摘要
[目的]建立检测猪蛔虫抗体的间接ELISA方法。[方法]用猪蛔虫重组AS16蛋白作为包被抗原,通过方阵滴定试验确定ELISA的最佳工作条件,包括抗原最佳包被浓度与血清最佳稀释度、最佳包被液、抗原封闭时间、血清反应时间、酶标二抗工作时间和间接ELISA判定标准,并通过交叉试验现场对1 200份猪血清样本采用该试验建立的ELISA和AGP同时进行蛔虫抗体的检测。[结果]ELISA的最佳工作条件是:抗原包被浓度为10μg/ml,37℃1 h,4℃过夜;血清稀释度为18∶0;酶标SPA稀释度为1∶10 000,37℃孵育45 min。该试验制备的ELISA酶标板的临界值为0.196。交叉试验检测表明,ELISA方法阳性检出率为45%,而AGP阳性检出率为30%。该方法具有特异高、快速、敏感等特点。[结论]研究结果证实重组AS16蛋白具有良好的反应原性,可以用来建立ELISA方法诊断猪蛔虫病。
[ Objective ] The study aimed to establishment of an indirect ELISA method for detection of antibodies to Ascaris suum. [ Method ] With recombinant AS16 protein Of Ascaris suum as the qoating antigen, the Optimal reaction conditions of ELISA including the optima/coating concn, of antigen and dilution degrees of serum, the optimal coating liquid, antigen closing time, reaction time of serum, working time of enzymelabeled second antibody and the judging standard of indirect ELISA were determined through the matrix titration test. 1 200 serum samples of pigs were detected on-site by the AGP and ELISA developed in this test for the detection of antibodies to A. suum through the cross test. [ Re- sult] The optimal reaction conditions of ELISA were as follows：the coating concn, of antigen was 10 μg/ml at 37 ℃ for 1 h and 4 ℃ overnight; the serum samples were diluted to 1：80; the peroxidase-labeled SPA were diluted to 1：10 000 and was incubated at 37 ℃ for 40 min. The critical OD value for the ELISA Plate was 0. 196. The cross test showed that 45% were positive anti-A, suum antibody by an indirect ELISA and 30% were by AGP. The method had high specificity, rapid and sensitive features. [ Conclusion ] The study result confirmed that the recombinant AS16 protein had good reaetionogenicity and could be used to establishment of an indirect ELISA method for diagnosing the disease of Ascaris suum.
出处
《安徽农业科学》
CAS
北大核心
2010年第31期17710-17712,共3页
Journal of Anhui Agricultural Sciences
