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目的建立A群脑膜炎球菌荚膜多糖(Group A meningococcal polysaccharide,GAMP)排溢法ELISA,并进行验证及初步应用。方法采用改良过碘酸盐氧化法制备抗GAMP-HRP。以抗GAMP单克隆抗体包被酶标板,抗GAMP-HRP作为酶标抗体,建立检测GAMP的排溢法ELISA。采用棋盘滴定法确定包被抗体及酶标抗体的最佳工作浓度,并对其特异性、敏感性及重复性进行验证。采用建立的排溢法ELISA检测GAMP-TT系列层析样品中的GAMP抗原活性,并与传统一步法ELISA及二步法ELISA进行比较。结果建立的排溢法ELISA的包被抗体最佳浓度为20μg/ml,酶标抗体最佳工作浓度为1∶128。该方法的特异性良好,敏感性与一步法和二步法相当,批内和批间变异系数与一步法相当;该方法检测GAMP-TT层析样品中的GAMP抗原活性的结果与二步法ELISA基本一致。结论排溢法ELISA的操作步骤与一步法ELISA大致相似,但较二步法ELISA显著简化,其对一步法ELISA中Hooks效应的纠正效果与二步法ELISA相同。
Objective To establish a Group A meningococcal polysaccharide (GAMP) ELISA for overflow and validation and preliminary application. Methods Anti-GAMP-HRP was prepared by modified periodate oxidation method. Anti-GAMP monoclonal antibody coated ELISA plate, anti-GAMP-HRP as an ELISA antibody, the establishment of the detection of GAMP by overflow ELISA. The optimum concentration of coating antibody and enzyme-labeled antibody was determined by chessboard titration. The specificity, sensitivity and reproducibility of the antibody were detected. The established GAMP-ELISA was used to detect GAMP antigen activity in GAMP-TT series of samples, and compared with the traditional one-step ELISA and two-step ELISA. Results The optimal concentration of coated ELISA for ELISA was 20μg / ml, and the optimum working concentration of enzyme-labeled antibody was 1:128. The specificity of the method was good and the sensitivity was comparable to the one-step and two-step methods. The coefficient of variation (CV) within the batch and the batch was comparable to that of the one-step method. The method was used to detect the GAMP antigen activity in the GAMP- ELISA basically the same. Conclusions The ELISA procedure of the overflow ELISA is similar to that of the one-step ELISA, but it is significantly simplified compared with the two-step ELISA. The correct effect of the Hooks effect in the one-step ELISA is the same as that of the two-step ELISA.