摘要
用纯化的融合蛋白p GEX-6p-1-VP1作为包被抗原,4E12单抗为检测抗体与待检血清竞争固相抗原,建立了单抗竞争ELISA方法来检测口蹄疫A型抗体,并进行了反应条件的优化,确定阴阳临界值。结果表明,抗原最适包被浓度为0.625μg/m L,待检血清稀释度为1∶2,单抗最大稀释度为1∶400,酶标抗体工作浓度为1∶5000,封闭液、待检血清和单抗作用时间分别为60、60、45 min;阴阳性判断抑制率(PI)≥30%为阳性。与液相阻断ELISA检测试剂盒比对,符合率为84.8%。试验表明,建立的单抗竞争ELISA检测方法具有特异性强、稳定性好等优点,可用于口蹄疫A型血清抗体的检测,这为口蹄疫A型免疫抗体水平检测、疫情监控及流行病学调查提供了技术平台。
Competitive ELISA was established based on purified protein p GEX-6p-1-VP1 as coating antigen and 4E12 as competitive antibody. And optimal reactive conditions and cut-off value were determined. The results showed that the working concentrations of coating antigen,serum,Mc Ab,and HRP-Ig G were 0. 625 μg / m L,1∶ 2,1∶ 400,1∶ 5000,respectively. The optimal reaction time of blocking solution,serum sample and Mc Ab were 60,60,45 min. The cut-off PI % was more than 30 % as positive sample. The agreement ratio between the competitive ELISA and a liquid phase blocking ELISA test was 84. 8 %. In summary,a competitive ELISA was established for detection Foot-and-mouth disease virus type A antibodies. The test has advantages in specificity and stability. It is a simple and rapid test and provided technical platform for immune antibody detection,monitoring and epidemiological investigation.
出处
《西南农业学报》
CSCD
北大核心
2015年第5期2268-2272,共5页
Southwest China Journal of Agricultural Sciences
基金
广西科技攻关项目(桂科攻0779001)
关键词
A型口蹄疫病毒
VP1蛋白
竞争ELISA
Foot and mouth disease virus serotype A
VP1 protein
Competitive ELISA
