Introduction. We have previously used a synthetic phage display library to derive single chain (ScAbs) antibodies which bind GnSAF bioactivity in-vitro. However, recovery of GnSAF using the ScAbs, which tended to aggregate and become unstable, was limited. We have therefore engineered these ScAbs into full-length human IgG forms.
Methods. CDR regions from soluble expression plasmids for two different GnSAF-recognising ScAbs were amplified by PCR. Two unique cloning sites: PstI/BstEII for the heavy chain and SacI/XhoI for the light chain were then introduced. PCR products were separately subcloned into mammalian expression vectors for heavy and light chains and the constructs co-transfected into transient cells (CHO-K1). An ELISA utilising antibodies against both heavy and light chains was used to monitor production of intact IgG molecules. The IgGs were collected and tested for recognition of GnSAF bioactivity in human follicular fluid (hFF) by (1) incubating 2 micrograms of IgG with 250 microlitres of hFF for 2 hours at 37 degC in bioactivity-blocking experiments and (2) incubating 75 microlitres of hFF with 1 microgram IgG immobilised/well in 96 well plates for 2 hours at 37 degC in bioactivity binding-experiments. The hFF was then recovered and tested for GnSAF bioactivity using duplicate rat pituitary cell bioassays.
Results. GnSAF bioactivity was reduced by between 1.4 and 1.8 fold in hFF incubated with IgGs in blocking experiments. However, in repeated bioassay of hFF incubated with immobilised IgGs in binding experiments, GnSAF bioactivity remaining in the hFF was reduced between 2.1 and 3.6 fold, demonstrating significant recognition and depletion of GnSAF bioactivity.
Conclusions. Engineering the ScAbs into human IgGs improves the properties of the antibodies, making stable transfection for large-scale antibody production worthwhile. The human IgGs produced in this study recognised GnSAF bioactivity and should prove suitable for subsequent immunopurification of this elusive molecule.
08 - 11 Apr 2002
British Endocrine Societies