猪乳汁中抗猪传染性胃肠炎病毒IgA抗体间接ELISA检测方法的建立

Development of an indirect ELISA to detect IgA induced by porcine transmissible gastroenteritis virus

为建立检测猪乳汁中抗猪染性胃肠炎病毒(TGEV)Ig A抗体的间接ELISA方法,本研究以TGEV重组N蛋白为包被抗原,以辣根过氧化物酶标记羊抗猪Ig A为检测抗体,并采用方阵法确定包被抗原和待检乳汁的最佳工作浓度,对各种反应条件进行优化,建立了猪乳汁中TGEV Ig A抗体的间接ELISA检测方法。该方法检测稀释160倍的阳性乳清仍呈阳性;与猪流行腹泻和猪轮状病毒感染阳性乳清无交叉反应;批内和批间变异系数均小于10%。利用建立的ELISA方法与间接免疫荧光方法分别对134份临床样品进行检测,阳性检出率分别为58.2%(78/134)和59.7%(80/134),总符合率为85.6%。本研究建立的检测方法能够有效评估乳汁中TGEV Ig A抗体的水平。

To develope a rapid method to detect the IgA against transmissible gastroenteritis virus （TGEV） in milk, an indirect ELISA was established with the recombinant N protein of TGEV as coating antigen. The assay was specific for detection of TGEV, but had no cross reaction with positive milk samples collected from pigs infected with porcine epidemic diarrhea virus and rotavirus. The assay was also highly sensitive, and the intra- and inter-assay variations was less than 10%. Tested on 134 clinical samples by both indirect ELISA and indirect immunofluorescence assay, the positive rates were 58.2% （78/134） and 59.7 （80/134） and the total coincidence rate was 85.6%. This method could be applied to evaluate effectively the of IgA levels in milk.