肺炎球菌调理吞噬杀菌实验方法建立与应用  

Establishment and application of opsonophagocytic killing assay for detection of antibodies against streptococcus pneumoniae

作　　者：王建 白霜[1,2] 周珊珊 赵伟 张鹏[1,2] 吴疆 吕敏[1,2] 郑群 WANG Jian;BAI Shuang;ZHOU Shan-shan(Institute for Immunizations and Vaccines,Beijing Center for Disease Prevention and Control,Beijing 100013,China;不详)

机构地区：[1]北京市疾病预防控制中心免疫预防所,北京100013 [2]北京市预防医学研究中心 [3]首都医科大学基础医学实验教学中心

出　　处：《中国公共卫生》2021年第4期598-602,共5页Chinese Journal of Public Health

基　　金：北京市属医学科研院所公益发展改革试点项目(京医研2018–1)。

摘　　要：目的建立肺炎球菌多重调理吞噬杀菌实验,并在此基础上评价肺炎球菌疫苗免疫后效果。方法参照美国阿拉巴马大学多重调理吞噬杀菌实验方法(MOPA),构建7价肺炎球菌结合疫苗血清型的肺炎球菌的工作菌种库和HL-60细胞库,测定工作菌种库抗生素耐药性及稀释度,HL-60分化5~6 d后的表面标志表达量及活性,同时测定标准血清007SP和参考血清09CS的调理杀菌值;在方法建立的基础上,对疫苗免前免后获得的6对血清样本的功能性抗体进行检测。结果工作菌种稀释度均> 300倍;HL-60分化5~6 d的表面标志表达量CD11b> 80%,CD35> 70%,CD71 <15%,活细胞比例> 70%,凋亡细胞比例<21%;补体的非特异杀伤均<30%。标准血清007SP和参考血清09CS的调理杀菌值稳定在质控范围内;疫苗接种后,血清样本的杀菌曲线为S型曲线,并且调理杀菌值保持在较高水平。结论成功建立MOPA实验方法,为肺炎球菌疫苗免疫后的效果评价提供了方法依据。Objective To establish a multiplexed opsonophagocytic killing assay(MOPA) for detection antibodies against Streptococcus pneumoniae and to evaluate the immune effect of pneumococcal vaccines. Methods Assay stocks containing seven vaccine-related serotypes of Streptococcus pneumoniae and HL-60 cell bank were produced according to the MOPA method published by University of Alabama at Birmingham, USA. The antibiotic sensitivity, resistance and optimal dilution of assay stocks were determined. The cell surface markers and cell viability of HL-60 cells were monitored after differentiated for 5–6 days by using flow cytometer. Meanwhile, the opsonization indices(OI) of standard reference serum 007 SP and reference serum 09 CS against seven serotypes were measured with MOPA method. Then MOPA method was employed to detect functional antibody of 6 pairs of sera samples obtained before and after vaccination. Results The dilution factors of all assay stocks were > 300. The expressions of cell surface markers CD11 b, CD35 and CD71 were > 80%,> 70% and < 15%, respectively after differentiated for 5–6 days. The proportions of live cells and apoptotic cells were >70% and < 21%. The nonspecific killing rates of complement were < 30%. The OI of standard reference serum 007 SP and reference serum 09 CS were stable and were within the quality control ranges. After vaccine immunization, high antibody levels were detected in sera samples and the killing curves were of general sigmoid shape. Conclusion MOPA method was successfully established in this study, which provide the basis for evaluating the efficiency of pneumococcal vaccines.

关 键 词：调理吞噬杀菌试验 肺炎球菌 HL-60细胞 

分 类 号：R186[医药卫生—流行病学]