猪源肠外致病性大肠杆菌抗体间接ELISA检测方法的建立  

Indirect Enzyme-linked Immuno Sorbent Assay(ELISA) Detection Method for Porcine ExPEC Antibody

作　　者：程家园 李亮 邢刚 刘雪兰[1] 孙裴[1] 魏建忠[1] 李郁[1] CHENG Jiayuan;LI Liang;XING Gang;LIU Xuelan;SUN Pei;WEI Jianzhong;LI Yu(College of Animal Science and Technology,Anhui Agricultural University,Hefei 230036,China;Ma'anshan Shiji Animal Health Management Co.,Ltd.,Ma'anshan 238251,China)

机构地区：[1]安徽农业大学动物科技学院,安徽合肥230036 [2]马鞍山史记动物健康管理有限公司,安徽马鞍山238251

出　　处：《养猪》2022年第2期89-94,共6页Swine Production

基　　金：国家星火计划重点项目(2014GA710002);安徽省重点研究与开发计划(面上攻关)项目(201904a06020013);安徽省长三角联合科技攻关项目(1101c0603065);安徽省生猪产业体系基金[皖农科(2016)84号]。

摘　　要：利用制备的猪源肠外致病性大肠杆菌(ExPEC)全菌体蛋白建立一种检测猪源ExPEC抗体的间接ELISA方法。将2株猪源ExPEC SDjie18-10(O;)、HByan18-2(O;)超声裂解的全菌体蛋白作为包被抗原,对反应条件进行优化,建立检测猪源ExPEC抗体的间接ELISA方法,并进行临床应用与评价。该方法的最佳条件:包被抗原浓度为15μg/m L,37℃1 h+4℃过夜;2%BSA于37℃封闭2 h;血清稀释度为1∶800,37℃孵育30 min;酶标二抗稀释度为1∶5 000,37℃作用30 min;显色时间为37℃15 min。该方法可特异性检测猪源ExPEC抗体,阳性血清稀释至1∶6 400仍可检出,与其他猪病原阳性血清均无交叉反应,批内及批间变异系数均小于10%。应用间接ELISA与MAT进行符合性比较及临床猪血清样品感染抗体的同步检测,两种方法的符合率分别为93.33%和94.50%且无显著性差异(P>0.05),具有较高的均一性,同时间接ELISA的感染抗体阳性检出率(19.00%)略高于MAT(17.50%),两者检测结果高度一致(K=0.816)。基于猪源ExPEC全菌体蛋白建立的猪源ExPEC抗体间接ELISA检测方法,具有良好的特异性、敏感性和重复性以及临床应用的可靠性,可用于猪源ExPEC的免疫监测和流行病学调查。Using the enomome protein of porcine ExPEC to establish an indirect ELISA method for detecting porcine ExPEC antibodies. Two strains of porcine ExPEC strains SDjie18-10(O;) and HByan18-2(O;) were screened out by antigenicity tests, and the sonicated protein was used as coated protein. The indirect ELISA method for detecting porcine ExPEC antibody was established through the optimization of a series of reaction conditions,and its clinical application and evaluation were carried out. The optimum reaction conditions of this method were as follows: 15 μg/mL, 37 ℃ 1 h+4 ℃ overnight concentration of coated antigen;2% BSA was sealed at 37 ℃ for 2 h.The serum dilution was 1∶800 which was incubated at 37 ℃ for 30 min. The dilution of HRP was 1∶5 000, and it was treated at 37 ℃ for 30 min;the optimal reaction time of TMB was 15 min. This method can specifically detect the porcine ExPEC antibody. The positive serum is still positive after 1∶6 400 dilution, and nocross-reactivity with other porcine pathogen positive serum. The coefficient of variation between in the batch and batches all less than 10%. Idirect ELISA and MAT were used for compliance comparison and simultaneous detection of infection antibodies in clinical swine serum samples, the two methods were 93.33% and 94.50% with no significant difference(P>0.05), while the infection antibody positive detection rate(19.00%) was slightly higher than MAT(17.50%) and the detection results of the two methods were highly consistent(K=0.816). This study based on the enomome protein of porcine ExPEC established indirect ELISA detection method for porcine ExPEC antibodies with good specificity,sensitivity and repeatability, as well as the reliability of clinical applications, provides atechnical means for porcine ExPEC immune surveillance and epidemiological investigation.

关 键 词：猪源ExPEC 全菌体蛋白 间接ELISA 抗体检测 

分 类 号：S858.28[农业科学—临床兽医学]