Anti-idiotypic Antibodies against Bovine Growth Hormone

Anti-antibodies against three mouse monoclonal antibodies viz. IIB5D6, VIA6E8 and VIC1F9 (specific to bovine growth hormone) in rabbits have been generated and characterized. Ammonium sulfate fractionated and affinity-purified monoclonal antibodies were used for producing anti-antibodies. The generated anti-antibodies were against common as well as uncommon antigenic determinants present in mouse monoclonal antibodies. The raised anti-antibodies replaced ( I 125 )bGH bound to goat liver microsomes indicating production of anti-idiotypic antibodies against bovine growth hormone. These antibodies can have profound implications in vivo in lactating bovines for enhancing milk yield. (Asian-Aust. J. Anim. Sci. 2003. Vol 16, No. 5 : 732-737)


INTRODUCTION
Growth hormone (GH) is a protein hormone produced in somatotrophs of pituitary gland (Wallis, 1988).It comprises of about 191 amino acids with a molecular weight of 22 kDa.The major immunologically active zones are confined to 87-124 and 125-149 regions (Ferrara et al., 1979).Somatogenic as well as lactogenic activities are present in bovine GH (bGH).There are atleast three different conformational epitopes in bovine growth hormone (bGH) (Kumar and Rajput, 1999).The first in action of GH is the binding of GH to its receptor present in cell membrane of target tissues followed by dimerization of receptor, a known feature in the action of type I cytokines (Wells and deVos, 1996;Sodhi and Rajput, 2001).Jerne (1974) proposed anti-idiotypic network theory and since then several anti-idiotypic antibodies against hormones have been raised.These include thyroid stimulating hormone (Baker et al., 1984), luetinizing hormone (Sairam et al., 1992), prolactin (Amit et al., 1986), follicle stimulating hormone (Udupa and Sheth, 1987), oestrogen (Mor et al., 1992) and growth hormone (Gardner et al., 1990;Schalla et al., 1994).In such studies, first polyclonal antibodies were raised and these antibodies were used for anti-idiotypic response.
bGH is known to enhance milk production in lactating animals and therefore it is interesting to know whether antiidiotypic antibodies against this molecule can lead to enhanced milk production.Contrary to expectations, active immunization of lactating cows with bGH polyclonal antibodies had no effect on milk yield (Schalla et al., 1994).Perhaps MAbs against bGH, when used as immunogen in lactating bovines can enhance milk yield.However, before this concept is finally tested in bovines or buffalo, the generation of anti-idiotypic antibodies must be established in laboratory animals.In the present investigation, results on generation of anti-idiotypic antibodies against bGH are presented.

MATERIALS AND METHODS
Three hybridoma cell lines viz.IIB5D6, VIA6E8 and VIC1F9 generated earlier and producing IgM, IgG 1 and IgG 2b antibodies against bGH were used ( Kumar and Rajput, 1999).

Culturing of hybridomas
Frozen cells were thawed at 37°C and routinely cultured in RPMI-1640 medium containing 15% FCS.Cell lines growing poorly after thawing were rescued by supplementing medium with OPI mix and if necessary feeder cells were also provided (Harlow and Lane, 1988).100 X OPI-mix was prepared by dissolving 1.5 g oxaloacetate, 0.5 g sodium pyruvate and 2000 I.U.insulin in 100 ml water.Culture supernatants from growing hybridoma cell lines were collected and supplemented with sodium azide (0.05% final concentration) and 1 ml of 1M Tris (pH 8.0) for every 20 ml supernatant.The supernatant was stored at -20°C till further use.

ELISA
ELISA was used to ascertain production of MAbs by hybridoma cell lines and method described by Engvall and Perlman (1971) with some modifications was followed.Briefly, multiwell plates were coated with bGH (1 µg/100 µl PBS, pH 7.4/well) by overnight incubations at 4°C.All subsequent incubations were carried out at room temperature with mild shaking on orbital shaker.Plates were washed with PBS-Tween 20 (0.05%) and then blocked by completely filling wells with blocking solution (1% BSA-PBS-Tween 20) for 2 h.After four times washing with PBS-Tween20, 100 µl of hybridoma supernatant (1:1 diluted with PBS) were added to different wells of ELISA plate.For negative and positive controls, 100 µl RPMI-1640 (15% FCS), 100 µl of mouse pre-immune (1:3,000 diluted with PBS) and mouse immune (1:3,000 diluted with PBS) were added.The plate was incubated for 2 h and then washed four times with PBS-Tween 20. 100 µl of 1:1,000 diluted rabbit anti-mouse IgG peroxidase conjugate (Bangalore, Genei) in 1% BSA-PBS-Tween 20 was added to individual well and the plate was incubated for 2 h.The plate was washed five times with PBS-Tween 20.Subsequently, 100 µl of substrate solution (4 mg ophenylenediamine hydrochloride dissolved in 10 ml of 50 mM sodium citrate buffer, pH 5.0 containing 0.01% H 2 O 2 ) was added to individual well.After 30 min., the reaction was terminated by addition of 100 µl 4N H 2 SO 4 to each well.Absorbance was recorded at 490 nm in ELISA plate reader (Toyo, Japan).

Purification of monoclonal antibodies
Antibodies were purified using ammonium sulfate precipitation and affinity chromatography techniques (Harlow and Lane, 1988).
Ammonium sulfate precipitation : To 100 ml tissue culture supernatants collected from hybridomas IIB5D6, VIA6E8 and VIC1F9, 31.3 g ammonium sulfate (providing 50% final saturation) was slowly added with constant stirring.The resultant precipitate was kept overnight at refrigerated temperature and centrifuged at 10,000×g for 20 min at 5°C.The pellet so obtained was dissolved in 10 ml of 10 mM Tris-HCl, pH 7.5 and dialyzed against 10 mM Tris-HCl, pH 7.5 overnight at 5°C with three changes.The dialyzed solution was centrifuged (10,000×g, 20 min) to remove any insoluble material.These antibody solutions were stored at -20°C after addition of sodium azide (0.05% final concentration).
Immunoaffinity purification of antibodies : Ammonium sulfate precipitated antibodies were further purified on goat anti-mouse immunoglobulin-agarose affinity column.These antibodies were loaded to affinity column (1.3×2 cm) equilibrated with 10 mM Tris-HCl buffer pH 7.5 by passing them thrice to the column to ensure binding of antibodies to the column.The unbound proteins were then removed by washing the column with 20 ml 10 mM Tris-HCl, pH 7.5.The bound proteins were then eluted by 100 mM glycine-HCl, pH 2.5.The column was again washed with 10 mM Tris-HCl, pH 7.5.Antibodies, which could not be eluted with glycine-HCl, were eluted by application of 0.1 M triethylamine buffer pH 11.5.Two ml fractions at a flow rate of 20 ml/h were collected.Fractions eluted with glycine-HCl and triethylamine were collected in tubes containing 200 µl 1 M Tris, pH 8.0.The eluted fractions were immediately mixed to bring the pH of eluted antibody to neutrality.Fractions eluted with glycine-HCl (elution volume between 22 to 26 ml, Figure .1)and triethylamine (elution volume between 64 to 68 ml, Figure .1)were pooled.These pooled fractions were concentrated by precipitating antibodies with ammonium sulfate (50% final saturation).The precipitate was dissolved in 10 mM Tris-HCl, pH 7.5 and dialyzed against the same buffer.These antibodies were either stored without sodium azide (for immunization of rabbits) or with 0.05% sodium azide (for hormone-receptor assay) at -20°C till further use.

Estimation of protein
The dye binding method of Bradford (1976) was used for estimation of protein.

SDS-PAGE
SDS-PAGE was carried out as per method of Laemmli (1970).Protein bands were visualized either by coomassie brilliant blue or ammonical silver stain (Harlow and Lane, 1988).

Immunization of rabbits
Each of the three MAbs viz.IIB5D6, VIA6E8 and VIC1F9 was administered to two rabbits (at least 2 months old).Primary injection (500 µg Mab emulsified with FCA) and three boosters (500 µg Mab in FIA) were given intramuscularly in hind legs.All immunizations were carried out after a gap of 21 days from each other.Ten days after last booster dose, blood was collected from ear vein and serum separated.Sodium azide (0.05%) supplemented serum was stored at -20°C till further use.For hormone receptor assay, antibodies from sera were precipitated with 50% saturated ammonium sulfate, dialyzed against PBS containing 0.05% sodium azide and then stored in small aliquots at -20°C.

Immunodiffusion
The presence of antibodies in sera obtained from immunized rabbits was tested in immunodiffusion as described by Nilsson (1984).The precipitation lines were visualized by coomassie brilliant blue R (Nilsson, 1984).

Hormone -receptor assay
Labelling of bGH : bGH was labelled with iodine by the method of Fraker and Speck (1978) as described by Cadman and Wallis (1981) with some modifications.Iodogen coated eppendorf tubes (0.5 ml) were prepared as follows. 2 µg iodogen (dissolved in 40 µl chloroform) was added to eppendorf tube.The tube was held in palm and continuously rotated in horizontal position to enable uniform coating of iodogen at maximum surface area.bGH ( 20 µg/35 µl 0.5 MPO 4 , pH 7.4) was added to iodogencoated eppendorf tube.Immediately, 5 µl NaI 125 (500 µCi) was added.Reaction was stopped by adding 200 µl 25 mM Tris -HCl, pH 7.5 containing 0.05% BSA and 0.05% sodium azide (TBA-buffer).Contents were mixed and loaded on sephadex G-50 column equilibrated with TBAbuffer.Fractions containing iodine bound to bGH were pooled and appropriately diluted with TBA-buffer to give 150,000 cpm per 100µl.
Microsome preparation : Liver microsomes were prepared as per the method of Haro et al. (1984) with some modifications.Liver from freshly slaughtered goat was collected, immersed in cold saline and brought to the laboratory.A piece (~20 g) of liver was placed in the petri dish and rinsed several times with cold saline.About 50 ml chilled Tris-buffer (25 mM), pH 7.0 containing 10 mM EDTA, 10 mM EGTA, 300 mM sucrose, 0.6 mM PMSF, 1 µM leupeptin and 1 µM pepstatin A was added, tissue was cut into small pieces and homogenized for one minute by giving five strokes.Homogenized tissue was filtered through eight layers of cheese cloth.The filtrate was centrifuged at 10,000×g for 15 min.at 5°C.Pellet was discarded and supernatant was ultracentrifuged (100,000×g, 90 min., 5°C).The pellet was resuspended in 25 mM Tris, pH 7.6 containing 10 mM EDTA, 10 mM EGTA, 0.6 mM PMSF, 1 µM leupeptin and 1 µM pepstatin A (Buffer A).Suspended pellet was uniformly mixed by homogenizing it for one minute by giving five strokes.Microsome preparation was diluted with buffer A to give protein concentration of 3 mg/ml and stored in small aliquots at -70°C.Assay : Hormone -receptor assay was performed as per the method of Cadman and Wallis (1981) with some modifications.This assay was carried out in 2 ml microcentrifuge tubes.Reaction mixture (500 µl) contained 100 µl microsomes (300 µg protein ), 100 µl [I 125 ] bGH (1,50,000 cpm) and appropriate volume of assay buffer (25 mM Tris, pH 7.6 containing 10 mM EDTA, 10 mM EGTA, 0.6 mM PMSF, 1 µM leupeptin, 1 µM pepstatin A, 0.1% BSA and 0.05 % Na-azide).The replacement of bound I 125 bGH with cold hormone or rabbit anti-(mouse Mabs) antibodies was studied by adding 10 µg bGH or 1,8 and 50 µg antibodies dissolved in µl assay buffer.
The reaction mixture was incubated at 25°C in water bath for 5 h.Then, 1.5 ml 25 mM chilled Na-acetate, pH 5.4 was added to stop the reaction.After 10 min, tubes were centrifuged at 4,000×g for 25 min at 5°C.Supernatant was carefully removed.Traces of supernatant attached to walls were removed with help of filter paper without disturbing the pellet.Counts in pellet were recorded in γ-counter.

Purification of antibodies
Culture supernatants from hybridoma cell lines IIB5D6, VIA6E8 and VIC1F9 were the source of Mabs against bGH.These cell lines were secreting Mabs as clear from signal in ELISA (Table 1).The culture supernatants from hybridoma cells grown in vitro contain antibodies of mouse origin and immunoglobulins of bovine origin.Ammonium sulfate fractionation separates immunoglobulins from other serum proteins present in culture supernatant.Subsequent purification of immunoglobulins on anti-mouse IgG antibodies affinity column will separate mouse immunoglobulins from bovine immunoglobulins and elution profile is shown in Figure1.The SDS-PAGE pattern of IIB5D6, VIA6E8 and VIC1F9 (Figure .2a) indicated that in all antibody preparations, a protein band corresponding to light chain (25 kDa) was distinct.In case of IIB5D6 (lane 2), a intense band corresponding to µ chain was noticed.Minor contaminants of higher or few lower molecules weight proteins than µ chain were also seen but overall preparation of IIB5D6 appeared to be reasonably pure (>80%).Antibodies VIC1F9 (lane 3) and VIA6E8 (lane 4) showed two doublet bands (~60 kDa) and one major band of approximately 75 kDa.It is expected that intensity of heavy chain should be atleast twice of light chain but this was not found with any of the three antibodies.There are few possibilities.Heavy chain is not completely dissociated from light chain and this can result in bands of higher molecular weight.Second possibility is that heavy chain is degraded by proteolytic enzyme since presence of one such enzyme viz.cathepsin D has been established in hybridoma culture supernatants (serum-free) collected from dead or lyzed cells (Erp et al., 1991a).Possibility also exists that clone specific proteolytic enzyme is present (Erp et al., 1991b).Other workers also observed degradation of heavy chain (Chen et al., 1984) and some workers referred as extra band as slow moving light chain (Lee, 1987).The present observation and observations of few other workers suggest the need for working out SDS-PAGE protocol for immunoglobulins where artifacts can be eliminated or minimized.Because of possible artifacts in movement of heavy chain, it is difficult to predict the purity of preparations.Nevertheless, it is also very unlikely that the preparations were without any contaminants.Emtner et al. (1989) could obtain purity greater than 75% using protein A affinity column and in present case also electrophoretically homogeneous preparation could not be obtained.
Rechromatography of VIA6E8 on antimouse immunoglobulin was done in order to improve purity of preparation and SDS-PAGE pattern is shown in Fig. 2b.There is improvement in purity but bands higher or lower than γ-chain could also be detected.In general, from 100 ml hybridoma culture supernatant, about 5 to 9 mg of antibodies were obtained.

Production of anti-(bGH antibodies) antibodies in rabbits
Three MAbs viz.VIC1F9, VIA6E8 and IIB5D6 were used for raising antibodies in rabbits and the presence of

Figure 1 .
Figure1.Affinity purification of mouse monoclonal antibodies using anti-mouse IgG agarose column.Ammonium sulfate fractionated proteins from hybridoma supernatants were loaded on affinity column.Bound proteins were eluted by 0.1 M glycine-HCl, pH 2.5 and 0.1M triethylamine, pH 11.5.Absorbance was recorded at 280 nm.

Figure 3 .
Figure 3. Testing presence of anti-antibodies using immunodiffusion technique.For each antibody, two rabbits were immunized.Column1 and column 3 contained serum from different rabbits immunized with Mabs.Column 2 contained Mabs.Row 1 contained VIC1F9 antibody and antisera against it.Row 2 contained VIA6E8 antibody and antisera against it.Row 3 contained IIB5D6 antibody and antisera against it.

Table 1 .
Relative antibody signals produced by hybridomas

Table 2 .
Replacement of bound [I 125] bGH to goat liver microsomes by bovine growth hormone and different antiantibodies