建立一种用重组蛋白检测抗口蹄疫病毒抗体的间接ELISA方法

Establishment of an indirect ELISA method with recombinant FMDV antigen for detection of specific antibodies

目的研究以重组蛋白作为包被抗原检测口蹄疫病毒(FMDV)感染后的动物血清中特异性抗体的可能性,为建立一种非病毒颗粒的ELISA检测试剂盒提供实验依据。方法利用自行构建表达的O型口蹄疫病毒VP1表位肽重组蛋白(VP1epi)作为包被抗原,采用间接ELISA方法确定抗原的最佳工作浓度和包被方法,优化各项实验条件,并以FMDV感染后的豚鼠血清作为标准阳性血清确定ELISA方法的特异性和灵敏度。结果FMDV感染后的阳性豚鼠血清可以很好地识别VP1epi重组蛋白,用此蛋白包被检测抗FMDV抗体的灵敏度可达1∶3200,并证明所检测的抗体是FMDV特异性的。结论VP1epi重组蛋白可以替代FMDV颗粒用于建立检测抗FMDV抗体的ELISA试剂盒。

Objective：To provide some experiment data for establishing an ELISA kit without virus particle as coating antigen, the possibility of recombinant protein as coating antigen in detecting specific antibody from sera of animals infected by foot-and-mouth disease virus（FMDV） was studied. Methods：Coating antigen of self-constructed type O FMDV derived VP1 epitope recombinant protein（ VP1 epi） was optimized for its optimal concentration, coating manner and other experimental conditions by indirect ELISA. Meanwhile, FMDV-infected Guinea pig sera were as standard positive sera for determining the specificity and sensitivity of the ELISA method.Results ：The positive sera from Guinea pig infected by FMDV could well recognized VP1 epi protein. The sensitivity of detecting anti-FMDV antibody with this recombinant protein reached 1 ： 3 200. The antibody detected by this protein was confirmed to be FMDV specific. Conclusion ：The VP1 epi protein can replace FMDV particle to set an ELISA kit in detecting anti-FMDV antibody.