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 biospecific adsorbents suitable for affinity chromatography to fractionate antibodies of different affinity and specificity from a polyclonal antiserum to progesterone-11 alpha-hemisuccinate-BSA. From an affinity column of progesterone-11 alpha-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)-11 alpha-ol-11 alpha-hemisuccinate; 4-pregnene-11,20 beta-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 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.
Parini
C
C
Istituto di Chimica degli Ormoni-CNR, Milan, Italy.
Bacigalupo
M A
MA
Colombi
S
S
Corocher
N
N
Baggiani
C
C
Giraudi
G
G
D000042:Absorption; D000906:Antibodies / Q000032:analysis*; D002846:Chromatography, Affinity; D006801:Humans; D006863:Hydrogen-Ion Concentration; D011374:Progesterone / Q000276:immunology*; D012997:Solvents
D000906:Antibodies; D012997:Solvents; D011374:Progesterone