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Characterization and Antibody-Binding Capacity of Streptococcal Group a Carbohydrate Haptens¹

From the Department of Pediatrics, University of Florida College of Medicine, Gainesville, Florida 32601

Abstract

Tritium-labeled group A streptococcal carbohydrate haptens have been prepared by incubation of acid hydrolysates of group A carbohydrate with ³H-borohydride. Isolation of hapten was achieved by column and thin layer chromatography. These procedures yielded an antigenically univalent hapten, " hapten," with an estimated molecular weight of 1100 daltons and a rhamnose: glucosamine molar ratio of 6:1. The hapten inhibited the binding of A-carbohydrate with homologous antiserum. Comparative antibody-binding studies were performed with group A streptococcal rabbit antibody preparations by using the hapten and a synthetic ³H-p-nitrophenyl--N-acetylglucosamine (³H-pNøNAG) hapten. These studies demonstrate that average association constants (K₀) obtained with the hapten are one to two orders of magnitude higher than the K₀ determined with ³H-pNøNAG. Significantly less nonspecific binding was observed with the hapten. Binding studies with the hapten enabled the determination of K₀ for a number of antibody preparations which failed to evidence specific binding with ³H-pNøNAG. The availability of the hapten makes more feasible the reliable determination of K₀ for antibody prepared under various conditions.

Footnotes

¹ This work was supported in part by National Institutes of Health Grants AI50428 and AI09645; by the Developmental Physiology Training Grant, NIH T1-HD0054; and by the Heart Association of Palm Beach County, and by the Kirkegaard Fellowship of the Broward County Heart Association, chapters of the Florida Heart Association.