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Fractionation of Antibodies to the Pneumococcal Polysaccharides by Affinity Chromatography¹

From the Cardiac Unit, Massachusetts General Hospital and the Department of Medicine, Harvard Medical School, Boston, Massachusetts 02114

Abstract

Antibodies to pneumococcal polysaccharides type III and VIII were fractionated by use of cross-reacting immunoadsorbents and gradient elution with cellobiose and NaCl. The polysaccharides were first derivatized with p-nitrobenzyl bromide. After reduction of the nitro group, they were linked to bovine -globulin by diazotization and coupling. The protein-polysaccharide complex was then linked to activated sepharose to form an immunoadsorbent. In other experiments, immunoadsorbents were synthesized from polysaccharides which had first been either subject to aminoethylation or to amidation. In some instances, partial acid hydrolysis was carried out on the completed immunoadsorbent. These adsorbents had a high capacity for antibody. By use of several different immunoadsorbents, as well as varying elution programs with cellobiose and NaCl, complex antibody mixtures could be resolved with the isolation of components of unique electrophoretic mobility. Light chains isolated from these preparations were shown to have a single amino acid sequence at the N-terminus. It is apparent that many antibodies to differing determinants on the relatively simple polysaccharide antigens may be elicited and separated on the basis of these properties.

Footnotes

¹ This work was supported by Grants AI04967 and HE06664 from the National Institutes of Health.

² Present address: University of California, Berkeley, School of Public Health, Berkeley, California 94720.