Heterogeneity of Antisera to Human Growth Hormone,Demonstrated by the Immunofluorescence Technique ∗

Abstract

Earlier studies (1) showed the fluorescein-conjugated antisera to human growth hormone (HGH) localized in acidophils of human, rat, and bovine pituitary glands, and that the fluorescence, which was inhibited when the specific antigen or the unconjugated antisera was applied before staining, was specific.

During inhibition studies, one batch of anti-HGH (titer, 1/12,000: batch HGH-5) inhibited fluorescence caused by another batch of fluorescein-conjugated anti-HGH (titer 1/25,000; Hg-1) in the human but not the rat pituitary gland. Inhibition was repeated with anti-HGH batch HGH-5 and followed by 1:2 and 1:3 dilution of fluorescein-conjugated anti-HGH Hg-1 (which still produced fluorescence); again, fluorescence was not inhibited in the rat pituitary. However, when conjugated and unconjugated anti-HGH Hg-1 from the same batch were used, staining was completely inhibited in both rat and human pituitaries. The present studies were carried out to determine the cause of this discrepancy.

Materials and Methods. The immunofluorescent-staining method used was described previously (2). Preparation of antisera to HGH from 9 rabbits and 1 guinea pig were used for inhibition experiments. Fluorescein-conjugated anti-HGH Hg-1 and Hgh-5, which were used as staining antisera, produced bright fluorescence in rat, human, and bovine pituitaries. The titers were measured by the bis-diazotized benzidine (BDB)-hemagglutination method.

Results. Two antisera did not inhibit staining in any experiment (Table I and II). In human pituitaries, all others produced inhibition of variable degree. In rat, complete inhibition occurred with only two antisera (Table I). In bovine pituitaries, complete inhibition occurred with only one antiserum. Six antisera were conjugated with fluorescein (Table III); since the titer of antisera that failed to stain was unchanged after conjugation, activity was not destroyed during this procedure. Indirect staining, using fluorescein-conjugated sheep anti-rabbit gamma globulin, showed some degree of fluorescence with all eight antisera (Table IV).

These experiments indicate that different rabbits may produce different antisera to a single antigen.