人血清抗肺炎球菌荚膜多糖的血清特异性IgG抗体ELISA检测方法的适用性验证

Validation study on the applicability of detecting anti-pneumococcus capsular polysaccharide IgG antibodies in human serum

目的 验证ELISA中使用国产酶标板和国产细胞壁C多糖(cell wall polysaccharides, CWPS)替代现用的进口酶标板(Greiner 655080)和丹麦国家血清研究所(Statens Serum Institut, SSI)的CWPS,用于检测肺炎球菌疫苗免疫人血清中抗荚膜多糖IgG抗体的可行性和适用性。方法 执行PnPS ELISA,以现用SSI的CWPS和Greiner 655080酶标板为对照,分别用国产CWPS和国产酶标板(96BTA),检测相同样品中24个肺炎球菌血清型别的抗荚膜多糖IgG抗体,对检测结果进行系统分析。结果 (1)国产酶标板的适用性验证结果:国产及进口酶标板检测质控血清09CS的24个血清型的荚膜多糖IgG抗体含量完全一致,林氏一致性相关系数(lin's concordance correlation coefficient,r_(c))为0.990 3;20份实验室内质控血清的结果为高度一致,r_(c)为0.958 0;24个血清型的样本最低检测限(lower limit of detection, LLQ)在0.008~0.072μg/mL,进口酶标板为0.010~0.079μg/mL之间。(2) CWPS的适用性验证结果:国产CWPS的生化检测结果和~1H核磁共振波谱图(nuclear magnetic resonance spectroscopy, NMR)与现用SSI的CWPS基本一致;国产和进口CWPS吸收后检测质控血清09CS的24个肺炎球菌血清型特异性荚膜多糖抗体值结果完全一致,r_(c)为0.999 0;国产及进口CWPS检测所选10对免前及免后血清的检测结果为高度一致,免前血清的r_(c)为0.953 0,免后血清的r_(c)为0.972 0。结论 经验证,国产CWPS可替代进口CWPS,国产酶标板可替代进口酶标板,用于肺炎球菌疫苗临床血清特异性抗体含量的检测。

Objective To verify the feasibility of using domestic microplate and domestic cell wall polysaccharides(CWPS) to replace currently imported enzyme plates(Greiner 655080) and the CWPS of Statens Serum Institut(SSI) for detecting anti-capsular polysaccharide IgG antibodies in human serum of pneumococcal vaccines. Methods Domestic microplate and CWPS were used separately to test anti-capsular polysaccharide IgG antibodies of 24 pneumococcal serotypes by PnPS ELISA method, then analyze the results with currently used CWPS of SSI and Greiner 655080 microplate as the control group. Results(1) The content of anti-capsular polysaccharide IgG antibodies of 24 serotypes was completely consistent with which tested by imported microplate, and the lin's concordance correlation coefficient(r_(c)) was 0.990 3;the consistency was strong when testing the same 20 laboratory quality-control sera, with r_(c) of 0.958 0;the lower limit of detection(LLQ) of the 24 serotypes was between 0.008 and 0.072 μg/mL,while the LLQ of currently used microplate was 0.010-0.079 μg/mL.(2) The biochemical test results and ~1H Nuclear Magnetic Resonance Spectroscopy(NMR) of domestic CWPS were basically consistent with the current SSI CWPS;the test results of quality-control serum 09CS were completely consistent, the r_(c) was 0.999 0. The test results of 10 pairs of sera(before and after immunization)detected by SSI CWPS and domestic CWPS,the consistency was strong, the r_(c) of pre-immunization serum was 0.953 0,and the r_(c) of post-immunization serum was 0.972 0. Conclusion Domestic CWPS and ELISA plates can replace imported ELISA plates and SSI's CWPS for detecting anti-capsular polysaccharide IgG antibodies in human serum of pneumococcal vaccines.