A群脑膜炎球菌荚膜多糖排溢法ELISA的建立及初步应用

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。

Development and preliminary application of excluded ELISA for group A meningococcal polysaccharide

Objective To develop,verify and preliminarily apply an excluded ELISA method for group A meningococcal polysaccharide(GAMP).Methods Anti-GAMP-HRP was prepared by modified periodate oxidation method.An excluded ELISA method was developed using anti-GAMP monoclonal antibody as coating antibody,and anti-GAMP-HRP as enzyme-labeled antibody.The working concentrations of coating and enzyme-labeled antibodies were optimized by checkerboard titration,and the developed method was verified for specificity,sensitivity and reproducibility.A series of GAMP-TT samples purified by chromatography were determined for GAMP antigen activity by the developed method,and the results were compared with those by traditional one-step and two-step ELISA methods.Results The optimal concentrations of coating and enzyme-labeled antibodies for development of excluded ELISA were 20 μg / ml and 1 ∶ 128 respectively.The developed method showed high specificity,of which the sensitivity was equivalent to those of one-step and two-step ELISA methods,while the intra-and inter-coefficients of variation were equivalent to those of one-step ELISA method.The determination results of GAMP antigen activities of GAMP-TT samples by the developed method were basically consistent with those by two-step ELISA method.Conclusion The procedure for excluded ELISA was similar to that for one-step ELISA method,while was significantly simplified as compared with that for two-step ELISA method.The correction efficiency of Hooks effect in one-step ELISA by the developed method was identical to that by two-step ELISA method.