检测牛边缘无浆体抗体的竞争抑制ELISA方法的建立  

Establishment of a competitive inhibition ELISA for detection of antibodies against bovine Anaplasma marginale

作　　者：倪宏波[1,2] 王冰[3] 姜海芳[2] 周玉龙[2] 王春仁[2] 宫大庆[2] 钱爱东[1] 

机构地区：[1]吉林农业大学动物科技学院,吉林长春130118 [2]黑龙江八一农垦大学动物科技学院,黑龙江大庆163319 [3]黑龙江农垦职业学院食品工程系,黑龙江哈尔滨150025

出　　处：《中国预防兽医学报》2011年第4期289-292,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：吉林省科技发展计划项目(20100220);黑龙江省科技厅实验动物青年基金项目(PC07S09)

摘　　要：为建立检测牛边缘无浆体(Anaplasma marginale)抗体的方法,本研究以牛A.marginale膜表面重组MSP5蛋白作为包被抗原,抗MSP5单克隆抗体(MAb)作为竞争抗体,建立一种用于检测牛A.marginale抗体的重组MSP5蛋白竞争抑制ELISA(CI-ELISA)方法。经优化确定CI-ELISA的最佳反应条件为:抗原包被浓度为2μg/孔,封闭液为2%脱脂乳,MAb的稀释度为1∶400,酶标二抗的稀释度为1∶1000,阴性和阳性血清临界值分别为33%和40%;该方法具有良好的特异性和重复性;2348份临床血清样品的检测结果表明,217份为阳性,阳性率为9.2%,与IDEXX A.marginale抗体检测试剂盒的阳性符合率为95.3%,阴性符合率为100%。本实验建立的ELISA方法具有较高的特异性和重复性,可用于流行病学调查研究。An competitive inhibition ELISA （CI-ELISA） for detection of antibodies against bovine Anaplasma marginale was developed by coating with the recombinant MSP5 protein expressed in E.coli and inhibiting with the monoclonal antibody of 1D8 against the MSP5 protein. The CI-ELISA was specific to inhibit A. marginale positive serum and no crossing inhibitions for sera to Eperythrozoon, Toxophasma, Theileria annulata, and Babesia, respectively. A total of 2,348 bovine serum samples from Heilongjiang area were tested by the CI-ELISA, comparing with IDEXX A. marginale-Ab ELISA kit. The results showed that 217 sera was positive （9.2%）, and the positive coincidence was 95.3%, indicating the CI-ELISA was specifically and reduplicative for detecting the antibody against A. marginale in naturally infected cattle, and would be useful for epidemiological investigation.

关 键 词：牛边缘无浆体 竞争抑制ELISA 重组MSP5蛋白 流行病学调查 

分 类 号：S852.4[农业科学—基础兽医学]