Figure 4

Immunopurification of GnSAF from granulosa/luteal cell-conditioned medium (G/LCM) using immobilised phage-derived monoclonal and rat polyclonal antibodies. (A) GnSAF bioactivity in G/LCM treated with (open circles) and without (closed circles) 2 M NaI elution buffer. GnSAF (b) and inhibin (e) bioactivities in quality control human follicular fluid (QC hFF, closed circles) but not in proteins non-specifically bound by the immunopurification media without immobilised antibody (non-specific binding: NSB, open circles). GnSAF (c) but not inhibin (f) bioactivity detected in G/LCM immunopurified using immobilised rat polyclonal (closed circles) or immobilised phage-derived antibody (3-c4b, open circles). GnSAF bioactivity remaining in excess protein not bound by immobilised phage-derived antibody (3-c4b, d). Data is from triplicate cell cultures, results are mean ± SE. The shaded dose-response curves (a-j) indicates the mean ± SE range for hFF incubated with culture medium (a, closed circles). The effects of unpurified G/LCM on basal FSH are not shown because of the high concentration of recombinant human FSH content which interferes with the rat FSH immunoassay.