Purification of inhibin from human follicular fluid using monoclonal antibody

Inhibin is a protein synthesized by granulosa and sertoli cells which preferentially inhibit pituitary secretion of FSH through a negative-feedback. Studies have showed that measurement of inhibin has a clinical role in understanding the fertility status of men and women and could also be used as a prognostic marker for pre-eclampsia and Down syndrome in fetus. There are many different multi-step procedures for the purification of inhibin. In this study we attempted to purify inhibin from human follicular fluid using immunoaffinity techniques. Follicular fluid (FF) collected from women referring to Avicenna Infertility Clinic, was filtrated and subsequently concentrated by ammonium sulfate. The presence of inhibin in follicular fluid was detected using enzyme-linked immunosorbent assay. In order to purify inhibin, an affinity chromatography column using anti-inhibin monoclonal antibody was prepared. The purified protein was analyzed through SDS-PAGE and western blotting after a protein solution load of human inhibin into the column. The SDS-PAGE results of affinity proved the presence of inhibin following silver staining appeared as a single 32 kDa band. Western blotting revealed that specific anti-inhibin antibody was able to recognize inhibin epitopes. The present protocol for purification of inhibin is a technique with 92% yield. This method is a sensitive procedure for high yielded productions of inhibin compared to the previously described methods, using HPLC and gel filtration. However, since preparing and obtaining follicular fluid, as a main source of inhibin, is rather difficult but suitable for laboratories, recombinant inhibin is recommended for mass production.