骆驼斯氏副柔线虫病间接ELISA检测方法的建立

doi:10.16431/j.cnki.1671-7236.2019.06.023

摘要/Abstract

摘要：

为建立骆驼斯氏副柔线虫病间接ELISA （iELISA）诊断方法，本试验对骆驼斯氏副柔线虫半胱氨酸蛋白酶CPR基因进行重组表达，将获取的重组蛋白（rCPR）进行纯化和Western blotting检测，然后以纯化好的重组蛋白为抗原，通过棋盘滴定试验优化了抗原包被浓度、包被条件、抗体稀释度、酶标二抗稀释度、封闭液和封闭时间等，建立了骆驼斯氏副柔线虫病iELISA诊断方法，并对建立的iELISA检测方法进行了重复性、敏感性、特异性试验和临床检测。结果显示，抗原最佳包被浓度为8 μg/孔，血清最佳稀释度为1：50，酶标二抗最佳稀释度为1：5 000，最佳包被条件为4℃包被过夜，最佳封闭条件为3% BSA封闭2 h。临界值为0.235，待检血清D_{450 nm}值>0.235则确定为阳性。重复性试验中变异系数均<10%，重复性较好；用该方法检测阳性血清的敏感性为96.3%；此方法仅与骆驼斯氏副柔线虫病阳性血清发生特异性反应，与感染了其他寄生虫的阳性血清无交叉反应，特异性为100%；对140份临床血清进行检测，阳性率为86.4%。综上可知，本试验成功建立了一种快速有效诊断骆驼斯氏副柔线虫病的iELISA方法。

关键词: 骆驼; 斯氏副柔线虫; 半胱氨酸蛋白酶; 间接ELISA

Abstract:

In order to establish an indirect ELISA (iELISA) diagnostic method for detection of camel parabronemosis,in this experiment,the CPR gene of cysteine protease was recombinant expression,and the obtained recombinant protein (rCPR) was purified and detected by Western blotting.Using the purified recombinant protein as an antigen,the conditions of coating antigen,coating conditions,antibody dilution,dilution of enzyme-labeled secondary antibody,blocking solution and blocking time were optimized by checkerboard titration test,and the iELISA diagnostic method for detection of Parabronema skrjabini was established.Repeated,sensitive,specific and clinical tests were performed on the established iELISA assay.The results showed that the optimal coating concentration of the antigen was 8 μg/well,the optimal dilution of the serum and the enzyme-labeled secondary antibody were 1:50 and 1:5 000,respectively,and the optimal coating condition was 4℃ overnight.The optimal blocking condition was 3% BSA for 2 h.The cut-off value was 0.235,and the serum D_{450 nm} value>0.235 was determined to be positive.The coefficient of variation in the repeatability test was <10%,and the repeatability was good;The sensitivity of detecting positive serum by this method was 96.3%;This method only specifically reacted with the positive serum of Parabronema skrjabini,and did not cross-react with other parasite-positive sera,the specificity was 100%;140 clinical sera were tested and the positive rate was 86.4%.In summary,this experiment successfully established an iELISA method for rapid and effective diagnosis of camel parabronemosis.

Key words: camel; Parabronema skrjabini; cysteine proteases; iELISA

中图分类号:

S855.9

苏倩, 冯陈晨, 赵学亮, 王金玲, 王文龙, 呼和巴特尔. 骆驼斯氏副柔线虫病间接ELISA检测方法的建立[J]. 《中国畜牧兽医》, 2019, 46(6): 1756-1763.

SU Qian, FENG Chenchen, ZHAO Xueliang, WANG Jinling, WANG Wenlong, HUHE Bateer. Establishment of Indirect ELISA Method for Detecting Parabronemosis of Camel[J]. , 2019, 46(6): 1756-1763.

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