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目的建立人血清中抗A、C、Y、W135群脑膜炎球菌荚膜多糖Ig G抗体含量的ELISA定量检测方法,并进行验证。方法筛选适用的酶标板,优化抗原包被浓度,确定ELISA检测过程的关键条件,对建立的ELISA方法的特异性、线性、准确性、精密度、最低检测值和稳定性进行验证,并建立实验室质控血清的质控检测范围。结果酶标板的板内CV为9.8%,板间CV为16.4%,符合检测使用要求。免疫血清经多糖吸收后,群特异性抗体含量与同群多糖吸收剂量呈明显的剂量依赖关系。12份美国CDC质控血清中各群Ig G抗体含量与血清的稀释度呈负相关,相关系数(r)和斜率(slope)均接近-1,线性关系良好;检测结果与CDC公布数据一致性良好,一致性相关系数A、C、W135、Y群分别为0.912 5、0.939 6、0.963 7和0.920 6,准确度较好。实验室内部质控血清Men20中各群Ig G抗体含量试验内和试验间精密度分别小于10%和20%,精密度较好;各血清群别的CV值均<30%,稳定性较好;A、C、Y、W135群多糖Ig G抗体含量最低检测值分别为0.092、0.118、0.067和0.035μg/ml,质控血清的检测范围分别为:92.49~36.53、37.29~17.69、66.18~25.54和50.15~23.31μg/ml。结论建立了标准化的检测人血清中抗A、C、Y、W135群脑膜炎球菌多糖Ig G抗体含量的定量ELISA方法,并确定了实验室日常用质控血清Men20的质控检测值范围。
Objective To establish a quantitative ELISA method for the quantitative determination of anti-IgM antibodies against serogroup A, C, Y and W135 in human serum. Methods The ELISA plates were screened, the concentration of antigen coating was optimized, the key conditions of ELISA test were determined, and the specificity, linearity, accuracy, precision, minimum detection value and stability of the established ELISA method were validated and established Laboratory quality control serum control testing. Results The intraplate CV of the ELISA plate was 9.8%, and the inter-plate CV was 16.4%, which met the testing requirements. Immune serum after polysaccharide absorption, the group specific antibody content with the same group polysaccharide absorbed dose was a significant dose-dependent manner. The concentrations of Ig G antibody in each of the 12 American CDC-controlled sera were negatively correlated with the dilution of serum, and the correlation coefficients (r) and slopes were all close to -1 with good linearity. The results were consistent with those published by CDC The good and consistent correlation coefficients A, C, W135 and Y were 0.912 5, 0.939 6, 0.963 7 and 0.920 6, respectively. The accuracy was good. The intra-laboratory quality control serum Men-20 IgG antibody content within the test and the test precision were less than 10% and 20%, respectively, with good precision; the CV values of all serogroups were <30%, the stability was good ; The minimum detectable value of Ig G antibody of groups A, C, Y and W135 was 0.092,0.118,0.067 and 0.035μg / ml respectively, the detection range of quality control serum were 92.49 ~ 36.53, 37.29 ~ 17.69 and 66.18 ~ 25.54 And 50.15 ~ 23.31 μg / ml. Conclusion A standardized ELISA method was developed for the detection of anti-IgM antibodies against serogroup A, C, Y and W135 in human serum and the range of quality control tests for routinely controlled Men20 in the laboratory was established.