A、C、Y、W135群脑膜炎奈瑟菌血清抗体特异性体外杀菌试验方法的建立及其验证  

Development and validation of serum bactericidal assay on Neisseria meningitides of serogroups A,C,W135 and Y

作　　者：乔瑞洁[1] 赵志强[1] 王浩[1] 王欣茹[1] 刘佳[1] 刘方蕾[1] 吴兵[1] 于旭博 林纪胜[2] 李亚南[3] 叶强[3] 谢贵林 

机构地区：[1]兰州生物制品研究所有限责任公司甘肃省疫苗工程技术研究中心,甘肃兰州730046 [2]美国阿拉巴马大学伯明翰分校病理系NIH细菌性呼吸道病原参考实验室,伯明翰35294 [3]中国食品药品检定研究院,北京100050 [4]中国生物技术集团公司,北京100029

出　　处：《中国生物制品学杂志》2014年第11期1435-1442,共8页Chinese Journal of Biologicals

基　　金：国家科技支撑计划(2008BAI66B01)资助

摘　　要：目的建立A、C、W135和Y群脑膜炎奈瑟菌血清抗体特异性体外杀菌试验方法 (serum bactericidal assay,SBA),并进行验证。方法对SBA中活菌收获时间、靶菌的制备、反应时间及补体进行优化,并确定质控血清Men10的质控范围。对优化后的方法进行特异性、线性、精密度验证。结果确定A600值为0.5~0.8时为收获细菌最佳收获时间;冻存菌种可替代新鲜制备菌种作为靶菌;最佳杀菌反应时间为60 min;17830和84321N批补体均可用于试验。质控血清Men10的质控范围A群为521~4 689,C群为1 047~9 423,W135群为1 779~16 008,Y群为1 416~12 741。A、C、W135和Y同群多糖吸收血清样品后能够抑制≥80%的同群SBA滴度,而异群多糖则<20%;血清样品稀释倍数与对应的SBA滴度呈显著负相关,斜率在-0.90^-1.10之间,Pearson相关系数在0.90~1.0之间,P均<0.001;同一实验内相同样品检测结果 CV≤25%,实验间相同样品的95%的检测结果落在检测值中位数3倍(一个稀释度)的范围内,CV≤40%,不同实验室间相同样品的检测结果至少有70%落在美国CDC公布结果的3倍(一个稀释度)范围内。结论建立了标准化的具国际间可比性的A、C、W135和Y群脑膜炎奈瑟菌SBA,该方法特异性、线性和精密度良好。Objective To develop and validate a serum bactericidal assay（SBA）on Neisseria meningitides of serogroups A,C,W135 and Y. Methods Harvest time,preparation of target bacteria,reaction time and complement in SBA were optimized,based on which the quality control range of Men10 was determined. The optimized method was verified for specificity,linearity and precision. Results The bacteria were harvested when the A600 value reached 0. 5 ~ 0. 8. Frozen bacteria could be used as target bacteria instead of fresh bacteria. The optimal reaction time was 60 min. Both the complements with lot No. of 17830 and 84321 N could be used for the assay. The quality control ranges were 521 ~ 4 689 for serogroup A,1 047 ~ 9 423 for serogroup C,1 779 ~ 16 008 for serogroup W135,and 1 416 ~ 12 741 for serogroup Y.The SBA titers of meningococcal polysaccharides of serogroups A,C,W135 and Y against homologous polysaccharides were not less than 80%,while those against heterologous polysaccharides were less than 20%. The dilution factor of serum was negatively related to the corresponding SBA titer,with slopes of-0. 90 ^-1. 10 and Pearson correlation coefficients of 0. 90 ~ 1. 0（each P 〈 0. 001）. The CVs of determination results of the same samples were not more than25% for intra-assay and not more than 40% for inter-assay. However,95% of determination results of the same sample in inter-assay were within 3 folds（one dilution）of median SBA titer,while at least 70% of determination results of the same sample by various laboratories were within 3 folds（one dilution） of results published by the US CDC. Conclusion Standard SBA for N. meningitides of serogroups A,C,W135 and Y was successfully developed,of which the determination result was internationally comparable,and the specificity,linearity and precision met the relevant requirements.

关 键 词：血清抗体杀菌试验 脑膜炎奈瑟菌 

分 类 号：R378.15[医药卫生—病原生物学] R392-33[医药卫生—基础医学]