Development of an inhibin alpha subunit ELISA with broad specificity

Abstract

Inhibin immunoassays with a sufficiently broad specificity to detect all alpha subunit-containing forms are of value in detecting and monitoring various ovarian cancers. Assays to date with this specificity are not readily amenable to wide diagnostic application. The objective of this study was to develop a sensitive two-site ELISA using alpha subunit-directed monoclonal antibodies (Mabs) able to detect all forms of inhibin to replace a previously described alpha subunit-directed immunofluorometric assay (IFMA). In this study, the major inhibin epitopes in the two polyclonal antisera used in the alphaC IFMA were initially identified and Mabs were raised to these regions. These Mabs in conjunction with the inhibin alpha subunit R1 Mab (Groome) were used to develop alpha subunit ELISAs with high sensitivity. Application of these assays to human serum and human follicular fluid following fractionation by an immunoaffinity/preparative PAGE/electroelution procedure which separated inhibins according to their molecular weights, indicated that the specificity of the various ELISAs differed between Mab combinations with preferences noted for either the alpha subunit or dimeric forms. A combination of Mabs in an ELISA was identified which provided data which matched that obtained with the alphaC IFMA and which may be useful as a replacement inhibin assay in clinical studies.