Benzoquinone activation of Cryptococcus neoformans capsular polysaccharide for construction of an immunoaffinity column.
p-benzoquinone was used as a two-step coupling reagent for preparation of an immunoaffinity absorbent in which the capsular polysaccharide of Cryptococcus neoformans was linked to an agarose gel. Cryptococcal polysaccharide is a difficult subject for chemical modification because it contains immunogenic O-acetyl groups which are sensitive to alkaline hydrolysis. The polysaccharide was activated by treatment with benzoquinone. The 'activated' polysaccharide was reactive with amino groups on AH-Sepharose. A pH of 8-9 was optimal for activation of the polysaccharide. Once activated, the polysaccharide was reactive with a model substrate, L-alanine-4-nitroanilide, over a pH range of 6-10. Since the O-acetyl groups are hydrolyzed at pH above 8.0, an activation pH of 8.0 and a coupling pH of 7.5 were used to prepare the conjugated gel. The polysaccharide immunoaffinity column was used successfully for isolation of rabbit antibodies to cryptococcal polysaccharide.
D016227:Benzoquinones; D002846:Chromatography, Affinity / Q000379:methods*; D003454:Cryptococcus / Q000276:immunology*; D003455:Cryptococcus neoformans / Q000276:immunology*; D006863:Hydrogen-Ion Concentration; D005779:Immunodiffusion; D011135:Polysaccharides, Bacterial / Q000276:immunology; D011809:Quinones; D012685:Sepharose
D016227:Benzoquinones; D011135:Polysaccharides, Bacterial; D011809:Quinones; C004532:quinone; D012685:Sepharose