Cherniak R, Cheeseman M M, Reyes G H, Reiss E, Todaro F
Department of Chemistry, Georgia State University, Atlanta 30303.
Diagn Clin Immunol. 1988;5(6):344-8.
A sensitive enzyme-linked immunosorbent assay (ELISA) to measure antibodies against capsular polysaccharide was developed, based on the enhanced binding of polysaccharide to polystyrene microtitration plates. The wells of the microtitration plate were primed with an adipic acid dihydrazide derivative of bovine serum albumin (AH-BSA) (100 micrograms/mL, 0.01 M NaPO4-0.14 M NaCl, pH 7.2 (PBS]. Capsular polysaccharide, the glucuronoxylomannan of Cryptococcus neoformans serotype A, was oxidized with NaIO4 for 5 min; the reaction was then quenched with ethylene glycol. The partially oxidized polysaccharide was dialyzed vs. PBS, and its concentration was adjusted to 50 micrograms/mL with PBS. This solution (100 microL/well) was covalently bound to the AH-BSA primed microtitration plates through formation of a Schiff base between the hydrazide group on the AH-BSA and the aldehyde groups on the polysaccharide. Antimouse IgG-alkaline phosphatase conjugate was used in an indirect ELISA to measure captured murine monoclonal antibodies directed against glucuronoxylomannan. Mean absorbances, after 15 min, were 0.13 in negative control wells, and greater than 0.7 in test wells. No intermediate steps were required to block nonspecific binding of antibody.
基于多糖与聚苯乙烯微量滴定板结合能力的增强，开发了一种用于检测抗荚膜多糖抗体的灵敏酶联免疫吸附测定（ELISA）方法。微量滴定板的孔先用牛血清白蛋白己二酸二酰肼衍生物（AH-BSA）（100微克/毫升，0.01M磷酸钠-0.14M氯化钠，pH7.2（PBS））包被。新型隐球菌A血清型的荚膜多糖葡糖醛酸木甘露聚糖用高碘酸钠氧化5分钟；然后用乙二醇终止反应。将部分氧化的多糖对PBS进行透析，并用PBS将其浓度调整至50微克/毫升。该溶液（100微升/孔）通过AH-BSA上的酰肼基团与多糖上的醛基团之间形成席夫碱，共价结合到用AH-BSA包被的微量滴定板上。抗小鼠IgG-碱性磷酸酶偶联物用于间接ELISA中，以检测针对葡糖醛酸木甘露聚糖的捕获鼠单克隆抗体。15分钟后，阴性对照孔的平均吸光度为0.13，测试孔的平均吸光度大于0.7。无需中间步骤来阻断抗体的非特异性结合。
