chapter 3 - shodhganga : a reservoir of indian theses...

126

Chapter 3

Avian antibody (IgY) generation

against staphylococcal

enterotoxin B

and its characterization

Avian antibody (IgY) generation against staphylococcal enterotoxin B and its characterization

Chapter 3

127

3.1. INTRODUCTION

3.1.1. Avian immunoglobulins

Antibodies have become an indispensable tool in various forms of

research due to their unique ability to recognize and bind specific structures

on other molecules. They have received maximum attention in recent years

because of their high specificity and selectivity. Antibodies are globular

proteins belongs to the group serum glycoproteins called immunoglobulins.

Over the years, mammalian systems have been tried extensively for

generation of monoclonal or polyclonal antibodies. However, the production of

mammalian antibodies involves immunization, repeated bleeding and

sacrificing for spleen removal causes massive distress to the experimental

animal. In this direction, chicken egg yolk antibodies represent a refinement in

the sense of animal welfare issues to avoid painful and invasive blood

sampling steps associated with antibody production (Chalghoumi et al., 2009;

Chouhan et al., 2010). Though raising antibodies in chicken is well known

since decades, only recently they have become an alternate source for

polyclonal antibody production making bleeding of lab animals obsolete

(Michael et al., 2010). Klemperer first demonstrated the transfer of specific

antibodies from serum to the egg yolk by immunization in 1893. However,

Klemperer’s results on antibody generation in hens attracted a great attention

only after 1980s as the animal welfare became a matter of serious ethical

concern for the scientific community. It was reported that the antibody

productivity in laying hens is nearly 18 times greater than that in rabbits

(Schade et al., 1996) producing almost 100 mg of antibodies from one egg


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(Akita and Nakai, 1992). IgY scores over IgG in terms of simple isolation

protocols for antibodies, less antigen requirement to obtain specific IgY with

high titer and stability over temperature variations.

3.1.2. Avian immune system and antibody production

Antibodies are produced by B-lymphocytes in the embryonic liver, yolk

sack and bone marrow. Avian antibodies are distinguished into three

immunoglobulins classes such as IgA, IgM and IgY based on their

concentration, structure and immunochemical function (Leslie and Martin,

1973). Transfer of IgY from serum to egg is receptor mediated in hens

(Tressler and Roth, 1987; Mohammed et al., 1998; Morrison et al., 2001).

Serum IgY is selectively transferred from the hen’s circulatory system into the

maturing oocyte across the oolemma in the ovarian follicle (Rose and Orlans,

1981). Morrison et al. (2001) identified and demonstrated that Fc region of IgY

plays an important role in its uptake into the egg yolk. However, IgA and IgM

are transferred directly into the egg white in the oviduct along with egg

albumins. Rose et al. (1974) reports that IgA concentration is ~0.7 mg/mL and

IgM is ~0.15 mg/mL in egg white whereas IgY concentration is 8-25 mg/mL in

egg yolk.

3.1.3. Structural difference between IgY and IgG

Structurally IgY is similar to mammalian IgG consisting of a similar

basic 4-chain subunit structure with two identical heavy (H) chains and two

identical light (L) chains, which are linked by disulfide bridge. The light chain is


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129

made up of one variable (VL) and one constant domain (CL) (Shimizu et al.,

1993). However, IgY differs from mammalian IgG having an additional heavy

chain which is made up of one variable domain (VH), four constant domains

(CH1, CH2, CH3 and CH4). The Fc portion of IgY contains an additional

carbohydrate side-chain in contrast to only one in IgG as a result of the

presence of an extra constant domain. The hinge region which separates the

CH1 and the CH2 domains in IgG is absent in IgY (Fig. 3.1). VH- and VL-

domains pair to form two identical Ag-binding sites (Fab) per subunit

structure, whereas domains of the heavy chain C-regions pair to form a

segment of the molecule called Fragment of crystallization (Fc).

Hinge region

Fig. 3.1 General structure of IgG and IgY.


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130

3.1.4. Physico-chemical properties of IgY

IgY slightly differs from IgG in terms of its structure and property due to

the presence of an extra constant domain. IgY has a molecular mass of ~180

kDa in comparison to mammalian IgG which is ~150 kDa. The isoelectric point

(pI) of IgY is in the range of 5.7 to 7.6 which is lower when compared to that of

IgG whose pI falls in the range 6.1 to 8.5 (Polson et al., 1980; Hodek and

Stiborova, 2003; Chalghoumi et al., 2009). The presence of an extra constant

domain in Fc fragment made IgY more hydrophobic than the IgG molecule

(Davalos et al., 2000).

3.1.5. Advantages of IgY over IgG

IgY present in egg yolk can be stored over a long period of time. Laying

hens are highly cost-effective producers of IgY as it is cheaper to feed and

maintain domestic chickens than rabbits. Shimizu et al. (1992) and Hatta et al.

(1993) studied thermal stability of IgY and reported that IgY can retain its

functional affinity even after exposure to temperature ranging between 60 °C

and 70 °C for few minutes. Shimizu et al. (1988) also reported that freezing

and freeze-drying did not affect the activity of IgY unless repeated several

times. The stability and retention of functional affinity of IgY at room

temperature for months are advantageous as chickens have relatively high

core body temperature of 41 °C (Michael et al., 2010). Table 3.1 summarizes

the properties between the two important immunoglobulins.


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131

Table 3.1 Comparison of properties of IgY with IgG.

Features for

comparison

Avian (IgY) Mammalian (IgG) References

1. Source of antibody Egg yolks in

Birds, Reptiles and Amphibians

Mammalian serum

Klemperer, 1893

Warr et al., 1995

2. Sampling Collecting eggs Bleeding Schade et al., 1991

3. Structural

differences

Less flexible hinge region,

Longer Fc region with additional

constant domain possessing 2

pairs of carbohydrate groups

Flexible hinge region,

Shorter Fc region bearing 1 pair

of carbohydrate groups

Warr et al., 1995

4. Extraction and

purification

Fast and simple Relatively more complicated and

slow

Akita & Nakai, 1993

5. Antibody yield ~ 100 mg/egg,

~ 8 mg/mL of yolk

~ 200 mg/bleed,

~ 5 mg/mL of blood

Schade et al., 1994


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132

6. Molecular Weight

(by SDS-PAGE)

Total weight: ~ 180 kDa

Light chains: ~ 25 kDa x 2

Heavy chains: ~ 65-67 kDa x 2

Total weight: ~ 150 kDa

Light chains: ~ 22 kDa x 2

Heavy chains: ~ 50 kDa x 2

Hatta et al., 1988

7. Affinity purification Requires Protein L which binds to

kappa variable light (VL) chain or

target specific antigens

Proteins A or G which binds to Fc

region or target specific antigens

Nilson et al., 1992

Eliasson et al.,

1988

8. Specific antibody ~ 2 - 10% ~ 5% Schade et al., 1991

9. Immune response Enhanced by phylogenetic

differences

Adversely affected by phylogenetic

homology

Gassmann et al.,

1990

10. Stability Good,

Stable at pH 4-9, up to 65 °C

Good,

Stable at pH 3-10, up to 70 °C

Hatta et al., 1993

Lee et al., 2002a

11. Hydrophobicity More hydrophobic than IgG Less hydrophobic than IgY Lee et al., 2002a

12. Overview Relatively new in product

development and application

Relatively matured in technology

development and application

Warr et al., 1995


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3.1.6. Egg yolk composition and IgY extraction

Egg yolk is made up of 51.3% of dry matter and 48.7% of water which

is almost equal in proportions (Siewert and Bronch, 1972). The dry matter in

yolk constitutes 16.6% of proteins, 32.6% of fats and lipids, 1% of

carbohydrates and 1.1% of inorganic matter. Lipid fractions being major

constituent of yolk dry matter includes triglycerides, phospholipids and

cholesterol. These water insoluble fractions can be separated from water

soluble proteins by centrifugation (Stadeklman et al., 1977). The protein

fractions in egg yolk can be classified into two types. The granular proteins

which accounts for 22% of total yolk proteins, are composed of 70% high-

density lipoproteins (HDL, α- and β-lipovitellins), 16% phosvitin

(glycophospoprotein) and 12% low-density lipoproteins (LDL) (Burley and

Cook, 1961). However, the globular proteins called livetin fractions that

constitute immunoglobulins accounts for only 14% of the total plasma protein

concentration in yolk (McCully et al., 1962). These water soluble livetins are of

three types namely α-, β-, and γ-livetin wherein, IgY is the predominant

fraction of γ-livetin (Kovacs-Nolan et al., 2005).

Therefore, IgY extraction from egg yolk requires removal of water

insoluble fractions and lipoproteins followed by purification of the IgY from

other livetins (Polson et al., 1980). Several methods are reported in the

literature for the extraction and recovery of IgY from egg yolk. Some of these

methods involve ultracentrifugation, extraction with organic solvents and

precipitation of lipoproteins with polyethylene glycol, sodium dextran sulphate,

water dilution and ultrafiltration (Akita and Nakai, 1993; Svendsen et al., 1996;


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Hatta et al., 1997; Fichtali et al., 1992; Kim & Nakai, 1996). Akita and Nakai

(1993) compared different methods of IgY extraction such as water dilution,

polyethylene glycol, dextran sulphate, and alginate methods in terms of yield,

purity and activity. Akita and Nakai (1993) further reported that water dilution

method is the most appropriate technique for IgY extraction in terms of

obtaining IgY in the highest level (91%), purity (31%) and economical with

simple procedures.

3.1.7. General applications of IgY

Avian antibodies have been used in many areas of research in recent

years. IgY may be a potential agent for toxin neutralization. IgY has been

tried in preventing viral and bacterial infections in humans (Sarker et al., 2001;

Shin et al., 2002; Amaral et al., 2008; Nilsson et al., 2008), pigs (Yokoyama et

al., 1992, 1997; Kweon et al., 2000; Owusu-Asiedu et al., 2003), dairy cows

(Zhen et al., 2008), fish (Lee et al., 2000b) and rabbits (O'Farrely et al., 1992).

IgY has also been used for diagnostic purposes in laboratory assays such as

ELISA (Piela et al., 1984; Gast et al., 1997; Holt et al., 2000; Hagan et al.,

2004; Thomas et al., 2006a). There is also an added advantage of using

chicken egg yolk antibodies for passive immunization by oral administration

as an emerging and promising nutritional strategy (Chalghoumi et al., 2009) to

establish protective immunity against viral and bacterial pathogens and also

against organisms that are non-responsive to antibiotic therapy. Chicken

antibodies raised against ricin and botulinum neurotoxin were successfully

made use to neutralize ricin and botulinum toxicity (Pauly et al., 2009).


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135

Efficacy of salmonella specific IgY in preventing fatal Salmonellosis has been

studied in mice and calves by oral administration (Peralta et al., 1994;

Yokoyama et al., 1998a, 1998b). IgY has also been applied to control

infectious intestinal diseases due to E. coli strains (Jungling et al., 1991).

Moon & Bunn, (1993) have studied the efficacy of IgY in inhibiting diarrhea in

a castor oil mouse model. There were also reports of application of IgY as a

common antibiotic therapy in curing piglets with diarrhea (Wiedemann et al.,

1991; Kim et al., 1996).

3.1.8. Staphylococcal enterotoxin B (SEB)

Staphylococcus aureus is one of the major foodborne pathogens that

have drawn attention due to toxin-mediated virulence, invasiveness and

antibiotic resistance. S. aureus is a facultative anaerobic Gram-positive

coccus which is non-motile in nature. They generally form grape-like clusters.

They produce staphylococcal enterotoxins (SEs) and are the causative agents

of staphylococcal food poisonings. The SEs are enterotoxins secreted by S.

aureus into the medium. So far, 22 serologically distinct staphylococcal

enterotoxins have been identified (Argudin et al., 2010). They are of two types

namely (i) the classical ones such as SEA (Huang et al., 1987), SEB (Bergdoll

et al. 1959; Huang and Bergdoll, 1970), SEC (with the SEC1, SEC2 and

SEC3, SEC ovine and SEC bovine variants) (Bergdoll et al., 1965; Schmidt

and Spero, 1983), SED (Casman, 1960; Casman et al., 1967) and SEE

(Bergdoll et al., 1971), and (ii) the new types of SEs include SEG (Munson et

al., 1998), SEH (Su and Wong, 1995), SEI (Munson et al., 1998), SEJ (Zhang


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136

et al., 1998), SEK (Orwin et al., 2001), SEL (Orwin et al., 2003), SEM, SEN,

SEO, SEP (Omoe et al., 2005), SEQ, SER, SES, SET (Ono et al., 2008), SEU

(Letertre et al., 2003) and SEV (Thomas et al., 2006b).

SEB being one among the 22 serologically distinct SEs identified, is the

most potent toxin associated with food poisoning (Kamboj et al., 2006; Nema

et al., 2007). SEB is a monomeric protein made up of 239 amino acids with a

molecular weight of 28.4 kDa (Johns and Khan, 1988). It is basic in nature

with a pI of ~8.7. SEB being protein of 28.4 kDa is a most efficient immunogen

due to polymorphism of its structure. Previously researchers were successful

in generating polyclonal antibodies against SEB in rabbits (Silverman 1963;

Shinagawa et al., 1974; Wood et al., 1997), mice (Bamberger et al., 1986;

Leclaire et al., 2002; Kamboj et al., 2006). However, keeping advantages of

avian antibodies (IgY) over mammalian antibodies (IgG) in mind, an attempt

was made to generate antibodies in avian system against SEB in this study.

Until now, reports on production of antibodies against bacterial enterotoxins in

avian system are very much limited. As far our knowledge is concerned,

protocols and methodology for generating IgY antibodies against SEB is not

available. LeClaire et al. (2002) made an attempt to examine the effect of

passive transfer of chicken anti-SEB antibodies raised against SEB toxin in

protecting Rhesus monkeys challenged with aerosolized SEB. Nevertheless,

LeClaire et al. (2002) used a very high dose (250-500 g) of SEB for

immunization. They have not reported extraction and affinity characterization

of IgY from immunized eggs. In this direction, present chapter deals with in-


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137

depth studies on generation of IgY antibodies against SEB, its extraction,

purification, affinity determination and overall characterization.

3.2. EXPERIMENTAL

3.2.1. Materials

Staphylococcal enterotoxin B, Sephadex G-75, Silver nitrate,

Acrylamide, Sodium dodecyl sulfate (SDS), N, N’-(1,2-Dihydroxyethylene)

bisacrylamide, Freund’s adjuvant complete (FCA), Freund’s adjuvant

incomplete (FICA), 3,3’,5,5’-Tetramethylbenzidine (TMB), Dimethyl sulfoxide

(DMSO), -Cyclodextrin, Urea hydrogen peroxide (U-H2O2), Carbonic

anhydrase, Trifluoroacetic acid (TFA), Acetonitrile solution (HPLC grade) and

Coomassie blue G-250 were procured from Sigma-Aldrich India Pvt. Ltd.,

(Bangalore-560 058, India). Rabbit anti-chicken IgY-HRP secondary antibody

and broad range protein molecular weight marker for SDS-PAGE were

procured from Bangalore Genei (Bangalore-560 058, India). Staphylococcus

aureus (ATCC 14458) was procured from American Type Culture Collection

(Manassas VA 20108, USA). Brain Heart Infusion (BHI) broth and sterile petri

dishes were procured from Himedia laboratories Pvt. Ltd. (Mumbai-400 086,

India), Dialysis membranes having 6-8 kDa molecular weight cut off was

procured from Spectra/Por, USA. Maxisorp enzyme-linked immunobsorbant

assay (ELISA) microtiter plates (flat bottom) were a product of Nunc

(Roskilde, Denmark). All reagents used were of analytical grade and acquired

from standard suppliers.


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3.2.2. Instruments

The instruments used were Synergy ultrapure water systems

connected with Elix-10 water purification system from Millipore (Millipore

(India) Pvt. Ltd., Bangalore-560058, India), Bioklenz vertical laminar air flow

system (Klenzaids contamination controls Pvt. Ltd., Valsad, India), Ecotron

incubator shaker (Infors AG, CH-4103, Bottmingen), Cooling centrifuge (Remi

laboratory instruments, Mumbai-400063, India), Alitea-XV peristaltic pump

(Sweden), Lyophilizer (Scanvac, Labogene Aps, DK-3450, Lynge, Denmark),

UV–Vis Spectrophotometer (UV-1601, Shimadzu, Japan), VERSAmax

tunable microplate reader (Molecular devises, California, USA) and High

performance liquid chromatography system equipped with SCL-10A system

controller, SPD-M10A diode array detector and LC-10AT pumps (Shimadzu,

Japan), Reverse phase C8 HPLC column of size 4.6 x 150 mm having 5 m

internal diameter (I.D) was procured from Grace Vydac (17434, Mojave street,

CA 92345, USA), Vertical slab gel electrophoresis system, Digital model

electrophoresis power pack operating at constant voltage and constant

current were supplied by Bangalore Genei (India) Pvt. Ltd., (Bangalore-560

058, India).

3.2.3. Growth conditions for S. aureus and extraction of SEB

SEB was extracted from S. aureus (ATCC 14458) grown in BHI broth

for 24 hrs at 37 °C and 350 RPM for three generations. After 24 hrs of

incubation, culture broth was centrifuged at 10,000 RPM at 4 °C for 15

minutes and supernatant was collected. Culture supernatant was subjected to


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ammonium sulfate precipitation at 20%, 40%, 60% and 80% sequentially.

Culture supernatant was centrifuged as above after each level of precipitation

and pellet was collected. Pellets were analyzed for SEB by SDS-PAGE run on

12% separating gel and 6% stacking gel by the protocol described by

Laemmli. Gel was stained with silver nitrate stain to observe the level of purity

of desired protein as described previously (Section 2.2.6, Chapter 2).

3.2.4. Purification and characterization of SEB

Crude extract of SEB that was precipitated from culture supernatant at

80% ammonium sulfate saturation was further purified by size exclusion

chromatography. Sephadex G-75 gel with bead diameter 40-120 m having

fractionating range 3,000 Da to 80,000 Da was swollen in milli-Q water for

overnight and packed in a glass column of internal diameter (I.D) 1.5 cm (bed

length was 57 cm). Column was repeatedly saturated with phosphate buffer

saline (PBS, 50 mM, pH 7.5) and loaded with 30 mg of crude protein. The

protein was eluted with PBS at a flow rate of 12 mL/hr and eluent was

collected sequentially in a micro-centrifuge tube of 1 mL capacity. Elution

profile was monitored at 280 nm in a spectrophotometer and eluted proteins

were analyzed by SDS-PAGE to determine the purity and molecular weight of

SEB as described previously (Section 2.2.6, Chapter 2).

Column purified SEB extract was confirmed by reverse phase HPLC in

a C8 column by comparing the retention time with standard SEB. Mobile

phase employed was 0.1% TFA in water (mobile phase A) and 0.1% TFA in

90% acetonitrile (mobile phase B) according to Callahan et al. (2006) at a


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gradient flow of 100% B for 5 minutes, 0-50% B in 40 minutes, 50-90% B in

next 20 minutes, 90% hold for another 5 minutes and flow back to 0% B in

next 20 minutes at a flow rate of 0.5 mL/minute. 20 L of standard SEB and

SEB extract from 2 mg/mL sample stock was injected separately and

respective retention time was monitored at 280 nm.

SEB extract was further confirmed by isoelectric focusing in a broad pI

calibration kit run on a 5% polyacrylamide gel containing pharmalyte with 3-

9.5 pI range pre-casted from Amersham biosciences. Focusing condition was

set at maximum power supply of 1500 V, 50 mA current, coolant temperature

at 16 °C under nitrogen atmosphere. The gel was fixed after focusing for 30

minutes in aqueous solution of 20% trichloroacetic acid followed by

equilibration in 25% methanol and 5% acetic acid for 30 minutes. Gel was

stained with 0.1% Coomassie blue G-250 solution in 25% methanol and 5%

acetic acid for 30 minutes.

3.2.5. Immunization profile

28 weeks old white leghorn hens were used to generate antibodies

against SEB. Single comb white leghorn layers were purchased from local

hatchery and individually maintained in a steel cage having free access to

water and poultry feed. Birds were observed for stable egg laying for 4 weeks

before immunization. Two hens were immunized intramuscularly to 4 sites in

the breast muscle with 1 mL of purified SEB (0.25 g) emulsified with an

equal volume of FCA (Table 3.2).


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Table 3.2 Immunization protocol.

1. Adjuvant used FCA for primary immunization

FICA for booster immunizations

2. Antigen dose As described in Table 3.3

3. Injection site Intramuscular

4. Injection volume 0.5 mL of distilled water containing antigen

emulsified with 0.5 mL of adjuvant (1 mL in total)

5. Injection frequency 10 times

6. Vaccination interval 15 days

7. Usage of chicken 210 days

Hens were administered subsequent booster injections of SEB with

FICA at 2, 4, 6, 8, 10, 12, 14 and 16 weeks after first immunization using the

same route. Hen A (Profile 1) was immunized with 0.5 g of SEB during each

booster injection and hen B (Profile 2) was immunized with 0.5, 1, 2, 5, 20, 50,

100, 250 and 500 g of SEB during respective boosters (Table 3.3). Eggs

were collected every day separately for both hens after first immunization till

26th week and stored at 4 °C for further analysis. Egg yolks were pooled

between each immunization separately for both hens as analysis samples.


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Table 3.3 Immunization schedules of SEB to hens.

Immunizations Schedule

(days)

Antigen immunized

(in µg)

Profile 1 Profile 2

Primary dose (FCA) 1 0.25 0.25

I booster (FICA) 15 0.5 0.5

II booster 30 0.5 1

III booster 45 0.5 2

IV booster 60 0.5 5

V booster 75 0.5 20

VI booster 90 0.5 50

VII booster 105 0.5 100

VIII booster 120 0.5 250

IX booster 135 0.5 500

Total

4.75 928.75

3.2.6. Extraction and purification of IgY from egg

Anti-SEB IgY antibody was extracted and purified according to water

dilution method (Akita and Nakai, 1993). Egg yolks were separated, washed

with double distilled water and rolled on a paper towel to remove adhering egg

white. Pooled yolks were processed by diluting 9 times with acidified water

(pH 5.3-5.5) and incubated for 30 minutes at room temperature. Diluted yolk

solution was centrifuged at 10,000 RPM and 4 °C for 15 minutes to separate


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fatty materials and supernatant was collected as water soluble fraction (WSF).

Yolk supernatant (WSF) was subjected to 19% sodium sulfate precipitation

and centrifuged as above after 2 hrs of incubation at RT. Supernatant was

discarded and pellet collected was re-dissolved in PBS (50 mM, pH 7.4) to the

original pooled yolk volume. This IgY extract was again subjected to 14%

sodium sulfate precipitation and centrifuged as above after 2 hr of incubation

at room temperature. Pellet collected was re-dissolved in minimal volume of

PBS and desalted by dialysis against PBS with four changes for 24 hr. The

IgY solution was lyophilized and preserved at -20 °C till further use. Purity of

IgY was confirmed by SDS-PAGE as described previously (Section 2.2.6,

Chapter 2).

Fig. 3.2 Schematic representation of IgY extraction from egg yolk.


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3.2.7. Indirect non-competitive ELISA and checkerboard analysis to

determine antibody titer

IgY extracted after each booster immunization was checked for titer by

checkerboard analysis by non-competitive ELISA using the coated antigen

format (Sreenath and Venkatesh, 2007). SEB was diluted and coated on to

flat bottom polystyrene microtiter plates with coating buffer (0.1 M carbonate

bicarbonate buffer, pH 9.4) overnight. Microtiter plates were incubated at 4 °C

overnight with 100 L of SEB/well. Coating antigen concentration was varied

from 2 g to 0.0625 g in different rows (2, 1, 0.5, 0.25, 0.125 and 0.0625) per

well. Plates were washed with PBS-T (PBS with 0.05% Tween-20) twice and

once with PBS. Plates were blocked with 1% BSA (200 L per well) and

incubated for 2 h at 37 °C followed by washing as above. Anti-SEB antibody

dilutions made in PBS (50 mM, pH 7.4) such as 1:2K, 1:4K, 1:8K, 1:16K,

1:32K, 1:64K, 1:128K, 1:256K were loaded sequentially to different columns

(100 L per well) and incubated for 1 hr at 37 °C followed by washing as

above. 100 L of rabbit anti-chicken IgY-HRP secondary antibody (1:12K)

was loaded to each well and incubated for 45 minutes at 37 °C followed by

washing as above. TMB substrate for HRP was prepared according to Wang

et al. (2011). Substrate solution (pH 5.0) was prepared by dissolving 410 mg

of sodium acetate, 125 mg of -Cyclodextrin, 125 mg of citric acid and 7.5 mg

of U-H2O2 in 50 mL of distilled water. Finally substrate solution was mixed with

1% TMB prepared in DMSO in the ratio 97:3 respectively just before use.

Freshly prepared TMB substrate for HRP was loaded onto each well (50 L

per well) and incubated for 15-20 minutes at room temperature. The reaction


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was stopped by adding 1.25 mM H2SO4 (50 L per well). The plate was read

on a tunable microplate reader at 450 nm.

3.2.8. Estimation of affinity constant

Affinity constant of anti-SEB antibodies extracted from different peak

titers were estimated by using Scatchard plot (Scatchard, 1949; Larrivee et

al., 2000; Rathanaswami et al., 2008) separately. Initially, the percentage of

antigen bound in indirect non-competitive ELISA of all peak titers at optimized

conditions were calculated separately. A graph was plotted between the ratios

of bound antigen to unbound antigen versus the molar concentrations of SEB

bound to the antibody. The slope was calculated separately for all peak titers.

3.2.9. Disposal of SEB toxin, S. aureus and toxin immunized

experimental hens

SEB was treated with 0.25 N NaOH and 2.5% NaOCl for 20 minutes

followed by autoclaving for 20 minutes at 15 lbs pressure and 121 °C.

S. aureus cultures were autoclaved at 15 lbs pressure and 121 °C for 20

minutes before disposing. SEB immunized hens were disposed according to

institutional animal ethical policy. Immunized hens were anaesthetized and

incinerated in an incineration chamber.


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3.3. RESULTS AND DISCUSSION

3.3.1. Extraction and purification of SEB

SEB is a 28.4 kDa monomeric extracellular protein made up of 239

amino acids secreted from S. aureus (Johns and Khan, 1988). S. aureus

strain ATCC 14458 is well known for the production of SEB (Stark and

Middaugh, 1969; Altenbern, 1977; Kamboj et al., 2006; Rajkovic et al., 2006;

Rall et al., 2008). It has also been reported that strain ATCC 14458 possess

gene responsible for the production of SEB only and not for other class of SEs

(Rajkovic et al., 2006). Therefore, strain ATCC 14458 was selected for this

work specifically for the production of SEB. Generally, growth conditions

significantly influence the production of any secondary metabolite. In this

direction, composition of nutrients in the media, temperature, pH of the

medium, aeration and incubation period have to be critically monitored to

obtain a better yield. Dietrich et al. (1972) have studied the influence of

shaking speed, flask size, ratios of media volume to flask volume,

temperature and pH on the production of SEB in detail. They have reported a

highest yield of 200 g/mL of SEB in ATCC 14458 strain at 350 RPM

(revolution per minute) after 24 h of incubation at 37 °C. Hence, similar

growth conditions were adopted in this study for the production of SEB from

S. aureus (ATCC 14458).

Ammonium sulfate saturation (80%) of cell free extract precipitated

SEB protein which was confirmed by SDS-PAGE in comparison with standard

SEB (Fig. 3.3). Size exclusion chromatography with Sephadex G-75 column

further purified the protein from crude extract (80% ammonium sulfate


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147

precipitate) based on its mobility against molecular weight under controlled

elution with PBS.

Fig. 3.3 SDS-PAGE showing ammonium sulfate precipitates of cell free

extract. Lane M is standard SEB and lane 80% is SEB extract at

80% ammonium sulfate saturation.

Reverse phase HPLC confirmed the purity based on retention time for

extracted SEB and its standard. Purified toxin showed a single major peak at

50th minute in HPLC analysis (Fig. 3.4) that is comparable to retention time of

standard SEB (Fig. 3.5).


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Fig. 3.4 RP-HPLC of SEB extracted and purified.

Fig. 3.5 RP-HPLC of SEB standard.


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149

Isoelectric point of the purified toxin was at 8.7 as observed under

isoelectric focusing (Fig. 3.6) that was in accordance with previous reports

(Jones and Khan, 1986). The yield of SEB was ~130 g/mL from 24 hrs

incubated culture broth, which was calculated as a function of total protein

concentration after column purification.

Fig. 3.6 Isoelectric focusing of SEB. A single band at 8.7 pI confirmed its

purity.

3.3.2. Immunization of SEB

Immunization profiles employed were modified method of Shinagawa

et al. (1974) to ensure optimal amount of toxin required for high affinity

antibody generation in avian system. Generally high affinity antibody

generation with better titer relies upon various factors such as the dose and

molecular weight of antigen, the nature of the antigen, the immunization

frequency and intervals, type of adjuvant used and route of antigen


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administration (Schade et al., 1996; Chalghoumi et al., 2009). Both high and

too low antigen doses may results in immune suppression, sensitization and

tolerance (Hanly et al., 1995; Chalghoumi et al., 2009). SEB being protein of

28.4 kDa is a most efficient immunogen due to polymorphism of its structure.

However, reports on production of antibodies against bacterial enterotoxins in

avian system are very much limited. Hence, SEB dose required for

immunization in this study was fixed based on the general recommendations

for protein antigens. Schade et al. (1996) in their report and recommendations

of ECVAM (European Center for the Validation of Alternative Methods)

workshop-21 have recommended that antigen dose can be 10 ng to 1 mg for

immunization in avian system. Therefore, 0.5 g of SEB was administered as

an antigen dose initially, as SEB is a toxic protein.

The titer of antibody response also depends on the type of adjuvant

used for immunization. Out of many adjuvants known, Freund’s complete

adjuvant (FCA) remains the most effective adjuvant for antibodies production

in laboratory animals. Gassmann et al. (1990) and Svendsen et al. (1996)

suggested that chickens show higher resistance to tissue damaging potency

of FCA than rabbits. However, FCA found to have local tissue damaging

potency due to inflammation at the site of injection in mammals (Chalghoumi

et al., 2008). Therefore, to avoid this local tissue damaging reactions the

Freund’s incomplete adjuvant (FICA) has been introduced as the most

effective substitute. However, as FICA is less efficient than FCA, researchers

preferred the use of a combination of the two adjuvants (Kapoor et al., 2000;

Li et al., 2006; Chalghoumi et al., 2008). Based on the above reports, in this


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151

study, a dual adjuvant system comprising FCA for primary immunization and

FICA for subsequent booster immunizations was used for antibody generation

in hens.

3.3.3. Generation and characterization of anti-SEB antibody

Purified SEB was immunized to generate polyclonal antibody in white

leghorn hen. A gradual increase in the protein (IgY) concentration from 15.32

mg IgY in preimmune egg to 51.1 mg IgY in hyperimmune egg after booster

immunizations indicated successful anti-SEB antibody generation in white

leghorn hen (Fig. 3.7). Peak titers were observed for II, IV and VIII boosters in

profile 1 whereas in profile 2 peak titers were observed for IV and VII

boosters.

10

15

20

25

30

35

40

45

50

55

0 30 60 90 120 150 180 210

IgY

co

nc

en

tra

tio

n i

n m

g/e

gg

Days

Profile 1

Profile 2

II

IV

VIII

IV

VII

Fig. 3.7 Antibody response shown by hens against SEB immunized with

profile 1 and profile 2. Maximum IgY yield was obtained with profile 1

immunization after VIII booster immunization.


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152

SDS-PAGE confirmed purity of IgY extract as electrophoretic analysis

exhibited a single protein band under non-reducing condition having

molecular weight of ~180 kDa. Under reducing condition IgY split into two

bands of ~65-67 kDa for heavy chain and 25 kDa for light chain further

confirming its generation and purity after purification steps (Fig. 3.8).

a

Fig. 3.8 SDS-PAGE of IgY confirming its extraction and purity. Lane M is

broad range molecular weight marker, lane 1 is IgY under reducing

condition and lane 2 is IgY under non-reducing condition.

Water dilution method was employed to extract IgY from egg yolk as

IgY extraction requires the removal of lipoprotein and purification from other

livetins in the WSF. It was reported by Akita and Nakai, (1993) that water

dilution method yields highest IgY (91%) with purity (31%). Thus, extraction of

IgY from egg yolks by water dilution method resulted in highly purified

antibodies with better yield without any IgA or IgM contamination. This is


Chapter 3

153

because transfer of both IgA and IgM from oviduct into the egg white together

with other proteins made its possibility almost impossible in WSF. On the

other hand selective transfer of serum IgY into the oocyte in the ovarian

follicle is receptor mediated and therefore they were reportedly present in egg

yolk in large quantities (Chalghoumi et al., 2009).

Antibody response graph indicated the effect of SEB concentration and

immunization profile on specific antibody generation. The preimmune and

hyperimmune eggs collected unveiled significant difference in their IgY

concentration. The yield of anti-SEB antibody was more and better with

immunization profile 1 where hen A was immunized constantly with 0.5 g of

SEB compared to immunization profile 2 (Table 3.3) where antigen

concentration was gradually increased with each booster immunization. A

highest yield of 51.1 mg IgY/egg was obtained with profile 1 after VIII booster

whereas profile 2 yielded only 34.31 mg IgY/egg after VII booster (Fig. 3.7).

The yield of IgY was increased from 15.32 mg in preimmune egg to 51.1 mg

in hyperimmune egg in present study. Almost similar yield for IgY were

obtained by Meenatchisundaram et al. (2011) and Sankareswaran et al.

(2011) for immunized Streptococcus mitis and Canine parvovirus respectively

in white leghorn chicken. However, SEB concentration used for immunization

in both profiles have not shown any obvious influence on egg laying capacity

or the bird’s health (physical observations only) after immunization. It was

reported that chickens tend to be more resistant against plant and microbial

toxins than other species which could be the reason behind this (Pauly et al.,

2009).


Chapter 3

154

3.3.4. Indirect non-competitive ELISA and affinity constant estimation

Antibody titer is defined as the reciprocal of the highest dilution that

gave a positive value in ELISA. Checkerboard analysis carried out for IgY

extracts with highest peak titer determined the sensitivity of these antibodies.

There was a variation in the sensitivity of anti-SEB antibody accordingly with

booster immunizations and also with immunization profile. The titration curves

show the sensitivity of anti-SEB antibodies from various boosters at different

dilutions as given below (Table 3.4, Fig. 3.9).

Table 3.4 Optimized dilutions of anti-SEB antibodies by checkerboard

analysis.

Immunization

boosters

Anti-SEB antibody

dilutions

(1 mg/mL stock)

Limit of detection

(LoD)

(in ng of SEB)

1. P1- VIII (a) 1:64,000 8.74

2. P1-VIII (b) 1:32,000 12.94

3. P1-IV 1:16,000 21.2

4. P2-VII 1:32,000 11.72

5. P1-II 1:8,000 9.12

6. P2-IV 1:4,000 42.07


Chapter 3

155

0

0.5

1

1.5

2

2.5

1 10 100 1000

Ab

so

rba

nce

@ 4

50 n

m

SEB concentration in ng

P1-VIII (a)

P1-VIII (b)

P1-IV

P2-VII

P1-II

P2-IV

Fig. 3.9 Comparison of binding curves of antibodies from different boosters

analyzed by checkerboard analysis.

Highest sensitivity was observed for anti-SEB antibody from VIII

booster of immunization schedule profile 1 between 1:32,000 and 1:64,000

dilutions (Table 3.4). Subsequently, the antibody dilution at 1:64,000 were

chosen as an optimum titer for further investigation. Titer peaks were almost

similar for both hens and reached up to 1:125,000 with considerable

differentiating ability against different concentrations of SEB as revealed by

indirect non-competitive ELISA.

Affinity constant was estimated for all peak titers. Affinity is the strength

with which an antibody binds to an epitope on an antigen molecule against

which it has been raised. In order to quantify the interactions between antigen

and antibody molecules, it is essential to determine the association constant

or affinity constant (Ka). Scatchard plot (Scatchard, 1949) is one of the


Chapter 3

156

several methods known for calculating affinity constant, which is based on

linearization procedure. Scatchard plot is the ratio of concentrations of

bound antigen to free antigen versus the bound antigen concentration. The

plot yields a straight line where inverse of the slope of the line is the affinity

constant [Slope = -(1/Ka)] for antigen binding. Affinity constant (Ka)

determines the strength of non-covalent interaction between antigenic

determinant (epitope) and variable region of both the heavy and light chains

(paratope) in an immunocomplex. Therefore, greater the Ka, stronger will be

the affinity between antigen and antibody. Affinity constant assessed by

Scatchard plot for titer peaks of both immunization profiles showed minor

variations (Fig. 3.10).

y = -55.04x + 14.69

y = -59.27x + 16.11

y = -75.82x + 20.04

y = -85.78x + 22.39

-10

-5

0

5

10

15

20

25

-0.05 0.05 0.15 0.25 0.35

Bo

un

d / F

ree

SEB concentration in pM

P1-VIII

P1-IV

P2-VII

P1-II

Fig. 3.10 Scatchard plot for anti-SEB antibody titer peaks of both

immunization profiles for determining affinity constant.


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157

Affinity constant for VIII booster titer peak of profile 1 that was having highest

yield was 1.81 x 1010 M-1 and for VII booster titer peak of profile 2 was

1.31 x 1010 M-1 (Table 3.5). This further confirms the successful generation of

anti-SEB antibody with high affinity towards native SEB.

Table 3.5 Affinity constant of anti-SEB antibodies against booster.

Si No. Booster name Ka

1. P1: II 1.16 x 1010 M-1

2. P1: IV 1.68 x 1010 M-1

3. P1: VIII 1.81 x1010 M-1

4. P2: VII 1.31 x 1010 M-1

3.4. CONCLUSIONS

In summary, avian system employed here successfully demonstrated

its efficacy towards polyclonal antibody generation against a bacterial toxin

with high affinity and better yield. SEB was successfully extracted and purified

at 80% ammonium sulfate saturation and Sephadex G-75 size exclusion

chromatography respectively. HPLC analysis confirmed the purity of extracted

SEB where a single major peak was observed at 50th minute comparable to

standard SEB. Isoelectric point of the purified toxin was at 8.7 that were in

accordance with previous reports. The yield of SEB was ~130 g/mL

calculated as a function of total protein concentration after column purification.

Immunization of 0.5 µg of SEB for multiple boosters successfully generated

anti-SEB antibodies in white leghorn hens. Indirect non-competitive ELISA


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158

unveiled the antibody yield, titer and affinity constant against each peak titer.

VIII booster gave the highest yield of 51.1 mg IgY per egg with affinity

constant of 1.81 x 1010 M-1. Titer reached up to 1:125,000 dilutions with

considerable differentiating ability against different concentrations of SEB.

Highest sensitivity was observed for anti-SEB antibody from VIII booster of

immunization schedule profile 1 at 1:64,000 dilutions. The yield of IgY

obtained was also considerably higher than mammalian system suggesting

avian model could be an alternative system for polyclonal antibody generation

with cost effectiveness. Though raising antibodies in chicken is well known

since decades, reports on production of antibodies against bacterial

enterotoxins in avian system are very much limited. As per our knowledge is

considered, protocols and methodology for generating IgY antibodies against

SEB is not available. Keeping advantages of avian antibodies (IgY) over

mammalian antibodies (IgG) in mind, an attempt was made to generate

antibodies in avian system against SEB in this study. In this direction, present

chapter provides a thorough knowledge over generation of IgY antibodies

against SEB, its extraction, purification, affinity determination and overall

characterization.


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159

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