Development of an inhibin alpha subunit ELISA with broad specificity

Inhibin immunoassays with a sufficiently broad specificity to detect all al pha subunit-containing forms are of value in detecting and monitoring vario us ovarian cancers. Assays to date with this specificity are not readily am enable to wide diagnostic application. The objective of this study was to d evelop a sensitive two-site ELISA using alpha subunit-directed monoclonal a ntibodies (Mabs) able to detect all forms of inhibin to replace a previousl y described alpha subunit-directed immunofluorometric assay (IFMA). In this study, the major inhibin epitopes in the two polyclonal antisera used in t he alphaC IFMA were initially identified and Mabs were raised to these regi ons. These Mabs in conjunction with the inhibin alpha subunit RI Mab (Groom e) were used to develop alpha subunit ELISAs with high sensitivity. Applica tion of these assays to human serum and human follicular fluid following fr actionation by an immunoaffinity/preparative PAGE/electroelution procedure which separated inhibins according to their molecular weights, indicated th at the specificity of the various ELISAs differed between Mab combinations with preferences noted for either the alpha subunit or dimeric forms. A com bination of Mabs in an ELISA was identified which provided data which match ed that obtained with the alphaC IFMA and which may be useful as a replacem ent inhibin assay in clinical studies. (C) 2001 Elsevier Science Ireland Lt d. All rights reserved.