pH gradient elution of human IgG1, IgG2 and IgG4 from protein A-sepharose

Pooled human serum having a normal IgG subclass content was chromatographed on a column of protein A-Sepharose. The immunoglobulins that bound to the column at pH 7.0 were eluted with a pH gradient generated by 3 equal volumes of citrate/phosphate buffer at pH 5.0, 4.5 and 2.2. The elution pattern consisted of two major overlapping peaks centered 0.4 pH units apart in the pH gradient; on average the first peak centered at pH 4.7, and the second centered at pH 4.3. Upon second passage of each component, single major peaks centered at the appropriate pH were seen. The subclass distributions of the re-chromatographed peaks were as follows: the high pH-eluting IgG contained less than 1% IgG1, 95% IgG2 and 5% IgG4; the low pH-eluting IgG contained 90% IgG1, 6% Ig2 and 5% IgG4. IgG3 does not bind to protein A and was thus absent from the pH gradient fractions. Chromatography on protein A-Sepharose provides a means for separating normal human IgG1 from IgG2 and may therefore prove useful as an additional tool for studying the relative biological role of these IgG subclasses.