Separation of Specific from Nonspecific Anti-FSH Antibody by Affinity Chromatography on Sepharose-HCG

Abstract

To enhance the specificity of a pool of anti-FSH sera with respect to HCG and LH crossreactivity, affinity chromatography was performed on a Sepharose-HCG column. In serial studies anti-FSH pool #9 was added to the column and eluted first with buffer and then with guanidine HC1. In each study a protein peak (I) was evident in the first fraction collected after application of the serum to the column and this antibody was capable of binding FSH-131I tracer but not LH-131I in the double antibody radioimmunoassay. Following application of guanidine HC1, a second protein peak was eluted: this antibody was capable of binding both FSH-131I and LH-131I. Dose response curves of IRP #2 HMG and HCG obtained with FSH-131I tracer and unprocessed pool #9 antibody showed high potency and sensitivity to FSH but marked nonspecificity when tested with HCG. In contrast, peak (I) antibody under comparable conditions proved to be low in potency and relatively insensitive to FSH but highly specific. These results were similar to those obtained with the unabsorbed pool #9 antibody when HCG was added to each assay tube in the conventional manner of absorption. These data provide a direct demonstration of the existence of two different populations of anti-FSH antibodies: one specific for only FSH and the other nonspecific, binding both FSH and LH. (Endocrinology90: 302, 1972