Objective We aimed to immunopurify GnSAF, using antibodies with demonstrated GnSAF bioactivity-blocking/binding activity, in order to confirm 2 new candidate amino acid sequences for human ovarian GnSAF (Fowler et al. Mol Human Reprod 2002;8:823-832).
Methods Rat antiserum, raised against a 60-70 kDa band of partially purified GnSAF, was immobilised on anti-rat IgG Dynabeads. In addition, a human antibody, derived from a phage display antibody library and expressed by CHO-K1 cells, was immobilised on protein L agarose beads. Both antibodies were then used for 15 consecutive immunopurification steps with excess human granulosa-luteal cell-conditioned medium (GCM). In both cases 2 M NaI was used to elute the bound GCM proteins. The eluted fractions were bioassayed for GnSAF and inhibin bioactivities and the proteins separated by 1-D and 2-D gel electrophoresis. Spots and bands of interest were excised from the gels and identified by mass spectroscopic peptide mass mapping.
Results Both antibodies yielded preparations containing GnSAF (GnRH-induced LH secretion reduced by up to 74 ± 14% of control) but not inhibin (no significant suppression of basal FSH secretion). Peptide mass mapping of the 68 kDa band immunopurified with the rat polyclonal suggested up to 8 further candidate GnSAF molecules. However, peptide mass mapping of the 2-D gels only identified serum albumin, while protein content was too low to allow positive identification of minor protein spots.
Conclusions The immobilised antibodies were successfully used to immunopurify GnSAF, but further work is required to increase yields in order to allow identification of minor GCM proteins separated by 2-D gel electrophoresis. Funded by BBSRC.