Immunoassays for inhibin and its subunits Further applications of the synthetic peptide approach

Abstract

We describe the preparation of a new rat monoclonal antibody (CRC1) to the N-terminal sequence of the 43 kDa subunit of human ovarian inhibin, and its use together with other anti-peptide monoclonal antibodies, in two-site immunoassays for the detection of inhibin-related material in biological fluids. The Fab fraction of a mouse monoclonal antibody (R1) to the N-terminal portion of the 20 kDa α subunit, coupled to alkaline phosphate, was used for detection, and either CRC1 or a monoclonal antibody (E4) to the β-A subunit were used as capture antibodies. The E4/R1 combination, expected to measure dimeric bioactive inhibin, could detect less than 2 pg/ml of recombinant inhibin in diluent, gave good recovery of activity spiked into human blood, and could measure significant levels of immunoreactivity in sera from women undergoing ovulation induction, and in some normal women. Sera from post-menopausal women contained undetectable levels. Apparent inhibin levels in human follicular fluid were increased six-fold by pretreatment with 8 M urea, suggesting masking of epitopes in this fluid. Activin cross-reactivity in the assay was 0.05%. The R1/CRC1 assay, expected to measure only large molecular weight forms of inhibin or its α subunit, could detect immunoreactivity in human FF diluted 50,000-fold, and in all sera tested, although the levels in the hyperovulated women were higher. By contrast to the E4/R1 assay much of the immunoreactivity was labile during the clotting process, or subsequent assay, and reliable measurements on blood with this assay will require special sample collection procedures. These results demonstrate the value of anti-peptide monoclonal antibodies in the study of inhibin, and the results obtained with CRC1 show that antibodies useful for immunoassays can sometimes be obtained without the purified target molecule being available for immunization or screening.