抗副溶血弧菌OMPK单克隆抗体的制备及其ELISA双抗体夹心检测方法建立 王晓瑞1，邱红玲2，王寿利3，朱海华1，周?莉1，平?洋1，谭?静1，王?永1，王?慧1,2,* （1.河南省商业科学研究所有限责任公司，河南?郑州 450002；2.上海交通大学医学院公共卫生学院，上海 200025；3.中国科学院上海生命科学研究所，上海 200233） 摘?要：制备全长和截短的弧菌外膜蛋白K（outer membrane protein K，OMPK），纯化后的OMPK免疫6～8 周雌性BALB/c小鼠，间接夹心酶联免疫吸附测定（enzyme linked immunosorbent assay，ELISA）法检测小鼠的血清效价，结果表明全长OMPK重组蛋白制备的血清效价是截短OMPK1重组蛋白制备血清效价的128?倍。通过全长的OMPK重组蛋白制备多株用于检测副溶血弧菌的单克隆抗体，用生物素（biotin）和辣根过氧化物酶对单克隆抗体进行标记。间接夹心ELISA实验结果表明：只有VP3-biotin和VP16-biotin与其他单克隆抗体配对检测不同的副溶血弧菌菌株表现出较高的灵敏度及良好的特异性，并且在检测其他种属20?株菌时无交叉反应。VP16(Fab)-biotin/VP3检测副溶血弧菌时较VP16-biotin/VP3具有更高的灵敏度，该方法用于检测海鲜样品时，VP16-biotin/VP3检测三文鱼（5～10?CFU/25?g）和血蚶样品为副溶血弧菌阳性。本研究开发了一种快速、灵敏、简便的副溶血弧菌间接夹心ELISA检测方法。 关键词：副溶血弧菌；弧菌外膜蛋白K；单克隆抗体；间接夹心ELISA Preparation of Monoclonal Antibodies against Outer Membrane Protein K of Vibrio parahaemolyticus and Development of a Double Antibody Sandwich ELISA WANG Xiaorui1, QIU Hongling2, WANG Shouli3, ZHU Haihua1, ZHOU Li1, PING Yang1, TAN Jing1, WANG Yong1, WANG Hui1,2,* (1. Henan Commercial Science Research Institute Co. Ltd., Zhengzhou 450002, China; 2. School of Public Health, Shanghai Jiao Tong University of Medicine, Shanghai 200025, China; 3. Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200233, China) Abstract: The full length and truncated recombinant outer membrane protein K (OMPK) of Vibrio parahaemolyticus were purified before being used to immunize 6–8-week-old female BALB/c mice. The titer of antisera was measured by an indirect sandwich enzyme linked immunosorbent assay (ELISA). The results showed that the titer of the antiserum against the full-length OMPK recombinant protein was 128-fold higher than that of the antiserum against the truncated OMPK1 recombinant protein. Several monoclonal antibodies (mAbs) were successfully generated by using the full-length OMPK recombinant protein and were used to detect V. parahaemolyticus after being conjugated with biotin and horseradish peroxidase (HRP). Indirect sandwich ELISA results indicated that only biotin conjugated VP3 and VP16 were paired with other mAbs, showing high sensitivity and good specificity to detect various V. parahaemolyticus strains. They showed no cross reactivity with 20 strains of other bacteria. Biotinylated VP16 Fab fragment-VP3 pair showed higher sensitivity to detect V. parahaemolyticus than biotinylated VP16-VP3 pair. By this method with biotinylated VP16-VP3 pair, salmon (about 5–10 CFU/25 g) and blood clam samples were found to be positive for V. parahaemolyticus. Thus, our research successfully developed a rapid, sensitive, and convenient method for V. parahaemolyticus detection by indirect sandwich ELISA. Keywords: Vibrio parahaemolyticus; outer membrane protein K; monoclonal antibody; indirect sandwich ELISA DOI:10.7506/spkx1002-6630-20191219-220 TS207.4