摘要
本研究旨在建立一种简便、快速的检测猪源致病性由携带脱皮毒素(Exh)C的猪源松鼠葡萄球菌引起的猪渗出性皮炎抗体的间接ELISA方法。首先利用本实验室保存的致病性松鼠葡萄球菌rExhC重组蛋白和阳性血清,对ELISA各项反应条件进行摸索,确定了最佳工作条件。结果表明,rExhC蛋白抗原最佳包被浓度为0.5μg/mL,血清的最佳稀释度为1:100,抗原抗体最佳反应条件为37℃,1 h,酶标二抗最佳工作浓度为1:1 000,酶标二抗最佳反应条件为37℃30 min,封闭条件为5%脱脂乳37℃,2 h,底物显色条件为室温30min,阴阳性临界值为0.102。检测证实所建立的ELISA方法具有较好的特异性、敏感性和准确性。用所建立的ELISA方法检测了2011年在江苏省张家港市采集的335份猪血清样本,结果阳性率为31.94%,与公开发表的猪渗出性皮炎的阳性率相符。综上所述,本研究建立的间接ELISA方法具有较好的特异性和敏感性,可用于猪源松鼠葡萄球菌引起的渗出性皮炎的检测。
This studye aim of this study iss to establish a simple and rapid indirect ELISA detection method for the detection of antibodies against exudative epidermitis caused by S. sciur by indirect ELISA, using the recombinant protein rExhC as coating antigen. Through a series of optimization tests, the optimal coating concentration of rExhC protein was set to be 0.5 ~g/mL, with the dilution of the serum being 1：100 and the dilution of the secondary antibody being at 1：1000. The proper blocking condition was 5% skim milk and incubated at 37 ~C for 2 h. The best condition for the primary antibody incubation was 37 ~C for 1 h, with the secondary antibody reaction condition being 37 % for 30 rain and the substrate reaction condition being for 30 rain at room temperature. Then we determined the threshold value of ELISA to be 0.102. We also confirmed that the indirect ELISA had a good specificity since the percentage of positive samples was the same with those determined by Western blot and there were no cross-reactions with the antibodies against PRRSV, HCV and PCV2. We used this method to detect 335 samples from Jiaug Su Province. The percentage of the positive results is 31.94%., which is the similar to those of other investigationors. In conclusion, we have succeeded successfully in establisheding an indirect ELISA method with good specificity and sensitivity and this method can be used to detect exudative epidermitis caused by a S. sciur.
出处
《中国兽医杂志》
CAS
北大核心
2013年第12期7-9,13,共4页
Chinese Journal of Veterinary Medicine
基金
国家自然科学基金项目(31072117 to SJ Zheng)
