肺炎链球菌型特异性IgG抗体定量ELISA方法建立与验证

白霜; 王建; 周珊珊; 赵伟; 张鹏; 吴疆; 吕敏; 郑群

doi:10.11847/zgggws1132585

肺炎链球菌型特异性IgG抗体定量ELISA方法建立与验证

Establishment and verification of a quantitative ELISA assay for detection of human serotype specific IgG against Streptococcus pneumoniae

摘要

摘要:
目的建立WHO推荐的肺炎链球菌血清型特异性IgG抗体定量ELISA检测方法。
方法参照WHO Pn PS ELISA操作手册，采用ATCC来源肺炎链球菌荚膜多糖4、6B、9V、14、18C、19F、23F型为包被抗原，以007sp为标准血清建立本实验室的人血清中肺炎链球菌血清型特异性IgG抗体定量ELISA检测方法。并用WHO校准血清盘（12/278）和内部质控血清PnG对所建方法准确性、稳定性进行验证。
结果确定了4、6B、9V、14、18C、19F、23F共7个血清型荚膜多糖的抗原包被浓度；建立了适用于本实验室的人血清中肺炎链球菌血清型特异性IgG抗体定量ELISA检测方法。12份WHO校准血清的验证结果显示，所测7个血清型IgG抗体中，均有 > 75 %的检测值落在WHO参比实验室赋值的 ± 40 %范围之内，总符合率达到86.9 %，相关性曲线斜率（slope） = 0.94，R² = 0.99，达到了WHO规定的室间评价要求。此外，连续20次的内部质控血清PnG的检测结果显示，不同批次检测值的变异系数（CV）均 ≤ 15 %，达到了WHO规定的室内评价要求。
结论本实验室成功建立WHO推荐的检测人血清中肺炎链球菌血清型特异性IgG抗体定量ELISA检测方法，并经验证所建方法结果准确，方法稳定，具备了开展疾病监测与疫苗效果评价应用要求。

Abstract:
Objective To establish a quantitative enzyme-linked immunosobent assay (ELISA) recommended by World Health Organization (WHO) for the detection of human serotype specific immunoglobulin G (IgG) against Streptococcus pneumoniae (Sp).
Methods According to WHO′s operation manual for Enzyme linked immunosobent assay ELISA for the quantitative of Streptococcus pneumoniae serotype specific IgG (Pn PS ELISA), we established a ELISA method to detect serotype specific IgG against Sp in human serum specimens by using Sp capsular polysaccharide 4, 6B, 9V, 14, 18C, 19F, and 23F (purchased American Type Culture Collection, ATCC) as coating antigens and pneumococcal capsular polysaccharide antibody standard serum 007 sp (from National Institute for Biological Standards and Control, NIBSC) as standard serum. The accuracy and stability of the established method were evaluated using WHO′s calibration serum panel and internal quality control serum PnG.
Results The concentration of 7 serotype specific coating antigens of Sp capsular polysaccharide (4, 6b, 9V, 14, 18C、19F and 23F) were determined and serotype specific IgG against Sp in human serum specimens were detected with the quantitative ELISA method established. More than 75% of measurements for 7 serotype specific Sp capsular polysaccharide IgG antibodies for 12 WHO calibration sera were within ± 40% of the measurement values assigned by reference laboratories of WHO, with an overall consistency rate of 86.9%, a correlation curve slope of 0.94, and an R² of 0.99, respectively. In addition, the results of 20 consecutive internal quality control serum test showed that the coefficient of variation (CV) of different batches were less than 15%, which met the requirements of WHO intra-laboratory evaluation.
Conclusion A quantitative ELISA method recommended by WHO for detection of serotype-specific Streptococcus pneumoniae IgG antibody in human serum was successfully established and verified. The accurate and stable method meet the requirements of disease surveillance and vaccine effect evaluation.

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