Fractionation of an antiserum to progesterone by affinity chromatography: effect of pH, solvents and biospecific adsorbents

Analyst Pub Date: DOI: 10.1039/AN9952001153

Abstract

Several progesterone–AH Sepharose 4B matrices were prepared as biospecific adsorbents suitable for affinity chromatography to fractionate antibodies of different affinity and specificity from a polyclonal antiserum to progesterone-11α-hemisuccinate–BSA. From an affinity column of progesterone-11α-hemisuccinate–AH Sepharose 4B no antibodies can be eluted, even with glycine buffer (pH 2.6) and 30% of 2-methoxyethanol. The use of biospecific adsorbents, prepared by coupling with AH Sepharose 4B progesterone derivatives [5-pregnene-3,20-dione di(ethyleneacetal)-11α-ol-11α-hemisuccinate; 4-pregnene-11,20β-diol-3-one-11α-hemisuccinate 20β-benzoate; progesterone-3-carboxymethyloxime] having a low cross-reactivity with the antiserum, makes the elution of various antibody fractions of variable affinity and specificity possible. 2-Methoxyethanol or N,N-dimethylformamide gradients, in acetate or TRIS buffer, were equally efficient for fractionating the antiprogesterone serum, while a decreasing pH gradient was less effective and eluted antibody fractions that were further separated into various binding components by a solvent gradient. Antibodies eluted from the affinity columns by an eluent containing a high solvent concentration have affinities higher than antibodies eluted at lower solvent concentration.

Recommended Literature