13价肺炎球菌多型调理吞噬试验方法的建立及验证

Establishment and validation of multiplexed opsonophagocytic killing assay for 13-valent pneumococcus

目的建立标准化的针对肺炎链球菌荚膜多糖特异性抗体的多型调理吞噬试验(multiplexed opsonophagocytic killing assay,MOPA)方法,并进行验证。方法参照有关组织研究和发布的MOPA实验操作规范(UAB-MOPA),通过菌种鉴定、HL60细胞分化规律的确定、补体筛选、质控血清质控范围的确定,建立本实验室的MOPA实验方法,并对方法进行特异性、线性、精密性、准确性以及耐受性验证。结果工作菌种的所有指标均符合UAB-MOPA操作规范的要求;HL60细胞在分化后3~6 d均可作为工作细胞使用;27531和84321N两批补体可作为工作补体使用;建立了09CS、QC2、B、C和F 5个质控血清的质控范围。方法的特异性、线性、精密性、准确性及耐受性均符合预定指标。结论成功建立了标准化的13价肺炎球菌多型MOPA方法,并进行了验证。

Objective To establish and validate a standardized multiplexed opsonophagocytic killing assay（MOPA） for measuring functional antibody specific to 13-valent pneumococcal capsular polysaccharide. Methods A standardized MOPA was established by preparation of working bacterial seeds, identification of serotype, optimizing culture and differentiation of HL60 cells, complement screening and establishment of quality controls based on the protocol published by WHO and US NIH Bacterial Respiratory Pathogen Reference Laboratory, Department of Pathology, University of Alabama at Birmingham（UAB）（UAB-MOPA for short）, and verified for specificity, linearity, precision, accuracy as well as robustness according to the guidance of International Conference Harmonization（ICH）. Results All the indexes of prepared working bacterial seeds met the requirements in UAB protocol. The HL60 cells after differentiation for 3 - 6 d could be used as effector cells for MOPA. The complements with Lot No. of 27531 and 84321 could be used as working complements. The quality control ranges of five control serum samples, i. e. 09 CS, QC2, B, C and F, were confirmed. The specificity, linearity, precision, accuracy and robustness of MOPA met the specified criteria. Conclusion A standardized MOPA on 13-valent pneumococcus was successfully established and validated.