SPECIFICITY IMPROVEMENT OF A RECOMBINANT ANTITESTOSTERONE FAB FRAGMENT BY CDRIII MUTAGENESIS AND PHAGE DISPLAY SELECTION

The monoclonal antibodies so far developed by hybridoma technology hav e not had high enough specificity or affinity to distinguish the close ly related steroid hormones in routine clinical assays, We have employ ed random mutagenesis and phage display approaches to improve the spec ificity of one anti-testosterone monoclonal antibody (3-C4F5), The aff inity of the antibody is 0.3x10(9) M-1 and the cross-reactivities with most of the related steroids are low. However, the antibody cross-rea cts about 1% with dehydroepiandrosterone sulfate (DHEAS) and owing to the high DHEAS serum concentration this is about 1000-fold too high fo r clinical immunoassays, The complementarity-determining regions (CDRs ) of the heavy and light chains, which were predicted by molecular mod elling to be in close contact with the testosterone (TES) ligand, were randomized and mutant Fab libraries were cloned into a phagemid vecto r. Binders were selected by a competitive panning procedure. By combin ing the identified light and heavy chain CDRIII mutations the TES affi nity was preserved at the wad-type level but DHEAS cross-reactivity wa s decreased to 0.03%, An important finding was that by the competitive panning procedure the overall binding specificity of the 3-C4F5, anti body was refined, since the cross-reactivities to related steroids wer e also significantly decreased in the combined mutant.