chapter 3 - shodhganga : a reservoir of indian theses...
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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
Avian antibody (IgY) generation against staphylococcal enterotoxin B and its characterization
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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
Avian antibody (IgY) generation against staphylococcal enterotoxin B and its characterization
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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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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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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;
Avian antibody (IgY) generation against staphylococcal enterotoxin B and its characterization
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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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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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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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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
Avian antibody (IgY) generation against staphylococcal enterotoxin B and its characterization
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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
Avian antibody (IgY) generation against staphylococcal enterotoxin B and its characterization
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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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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).
Avian antibody (IgY) generation against staphylococcal enterotoxin B and its characterization
Chapter 3
148
Fig. 3.4 RP-HPLC of SEB extracted and purified.
Fig. 3.5 RP-HPLC of SEB standard.
Avian antibody (IgY) generation against staphylococcal enterotoxin B and its characterization
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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
Avian antibody (IgY) generation against staphylococcal enterotoxin B and its characterization
Chapter 3
150
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
Avian antibody (IgY) generation against staphylococcal enterotoxin B and its characterization
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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.
Avian antibody (IgY) generation against staphylococcal enterotoxin B and its characterization
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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
Avian antibody (IgY) generation against staphylococcal enterotoxin B and its characterization
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).
Avian antibody (IgY) generation against staphylococcal enterotoxin B and its characterization
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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
Avian antibody (IgY) generation against staphylococcal enterotoxin B and its characterization
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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
Avian antibody (IgY) generation against staphylococcal enterotoxin B and its characterization
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.
Avian antibody (IgY) generation against staphylococcal enterotoxin B and its characterization
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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
Avian antibody (IgY) generation against staphylococcal enterotoxin B and its characterization
Chapter 3
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.
Avian antibody (IgY) generation against staphylococcal enterotoxin B and its characterization
Chapter 3
159
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