Materials and Methods - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/17028/7/07_materials...

Materials and Methods


REAGENTS

Sephacryl S-400 was from Pharmacia Biotech, Uppsala, Sweden; endo-(3-galactosidase

was obtained from Seikagaku Kogyo Co., Japan; bicinchoninic acid was from Pierce

Chemical Co., Rockford, USA. Hydroxylapatite, Tris, glycine, acrylamide, N' N'

methylene bisacrylamide, sodium dodecyl sulfate (SDS), N', N', N', N'-tetramethylethylene

diamine (TEMED), ammonium persulfate, 2-mercaptoethanol and molecular weight

standards were obtained from Sigma Chemical Co., St. Louis, USA. Reagents for peptide

synthesis viz., piperidine, dimethyl formamide (DMF), diisopropylethylamine (DIPEA),

diisopropylcarbodiimide (DIPCDI), 1-hydroxybenzotriazole (HOBt), tritluoroacetic acid

(TF A), tritluoromethanesulfonic acid (TFMSA), anisole, thioanisole, ethane dithiol (EDT),

trimethylamine, were also obtained from Sigma Chemical Co., St. Louis, USA. Resin and .

aa for Fmoc solid phase synthesis were obtained from Millipore, Bedford, USA, while aa

for multipin peptide synthesis were procured from Chiron Technologies, Clayton,

Australia. Resin as well as aa for tBoc synthesis were obtained from NovaBiochem,

Laufelfingen, Switzerland. Reagents for enzyme immunoassays viz., bovine serum

albumin (BSA), orthophenylene diamine (OPD) and diammonium 2,2'-azino-bis[3-

ethylbenz-thiazoline-6-sulfonate] (ABTS) was procured from Sigma Chemical Co., St.

Louis, USA while Tween-20 was obtained from Amresco, Solon, U.S.A. Goat anti-mouse

IgG-HRPO for screening of antibody to pin bound peptides as well as in ELISA was from

BioRad Laboratories, California, USA. Reagents used in conjugation viz., DT was from

Serum Institute, Pune, India while glutaraldehyde and L-lysine were procured from Sigma

Chemical Co., St. Louis, USA. Reagents used in immunization viz., complete Freund's

adjuvant (CF A) and incomplete Freund's adjuvant (IF A) were obtained from Difco

Laboratories, Detroit, USA while 2, 6, 10, 15, 19, 23-hexamethyl-2, 6, 10, 14, 18, 22-

tetracosa-hexaene (Squalene) and mannide monooleate (Arlacei'A') from Sigma Chemical

Co., St. Louis, USA. Secondary revealing antibodies viz., goat anti-rabbit IgG-HRPO

(Pierce Chemical Co., Rockford, USA), goat anti-monkey IgG (whole molecule)-HRPO

26


•o (Sigma Chern. Co., St. Louis, USA), anti-rabbit-FITC (Reagent Bank, Nil, New

ti) and anti-mouse-FITC (Pierce Chemical Co., Rockford, USA) were also procured.

~ents used in the estimation of steroid hormone such as gelatin, charcoal, dextran, 3H-.

~esterone and anti-progesterone antibody were provided by the WHO Matched

gent Assay Programme while diphenoxazole (PPO), 1-4 bis (5-phenyl-2-oxazolyl)

~ene (PO POP), and mercury-[( o-carboxyphenyl)thio ]ethyl sodium salt (Thimerosal)

e obtained from Sigma Chemical Co., St. Louis, USA.

27


MATERIALS AND METHODS

l Porcine Zona Pellucida Antigens

The porcine ZP glycoproteins were prepared as described (Yurewicz et al., 1987). Briefly,

porcine ovaries were collected from an abattoir and stored at -20°C until further

processing. Solubilized isolated zona pellucida (SIZP) was treated with SDS, pZPl

removed from pZP3a and pZP3 0 by chromatography on Sephacryl S-400, and

hydroxyapatite resins. Following removal of SDS and renaturation, the mixture of pZP3a

and pZP30 was digested with endo-0-galactosidase (Escherichia freundii). The pZP3a

and pZP3 0 glycoproteins were separated by reverse phase HPLC, lyophilized, redissolved

in water and stored at -20°C. Protein concentration was quantitated with bicinchoninic

acid as per the manufacturers instructions.

ll Monoclonal Antibodies

A panel of MAbs were raised earlier by immunizing BALB/c mice with either pZP3 or

pZP3 0 and following fusion of splenocytes with mouse myeloma cells, positive hybrid

cells were cloned to obtain cell lines secreting antibodies (Bagavant et al., 1993; Gupta

and Gupta, 1994). For the purpose of our experiments, hybrid cell clones which were

kept frozen in liquid nitrogen were revived and maintained using standard tissue culture

techniques and culture supernatants were harvested. Hybrid cell clones were also grown as

ascites in the peritoneal cavity of Pristane (Sigma Chemical Co., St. Louis, USA) primed

BALB/c mice. Ascites fluid tapped from the peritoneal cavity was made cell free by

centrifugation at 800 x g for 15 min at 4°C. Subsequently, immunoglobulins were purified

by 40% ammonium sulfate precipitation followed by purification on DEAE A-50 column.

Purified antibodies were concentrated using Amicon membrane (PM-30), followed by

dialysis against PBS and stored at -20°C until required.

28


Antibodies were shown to recognize eight epitopic domains (Gupta et al., 1993; Gupta

and Gupta, 1994). A listing of some these antibodies used in the present study along with

a few characteristics and arbitrary binding domains based on competitive inhibition assays

(Gupta et al., 1993) is given below:

MAb Isotype Bioefficacy Empirical Overlap Domain

451 IgG1 No DVIII

454 IgG2b Yes DVI DV

455 IgG2b Yes DVI DV

462 lgG1 No DVII

470 lgG1 No DVII

467 IgG1 Yes DV DVI

30 IgG1 Yes DV DVI

111. Epitope Mapping For MAbs By Mimotope Strategy

In order to precisely map the epitopes of the above MAbs, we have used the multipin

peptide synthesis approach.

1. Multipin Peptide Synthesis

The synthesis of overlapping 12-mer peptides with 6 aa overlap corresponding to the

pZP3~ precursor protein sequence (424 aa, excluding 1-22 aa signal sequence), peptides

having an increment of one aa at a time and glycine substituted analogues was

accomplished using 9-fluorenylmethyloxycarbonyl (F-moc )-f3-alanine group prederivatized

polypropylene plastic pins as solid-phase support (Geysen et al., 1987). Peptides were

synthesized in the carboxy- to amino- terminal direction as per the manufacturers

instructions and the protocol was as follows:

A. Fmoc Deprotection and Washing

1. The block of pins was placed in a bath containing 20% (v/v) piperidine in dimethyl

formamide (DMF) for 20 min. Care was taken to ensure that the crowns were fully

·Immersed in the bath.

29


2. The block was removed from the bath, excess liquid was shaken off and the pins were

washed in a DMF bath for 2 min at room temperature.

3. The excess DMF was shaken off and the block was immersed completely in a bath of

methanol for 2 min at room temperature.

4. The block was then placed in a second methanol bath to fully cover the crowns and

washed for 2 min. This step was repeated twice using fresh methanol each time.

5. The block was removed and air-dried in a fume-hood for 20 min.

All steps were carried out at room temperature.

B. Coupling of N-a-Fmoc Protected Amino Acids

Fmoc aa (60 mM) were weighed into glass tubes and dissolved in purified DMF

containing 96 mM 1-hydroxybenzotriazole (HOBt). The aa were activated with 240 mM

diisopropylcarbodiimide (DIPCDI) dissolved in purified DMF. The solution was

thoroughly mixed and activated aa were very carefully dispensed into the relevant reaction

tray wells and the block of pins was placed in the tray and sealed. Coupling reaction was

allowed to proceed for 4 hours at room temperature. For overnight coupling reaction, the

aa were used at a concentration of 30 mM, and HOBt and DIPCDI at 48 and 120 mM

respectively.

C. Post-Coupling Processing

i) Acetylation

Following coupling, the block of pins was placed in a methanol bath and washed

thoroughly with agitation for 5 min. Before the start of the next coupling cycle, the aa

attached to the pins were deprotected as mentioned above. Following the coupling of the

final aa, the block was washed as usual and the N-terminal group of each peptide was

acetylated in order to remove the charge associated with a free terminal amino group. For

acetylation, an acetylation mixture consisting of 150 ~LI of DMF: acetic anhydride:

triethylamine in a ratio of 50:5: I (v/v/v) were added to the wells of the reaction tray and

the block of pins was placed in the tray. The reaction was allowed to proceed for 90 min

30


at room temperature. Following acetylation, the block of pins was washed once m a

methanol bath for 15 min followed by air -drying for at least 15 min. The peptides were

then processed for side-chain deprotection.

ii) Side Chain Deprotection

Pin attached peptides were side chain deprotected by using a mixture of trifluoroacetic

acid (TFA): ethanedithiol (EDT): anisole in a ratio of 38:1:1 (v/v/v) for 2.5 hrs at room

temperature. On removal from the deprotection mixture, the block was immediately

immersed in methanol for 10 min. Excess methanol was removed and the block was next

soaked in methanol/distilled water (1: 1 v/v) with 0.5% glacial acetic acid for 2 min. The

block was then allowed to air dry overnight.

2. Enzyme Linked Immunosorbent Assay (ELISA)

For ELISA, peptide containing pins were first treated with a pre-coat buffer containing

2% (v/v) bovine serum albumin (BSA, Fraction V), 0.1% (v/v) Tween-20

(polyoxyethylene-20-sorbitan monolaureate), 0.1% (w/v) sodium azide and 0.01 M

phosphate buffered saline (PBS) pH 7.2, in order to reduce non-specific binding and

obtain a better signal-to-background ratio. Buffer (200 ~Ll/well) was dispensed into wells

of a microtitration plate and the pins were incubated for 60 min at room temperature with

agitation. Following pre-coating, excess buffer was flicked off and the pins were placed in

a microtitration plate containing 175 ~Ll of suitably diluted primary antibody (ascites fluid

1: 1 00; culture soup 1 :20) in pre-coat buffer and incubated at 4°C overnight on a shaker

table set at 100 RPM.

Pins were removed from the primary antibody solution and washed (X4) in a bath of

0.01M PBS (pH 7.2) for 10 min each at room temperature. This was followed by the

conjugate reaction. The washed pins were incubated for 60 min at room temperature with

agitation in the presence of 175 ~tl!well of goat anti-mouse immunoglobulin (IgG)

conjugated to horseradish peroxidase (HRPO) added at a dilution of 1: 1000 in a conjugate

diluent (1% normal goat serum, 0.1% Tween-20, 0.1% fetal calf serum, 0.01M PBS pH

31


7.2). Following the conjugate reaction, pins were washed (X4) in 0.01M PBS (pH 7.2)

for 10 min each. After washing, pins were incubated with substrate buffer ( 17.8 gm

Na2HP04.2H20; 16.8 gm citric acid monohydrate, pH 4.0) containing 0.5 mg/ml of

diammonium 2,2'-azino-bis[3-ethylbenz-thiazoline-6-sulfonate] (ABTS) and 0.06% (w/v)

hydrogen peroxide (H202) for 45 min at room temperature on a shaking platform.

Enzyme action was stopped by removing the pins from the substrate solution and intensity

of the colour at 405 (-) 490 nm was measured using a microtitre plate reader (Molecular

Devices Corp., California, USA).

3. Disruption of Bound Antibody

The block of pins was placed in a bath containing disruption buffer [0.1 M PBS containing

1% (w/v) sodium dodecyl sulfate (SDS), 2-mercaptoethanol (0.1% v/v), pH 7.2] at 60°C

and subjected to sonication for 10 min at approximately 30W energy output for disruption ..

of bound antibody (Branson Sonifier 450, Connecticut, USA). The temperature was

allowed to equilibrate between 55-65°C since higher temperatures damage the peptides

and lower temperatures may lead to ineffective antibody removal. Following this, the

block was rinsed twice in distilled water pre-heated to 60°C for atleast 30 min. Excess

water was removed and the blocks were immersed in hot methanol (60°C) for atleast 15

sec. The block was air-dried for 15 min and reused for the next screening. Removal of the

antibody was ascertained by repeating the conjugate reaction on the regenerated peptides.

IV. Synthesis of Peptides Corresponding to Epitopes Recognized by Monoclonal

Antibodies

A. Peptide Synthesis

i) General Scheme of Synthesis

In solid phase peptide synthesis, the deprotected amino terminus of a support bound aa is

reacted with the activated carboxyl terminus ofthe incoming aa, resulting in the formation

of a peptide bond. The process of deprotecting the amino terminus, activation of the

32


carboxyl terminus and performing the coupling reaction of the next aa is repeated until the

peptide synthesis is completed. The peptide chains are assembled from the COOH

terminus to the NH2-terminus. Cleavage of the support bound peptide then allows for

isolation of the product.

DEPROTECTION

(Removal of a-amino protecting group)

.!,

ACTIVATION

(incoming amino acid)

.!,

COUPLING

(to growing amino acid chain)

.!,

SIDE CHATN DEPROTECTION

(removal offunctional group protections)

.!,

CLEAVAGE

(from resin)

.!,

POST CLEAVAGE WORK-UP

(Lyophilization, HPLC, aa analysis)

Peptides corresponding to pZP3f3 viz., Pi, QPVWQDEGQRLRPSK (residues 23-37); Pii,

FSEEKL VFSLRLMEENC (residues 164-179 with an additional Cys at the C-terminus );

Piv, PVEGPA VICRCC (residues 310-321) were synthesized by standard 9-

fluorenylmethyloxycarbonyl (Fmoc) chemistry on an automated peptide synthesizer using

a PepSynKB resm and Fmoc protected aa, while peptide Piii,

33


ASSAFKAPRPGPETLQFTC (residues 246-263 with an additional Cys at the C-terminus)

was synthesized by tert-butyloxycarbonyl (tBoc) chemistry. Peptides corresponding to

bonnet monkey ZP3 viz., PI, KQPFWLLQGGASRAETSVQPVLVE (residues 23-45

with an additional Kat theN-terminus); P2, FSEEKL VFSLRLMEENC (residues 165-180

with an additional Cys at the C-terminus and T171 replaced by V); P3,

CSFSKSSNSWFPVEGPADICQCC (residues 300-322) and P4,

KGDCGTPSHSRRQPHVVSQWSRSA (residues 324-347) were synthesized by standard

Fmoc chemistry.

The list of peptides synthesized is summarized below:

p .d ept1 e p rot em R .d es1 ues s equence Pi Porcine ZP3 ~ 23-35 aa QPVWQDEGQRLRPSK

Pii -do- 164-179 aa FSEEKL VFSLRLMEEN(C) • Piii -do- 246-263 aa ASSAFKAPRPGPETLQFT(C) * Piv -do- 310-321aa PYEGPA VICRCC PI Bonnet ZP3 23-45 aa (K) • QPFWLLQGGASRAETSVQPVL VE P2 -do- 165-180 aa FSEEKL VFSLRLMEEN(C) * P3 -do- 300-322 aa CSFSKSSNSWFPVEGPADICQCC

P4 -do- 324-347 aa KGDCGTPSHSRRQPHVVSQWSRSA

* aa within parenthesis does not correspond to the native sequence but was added for

conjugation with carrier protein.

ii) Fmoc synthesis

Fmoc synthesis was accomplished usmg a Milligen 9050 Peptide Synthesizer and a

PepSynKB (polyamide kieselguhr composite support) resin with a substitution of 0.2

meq/g of resin to yield peptide amides. These supports consist of a polyacrylamide gel

held within the pores of an inert macro porous kieselguhr (diatomaceous earth) matrix. The

rigid framework of the kieselguhr particles provide a non compressible support that retains

the open channels necessary for rapid diffusion of reactants throughout the gel matrix.

34


A list of amino acids used in Fmoc synthesis is given below:

Amino Acid

Alanine Arginine Asparagine Aspartic acid Cysteine Glutamine Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine

Side-Chain Protection n/u Mtr/Pmc n/u OtBu Trt n/u OtBu n/u tBoc n/u n/u tBoc n/u n/u n/u tBu tBu n/u tBu n/u

Unprotected M. Wt. 89.1 174.2 132.1 133.1 121.2 146.0 147.1 75.1 155.2 131.2 131.2 146.2 149.2 165.2 115.1 105.1 119.1 204.2 181.2 117.1

Active Ester M. Wt. (OPfp) 477.3 774.7 520.4 577.5 751.8 534.4 751.7 463.4 634.6 519.5 519.5 634.6 537.5 553.5 503.4 528.6 (ODhBt) 542.6 (ODhBt) 592.5 625.5 505.4

Peptides were synthesized using orthopentafluorophenyl (OPfp) active esters with HOBt

as a catalyst on a 0.2 mM scale using a four fold excess of aa in a reaction column and a

loop size of 10 mi. An on-line spectrophotometer was used to monitor acylation and

deprotection was monitored at 365 nm for release of Fmoc groups from the peptide

chains. The N-terminal Fmoc group on the peptide chain was deprotected usmg a

solution of 20% piperidine in DMF and dried overnight in a dessicator.

iii) tBoc synthesis

Solid phase tBoc synthesis was performed manually in the batch mode following the

protocol outlined by Stewart and Young (1984). For this purpose, methyl

35


benzhydrylamine (MBHA) resin with a substitution of 0.4-0.6 mmole/gm resin, was used

as the solid phase support which is used to yield peptide amides (Matsueda and Stewart,

1981). Resin consists of a polystyrene bead (100-200 mesh, 1% divinyl benzene)

acylated with methylbenzoyl chloride to introduce an additional phenyl ring and yield a

ketone resin. This intermediate is then reductively aminated to yield MBHA resin. The

additional phenyl ring renders the peptide resin linkage labile to HF giving peptide

amides upon cleavage. The aa is attached directly to the amino group of the resin using

normal coupling methods.

Amino acid derivatives used in tBoc synthesis is given below

Amino Acid

Alanine Arginine Asparagine Aspartic acid Cysteine Glutamine Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine

Side-Chain Protection

n/u Mts n/u OBzl pMeOBzl n/u OBzl n/u Tos n/u n/u 2Cl-Z n/u n/u n/u Bzl Bzl Formyl Bzl n/u

Unprotected M. Wt.

89.1 174.2 132.1 133.1 121.2 146.0 147.1 75.1 155.2 131.2 131.2 146.2 149.2 165.2 115.1 105.1 119.1 204.2 181.2 117.1

36

Protected M. Wt.

189.2 456.6 232.2 323.4 341.4 246.3 337.4 175.2 409.5 240.3 249.3 414.9 249.3 265.3 215.3 295.3 309.4 332.4 371.4 217.3

JYlucenuts unu JYtecnous

Peptides were synthesized at a 0.05 mmole scale of synthesis using a 6-fold excess of

tBoc protected aa. Measured quantities of the aa were dissolved in dry distilled DCM and

activated using 1.5 mmole DIPCDI before addition to the resin taken in a reaction vessel

with a sintered disc. For Asn and Gin, DMF was used as the solvent and DIPCDI and

HOBt were added in an equimolar ratio. Synthesis essentially involved pre-swelling of the

resin in DCM followed by addition of activated tBoc protected aa and allowing the

reaction to proceed on an end-to-end shaker. Completion of coupling was monitored by

carefully drawing out few resin beads with a pasteur pipette into a test tube and thorough

washing with ethanol and detection of the presence or absence of free amino groups by

the method ofKaiser et al (1970) as described in the section below. Following a negative

Kaiser reaction indicative of absence of free amino groups and therefore of completeness

of the reaction, the solvents were drained out using suction and the resin was washed

once with DMF and then with DCM. This was followed by addition of the deprotection

mixture consisting of 25% TF A in DCM and allowed to react with shaking for a minute.

The mixture was drained out and fresh deprotection mixture was added and the reaction

was allowed to proceed for 30 min. Deprotection was confirmed by performing the

Kaiser-Ninhydrin test and appearance of a dark blue colour. This was followed by the

addition of a neutralization mixture consisting of 10% diisopropylethylamine (DIPEA) in

DCM for 2 min, draining out the same and addition of a fresh mixture for a further 3 min.

The resin was then washed with DCM (X6) for 1 min each. A further wash with DMF was

incorporated ifDMF was used in the dissolution of the next aa before initiation of the next

residue coupling.

iv) Kaiser-Ninhydrin Test

This is a qualitative test to determine the presence or absence of free amino groups and is

employed as a measure of estimation of completeness of coupling. Essentially, resin beads

were withdrawn into a clean glass tube and were thoroughly washed several times with

ethanol. Two drops each ofReagent A (5 g ninhydrin in 100 ml ethanol), Reagent B (80 g

37

Material.'> and Methods

of liquefied phenol in 20 ml of ethanol) and Reagent C (2 ml of 0. 001 M aqueous solution

of potassium cyanide in 98 ml pyridine) were added to the beads, mixed well and heated to

l 00°C for 4-6 min. Positive test is indicated by blue resin beads. Proline, a secondary

amino acid does not yield a positive reaction.

v) Cleavage Procedure

Following completion of coupling, the resin was dried overnight in a vacuum dessicator

and taken up for simultaneous side chain deprotection and cleavage. Standard cleavage

protocols used were as follows:

For Fmoc peptides, the dried resin was taken up for simultaneous side-chain deprotection

and cleavage. Peptide resin was placed in a round bottomed flask and thioanisole ( 5%,

v/v), anisole (2%, v/v) and EDT (3%, v/v) used as scavengers were added to the peptide

resin and stirred at room temperature for l 0 min. The flask was kept in an ice bath and

TF A (90%, v/v) was added with stirring. The flask was removed from the ice bath,

allowed to reach room temperature and the reaction was allowed to continue for 2 hrs.

However, where Arg (side chain protection, Mtr) was present in the peptide sequence, the

reaction was allowed to proceed for 8-12 hrs. A similar protocol was followed for tBoc

peptides and thioanisole and EDT (2: l, v/v) used as scavengers were added to the resin

and stirred at room temperature for 1 0 min. The reaction vessel was placed in an ice bath

and measured quantity of neat TF A was added and allowed to stir for 1 0 min. Following

this, TFMSA (trifluoromethanesulfonic acid) to a final concentration of 10% was added

drop wise with vigorous stirring. The flask was removed from the ice bath and the

contents were allowed to attain room temperature. Mixture was allowed to react for 1. 5-2

hrs.

vi) Post Cleavage Work-Up and Purification

The mixture was then transferred via a fritted glass funnel (fine porosity) into a flask

containing dry chilled ether into which the peptide was allowed to precipitate. The

contents in the funnel were washed further with a minimal volume of neat TF A and the

38


filtrate was collected for precipitation of the peptide into dry ether which was allowed to

incubate at 4°C overnight for maximum recovery. The peptide was transferred to

another fritted funnel and washed thoroughly with cold ether to remove scavengers. The

peptide was then dissolved in water and acetic acid was used to aid in dissolution of very

hydrophobic peptides and lyophilized.

Crude peptides were purified to more than 90% purity by reverse-phase HPLC (high

pressure liquid chromatography) on a C-18 column using a Waters HPLC system. Briefly,

peptides were dissolved in 0.1% TF A, filtered through a 0.4 ~Lm filter and loaded onto a

semi-preparative column. Elution was performed using a linear gradient of 60% CH3CN

(acetonitrile) in the presence of 0.1% TF A. Flow rate was maintained at 2 ml/min and

monitoring was done at 215 nm. Eluted fractions were taken up for aa compositional

analysis using a Waters Pico-Tag system after anaerobic hydrolysis for 24 hrs with 6 M

HCI (hydrochloric acid).

V. Recognition ofMAbs by Synthetic Peptides

In order to evaluate the ability of the synthetic peptides to recognize and bind to the

corresponding MAbs, two kinds of assays were employed.

i) Direct Binding Enzyme Immunoassay

Microtitration plates were coated with the respective peptides at a concentration of

1 ~Lg/well in 50 mM PBS, pH 7.4 for 1 hr at 37°C and then at 4°C overnight. Plates were

subsequently washed once with PBS and blocked with 1% BSA for 1 hr at 3 7°C in PBS

to reduce non-specific binding. Blocking was followed by three washes of 10 min each

with PBS containing 0.05% Tween-20 (PBST). MAbs (culture soups) were added at

appropriate dilutions and incubated at 37°C for 1 hr. Following this, plates were washed

with PBST and incubated with goat anti-mouse immunoglobulin conjugated to HRPO at

an optimized dilution of 1 :4000 in PBS. Estimation of enzyme activity was done with

0.1% orthophenylenediamine in 50 mM citrate phosphate buffer, pH 5.0 with 0.06% of

39


hydrogen peroxide as the substrate. The reaction was stopped by adding 50 j..tl/well of 5 N

H2S04 and the absorbance read at 490 nm in a microplate reader (Molecular Devices

Corporation, California, USA).

ii) Inhibition Assay

It was performed with a slight modification of the above procedure. Briefly, microtitration

plates were coated with pZP3~ at a concentration of 50 ng/well in 50 mM PBS, pH 7.4

for 1 hr at 37°C and then at 4°C overnight. The plates were subsequently washed once

with PBS and blocked with 1% BSA for 1 hr at 3 7°C in PBS to reduce non-specific

binding. Blocking was followed by three washes of 10 min each with PBS containing

0.05% Tween-20 (PBST). Dilutions of MAbs in PBS, giving an absorbance of 1.0 for

binding to pZP3 ~ in the absence of competitor in ELISA were determined. Subsequently,

respective MAbs at 50% of the above dilution were incubated with an equal volume of

increasing concentrations of competing peptide overnight at 4°C and incubating mixtures

were added in duplicates at 100 j.ll/well to the microtitration plates precoated with pZP3~.

Following an incubation period of 2 hrs at 37°C, plates were washed thrice with PBST,

incubated with goat anti-mouse immunoglobulin conjugated to HRPO at an optimized

dilution of 1:4000 in PBS and processed for colour development as described for Direct

Binding Enzyme Immunoassay. Concentration of peptides resulting in 50% inhibition in

the binding of MAb to pZP3 ~ was determined by regression analysis and was designated

IC5o·

Vl Generation of Anti-Peptide Antibodies

In order to test the efficacy of these peptides, it was essential to generate anti-peptide

antibodies. This was accomplished as follows:

i) Conjugation with Diptheria Toxoid

Synthesized peptides were conjugated to diptheria toxoid (DT) by the "one-step"

glutaraldehyde coupling procedure (A vrameas, 1969) which cross-links peptide and

40


carrier molecules through their amino groups. For conjugation, peptide and DT were used

in a 10:1 molar ratio. Briefly, peptide was dissolved in 100 mM PBS (pH 7.4) and

measured quantity of DT was added to it. The peptide-DT mixture was kept on ice and

glutaraldehyde was added to it with mixing to a final concentration of 0.1% (v/v). The

reaction was allowed to proceed overnight at 4°C with gentle end-to-end mixing.

Unreacted sites were blocked with 10 mM lysine for 3 hr at room temperature and

following this, the conjugate was dialyzed extensively against 10 mM PBS (pH 7.0) at 4°

C using a molecular weight cut-off of 12 kDa. The conjugate was aliquoted and stored at -

20°C until use.

ii) Induction of Anti-Peptide Antibodies

a) Mice

Female BALB/c mtce (8-1 0 weeks old) were immunized with individual peptide-DT

conjugates corresponding to peptides P 1, P2, P3 and P4 of the bonnet monkey ZP3. The

primary immunization (subcutaneously) consisted of conjugate equivalent to 20 ~Lg of

peptide along with Squalene and Arlacel 'A' ( 4:1) as adjuvants. The primary immunization

also contained 100 ~tg of SPLPS as an additional adjuvant. Animals were boosted,

intraperitoneally with an equivalent amount of peptide-DT conjugate as in primary

injection after 4 weeks and subsequently as and when required.

b) Rabbit

Female New Zealand White rabbits (Small Animal Facility, National Institute of

Immunology, New Delhi, India), 6-9 months of age were immunized intramuscularly (i.m.)

with peptide-DT conjugate equivalent to 200 ~tg of the peptide in 0. 9% saline emulsified

with an equal volume of complete Freund's adjuvant (CF A) as the primary injection. This

was followed by a booster i.m. after 4 weeks with an equal amount of peptide-DT

conjugate in incomplete Freund's adjuvant (IF A). Following determination of antibody

titres in a direct binding ELISA, an additional booster was given in the same manner after

15 days.

41


c) Bonnet monkey

Female bonnet monkeys (Macaca radiata) reared in the Primate Facility (Nil, New Delhi)

were used in the present study. Serum progesterone levels were estimated for atleast

three months in blood samples collected biweekly and animals showing irregular cyclicity

were grouped for immunogenicity experiments.

Animals (n=2/group) were immunized with P1-DT (MRA 769, 776), P2-DT (MRA 404,

560), P3-DT (MRA 653, 669) and P4-DT (MRA 771, 778). Respective conjugates

equivalent to 250 j.lg of the peptide were emulsified with Squalene and Arlacel "A" in a

ratio of 4:1 and administered intramuscularly (i.m.) at two sites. In addition, the primary

dose also contained 1 mg/animal of SPLPS as an additional adjuvant. The primary

immunization consisted of three injections of the above at monthly intervals. Following

this, bleeds ( 1-2 ml) were collected from the antecubital vein for antibody analysis and

boosters (same dose as in primary but without SPLPS) were administered as and when

required.

iii) Estimation of Antibody Titres

a) ELISA Procedure

Microtitration plates were coated as described above with optimized concentrations of the

following respective antigens depending on the rationale of the assay: Peptides (1 ~t

g/well), DT (500 ng/well), pZP313 (200 ng/well), solubilized isolated porcine zona

pellucida (pSIZP, 250 ng/well), recombinant bonnet ZP3 (Rec-ZP3, 200 ng/well),

solubilized isolated human zona pellucida (hSIZP, equivalent to the zona on 5

oocytes/well) in 50 mM PBS, pH 7.4. Plates were processed as described earlier and

incubated with varying dilutions of anti-peptide sera and bound antibody was revealed

with the appropriate second antibody-HRPO conjugate used at an optimized dilution of

1:3000 in PBS. The antibody titre was calculated by regression analysis and is represented

by antibody units as the reciprocal of the dilution of the antibody giving an absorbance of

1.0.

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Vlll Characterization of Anti-Peptide Antibodies

The anti-peptide antibodies raised in different species were variously characterized using

appropriate tests as described below:

i) SDS-PAGE

Porcine SIZP (2 ~tg/well), pZP3a ( 1 00 ng/well) and pZP3 ~ (1 00 ng/well) were resolved

by 0.1% SDS-12% polyacrylamide gel electrophoresis as per the procedure of Laemmli,

1970. Antigens were dissolved in reducing sample buffer (0.0625 M Tris, pH 6.8, 2%

SDS, 10% glycerol and 2% B-mercaptoethanol), boiled for 3 min and applied to the

polyacrylamide gel. The concentrations of stacking and resolving gels were 5% and 12%

respectively. Electrophoresis was carried out at 15 rnA constant current in running buffer

(0.125 M Tris, 0.192 M glycine, pH 8.3 with 0.1% SDS). The gels were stained with

Coomassie blue (R-250, 0.125% in methanol: acetic acid: water, 4:1 :5) (Hames 1990).

ii) Western Transfer and lmmunoblotting

The proteins were electrophoretically blotted to 0.45 ~tm nitrocellulose membrane

(BioRad, Hercules, USA) overnight at a constant voltage of 15 V in Tris glycine buffer

with 20% methanol (Towbin et al., 1979). For immunoblotting, non-specific sites on the

membrane were blocked by incubation in 5% BSA in 50 mM PBS, pH 7.2 for 2 hrs at

room temperature and washed 3 times in PBS with 0.1% Tween-20 (PBST) for 15 min.

The membrane was incubated for 2 hr at room temperature with appropriate dilution of

anti-peptide serum followed by washings in PBST. Anti-rabbit immunoglobulin conjugated

to horseradish peroxidase was used to reveal the bound antibody. A freshly prepared

solution of 4-chloro naphthol (0.6% w/v) in 50 mM PBS and 20% methanol with 0.06%

H202 was used as the substrate and the reaction stopped by 0.1% sodium azide.

iii) Indirect Immunofluorescence Assay on Porcine Oocytes

The porcine oocytes were obtained as per the protocol of Sacco et al., 1989. Briefly, zona

encased pig oocytes were acquired from follicular phase ovaries obtained at a local

slaughter house by mincing ovaries under 8-10 ml of0.1 M PBS. A portion ofthe ovarian

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suspension was viewed under a stereo microscope (Nikon Corp., Japan) and the oocytes

(representing various maturational stages) were collected using a fine glass pipette and

washed in three changes of PBS. Adhering cumulus cells were removed by shaking the

oocytes for 30 sec in 1 ml PBS in a 12 X 75 mm culture tube. Cumulus free zona encased

oocytes were incubated in 1 OOJ..ll volume of various anti-peptide antibodies (polyclonal

sera; 1:500 dilution) for 1 hr at 37°C after which they were washed in 4 changes of PBS.

Subsequently, the oocytes were incubated with anti-rabbit immunoglobulin conjugated to

fluorescein isothiocyanate for 1 hr at 37°C. After several washings in PBS, the oocytes in

glycerol : PBS (9: 1) were observed under fluorescence microscope (Nikon Optiphot,

Japan). Oocytes treated with normal rabbit serum served as the control.

iv) Indirect Immunofluorescence on Bonnet Monkey Ovarian Sections

Two female bonnet monkeys (3 and 1 0 years old) were ovarectomised and the ovaries

were snap frozen in liquid nitrogen. Ovarian sections of 5 ~Lm thickness were cut in a

cryostat (2800 Frigocut, Reichert-Jung, Germany) at -20°C and fixed for 20 min in chilled

methanol. Sections passing through a follicle were selected, washed in PBS and blocked

for 30 min in 5% normal rabbit serum. The sections were incubated at 37°C with 1:100

dilution of mouse anti-peptide preimmune and immune sera for 1 hr and washed with PBS.

Following this, the sections were incubated for 1 hr with rabbit anti-mouse

immunoglobulins conjugated to fluorescein isothiocyanate (FITC). Subsequently, slides

were washed with PBS, mounted in glycerol: PBS (9: 1) and examined under fluorescent

microscope (Nikon Optiphot, Japan).

IX. In vitro Contraceptive Efficacy of Anti-Peptide Antibodies

In order to evaluate the efficacy of anti-peptide antibodies, the following functional assay

was employed:

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i) Sperm-Egg Attachment Assay

The ability of anti-peptide antibodies to prevent binding of boar sperm to zona-encased pig

oocytes was assayed as described by Sacco et al., 1989a. Porcine oocytes were obtained

as described above. For boar sperm, testes were surgically removed and washed

thoroughly in normal saline. The epididymis was minced with a scalpel, spermatozoa

collected by washing with modified extender medium (Composition in Appendix 2) and

incubated at 37°C for 1 hr. Viable sperm were purified on a Percell gradient (45-90%) at

300 x g for 20 min at room temperature. The sperm pellet was washed twice by

resuspending in capacitation medium (composition in Appendix 3), containing BSA (20

mg/ml) and centrifuged for 10 min as above. Following the second centrifugation, the

sperm pellet was diluted with capacitation medium and incubated at 37°C in 5% C02 and

air for 4 hrs and assessed for count and motility. Alternately, boar sperm (Swine Genetics

International, Cambridge, USA) was also prepared by centrifuging 5 ml of the suspension

at 800 X g for 10 min and washing the sperm pellet twice in 5 ml volumes of Boar Sperm

Medium (Sacco et al., 1984). After the second centrifugation, the sperm were suspended

in 4 ml of Boar Sperm Medium, incubated at 37°C in 5% C02 and air for 60 min and

assessed for count and motility. For the assay, 5-10 zona encased oocytes were incubated

in 100 Jll volumes of various anti-peptide antisera (1:20 dilution) for 1 hr at room

temperature after which they were washed 4 times in PBS and transferred into 40 111 of

Boar Sperm Medium under oil. Five million motile sperm were added and the sperm-egg

mixtures were incubated at room temperature for 1 hr. Following incubation, the oocytes

were gently washed by repeated pipetting to remove loosely bound sperm and then

transferred to depression slides where sperm heads visible in one plane of focus were

counted. Oocytes pre-incubated with rabbit polyclonal antibodies against ZP3 served as

positive controls whereas incubation of oocytes in normal rabbit serum served as negative

control.

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X In vivo Contraceptive Efficacy of Anti-Peptide Antibodies

The efficacy of immunization with synthetic peptide to regulate fertility tn the animal

model was evaluated as follows:

i) Immunization of Female Bonnet Monkeys

Female bonnet monkeys (Macaca radiata) reared in the Primate Facility (NIT, New Delhi)

were selected and serum progesterone levels were estimated for atleast three months in

samples which were collected biweekly. Animals showing atleast two consecutive normal

ovulatory peaks (serum progesterone levels >2 ng/ml) (Bamezai, 1986) were selected for

fertility trials.

Four animals (MRA 665, 766, 772 and 774) were immunized with a physical mixture

(cocktail) of synthetic peptides derived from the primary aa sequence of bonnet monkey

ZP3 viz., Pl (residues 23-45 with an additional N-terminal Lys), P2 (residues 165-180

with an additional C-terminal Cys), P3 (residues 300-322) and P4 (residues 324-347),

individually conjugated to DT and mixed at a concentration equal to 1 00 ~Lg of peptide.

They were emulsified with Squalene and Arlacel "A" which are adjuvants permitted for

human use, in a ratio of 4:1 and administered intramuscularly (i.m.) at two sites. In

addition, the primary dose also contained 1 mg/animal of SPLPS as an additional adjuvant.

Following completion of the primary immunization comprising of three injections at

monthly intervals, bleeds ( 1-2 ml) were collected biweekly from the antecubital vein for

estimation ofhormonallevels and antibody titres. Boosters were administered as and when

required. Animals were put on continuous mating with males of proven fertility after

administration of the three primary injections and monitored for menstrual cyclicity and

conception.

ii) Estimation of Steroid Hormone

Progesterone levels were estimated from sera of peptide cocktail immunized animals

which were bled biweekly using a radioimmunoassay employing reagents and protocol as

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prescribed by the W.H.O. Matched Assay Reagent Programme (Sufi et al., 1983). Each

sample was run in duplicates. Steroid was extracted from serum (0.1 ml) by the addition of

2 ml of ice-cold ether in each tube and vortexing for 2 min. The tube was immersed in

liquid nitrogen in order to flash freeze the serum phase and the unfrozen ether phase which

contained the extracted steroid hormone was decanted into another tube. The ether was

allowed to evaporate overnight and 0.5 ml of steroid assay buffer (0.1 M PBS, pH 7.3,

0.1% thiomersal and 0.1% gelatin) was added to the tubes and the tubes were incubated at

40°C for 30 min. Steroid sticking to the walls of the tubes was recovered by vigorous

vortexing. Anti-progesterone antibody (0.1 ml) was added (at a dilution giving approx.

50% binding of tritiated progesterone in the absence of unlabelled competing

progesterone) was then added to the tubes followed by addition of 0.1 ml of 3H

progesterone (approx. I 0,000 cpm/tube). The mixture was incubated for at least 16 hrs at

4°C. Unbound progesterone was separated by addition of 0.2 ml of ice cold assay buffer

containing 0.625% activated charcoal and 0.0625% dextran and incubated for 30 min at 4

0 C. This was followed by centrifugation at 2500 RPM for 15 min at 4°C. The supernatant

was carefully decanted into scintillation vials and 4 ml of scintillation fluid (0.4% 2,5

diphenoxazole; 0.01% POPOP [1-4 bis(S-phenyl-2-oxazolyl)benzene] in sulfur free

toluene) was added and counted in a liquid scintillation beta counter (Beckman

Instruments, California, USA). The amount of progesterone per ml of serum was

calculated from a standard curve with known amounts of progesterone in each assay.

iii) Ovarian Histology

Individual peptide-DT immunized animals were taken up for histological examination of

the ovary. Animals were administered ketamine hydrochloride (5 mg/kg body weight) as a

general anesthetic. Ovaries were surgically removed, washed and fixed in buffered

formalin (1 0%) for 48 hrs. Tissue was then washed extensively in running tap water and

dehydrated through ascending grades of alcohol (50%, 70%, 90% and 1 00%) and xylene

which was followed by embedding in paraffin. Serial sections of 5 ~Lm thickness were cut

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and stained by haematoxylin and eosin using standard protocols (Luna, 1968). For each

ovary, 10 non adjacent sections were randomly chosen and examined for ovarian

pathology in the form of cystic changes or lymphocytic infiltration. The changes in the

morphology of follicles in different stages of development were observed.

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