人血清抗肺炎链球菌荚膜多糖抗体IgG定量ELISA方法的建立与验证

Establishment of a quantitative ELISA assay recommended by WHO for the detection of human IgG antibodies specific for Streptococcus pneumoniae capsular polysaccharides （ Pn PS ELISA）

目的建立WHO推荐的检测人血清抗肺炎链球菌 （ Streptococcus pneumoniae ）荚膜多糖抗体IgG定量ELISA（PnPSELISA）。方法依照WHO推荐的Pn PS ELISA标准操作要求，优化荚膜多糖的包被浓度，筛选合格的ELISA板和二抗；在检测WHO参考实验室提供的质量控制血清验证实验条件的基础上，分别在WHO参考实验室和兰州生物制品研究所有限责任公司（LIBP）实验室检测16份由LIBP制备的质控血清中抗13种肺炎链球菌血清型（1、3、4、5、6A、6B、7F、9V、14、18C、19A、19F、23F）的荚膜多糖IgG抗体浓度。同时，在LIBP实验室建立了实验室内部质控血清907的检测范围，并评价了LIBP实验室实验间检测值的精确度。结果在WHO参考实验室和LIBP实验室分别检测的16份LIBP质控血清的抗13种肺炎链球菌血清型荚膜多糖IgG抗体浓度具有良好的相关关系（slope=0．94，r=0．97，P〈0．05）。LIBP实验室的检测值有81％落在WHO参考实验检测值±40％范围内，符合评价实验室之间所用方法的75％的数据在±40％的检测值范围内的标准。建立了LIBP实验室内部质控血清907的抗13种肺炎链球菌血清型荚膜多糖抗体IgG的检测值范围。LIBP实验室质控血清的各血清型IgG抗体浓度的实验间检测值的变异系数（CV）均〈30％。结论LIBP实验室成功建立了WHO推荐的检测人血清中抗肺炎链球菌荚膜多糖抗体IgG定量ELISA方法，并确定了LIBP实验室日常用质控血清907的检测值范围。

Objective To establish a standardized quantitative enzyme-linked immunosorbent as- say （ELISA） recommended by WHO for the detection of human IgG antibodies specific for Streptococcus pneumoniae capsular polysaccharides （Pn PS ELISA）. Methods According to the WHO recommended standard Pn PS ELISA protocol, capsular polysaccharide concentrations of 13 serotypes （1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F） of Streptococcus pneumonia for coating were optimized; the ELISA plates and AP conjugated goat anti-human IgG antibody for the detection were experimentally selected. Using the established assay parameters assured by testing quality control standards （QCs） provided by WHO refer- ence laboratory, a panel of 16 LIBP （ Lanzhou Institute of Biological Products Co. Ltd. ） QCs were measured for comparison analysis between WHO reference laboratory and LIBP laboratory. In the meantime, the range of IgG concentrations of an internal QC panel 907 for 13 serotypes of pneumococcal capsular polysaccharide were established for routine QC work in LIBP laboratory, and inter-assay precision within LIBP laboratory was assessed as well. Results The standardized Pn PS ELISA assay established in LIBP laboratory met the criteria required by WHO. There was a high correlation between the data collected by WHO reference labora- tory and LIBP laboratory （ slope = 0.94, coefficient of correlation r = 0. 97, P〈0.05 ）. Eighty one percent ofIgG concentrations measured by LIBP laboratory were within the range of ＋40% of those measured by WHO reference laboratory, which met the criteria of 75% data falling within the ambit of ＋-40% of assigned values commonly used for comparison. The ranges of IgG concentration in LIBP QC 907 for 13 serotypes had been established. The inter-assay precision in LIBP laboratory was high with coefficiency of variation （CV） less than 30%. Conclusion LIBP laboratory has successfully established the standardized ELISA recommended by WHO for quantitative detection of human IgG antibodies against pneumococcal capsular polysaccharides （ Pn PS ELISA）. The range of IgG concentrations in LIBP QC 907 for 13 seretypes of pneumococcal capsular polysaccharide are also established for routine quality-control practice.