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carboxyl-terminal 15 amino acid residues may play a dominant role for the limitation of the antigenic recognition sites at the terminal 15 amino acid residues.

DEVELOPMENT OF MOUSE HYBRID CELL CLONES SFCRETING ANTI-LHRH ANTIBODY

S.K. Gupta and G.P. Talwar . National Institute of Immunology, Post Box No.4922, New Delhi-l10029, India.

Hybrid cell clones secreting monoclonal anti-LHRH (leuteinizing hormone release hormone) antibodies have been developed by fusing mouse myeloma (SP2/O) with splenocytes obtained from Balb/c mouse immunized with LHRH conjugated to tetanus toxoid. Some o~ these clones have now been propogated and adapted to mouse peritoneum. The antibody titre of the ascites fluid product of clone P81662 was 1 million (30 to 40% binding of iodinated LHRH at this dilution in radioimmunoassay). The association constant of the antibody was 2.86xi09 L/M; with a linear Scatchard plot. The antibody was IgG 2 and kappa. The antibody is highly specific for LHRH and does not recognize TRH and TSH. The recognition for LHRH agonists was I0 fold less with 6-D serine des glycine ethylamide and I00 fold less with Bzl-His6pro 9 LHRH.

DESIGN, CHEMICAL SYNTHESIS AND IMMUNOLOGY OF A CYCLIC PEPTIDE OF ~-hCG. D. Sahal, K.S.N. Iyer, & G.P. Talwar. Department of Biochemistry, All India Institute of Medical Sciences and National Institute of Immunology, New Delhi-l10029, India.

The importance of intrachain-S-S-linkages in ~-hCG for conformations recognised by the antibodies and the target tissue receptors is well recognised. The eicosapeptide 82-101 which contains an exposed disulfide bond of ~-hCG between Cys 93 and I00 and has six important aminoacid substitutions visa vis ~-hLH was chemically synthesized. The nonapeptide 82-90 with substitutions of Cys88, 90 by Gly was synthesized in solution phase while the undecapeptide 91-101 was assembled on the Merrifield resin. Fragment condensation of the nona and the undecapeptides in solution by azide coupling yielded the linear eicosapeptide. Iodine oxidation was done to form an intramolecular disulfide bond between Cys-Acm 93 and 100.SPDP was used to conjugate the cyclic peptide to tetanus toxoid. Antipeptide antibodies were elicited in rabbits on immunization with the conjugate. Immunobiological properties of these antibodies will be presented.

STUDIES ON ADJUVANTS FOR CONTRACEPTIVE VACCINES

Om Singh, Vined Singh and ~.P. Talwar. Department of Bioche~All India Institute-of Medical Sciences & National Institute of Immunology, New Delhi-l10029, India.

Contraceptive vaccines demand ~dequate potentiation of

106

antibody response. The objective of this study was to develop adjuvants which can enhance antibody resnonse a~ainst two antigens, B-hCG and LHR;~, linked to a carrier, tetanus toxoid. The antigen-carrier conjugates were adsorbed on alum. The adsorption increased the immunogenicity as compared with the antigen in saline. More than 30 substances were tested for their adjuvanticity to increase the antibody titres over and above the alum adsorbed Cormulation. Four of them were found promising for primary response. A lipidic non-toxic emulsion gave a good secondary immune response.

APPLICATION OF MONOCLO~AL Ah~IBODIES TO HUMAN CHORIONIC C/]NA[X)- TROPIN IN ENZYME-]ZgftrNOASSAYS.

Eric WCNG, Vinoent I]3M, Evelyn LGW, Albert F. CHEN and Chi- Yu Gregory LEE. Department of Obstetrics and Gynaecology, The --

University of British Columbia, Vancouver, Canada.

To develop sensitive enzyme-immunoassays for human chorionic gonado- tropin (HCG), we have raised monoclonal antibodies specific to ~ -subunit of HCG by an improved hybridoma technique. Following successive immunizations with ~ -subunit of HCG partially purified by sodium dodecylsulfate acrylamide gel electrophoresis, spleen cells of immunized mice were fused with NS-I myel- cma cells. The hybrid cells were cultured initially in a semi-solid selection mediu~n containing methylcellulose in petri-dishes. Visible colonies were removed and subcultured, seven to ten days after cell fusions. By microplate enzyme-ir~nunoassay and sodium dodecylsulfate gel/protein blot radioir~uno- binding method, about one dozen of hybrid cell lines were shown to produce antibodies that bind both HCG and ~-HCG and exhibit little cross-reactivity to LH and FSH. To perform a sandwich solid-phase enzyme-immunoassay, each

-HCG-specific monoclonal antibody was coated on nylon balls (2 mm in diameter), nitrocellulose filters or microtiter plates. A quantitative irm~m~ assay was proceeded by incubating HCG at proper concentrations and then horse radish peroxidase-labeled ~-HCG monoclonal antibodies as the second antibodie~ Pairs of menoclonal antibodies that bind nenoverlapping antigenic dc~ains of HCG were selected for this sandwich inmunoassay. This irmmnoassay procedure is highly specific to HCG and permits facile and quantitative determinations of 1 ng/ml HCG in biological fluids.