FRACTIONATION OF AN ANTISERUM TO PROGESTERONE BY AFFINITY-CHROMATOGRAPHY - EFFECT OF PH, SOLVENTS AND BIOSPECIFIC ADSORBENTS

Several progesterone-AH Sepharose 4B matrices were prepared as biospec ific adsorbents suitable for affinity chromatography to fractionate an tibodies of different affinity and specificity from a polyclonal antis erum to progesterone-11 alpha-hemisuccinate-BSA. From an affinity colu mn of progesterone-11 alpha-hemisuccinate-AH Sepharose 4B no antibodie s can be eluted, el en with glycine buffer (pH 2.6) and 30% of 2-metho xyethanol, The use of biospecific adsorbents, prepared by coupling wit h AH Sepharose 4B progesterone derivatives [5-pregnene-3,20-dione di(e thyleneacetal)-11 alpha-ol-11 alpha-hemisuecinate; 4-pregnene-11,20 be ta-diol-3-one-11 alpha-hemisuccinate 20 beta-benzoate; progesterone-3- carboxymethyloxime] having a low cross-reactivity with the antiserum, makes the elution of various antibody fractions of variable affinity a nd specificity possible. 2-Methoxyethanol or N,N-dimethylformamide gra dients, in acetate or TRIS buffer, Here equally efficient for fraction ating the antiprogesterone serum, while a decreasing pH gradient was l ess effective and eluted antibody fractions that were further separate d into various binding components by a solvent gradient. Antibodies el uted from the affinity columns by an eluent containing a high solvent concentration have affinities higher than antibodies eluted at lower s olvent concentration.