The pH-dependent binding of goat IgG1 and IgG2 to protein A-Sepharose

Abstract

Hjelm et al. (1972) introduced protein A-Sepharose to separate protein A-reactive from nonreactive immunoglubulins. In the conventional use of this procedure, all bound immunoglobulins are eluted in a single step with 1 M acetic acid. We used instead a pH gradient to elute column-bound goat immunoglobulins and resolved two major IgG components; one eluting with a peak at pH 6.7 and the other at pH 5.8. We investigated the relationship between these components and the known IgG subclasses which, in the goat, are separable by DEAE-cellulose chromatography. Protein A-Sepharose chromatography of DEAE fractions indicates that the second DEAE peak, in which IgG1 is known to predominate, corresponds to the IgG eluting at pH 6.7 and that the first DEAE peak, in which IgG2 predominates, corresponds to the IgG eluting at pH 5.8. Commercial anti-bovine IgG1 reacted strongly upon double immunodiffusion with the pH 6.7 IgG and only slightly with the pH 5.8 IgG; anti-bovine IgG2, however, failed to react with either sub-population of goat IgG.