Preparation of monoclonal antibodies to the beta A subunit of ovarian inhibin using a synthetic peptide immunogen.

Seven different synthetic peptides were prepared corresponding to regions of the beta A subunit of 32 KDa human ovarian inhibin predicted to contain possible continuous B cell epitopes. These were coupled to tuberculin as a carrier and used to immunize mice previously given a priming dose of human tuberculosis vaccine. Only one of these peptides, corresponding to sequence 82-114, consistently gave good titres of antibodies reactive with intact 32 KDa bovine inhibin by ELISA. From one of the mice immunized with this peptide, six stable hybridomas were prepared. Five of these secreted an IgM and the sixth (clone E4) secreted an IgG2b antibody. Immunoblotting experiments on follicular fluid concentrates, after treatment with sodium dodecyl sulphate and mercaptoethanol, showed strong reactivity of the antibody from clone E4 only with bands of about 13 kDa and 58 kDa corresponding to forms of the beta A subunit previously described. This monoclonal antibody, and an antibody to the alpha subunit previously made in this laboratory using synthetic peptides, should prove useful reagents for further study of inhibins and activins.