Isolation of antibodies of improved specificity after fractionation on antigen-sepharose affinity columns of antisera to bovine serum albumin conjugates of C-3- and C-7-linked (O-Carboxymethyl)Oximino- and hemisuccinamido derivatives of 5α-dihydrdtestosterone

Abstract

Rabbit antisera to bovine serum albumin (BSA) conjugates of 3-(O-carboxymethyl)oximino-, 7-(O-carboxymethyl)oximino- and 7β-hemi-succinamido derivatives of 5α-dihydrotestosterone (DHT) were applied to four affinity columns bearing respectively these three antigens and a fourth 3β-hemisuccinamido-5α-androstan-17β-ol-BSA antigen as ligands. The antibodies retained on the columns were totally desorbed by an excess of DHT, but in DHT-bound form, whereas 1M mh 4oh and electrophoretic elution allowed a recovery of 60% of the retained antibodies in unbound form. The antibody fractions (40%) remaining on the columns after NH 4OH or electrophoretic elution were totally recovered by addition of DHT following the electrophoretic elution only. All the DHT-bound fractions were dissociated by dialysis but with a 70% loss of binding activity. The association constants for DHT of most of the antibody fractions were similar to those of the crude antisera (Ka ~ 10 10M −1), with the exception of the antibodies recovered from the antibody fractions resistant to electrophoretic elution which had higher affinities (Ka ~ 2.0 to 30 × 10 10M −1). The specificity charts of the antisera were in some cases considerably modified after fractionation, according to the choice of the ligand employed in the affinity columns as well as of the elution methods. The lowest cross-reactions with testosterone were observed after elution with 1M NH 4OH (17–20%) or electrophoresis (23–25%) of the anti-7-(O-carboxymethyl)oximino-DHT antisera fractions retained on 3β-hemisuccinamido-5α-androstan-17β-ol-BSA-Sepharose columns.