Isolation of antibodies to steroid hormones by affinity chromatography on antigen-linked sepharose: An efficient electrophoretic elution procedure

An electrophoretic elution procedure of antibodies retained on affinity columns is described. It afforded a 60% recovery of the binding activity of a high affinity (Ka ～ 10¹⁰ M^?1) antiserum to 5α-dihydrotestosterone retained on antigen-linked Sepharose 4B affinity columns. These purified unbound antibodies, (Ka ～ 10¹⁰ M^?1) when applied again on identical antigen-linked affinity columns, were all retained and totally recovered after a new electrophoretic elution. Comparable results were obtained by elution with 1M NH₄OH.The residual 40% binding activity remaining on the antigen-linked Sepharose gel after electrophoretic elution was totally recovered by elution with an excess of 5α-dihydrotestosterone. It corresponded to antibodies of higher affinity (Ka ～ 10¹¹ M^?1). On the other hand the residual 40% fraction of antibodies resistant to NH₄OH elution was denaturated.