羊伪结核抗体间接ELISA检测方法的建立  

Establishment of indirect ELISA for detecting antibodies against Corynebacterium pseudotuberculosis

作　　者：霍宁宁[1] 朱伟英[1] 高洋[1] 杨雯昱[1] 岳进华 赵燕清[1] 陈德坤[1] 

机构地区：[1]西北农林科技大学动物医学院,陕西杨凌712100

出　　处：《西北农林科技大学学报（自然科学版）》2016年第10期12-17,共6页Journal of Northwest A&F University(Natural Science Edition)

基　　金：陕西省科技统筹创新工程计划项目(2015KTTSNY04-04)

摘　　要：【目的】建立羊伪结核棒状杆菌抗体的间接ELISA检测方法。【方法】分别用不同浓度的伪结核棒状杆菌菌体抗原和培养上清可溶性抗原包被酶标板,用脱脂奶粉封闭酶标板,设置封闭时间分别为1.0,1.5和2h,再用不同稀释度的血清(1∶100,1∶200,1∶400和1∶800)与之反应,以此优化间接ELISA反应条件,确定阳性结果判定的临界值。对间接ELISA方法的特异性和重复性进行考察,并在此基础上对采集的临床样本进行检测。【结果】选用伪结核棒状杆菌菌体为包被抗原,最佳包被抗原菌体密度为OD600=0.08,血清的最佳稀释度为1∶400,封闭时间为2h,阳性血清的临界值为0.329。该抗体检测方法可特异性地检测出伪结核阳性血清,羊口疮阳性血清、羊痘阳性血清、乳房炎阳性血清均呈阴性反应;组内,组间变异系数<10%。该方法对临床样本的检测结果与实际患病状况一致。【结论】成功建立了伪结核抗体间接ELISA检测方法,该方法特异性强、重复性好,可用于临床诊断和批量血清学检测。【Objective】This study established an indirect ELISA to detect antibodies against Corynebacterium pseudotuberculosis in serum.【Method】After the ELISA plates were coated with somatic antigen and soluble antigen,blocking buffer was added,blocking time（1,1.5,and 2h）was determined,and serums with different dilution times（1∶100,1∶200,1∶400,and 1∶800）were added.Then the optimal conditions were determined and positive criterion was obtained.The sensitivity and repeatability were detected and the assay was used to test clinical samples.【Result】The optimal conditions were：concentration of coating antigen OD600=0.08,dilution times of serum 1∶400,and blocking time 2h.The positive criterion was 0.329.No cross reaction with positive serum of orf,goat pox and mastitis was observed.The coefficient of variation（CVs）of intro-batch and inter-batch repetition tests was less than 10%.The ELISA results were consistent with clinical results.【Conclusion】The established indirect ELISA method can be used for epidemiologic survey and clinical tests with high sensitivity and specificity.

关 键 词：羊干酪性淋巴结炎 伪结核棒状杆菌 间接酶联免疫吸附试验 

分 类 号：S858.265[农业科学—临床兽医学]