High resolution affinity chromatography of an anti-steroid antiserum by gradient elution with propionic acid

Chromatographic resolution of polyclonal antibodies is a challenging analytical problem and a successful method may have many applications. We have resolved polyclonal antibodies against testosterone 3-(O-carboxymethyl)oxime on a homologous testosterone-Sepharose matrix by using a ternary gradient system of propionate-1 M propionic acid-2 M propionic acid. Nineteen peaks were detected, sixteen of which were characterized by steroid binding activity and IgG assay. The relative affinities of the pooled fractions, determined by ELISA and by thiocyanate elution, correlated well with their retention times. The slopes of the displacement curves in antigen-immobilized testosterone ELISA increased with retention times of the fractions; testosterone required for 50% displacement being, 90, 5.8 and 24 ng/well for fraction I, fraction XVI and total antiserum, respectively. Cross-reactivity of the fractions towards 5alpha-dihydrotestosterone varied from 31 to 74% compared to 47% in the total antiserum.