重组金黄色葡萄球菌疫苗免疫血清中功能性抗体OPK检测方法的建立  

Establishment of an OPK method for the detection of functional antibody in immune serum of Staphylococcus aureus vaccine

作　　者：周恒 杨柳扬 杨峰 章金勇 邹金桃 程平 曾浩 邹全明 

机构地区：[1]陆军军医大学药学与检验医学系微生物与生化药学教研室暨国家免疫生物制品工程技术研究中心,重庆400038

出　　处：《免疫学杂志》2018年第3期252-258,共7页Immunological Journal

基　　金：国家自然科学基金(31370932)

摘　　要：目的建立重组金黄色葡萄球菌疫苗免疫血清中功能性抗体检测方法。方法在参考美国阿拉巴马大学的多重调理吞噬实验(UAB-MOPA)方法的基础上,构建并鉴定HL-60效应细胞库及金黄色葡萄球菌工作种子批,完成补体浓度、调理和吞噬孵育时间及血清预稀释倍数等关键实验参数的优选。在此基础上对5种不同来源金黄色葡萄球菌进行调理吞噬杀菌(OPK)活性的检测。结果 HL-60细胞经0.8%DMF诱导分化4 d后,CD35表达量为87.1%,CD71表达量为2.82%,活细胞比例为75.7%;靶细菌工作种子批冻存复活率>90%,工作稀释度>1 000;体系中补体终浓度确定为6%;选择30 min、60 min作为调理和吞噬孵育时间;血清预稀释倍数为2倍;疫苗免疫血清对5种不同来源的金黄色葡萄球菌均表现出良好的OPK活性及标准的"S"型杀菌曲线。结论本研究优化建立的OPK检测方法,可有效评价重组金黄色葡萄球菌疫苗的体外功能性抗体水平,为疫苗临床试验样本检测及免疫有效性替代终点的确立提供了重要的技术支撑。The study is aimed to establish a method to detect functional antibodies in the immune serum of Staphylococcus aureus vaccine. According to the University of Albama at Birmingham Protocol for multiplexed opsonophagocytic killing assay（UAB-MOPA） method, we established the HL-60 effect cell cultures, S. aureus stocks, and selected the optimal complement concentration, serum pre-dilution fold and the incubation time of opsonization and phagocytosis phase. Then OPK assay was performed by using five different S. aureus strains. Data showed that after HL-60 cells were differentiated in 0.8% DMF culture for 4 days, the expression of CD35 was87.1%, the expression of CD71 was 2.82%, the percentage of living cells was 75.7%. Target bacteria work resurrection rate was 〉90%, while dilution degrees of bacterial stocks were 〉1 000; complement in the final concentration was 6%. The opsonization and phagocytosis phase times were confirmed as 30 minutes and 60 minutes. Serum was pre-diluted 2-folds, and vaccinated serum showed OPK activity against five different S. aureus strains, and all curves exhibited standard＂S＂shape. In conclusion, we established the OPK detection method for recombinant S. aureus subunit vaccine, which could effectively evaluate the serum functional antibody in vitro, thus provides an important technical support for theeffective alternative destination of clinical trials.

关 键 词：金黄色葡萄球菌 重组蛋白疫苗 功能性抗体 调理吞噬杀菌实验 

分 类 号：R-331[医药卫生]