4) sera after immunization of bomeol-LPH two times. Five different concentrations o f Ab (1/500, 1/1,000, 1/5,000, 1/1,0000 and 1/10,0000) were used as a primary Ab source and four different concentrations o f MIB-BSA (lOOpg/ml, 10ng/ml, lp-g/ml and O.lpg/ml) were used as a solid phase protein conjugates. Graph a) is checker board ELISA result o f mouse #3 and graph b) is checker board ELISA result o f mouse #4.

73


To assure that the polyclonal antibody was binding free MIB, a competitive

ELISA was performed. Figure 12 shows the competitive ELISA results using

mouse sera after two immunizations. Ab concentration and solid phase conjugate

concentration were predetermined using checkerboard ELISA. Ab from serum

recognized solid-phase MEB conjugate and was competitively removed by free

MTB in the ELISA even at 0.1 (ig/mL o f MIB concentration.

4.2.2. Fusion and screening o f mouse I and 2 immunized with MIB-LPH

As the production o f anti MIB polyclonal antibody in the mouse was

identified by ELISA, fusion of splenocytes with myeloma was made to prepare

monoclonal antibody against MIB. Myeloma cells were previously cloned and

tested in HAT media. Mouse 2 was immunized five times before the fusion and

fusion was performed after three days of the last fusion. As a 10 to 1 ratio is

generally recommended for cell numbers (splenocytes vs myeloma cells), a total

of 8.5 x 107 splenocytes were fused to 8.8625 x 106 myeloma cells and distributed

in four 96 well tissue culture plates. Each plate had 4 wells of control cells

(myeloma).

After 5 days, fusion o f splenocytes o f mouse 2 with myeloma produced 2

to 6 hybridomas per well totaling approximately 1000 hybridoma. The first

screening o f supernatant was made 6 days after fusion. In the first screening 7

wells showed positive response binding to the MIB-BSA solid phase conjugate

and Ao values were from 0.301 to 0.593. In the second screening (10 days after

fusion), most o f the wells showed affinity to the MIB-BSA and cells from 42


a)

Eeino•*r

0.7

0.6

0.5

0.3

0.2

0.1ooo

oo oo

o oo

1/500.5%MeOH

1/500,20%MeOH

b )

MIB concentration (ug/ml)

Cm

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0oooo

ooo

oo’

MIB concentration (ug/ml)

oo

1/5000,5%MeOH

1/5000,20%MeOH

Figure 12. Test o f mouse sera for the competitive inhibition o f binding to solid phase protein conjugate. Graph a) is the result o f serum from mouse # 2 that was immunized with MIB-LPH, two times. Serum is diluted 1/5,000 times in 0.005% BSA solution in PBS (pH7.4) and used as a 1st Ab source. MIB-BSA (0.5|ig/ml) was used as a solid phase protein conjugate. Graph b) is the result o f serum from mouse #3 that was im m u n iz e d two times with bomeol-LPH. Serum was diluted 1/5,000 times and used as a 1st Ab source and MIB-BSA (lpg/m l) was used as a solid phase protein conjugate. MIB solution was diluted from 100 jig/ml to 0 .0 0 0 lpg/ml by serially diluting 1/10 times and 10% methanol and 20% methanol was used for the dilution o f MIB solutions.

75


wells were selected and transferred to 24 well tissue culture plates. Appendix II

depicts the result o f screening after 6 days and 10 days.

Cells from 42 wells transferred to 24 well tissue culture plates were tested

for their ability to be competitively removed from the solid phase in the presence

of 10 pg/mL o f MIB. Any of the wells tested did not show competitive inhibition

by free MIB (Appendix HI). In order to avoid potential loss o f positive cells that

can show inhibition by free MIB, 5 wells were chosen and used for the cloning of

the cells.

Figure 13 is the result of competitive inhibition test o f the cloned cells. All

cloned cells showed binding o f Ab to the MIB-BSA but did not show competitive

inhibition o f binding to the solid phase conjugate by free MIB.

As antibody was made against MIB-LPH, too high an affinity against

MIB-hemisuccinate could restrict inhibition o f binding by free MIB. A different

solid phase conjugate was tested to avoid this effect. Bomeol-BSA was used as a

solid phase protein conjugate o f ELISA and showed no difference from MIB-

BSA. High nonspecific binding was responsible for this result and it was finally

shown that these antibodies were binding to the BSA itself. This may be

nonspecific binding or possibly because the mouse may have been exposed to

BSA during the immunization or during feeding o f food to the mouse.

Mouse 1 was immunized (6x) with the same MIB-LPH conjugate, used for

the fusion with myeloma cells to make a hybridoma cell line that produced anti

MIB antibody. The same procedure was followed as for the fusion o f mouse 2,

except for cell numbers. A total o f 1.2 x 108 splenocytes were fused to 1.1 x 107

76


a)

<0 CM tf)to .oCO CO 00*0 *0 *0CM CM CM

<sCO*0CM

No MIBMIB,1ppmMIB.10ppm

b)

No MIBMIB,1ppmMIB,10ppm

Figure 13. Cloning o f cells for the production o f Ab that specifically bind to free MIB after fusion o f splenocytes o f mouse 2 (immunized with MIB-LPH) with myeloma cells. Supernatants o f cell culture were diluted 1/15 with 0.05% BSA solution in PBS (pH7.4) and used as primary Ab source and MIB-BSA (lug/m l) was used as a solid phase protein conjugate. Goat anti mouse IgG-peroxidase (1/3000) was used as a secondary Ab. MIB solutions were diluted in 10% methanol solution.

77


myeloma cells with a 12 to 1 cell ratio (splenocytes vs myeloma). After screening

with 1 mg/L of isobomeol-BSA as a solid phase conjugate, L44 wells were

selected and tested for the competitive inhibition o f binding to the solid phase

conjugate in the presence o f 5 pg/mL of MIB solution. All tested cells showed no

inhibition of binding of Ab to the solid phase conjugate-

4.2.3. Fusion and screening o f mouse 3 that had been immunized with bomeol- LPH

Mouse 3 was immunized four times with bomeol-LPH conjugate. The

fusion of splenocytes o f mouse 3 with myeloma cells gave rise to between 5 and

10 hybridoma per well, for a total o f approximately 400 hybridoma. These

hybridomas actively grew in the presence of aminopterin in HAT medium while

control cells (myeloma) did not. Five days after fusion, screening o f supernatant

revealed that three wells were producing Ab specific for MIB. O f these, cells

from wells dl 1 and f6 retained their ability to make Ab, whereas cells from well

c3 did not produce antibody at the screening 9 days after fusion.

Figure 14 depicts the result o f screening o f cell culture supernatant at 5

days and 9 days after fusion. Cells were subcultured to 24 well tissue culture

plates and tested again for the ability to produce antibodies. Cells were expanded

in 5ml of cell culture media and used for the cloning.

4.2.4. Cloning of anti bomeol monoclonal antibody.

Cells were selected for cloning from positive wells. This was based on

both Ab production and sensitivity in a competitive El, and on cell growth.

Cloning was performed three times from the cells o f d l 1 and f6 . All wells that


Figure 14. Screening of cells for the production o f antibody after 5 days and 9 days fusion o f splenocytes (mouse 3 immunized with bomeol-LPH) with myeloma cells. Cell culture supernatant was collected (50 pi each) from the 96 wells of the cell culture plate at days 5 and 9 after fusion. MIB-BSA (10 pg/ml) was used as a solid phase protein conjugate. Cell culture supernatant (20 pi each) was used as a source o f 1st Ab. Goat anti-mouse IgG-peroxidase (1/4000) was used as a secondary Ab. ABTS was used as a substrate.

79


showed cell growth after limiting dilution, were screened for the production o f

anti-bomeol Ab.

Appendix IV is the result o f screening after subculturing o f cells to 24

well plates. Cell culture supernatant (20pl) was used for the ELISA. From this

result five wells (dl lb3. dl lf4, dl lg3, d l lh 8 and f6b4) were chosen based on the

A/Ao values and propagated to 5 mL o f cell culture media. Cells were cloned two

more times to ensure clonality. Appendix V shows the results o f the second

cloning. Cells were again cloned from cell line f6b4g7. Appendix VI depicts

results of the 3rd cloning. Ao values varied from well to well depending on the cell

number and condition.

In competitive ELISA using isobomeoI-BSA (0.3 pg/mL), 10 pg/mL of

MIB was able to reduce A/Ao values for all cells cloned this times. Cell line

f6b4g7b4 was selected and propagated to use as the 1st Ab source in further

experiment. Figure 15 shows the standard curves using dilutions o f cell culture

supernatant o f cell line f6b4g7b4 and two coating concentrations o f isobomeol-

BSA.

4.3. Effect of Ab and solid phase conjugate concentrations on the sensitivity of ELISA

4.3.1. Effect o f Ab concentration on the sensitivity of ELISA

The effect o f Ab concentration was investigated when the solid phase

protein conjugate, MIB-BSA, was used. Optimal dilution of Ab and concentration

of solid phase protein conjugate were determined using checker board ELISA.

Three different concentrations of antibodies and solid phase conjugates that

showed absorbance o f more than 1 were compared in competitive ELISA for their

80


a).

2.82.4

v 1-2

0.8

0.4

mo

MIB concentration

eCto

b)2.8

2.4

2

1.6

1.2

0.8

0.4

0CD2o

Q .Q .O

Q . AQ.a ..oQ.Q.o

Aa.a.£a.Q.

E EQ . Q.Q» Q-O Q

1/8

1/12

1/16

1/20

MIB concentrator!

Figure 15. Standard curves constructed using cell culture supernatants from three times cloned cell line f6b4g7b4. Cell culture supernatants diluted 1/8, 1/12, 1/16 and 1/20 times were used as a 1st Ab source and two concentrations o f isobomeol- BSA (0.5 {ig/ml and 0.2 pg/ml) were used as solid phase conjugates. Goat antimouse IgG-peroxidase (1/3000) was used as a secondary Ab and ABTS was used as a substrate. MIB solutions were serially diluted from 100 mg/L to 10 ng/L by 1/ 10 times in 10% methanol. Graph a) was coated with 0.5 |xg/ml o f isobomeol- BSA and Graph b) was coated with 0.2 pg/ml o f isobomeol-BSA.

81


effects on sensitivity (measured as I50) and precision. Figures 16, 17, and 18

depict results relative to Ab concentration effects. Four different concentrations o f

Ab (1/125 times, 1/250 times, 1/500 times, 1/1000 times) were compared at three

different concentrations o f MIB-BSA. When a high concentration o f solid phase

conjugate (MIB-BSA, 1 pg/mL) was used as a coating solution, the sensitivity o f

ELISA (measured as I50) increased almost 20 times when the Ab concentration

was changed from 1/125 to 1/250. The Ao value decreased from 3.536 to 2.218,

still showing a high absorbance. Correlation coefficient values (R2) were 0.997

and 0.993. Low concentrations o f Ab (1/500 and 1/1000) showed high sensitivity,

however the Ao value decreased as did the correlation coefficient.

Figure 17 depicts the effect o f Ab concentration when MIB-BSA was used

at 0.5 pg/mL. The effect of changing Ab concentration was least apparent at this

MIB-BSA concentration. An Ab concentration of 1/250 had the highest

sensitivity and a high Ao value with a high correlation coefficient value. This

graph showed a wide linear range from lng/L to 100 mg/L MIB.

Table 7 summarizes maximum absorbance without MIB (Ao), I50 values

and correlation coefficient values (R2). Depending on the concentrations o f solid

phase conjugate, the effect of Ab concentration on the sensitivity of ELISA varied

from 20 times to 2 times. The effect o f Ab concentration on the sensitivity o f

ELISA was greatest when Ipg/mL MIB-BSA was used for coating; this is

probably because Ab concentration is a limiting factor when there is adequate

solid phase conjugate. By lowering solid phase conjugate concentrations, the


a)

Ecin

4

3

2

1

0

•1_jca

O) o> o) o>O) 0>e a teoQ)

1/125

1/250

1/500

1/1000

MIB concentration

b)

m5o

1.2

0.8

0.2

oic d> a oi d «- O o T- O

o>

° IMIB concentration

0) 0) 0) E E E ■ < - 0 0 o

1/125

1/250

1/500

1/1000

Figure 16. Test of primary Ab concentrations effect when high concentration (1 ̂ g/ml) o f MIB-BSA was used as coating protein. Cell line, f6b4g7b4 was used as a 1st antibody source. Four different concentrations o f Ab (1/125, 1/250, 1/500 and 1/1000 concentrated by ammonium sulfate precipitation) were used as 1st Ab. Goat anti-mouse IgG-peroxidase conjugate (1/2000) was used as a secondary Ab. MIB was diluted in 10% methanol from 100 mg/L to 0.1 ng/L by serial dilution of 1/10 times. Graph a) is a result showed as a absorbance at 415nm and graph b) is a same result of graph a) expressed as a A/Ao.

83


a)

EcID

b)

4

3

2

1

0

-1GO o>a>o>cs305as 05

MIB concentration

1.2

0.8

0.6

0.2

- 0.2—i—iGO

0505 05 as 05as 05

1/125

1/250

1/500

1/1000

1/125

1/250

1/500

1/1000

MIB concentration

Figure 17. Test o f primary Ab concentrations effect when medium concentration (0.5|ig/ml) o f MIB-BSA was used as coating protein. Cell line, f6b4g7b4 was used as a 1st antibody source. Four different concentrations o f Ab (1/125, 1/250, 1/500 and 1/1000 concentrated by ammonium sulfate precipitation) were used as 1st Ab. Goat anti-mouse IgG-peroxidase conjugate (1/2000) was used as a secondary Ab. MIB was diluted in 1.0% methanol from 100 mg/L to 0.1 ng/L by serial dilution o f 1/10 times. Graph a) is a result showed as a absorbance at 415nm and graph b) is a same result o f graph a) expressed as a A/Ao.

84


a)

b)

4

3

2cin

1

0

•1_!GQ - j at OlaiO)e

1/125

1/500

1/1000

MIB concentration

1.2

0.8

0.2

- 0.2—iCD

2 O)05 05c o>

1/125

1/250

1/500

1/1000

MIB concentration

Figure 18. Test o f primary Ab concentrations effect when low concentration (0.25pg/ml) o f MIB-BSA was used as coating protein. Cell line, f6b4g7b4 was used as a 1st antibody source. Four different concentrations o f Ab (1/125, 1/250, 1/500 and 1/1000 concentrated by ammonium sulfate precipitation) were used as Ist Ab. Goat anti-mouse IgG-peroxidase conjugate (1/2000) was used as a secondary Ab. MIB was diluted in 10% methanol from 100 mg/L to 0.1 ng/L by serial dilution o f 1/10 times. Graph a) is a result showed as a absorbance at 415nm and graph b) is a same result o f graph a) expressed as a A/Ao.

85


Table 7. Effects o f Ab concentrations on the sensitivity o f ELISA when MIB- BSA was used as coating protein. These tables are results o f figure 16, 17, and 18 expressed as I50 values. Table a) is the result o f figure 16 and lpg/m l o f MIB- BSA was used as a coating solution. Table b) is the result o f figure 17 and 0.5 pg/ml o f MIB-BSA was used. Table c) is the result o f figure 18 and 0.25 ug/ml o f MIB-BSA was used.

a) solid phase conjugate: MIB-BSA lpg/m lAb concentration ■ A c a r

I 5 0 R -1/125 3.536 1 pg/L 0.9971/250 2.218 0.0426 pg/L 0.9931/500 1.047 0.125 pg/L 0.97

1/1000 0.61 0.0352 pg/L 0.972

b) solid phase conjugate: MIB-BSA 0.5pg/mlAb concentration Aw - ■ hom R2W

1/125 3.436 1 pg/L 0.9811/250 2.345 0.407 pg/L 0.9921/500 1.268 1 pg/L 0.996

1/1000 0.598 1 pg/L 0.988

c) solid phase conjugate: MIB-BSA 0.25pg/mlAb concentration ATar U P p2C9 - -

1/125 3.233 11 pg/L 0.9791/250 2.59 7 pg/L 0.9921/500 1.299 2 pg/L 0.964

1/1000 0.582 2 pg/L 0.962

a: absorbance when plate was saturated with Ab.b: the concentration o f MIB that shows 50% o f inhibition o f Ab binding to solid phase conjugate, c: correlation coefficient value.A, I50 and R were calculated by program soft max pro.

86


effect o f Ab concentration was smaller and the lower concentration o f solid phase

conjugate (0.25 pg/mL) gave more variation.

When 0.25 pg/mL of MIB-BSA was used as a solid phase protein

conjugate, an Ab dilution o f 1/125 may have provided excess Ab that restricted

sensitivity of ELISA. An Ab dilution o f 1/1000 was too low to yield enough

absorbance, resulting in high variance.

The use o f this MAb supernatant diluted to 1/250, was considered to be

optimum when MIB-BSA was used at 1 pg/mL or 0.5 pg/mL for this enzyme

immunoassay, because the Ao values were higher than 2.0, background

absorbances were minimal and sensitivities were maximized. Correlation

coefficients (R2) were 0.993 (MAb 1/250, MIB-BSA lpg/ml) and 0.992 (MAb

1/250, MIB-BSA 0.5 pg/ml) showing good correlation coefficients.

Effects o f Ab concentrations on the sensitivity of ELISA were also

investigated using bomeol-BSA as a solid phase conjugate. Four different

concentrations o f Ab (1/125 times, 1/250 times, 1/500 times, and 1/1000 times)

were tested as in MIB-BSA solid phase conjugate. Figure 19, 20, and 21 depict

the results of the test of Ab concentration effects at three different concentrations

of bomeol-BSA (lpg/mL, 0.5 pg/mL, and 0.25 pg/mL). All o f these results

showed similar patterns and the sensitivity o f ELISA was greatest when low

concentrations o f Ab were used.

Effects o f Ab concentration was greatest when high concentrations of

bomeol-BSA (I pg/mL) were used as a solid phase protein conjugate and

sensitivity of ELISA increased almost 30 times by lowering Ab concentration

87


a)

Ecto?

4

3

2

1

0COso

O)co

OJc O) o) d)C C S o o T—o

O) o> 3 35 8

o>E

O)Eo oo

MIB concentration

b)

1.2

1

0.8

< 0.6

0.4

0.2

0CQ2o2

O)co

O) o>coOi O) O)3o

o> 'ftE

o>I

a>Eoo

1/125

1/1000

1/125

1/250

1/500

1/1000

MIB concentration

Figure 19. Test of primary Ab concentrations effect when high concentration (lpg/ml) of bomeol-BSA was used as coating protein. Cell line, f6b4g7b4 was used as a 1st antibody source. Four different concentrations o f Ab (1/125, 1/250, 1/500 and 1/1000 concentrated by ammonium sulfate precipitation) were used as 1st Ab. Goat anti-mouse IgG-peroxidase conjugate (1/2000) was used as a secondary Ab. MIB was diluted in 10% methanol from 100 mg/L to 0.1 ng/L by serial dilution o f 1/10 times. Graph a) is a result showed as a absorbance at 415nm and graph b) is a same result o f graph a) expressed as a A/Ao.

88


A/A

o

oo £ §MIB concentration

b)1.2

1

0.8

0.6

0.4

0.2

0go2o

o>co'

o> o> O)c c c^ ° §

o>3o oT- O

OJ o>E Eo ioo

1/125

1/250

1/500

1/1000

MIB concentration

Figure 20. Test of primary Ab concentrations effect when medium concentration (0.5{j.g/mi) o f bomeol-BSA was used as coating protein. Cell line, f6b4g7b4 was used as a 1st antibody source. Four different concentrations o f Ab (1/125, 1/250, 1/500 and 1/1000 concentrated by ammonium sulfate precipitation) were used as 1st Ab. Goat anti-mouse IgG-peroxidase conjugate (1/2000) was used as a secondary Ab. MIB was diluted in 10% methanol from 100 mg/L to 0.1 ng/L by serial dilution o f 1/10 times. Graph a) is a result showed as a absorbance at 415nm and graph b) is a same result o f graph a) expressed as a A/Ao.

89


a)

Ecto

01

4

3

2

1

0

-1m

OJ o>OJOJOJcoo

1/125

1/250

1/500

1/1000

MIB concentration

b)

1.2

1

0.8

0.6

0.4

0.2

0

- 0.2m5o

OJco

OJc OJ "3j o jC C 3

5 § -O) O) O) O)3 3 £ £ E5 8 ^ 1 1

1/125

1/250

1/500

1/1000

MIB concentration

Figure 21. Test o f primary Ab concentrations effect when low concentration (0.25pg/ml) o f bomeol-BSA was used as coating protein. Cell line, f6b4g7b4 was used as a 1st antibody source. Four different concentrations o f Ab (1/125, 1/250, 1/500 and 1/1000 concentrated by ammonium sulfate precipitation) were used as 1st Ab. Goat anti-mouse IgG-peroxidase conjugate (1/2000) was used as a secondary Ab. MIB was diluted in 10% methanol from 100 mg/L to 0.1 ng/L by serial dilution o f 1/10 times. Graph a) is a result showed as a absorbance at 4l5nm and graph b) is a same result o f graph a) expressed as a A/Ao.

90


Table 8 . Effects o f Ab concentrations on the sensitivity o f ELISA when bomeol- BSA was used as coating protein. These tables are results o f figure 19, 20, and 21 expressed as I50 values. Table a) is the result o f figure 19 and 1 pg/ml o f bomeol- BSA was used as a coating solution. Table b) is the result o f figure 20 and 0.5 pg/ml of bomeol-BSA was used. Table c) is the result o f figure 21 and 0.25pg/ml of bomeol-BSA was used.

a) solid phase conjugate: bomeol-BSA lpg/mlAb concentration A(a) ' ' IsoW R 2CC)

1/125 3.569 914pg/L 0.9981/250 3.488 294pg/L 0.9941/500 3.08 81pg/L 0.989

1/1000 1.89 31pg/L 0.973

b) solid phase conjugate: bomeol-BSA 0.5pg/mlAb concentration A» - - IsoW R2*)" '

1/125 3.582 728pg/L 0.9951/250 3.616 341pg/L 0.9981/500 3.381 99pg/L 0.998

1/1000 2.473 35pg/L 0.984

c) solid phase conjugate: bomeol-BSA 0.25pg/mlAb concentration A® IsoW R2(C)

1/125 3.528 254pg/L 0.9951/250 3.489 72pg/L 0.9941/500 3.298 15pg/L 0.991

1/1000 2.131 25pg/L 0.985

a: absorbance when plate was saturated with Ab.b: the concentration o f MIB that shows 50% of inhibition o f Ab binding to solid phase conjugate.c: R2 is correlation coefficient value.A, I50 and R2 were calculated by program soft max pro.

91


from a dilution o f 1/125 to 1/1000. The effect of Ab concentration was least when

low concentrations o f bomeol-BSA (0.25 pg/mL) were used as a solid phase

protein conjugate and sensitivity o f ELISA increased by almost 10 times by

lowering Ab concentration from 1/125 to 1/1000. I50 values, however, for Ab

dilutions of l / l 000 were similar in all three tests using three different solid phase

conjugate concentrations. This is probably because Ab concentration became a

critical factor determining sensitivity of ELISA at low concentrations o f solid

phase protein conjugate (i.e. solid phase protein was limiting). When a high

concentration of solid phase protein conjugate was used as a coating protein, solid

phase conjugate also influenced the sensitivity of ELISA, decreasing sensitivity to

a great extent at a high concentration o f Ab.

When bomeol-BSA was used as a solid phase protein conjugate, the

ELISA was 10 to 1000 times less sensitive (measured as an increase in Iso),

depending on the concentrations of Ab and solid phase protein conjugate,

compared with MIB-BSA- Most ELISA (using bomeol-BSA as coating protein)

results showed detection limit s of ppb (parts per billion, ng/mL) when low

concentration o f Ab (1/1000 times) was used to obtain a maximum sensitivity.

4.3.2. Effect of solid phase conjugate concentrations on the sensitivity o f ELISA

Solid phase conjugate concentrations effect on the sensitivity o f ELISA

was investigated at three different concentrations o f 1stAb. Four different

concentrations of solid phase conjugates (MIB-BSA 1 pg/mL, 0.5 pg/mL, 0.25

pg/mL and 0.125 pg/mL) were compared using Ab dilutions o f 1/125, 1/250 and


1/500 (Figure 22, 23, and 24). All three results showed an increased sensitivity

when lower solid phase conjugate concentrations were used.

Table 9 summarizes the effects of solid phase conjugate concentration on

the sensitivity o f ELISA using MIB-BSA solid phase conjugate. Depending on

the concentration o f Ab, the sensitivity improved from 7 to 300 times. When Ab

was diluted 1/250, the effect o f solid phase concentration was greatest. By

lowering solid phase concentration from lpg/ml o f MIB-BSA to 0.25 pg/mL of

MIB-BSA, the sensitivity o f ELISA improved steadily. Ao values were higher

than I even at 0.25 pg/mL of MIB-BSA when Ab was diluted 1/125 and 1/250

were used. The sensitivity of ELISA did not increase by lowering concentrations

of solid phase conjugates from 0.25 to 0.125 pg/mL and Ao values were below

1.0 in all three tested results. When the concentration o f 1st Ab (1/500 dilutions)

was too low, Ao values were too low to be used to make standard curves at 0.25

and 0.125 pg/ml of MIB-BSA.

Solid phase conjugate concentration effect was also investigated using

bomeol-BSA as a solid phase conjugate. Figures 25, 26, and 27 are tests of solid

phase conjugate concentration effects comparing four different concentrations of

bomeol-BSA (1 pg/mL, 0.5 pg/mL, 0.25 pg/mL and 0.125 pg/mL) at three

different dilutions o f Ab (1/125 times, 1/250 times and 1/500 times). Table 10

summarizes the results o f Figures 28, 29 and 30 expressed as I50 values, Ao and

R2.

The sensitivity of the ELISA changed from 9 times to 30 times when the

bomeol-BSA concentration was changed from 1 pg/mL to 0.125 pg/mL,

93


a)

Ecm

b)

4

3

2

1

0

■1CD CJJco

Olo>o>c

0.5ug/ml

0.25ug/ml

0.125ug/ml

MIB concentration

1.5

01

-0.503 e»o> O) oi O)

1ug/ml

0.5ug/ml

0.25ug/ml

0.125ug/ml

MIB concentration

Figure 22. Test o f solid phase protein conjugate (MIB-BSA) concentrations effect when high concentration o f Ab (F6b4g7b4, 1/125 times) was used as a 1st Ab. Four different concentrations of solid phase protein conjugates (MIB-BSA, lpg/ml, 0.5pg/ml, 0.25pg/ml and 0.125pg/ml) were coated on the microtiter plate and compared. Goat anti-mouse IgG-peroxidase (1/2000) was used as a secondary antibody and MIB was diluted in 10% methanol from lOOmg/L to O.lng/L by serial dilution o f 1/10 times. Graph a) is a result showed as a absorbance at 415nm and graph b) is a same result o f graph a) expressed as a A/Ao.

94


a)

3.5

2.5

Ec

0.5

-0.5—iCQ o>O) Ui Oi O)O) O)O)5

b)

1ug/ml

0.5ug/ml

0.25ug/ml

0.125ug/ml

MIB concentration

1.2

0.8

0.2

- 0.2—im o> Ol

1ug/ml

0.5ug/ml

0.25ug/ml

0.125ug/ml

MIB concentration

Figure 23. Test o f solid phase protein conjugate (MIB-BSA) concentrations effect when medium concentration o f Ab (F6b4g7b4, 1/250 times) was used as a 1st Ab. Four different concentrations o f solid phase protein conjugates (MIB-BSA, lpg/ml, 0.5pg/ml, 0.25pg/ml and 0.125pg/ml) were coated on the microtiter plate and compared. Goat anti-mouse IgG-peroxidase (1/2000) was used as a secondary antibody and MIB was diluted in 10% methanol from lOOmg/L to O.lng/L by serial dilution o f 1/10 times. Graph a) is a result showed as a absorbance at 415nm and graph b) is a same result o f graph a) expressed as a A/Ao.

95


MIB concentration

o<

b)

1.4

1.2

1

0.6

0.4

0.2

0go2o

9 9e 9Co

9

Oo9 9OJ3 "3>E

lug/ml

0.5ug/ml

0.25ug/ml

0.125ug/ml

MIB concentration

Figure 24. Test o f solid phase protein conjugate (MIB-BSA) concentrations effect when low concentration o f Ab (F6b4g7b4, 1/500 times) was used as a 1st Ab. Four different concentrations o f solid phase protein conjugates (MIB-BSA, lpg/ml, 0.5pg/ml, 0.25pg/ml and 0.125pg/ml) were coated on the microtiter plate and compared. Goat anti-mouse IgG-peroxidase (1/2000) was used as a secondary antibody and MIB was diluted in 10% methanol from lOOmg/L to O.lng/L by serial dilution o f 1/10 times. Graph a) is a result showed as a absorbance at 415nm and graph b) is a same result o f graph a) expressed as a A/Ao.

96


Table 9. Effects o f solid phase conjugate (MIB-BSA) concentrations on the sensitivity o f ELISA. These tables are results o f figure 22,23 and 24 expressed as Iso values. Four concentrations of MIB-BSA (lpg/m l, 0.5pg/ml, 0.25pg/ml and 0.125pg/ml) were compared. Table a) is the result o f figure 22 and 1/125 concentration o f Ab was used. Table b) is the result o f figure 23 and 1/250 concentration o f Ab was used. Table c) is the result o f figure 24 and 1/500 concentration o f Ab was used.

a) Ab concentration: f6b4g7b4 1/l25MIB-BSA A<a> U F ...............

RiW

lpg/ml 3.472 37pg/L 0.9960.5pg/ml 3.121 2pg/L 0.993

0.25pg/ml 1.483 0.244pg/L 0.9930.125pg/ml 0.593 0.268pg/L 0.941

b) Ab concentration: f6b4g7b4 1/250MIB-BSA - Aw Iso00 R 2(C,


0.25pg/ml 1.923 0.0916pg/L 0.9910.125pg/ml 0.593 0.454pg/L 0.955

c) Ab concentration: f6b4g7b4 1/500MIB-BSA - - AGn- Uom Rm

lpg/ml 2.412 3pg/L 0.9840.5pg/ml 1.243 lpg/L 0.9920.25pg/ml 0.46 0.414pg/L 0.991

0.125pg/ml 0.082 lpg/L 0.917

a: absorbance when plate was saturated with Ab.b: the concentration o f MIB that shows 50% o f inhibition of Ab binding to solid phase conjugate, c: correlation coefficient value.A, I5o and R2 were calculated by program soft max pro.

97


depending on Ab concentration. Solid phase conjugate concentration effect was

highest at high concentrations o f Ab (1/125 dilutions) and was lowest at low

concentrations o f Ab (1/500 dilution). This is probably because high

concentrations o f Ab provide enough chance to bind to extra solid phase

conjugate and low concentration o f Ab can also be a limiting factor in Ab binding

for solid phase conjugate binding.

There seems to be greater propensity to increase the sensitivity o f ELISA

by lowering solid phase conjugate concentrations because Ao values o f ELISA

3.406, when 0.125 pg/ml of bomeol-BSA, and Ab dilutions of 1/125 were used.

With bomeol-BSA as coating, it was found that the sensitivity o f ELISA did not

increase by lowering solid phase conjugate concentrations from 0.25pg/ml to

0.125pg/ml showing the same Iso values, 17pg/ml, when MAb was diluted by

1/500. Correlation coefficient values decreased from 0.993 to 0.932. These results

show that there is a limit in increasing the sensitivity o f ELISA by lowering Ab

concentrations and solid phase conjugate concentrations.

4.4. Effect of solid phase conjugate protein structure on the sensitivity of ELISA

Since the assay depends on the competition o f the antibody binding to

either the immobilized conjugate or the soluble MIB from the sample being

tested, sensitivity will be greatest if the antibody affinity for the soluble MIB is

enhanced relative to the immobilized conjugate.

The nature of hapten linkage to carrier protein is critical to the specificity

and sensitivity o f the immunoassay. Immunogens prepared by haptenation o f

protein often generate antibodies against the linkage. Three types of heterologies

98


a)

Eeto

4

3

2

1

0mSo

cn o>c c1- o 8o>3O

o>3 O) o) '3)E E E- ? 8

MIB concentration

b)

1.2

1

0.8

0.6

0.4

0.2

0CQ - J2 go2 O

05 05c cT- o05c 05 05

3053

05 05 05E E E- £ 8

1ug/ml

0.5ug/ml

0.25ug/ml

0.125ug/ml

1ug/ml

0.5ug/ml

0.25ug/ml

0.125ug/ml

MIB concentration

Figure 25. Test o f solid phase protein conjugate (bomeol-BSA) concentrations effect when high concentrations o f Ab (F6b4g7b4, 1/125 times) was used as a 1st Ab. Four different concentrations o f solid phase protein conjugates (bomeol- BSA, lpg/ml, 0.5pg/ml, 0.25pg/ml and 0.125pg/ml) were coated on the microtiter plate and compared. Goat anti-mouse IgG-peroxidase (1/2000) was used as a secondary antibody and MIB was diluted in 10% methanol from lOOmg/L to O.lng/L by serial dilution o f 1/10 times. Graph a) is a result showed as a absorbance at 415nm and graph b) is a same result o f graph a) expressed as a A/Ao.

99


a)

Ecto5

4

3

2

1

0

•1_jGO

OJ OJOJOJe O) OJ3oOJ

MIB concentration

b)

1.5

0.5

-0.5_iCQ o>co>c o>o>c o>

3oS

1ug/ml

0.5ug/ml

0.25ug/ml

0.125ug/ml

1ug/ml

0.5ug/ml

0.25ug/ml

0.125ug/ml

MIB concentration

Figure 26. Test o f solid phase protein conjugate (bomeol-BSA) concentrations effect when medium concentrations o f Ab (F6b4g7b4, 1/250 times) was used as a 1st Ab. Four different concentrations o f solid phase protein conjugates (bomeol- BSA, lpg/ml, 0.5pg/ml, 0.25pg/ml and 0.125pg/ml) were coated on the microtiter plate and compared. Goat anti-mouse IgG-peroxidase (1/2000) was used as a secondary antibody and MIB was diluted in 10% methanol from lOOmg/L to O.lng/L by serial dilution o f 1/10 times. Graph a) is a result showed as a absorbance at 415nm and graph b) is a same result o f graph a) expressed as a A/Ao.

100


MIB concentration

b)

1.5

1.25

$ 0.75

0.5

0.25

—i—j _im o> OJOJ OJc OJ OJ

o

1ug/ml

0.5ug/ml

0.25ug/ml

0.125ug/ml

MIB concentration

Figure 27. Test o f solid phase protein conjugate (bomeol-BSA) concentrations effect when low concentrations of Ab (F6b4g7b4, 1/500 times) was used as a 1st Ab. Four different concentrations o f solid phase protein conjugates (bomeol- BSA, lpg/ml, 0.5pg/ml, 0.25pg/ml and 0.125pg/ml) were coated on the microtiter plate and compared. Goat anti-mouse IgG-peroxidase (1/2000) was used as a secondary antibody and MIB was diluted in 10% methanol from lOOmg/L to O.lng/L by serial dilution o f 1/10 times. Graph a) is a result showed as a absorbance at 415nm and graph b) is a same result o f graph a) expressed as a A/Ao.

101


Table 10. Effects o f solid phase conjugate (bomeol-BSA) concentrations on the sensitivity o f ELISA. These tables are results o f figures 25,26, and 27 expressed as I50 values. Four concentrations o f bomeol-BSA (lpg/m l, 0.5pg/ml, 0.25pg/ml and 0.125pg/ml) were compared. Table a) is the result o f figure 25 and 1/125 concentration o f Ab was used. Table b) is the result o f figure 26 and 1/250 concentration o f Ab was used. Table c) is the result o f figure 27 and 1/500 concentration o f Ab was used.

a) Ab concentration: f6b4g7b4 1/125Bomeol-BSA 'A® I 5 0 ”


0.25pg/ml 3.604 346pg/L 0.9980.125pg/ml 3.406 59pg/L 0.991

b) Ab concentration: f6b4g7b4 1/250Bomeol-BSA A» IsoW " - - r2 W

lpg/ml 3.454 25 lpg/L 0.9260.5pg/ml 3.49 347pg/L 0.999

0.25pg/ml 3.522 95pg/L 0.9990.l25pg/ml 2.899 19pg/L 0.976

c) Ab concentration: f6b4g7b4 1/500Bomeol-BSA 'A ® Uow '

- r2 (C)


0.25pg/ml 3.051 17pg/L 0.9930.125pg/ml 1.753 17pg/L 0.932

a: absorbance when plate was saturated with Ab.b: the concentration o f MIB that shows 50% o f inhibition of Ab binding to solid phase conjugate, c: correlation coefficient value.A, I 5 0 and R2 were calculated by program soft max pro.

102


can be used to improve detectability. Hapten heterology can be introduced by

attaching different but related haptens through the same site by the same linkage.

Bridge heterology can be introduced using different linkers for coupling the

haptens to carrier protein and solid phase conjugate. Site heterology can be

introduced by attaching the same linking group to different sites o f the hapten. It

is critical to use at least one o f these three methods to improve sensitivity o f

ELISA (Deshpande, 1996). Vallejo et al.(1982) found by using two different

linking arms sensitivity o f an assay for parathion was improved.

In this study, there was not a large difference in the titer o f antibodies

when high concentrations o f solid phase conjugates (Ipg/mL) was used as coating

solution and antibodies were serially diluted from 1/125 to 1/64,000 by I in 2

times (Figure 28). This is because solid phase conjugates were not a limiting

factor in antibodies binding to solid phase conjugates.

The affinities o f solid phase conjugates to antibodies were compared in

which high concentrations of antibodies (1/125) and serially diluted solid phase

conjugates (Figure 29) were used for the test. When high concentrations of solid

phase conjugates concentrations (5 and 1 pg/mL) were used, plates were saturated

with antibodies showing maximum absorbance. However, solid phase conjugates

became a limiting factor in binding of antibodies to solid phase conjugates at low

concentrations o f solid phase conjugates concentrations (0.2 and 0.04 pg/mL) and

showed large differences in affinity o f solid phase conjugates to antibodies.

Bomeol-BSA showed the highest affinity to the antibodies and MIB-BSA

showed the lowest affinity to the antibodies. This is reasonable because antibodies

103


were made using bomeol-LPH as an immunogen. The difference between the

isomers, bomeol and isobomeol, is the position o f hydroxyl: axial vs.equitorial.

As linking arms (hemisuccinate) are bound through these hydroxyl groups,

different position o f the same linking arm showed difference in affinity o f

antibodies and these difference in affinity provide position heterology effects in

competitive ELISA. The difference between isobomeol and 2-methylsiobomeoI

(MIB) was the presence of another methyl group in MIB. The presence o f another

methyl group resulted in a decrease in affinity o f MIB-BSA to the anti-bomeol

monoclonal antibody.

The effects o f these solid phase protein conjugates on the sensitivity o f

ELISA were investigated using three different combinations of antibodies and

solid phase protein conjugates. Figure 30 and figure 31 depict results o f the

comparison between these three solid phase conjugates and table 11 summarized

Iso values.

MIB-BSA provided approximately 550 times better sensitivity compared

to bomeol-BSA when MAb were diluted 1/125 and 1 pg/ml o f solid phase

conjugate were used. Depending on the concentrations of antibodies and solid

phase conjugates, the solid phase conjugate structure changed I50 values from 10

times to 550 times. All three tests showed maximum sensitivity when MIB-BSA

was used as the solid phase conjugate and minimum sensitivity when bomeol-

BSA was used as a solid phase conjugate.

These tests showed the same results with the tests o f solid phase conjugate

affinities to antibodies (Figure 29). This is mainly because o f the differences in

104


the affinity of antibodies against solid phase conjugates. These results indicate

that sensitivity o f ELISA can be improved by lowering the relative affinity of the

Ab to the solid phase material, because the sensitivity o f ELISA is determined by

the competition o f free MIB against MIB, bomeol, or isobomeol bound to solid

phase conjugates.

4.5. Specificity of antibody

The specificity o f MAb was determined by comparing the cross reactivity

of several compounds that have similar structures to MIB. Table 12 summarizes

the results of the cross reactivity studies. MIB had a cross reactivity of 100% by

definition and showed the highest cross reactivity.

These MAb were made using bomeol-LPH immunogen. It was therefore

expected that bomeol will show higher cross-reactivity compared to MIB.

However, bomeol showed only 19.9% cross reactivity compared to MIB, which is

probably because the presence of the methyl group in MIB increased affinity

against antibody. The methyl group in MIB is in the same position as the

hemisuccinyl o f bomeol-LPH.

Bomeol and isobomeol showed similar low cross reactivities (19.9% and

19.6%). These results indicate that the position o f the hydroxyl group in those

compounds did not influence the binding of antibody.

The difference between camphor and bomeol is the presence of a ketone

group in camphor instead o f a hydroxyl group in bomeol. Camphor has very low

cross reactivity showing approximately 4%. This low cross reactivity may be

attributed to the presence o f a double bond o f ketone group in camphor. The

105


5

4

1

0oo o o

§ sCNL r r

ooo00 CMCO

MIB-BSA

isobomeol-BSA

bomeol-BSA

Ab concentration

Figure 28. Comparison o f Ab titer using three different solid phase protein conjugates. Ab (f6b4g7b4) was serially diluted from 1/125 to 1/64000 by lin 2 times using 0.05% BSA solution in PBS (pH 7.4) buffer. Solid phase concentrations were lpg/ml. Goat anti mouse IgG-peroxidase (1/2000) conjugate as used as a secondary antibody and ABTS was used as a substrate.

Ecin•"T

5

4

3

2

1

0

MIB-BSA

isobomeol-BSA

bomeol-BSA

O J ”o ) 0 1 O J O JE E E E Ein CM

O 3O

GO

8O

o>Z3<o 3

CMCOO

O)3

Solid phase conjugate concentration

Figure 29. Comparison of Solid phase conjugate affinity to anti bomeol monoclonal antibody (F6b4g7b4). First Ab concentration was 1/125. Solid phase conjugate was serially diluted from 5 mg/L to 0.064 pg/L by 1 in 5 times. Goat anti mouse IgG-peroxidase (1/2000) was used as a secondary antibody and ABTS was used as a substrate.

106


a)

01

CD2O

2.5

1.5Eein

0.5

CD o> o>o»c O)O)c o>o>

MIB concentration

b)

1.2

1

0.8

0.6

0.4

0.2

0

- 0.2

o>c o>coen O)3 O) O)3 o>

Eo>E

° I * 2 8MIB concentration

MIB-BSA

isobomeol-BSA

bomeol-BSA

MIB BSA

isobomeolBSA

bomeol BSA

Figure 30. Test o f solid phase conjugate structure effect using 1/250 times o f 1st Ab (Cell line f6b4g7b4 ) and 0.5 pg/ml o f solid phase conjugates. Goat anti mouse IgG-peroxidase (1/2000) was used as a secondary Ab. Three solid phase protein conjugate MIB-BSA, isobomeol-BSA, and bomeol-BSA were coated on the microtiter plate and compared. Graph a) is a result expressed as absorbance at 415nm and graph b) is same result expressed as A/Ao.

107


MIB concentration

01

eg5o

b)1.2

1

0.8

0.6

0.4

0.2

0

- 0.2

O ) CD O ) O )c _ _ _O) O) O)c c c _ _ _ _s - 2 § S 8

o> *05 "5 i

- I 8

MIB-BSA

isobomeol-BSA

bomeol-BSA

MIB concentration

Figure 31. Test of solid phase conjugate structure effect using 1/125 times of 1st Ab ( Cell line f6b4g7b4 ) and 1 pg/ml of solid phase conjugates. Goat anti mouse IgG-peroxidase (1/2000) was used as a secondary Ab. Three solid phase protein conjugate MIB-BSA, isobomeol-BSA, and bomeol-BSA were coated on the microtiter plate. Graph a) is a result expressed as absorbance at 415nm and graph b) is same result expressed as A/Ao.

108


Table 11. Test o f solid phase structure effect. Three combinations o f Ab and solid phase conjugate were used. Each result was shown as Iso values those are the concentrations showed 50 % inhibition o f binding o f Ab to the solid phase protein conjugate in the ELISA.

Abconcentration

Solid phase conjugate concentration (pg/ml)

MIB-BSA(Iso)

isoborneol-BSA(Iso)

borneol-BSA(Iso)

1/250 0.50 0.284 pg/L 1 pg/L 16 pg/L1/125 1 0.041 pg/L 1 pg/L 22 pg/L1/250 1 4 pg/L 12 pg/L 42 pg/L

109


presence o f a double bond would influence in the bond angles and lengths in the

ring structure and this may be responsible for the decrease in the affinity of

antibody recognition. This result may also explain why the polyclonal antibody

produced by Chung et al. (1990) showed low affinity against MIB and showed

low sensitivity in the ELISA. They produced polyclonal antibody by immunizing

with camphor-BSA immunogen.

Camphorquinone has an additional carbonyl group as compared to

camphor and showed some cross-reactivity with the antibody and a decrease in

cross-reactivity comparing to camphor. All o f these results indicated that antibody

was most specific to MIB and recognized the presence o f the methyl group.

2-Methyleneborane and 2-methyl-2-borene are dehydration products o f 2-

methylisobomeol and do not cause off-flavor (Korth et al., 1992). Martin et al.

(1988) isolated these two compounds from chronically off-flavored catfish flesh.

The cross reactivity o f these two dehydration products was not investigated in this

research. Further studies will be needed to determine whether their presence could

potentially yield false positives.

4.6. S tandard curve

From previous results o f antibody concentration effects, solid phase

conjugate concentration effects and solid phase conjugate structure effects,

optimum concentrations of antibodies and solid phase conjugate were determined

that would show maximum sensitivity o f ELISA.

A standard curve was constructed using anti-bomeol monoclonal antibody

diluted 1/250 times and 0.5 pg/mL o f MIB-BSA as a solid phase conjugate

n o


Table 12. Cross reactivity o f compounds that have similar structure with MIB. Each result was shown as I50 values and cross reactivity was expressed as (I50

value of MIB/Iso value o f the compound tested) x 100 (%). Ab from cell line F6b4g7b4 was diluted 1/250 and used as 1st Ab and MIB-BSA (0.5 pg/ml) was used as a solid phase protein conjugate. Each result is a mean value o f three replicates o f ELISA.

Compound I50 Cross reactivityMIB 0.019ppm 100%Bomeol 0.094ppm 19.9%Isobomeol 0.095ppm 19.6%Camphor (R+) 0.396ppm 4.7%Camphor (R-) 0.496ppm 3.8%Camphorquinone 0.589ppm 3.2%Geosmin 1.090ppm 1.7%Bomylamine 301.719ppm 0%

i l l


(Figure 32). MIB was diluted in 10% methanol solutions from 100 mg/L to 0.1

ng/L by serially diluting I in 10 times. It showed a detection limit o f I ng/L when

the plate was incubated 30 min after adding substrate. Detection limit was defined

as the concentration of MIB showing A/Ao value o f 0.8. This value is the

concentration showing 20% inhibition in antibody binding to the solid phase

conjugate protein.

i l l


a)

ECto

2.5

2

1.5

1

0.5

0

-0.5tn at a»a»o> 01OJc

30min

MIB concentration

b)

1.2

0.8

0.2

-0.2a _i

'fts

MIB concentration

Fig 32. Standard curve constructed using Ab concentration o f 1/250 (f6b4g7b4) and 0.5 pg/ml o f MIB-BSA as a solid phase protein conjugate. Goat anti-mouse IgG-peroxidase (1/2000) was used as a secondary Ab. ABTS was used as a substrate. MIB solution was serially diluted from lOOmg/L to O.lng/L by 1/10 times in 10% methanol solution. Graph a) is a result o f absorbance after 30min o f incubation at RT with substrate. Graph b) is the same result expressed as A/Ao.

113


CHAPTER 5. SUMMARY AND CONCLUSION

In order to produce monoclonal antibody against 2-methylisobomeol, 2-

methylisobomeol (MHS)-Limulus poyphemus hemocyanin (LPH) conjugate and

bomeol-LPH were prepared as immunogens. Immunization with these two

compounds produced polyclonal antibodies in mice sera after two immunizations

and those antibodies specifically recognized MIB in indirect competitive ELISA.

Fusions o f splenocytes from mice (mouse I and 2) immunized with MIB-

LPH to myeloma cells did not produce any cell line that specifically recognized

free MIB in the competitive ELISA. The antibodies from those hybridomas

showed high background and non-specific binding. Fusion o f splenocytes from

mouse 3 immunized with bomeol-LPH to myeloma cells yielded a cell line

producing monoclonal antibody that specifically recognized free MIB in the

indirect competitive ELISA.

After three clonings, cell line f6b4g7b4 was established and used as a

primary antibody source in the ELISA. The same methodology was used for the

preparation o f solid phase conjugates and three different kinds of solid phase

conjugates: MIB-BSA, isobomeol-BSA and bomeol-BSA, were prepared.

Tests o f Ab and solid phase conjugate concentrations showed that

sensitivity of ELISA could be improved by lowering concentrations o f Ab and

solid phase protein conjugates. However, use o f low amounts o f Ab and solid

phase protein conjugate also decreased maximum absorbance and increased

variation within samples. This indicates that there should be a compromise

between sensitivity o f assay and maximum absorbance.

114


The bridge heterology effect can improve the valence o f antibody affinity

for the solid phase protein conjugate and soluble analytes. In this research, the

same conjugation methods were used for the introduction o f bridge to the

immunogen and solid phase protein conjugates by using the same

hemisuccination reaction. However, by using an isomer (isobomeol) of hapten

(bomeol) in immunogen for the preparation of solid phase conjugate, a site

heterology effect was introduced, and a hapten heterology effect was introduced

by using similar structure o f compound (MIB) with immunogen. Comparison of

these three different solid phase protein conjugates showed this anti-bomeol MAb

recognized the linking arm and distinguished the presence o f a methyl group in

the epitope.

In the test o f specificity o f antibody, the Ab showed highest cross

reactivity to MIB. Bomeol and isobomeol showed only 20% cross-reactivity

compared to MIB. The presence o f a methyl group in MIB increased affinity

against Ab. The methyl group in MIB is in the same position with hemisuccinate

of bomeol-LPH (immunogen) and this helped to increase the affinity against

antibody.

Standard curve from 0.1 ng/L of MIB solution to 100 mg/L of MIB

solution diluted in 10% methanol was constructed using prediluted MAb of

F6b4g7b4 (1/250 dilution) with a solid phase conjugate (MIB-BSA 0.5 pg/ml) in

indirect competitive ELISA. This result showed that the MAb could detect MIB

down to 1 ng/L (parts per trillion) levels. This result indicates that the assay has


sufficient sensitivity for direct application to samples without any previous

concentration steps.

Regardless o f the extremely small size o f hapten, it was possible to

produce a monoclonal antibody that is highly sensitive to MIB. The affinity

characteristic o f this antibody and its supply will remain constant, because it was

produced as a monoclonal antibody. By using monoclonal antibody for this

compound, MIB can be easily tested from field samples.

116


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Aoyama, K., 1990. Studies on the earthy-musty odors in natural water IV. Mechanisms o f earthy-musty odor production o f Actinomycetes. Journal of Applied Bacteriology, 68: 405-410.

Arganosa, G.C. and Flick, JR.G.J. 1992. Off-flavors in fish and shellfish, In: Off-flavors in Food and Beverages. (G. Charlambous ed.) pp. 103-126. Elsevier Science Publishers B.V. New York, USA.

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APPENDIX I. EPITOPE DENSITY DETERMINATION

Epitope density determination by trinitrobenzene sulfonate (TNBS) method, a) is the result o f comparison between BSA and MIB-BSA expressed as absorbance at 335nm. b) is the result o f comparison between LPH and MIB-LPH expressed as absorbance at 335nm..

a )

Concentration(mg/ml)

BSA MIB-BSA

0.2 0.4793 0.18530.4 0.9629 0.32940.6 1.1914 0.57980.8 1.4603 0.76021.0 1.6064 0.8164

b)Concentration

(mg/ml)LPH MIB-LPH

0.2 0.2515 0.12790.6 0.5793 0.17831.0 0.783 0.4339

130


APPENDIX n . SCREENING (1)

Screening o f fused cells after fusion o f splenocytes (mouse 2, immunized with MIB-LPH) with myeloma cells in the days o f 6 and 10.20 |xl o f supernatant (6 days) and 50 p i o f supernatant (10 days) were used as 1st Ab source and MIB- BSA (1 pg/ml) was used as solid phase conjugate. Anti mouse IgG-peroxidase (1/2000) was used as secondary Ab.

6 days 10 days 6 days 10 daysIc2 0.174 1.865 3a6 0.228 0.705lc6 0.246 1.678 3a7 0.428 0.427

lclO 0.175 0.651 3b3 0.201 2.403le3 0.175 0.839 3bI0 0.194 2.247

le l l 0.201 1.256 3e7 0.19 0.871lf7 0.192 1.736 3f6 0.189 2.238lg8 0.172 0.618 3h3 0.208 2.431lh3 0.173 0.885 4a3 0.177 0.4732a3 0.192 1.319 4a8 0.19 0.3072a8 0.228 1.59 4al0 0.392 0.4022b6 0.173 0.693 4b6 0.575 0.7052 c ll 0.202 1.596 4b9 0.364 0.4472d8 0.196 2.199 4 c ll 0.179 0.332e7 0.224 2.056 4d6 0.575 0.705

2el0 0.221 0.819 4d9 0.2 0.442fl0 0.593 0.562 4e7 0.183 0.3512g6 0.183 0.712 4el0 0.203 0.66

2g ll 0.196 0.904 4fl0 0.301 2.2972h6 0.274 2.119 4a9 0.448 0.509

131


APPENDIX III. SCREENING (2)

Screening o f fused cells (mouse 2) by competition with free MIB (10 fig/ml). Supernatants (25pi each ) o f cell culture were used as 1st Ab and MIB-BSA (lpg/ml) was used as solid phase conjugate. Anti mouse IgG-peroxidase (1/2000) was used as secondary Ab.

No MIB MIB 10 pg/ml

No MIB MIB10 pg/ml

Lc2 1.049 0.788 2g6 0.728 0.7lc6 0.792 0.642 2 g ll 0.279 0.303lclO 0.791 0.588 2h6 0.73 0.684le3 0.291 0.286 3a6 0.176 0.193

le l l 1.272 0.919 3b3 1.409 1.808lf7 0.5 0.62 3bl0 0.274 0.319

l f l l 1.103 1.114 3e7 0.323 0.495lg8 0.515 0.465 3f6 1.062 0.929lh3 0.648 0.616 3h3 1.206 1.1282a3 0.475 0.531 4al 0.156 0.2022a8 0.579 0.8 4b4 0.519 0.7242b6 0.248 0.299 4c7 0.478 0.5432 c ll 0.529 0.44 4d6 0.131 0.1432d8 0.783 0.561 4d8 0.594 0.7132e7 0.627 0.672 4fl0 0.785 1.02

2el0 0.286 0.441

132


APPENDIX IV. RESULT OF 1st CLONING

2

IDO

<8

■ NO bomeol

~ bomeol, lOppm

□ bomeol, lOOppm

Result of 1st cloning o f the cells from dl 1 and f6. Cell culture supernatants (20jal each) from cells subcultured in 24 well cell culture plate were used as an Ab source. Two concentrations o f bomeol (lOppm and lOOppm) were used for the competitive inhibition o f Ab binding to the solid phase conjugate (MIB-BSA, lppm).

133


APPENDIX V. RESULT OF 2nd CLONING

| No MIB u MIB. lOppm

b)

1.4

1.2

1

E 0.8inu 0.6

0.4

0.2

0

No MIBMIB.10ppm

d11f4h6 d11f4h9 d11f4g4 d11f4g6

Result of 2nd cloning o f the cells from d l lb3 (graph a) and d l lf4 (graph b). Cell culture supernatants (5pl each) from cells subcultured in 24 well cell culture plate were used as an Ab source. Ten ppm o f MIB concentration was used for the competitive inhibition o f Ab binding to the solid phase conjugate (MIB-BSA,lppm).

134


a)

1.2

0.8

mo■'j-0.4

0.2

wo

d11g3h4 d11g3g2 d11g3g8 d11g3g9 d11g3f3

b)

1.2

1

0.8

0.4

0.2

No MIBMIB.10ppm

No. MIBMIB.10ppm

d11h8h6 d11h8h10 d11h8g7 d11h8g10 d11h8f3

Result o f 2nd cloning of the cells from dl lg3 (graph a) and d l lh8 (graph b). Cell culture supernatants (5ul each) from cells subcultured in 24 well cell culture plate were used as an Ab source. Ten ppm o f MIB concentration was used for the competitive inhibition of Ab binding to the solid phase conjugate (MIB-BSA, lppm).

135


1.2

0.8

ino

0.4

0.2

f6b4h5 f6b4g2 f6b4g7 f6b4f1 f6b4f2

No MIBMIB.10ppm

Result o f 2nd cloning of the cells from f6b4. Cell culture supernatants (5pl each) from cells subcultured in 24 well cell culture plate were used as an Ab source. Ten ppm o f MIB concentration was used for the competitive inhibition o f Ab binding to the solid phase conjugate (MIB-BSA, I ppm).

136


APPENDIX VL RESULT OF 3rd CLONING

5

■ No MIB ~ MIB,

1ppma Mlb,

10ppm

Result of 3 rd cloning o f cells from cell line f6b4g7. Cell culture supernatants (20pl each) from cells subcultured in 24 well cell culture plate were used as an Ab source. Isobomeol-BSA (0.3ppm) was used as solid phase protein conjugate. Two concentrations o f MIB solutions (lppm and lOppm) were used for the competitive inhibition of Ab binding to solid phase conjugate.

137


VITA

The author was bom in Jeju, Korea, on July 20, 1966. He entered Korea

University in March. 1985. and was graduated in February, 1991, with a bachelor

o f science degree in agricultural chemistry.

The author was admitted by the Graduate School o f Korea University in

August, 1991, and received a master o f science degree in agricultural chemistry in

July, 1993.

The author was accepted to the Graduate School of Louisiana State

University through the Department of Food Science in August. 1995. He worked

as a graduate research assistant for Dr. Leslie C. Plhak.

The author is currently a candidate for the degree o f Doctor in Philosophy

in Food Science, which will be conferred in December, 1999.

138


DOCTORAL EXAMINATION AND DISSERTATION REPORT

Candidate; Eun Sung Park

Major Field: Food Science

Title of Dissertation: Development of Monoclonal Antibody and EnzymeLinked Immunosorbent Assay for Detection of Off-flavor Compound 2-Methylisobomeol

Approved:

Professor

iduate ScnoolDean of

EXAMINING COMMITTEE:

Zt- v m/ f i .

May 7, 1999


development of monoclonal antibody and enzyme linked

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