摘要
为建立伪结核棒状杆菌(Cp)血清抗体间接ELISA检测方法,以热灭活处理的不同的浓度Cp菌株作为固相抗原包被酶标板,用不同封闭液封闭,设置不同的封闭时间,再用不同稀释度的待检血清和不同稀释度的酶标二抗与之反应,以此优化间接ELISA反应条件,确定阳性临界值。对间接ELISA方法的特异性和重复性进行试验,并在此基础上对采集的临床样本进行检测。结果显示,最佳抗原包被量为OD600 nm=0.084的菌悬液100μL,10 g/L BSA封闭时间2 h,一抗血清稀释度为1∶400,酶标二抗的稀释度为1∶5000,血清阴阳性的OD450 nm值临界值为0.352。该方法仅对Cp阳性血清呈特异性反应,与6种常见病原阳性血清均无交叉反应,特异性较强。批内试验和批间试验的变异系数均小于9.5%,重复性较好。敏感性试验结果表明,当伪结核标准阳性血清进行1∶1280稀释时检测仍为阳性,敏感性较强。用该方法对10份经细菌分离鉴定为阳性的血清进行Cp抗体检测,结果均为阳性。用建立的方法对临床随机采集的423份血清进行检测,结果显示阳性率为35%。研究建立的抗体间接ELISA方法为Cp抗体检测及血清学调查奠定了基础。
In order to establish an ELISA method for indirect detection of Pseudotuberculosis serum antibodies,this experiment used heat-inactivated Cp strain as the antigen to coat ELISA plate with different concentrations of antigen respectively.The different blocking solutions were used for blocking,the different blocking time were set,and then the different serum dilutions and the different the second antibody dilutions were used optimize the indirect ELISA reaction conditions and determine the positive cutoff value.The specificity and reproducibility of the indirect ELISA method were studied,and the clinical samples collected were tested on this basis.The results showed that the best antigen coating volume is 100μL of bacterial suspension,OD600 nm=0.084,10 g/L BSA blocking time is 2 h,primary antibody dilution is 1∶400,and enzyme-labeled secondary antibody dilution is 1∶5000.The critical value of OD450 nm for negative and positive is 0.352.This method only showed specific reaction to Cp-positive sera,and did not cross-react with positive sera of 6 common pathogens,with strong specificity.The coefficient of variation of intra-assay and inter-assay are both less than 9.5%,and the repeatability is good.The sensitivity test showed that when the Corynebacterium pseudotuberculosis standard positive serum is diluted 1∶1280,the test is still positive and the sensitivity is strong.This method was used to detect Cp antibodies that were identified as positive by bacterial isolation in 10 sera,and the results were all positive.The established method was used to test 423 sera randomly collected in the clinic,and the positive rate was 35%.The antibody indirect ELISA method established in this study laid the foundation for Cp antibody detection and serological research.
作者
刘刚
尹峥
李晨露
徐海玲
王晶晶
张琪
许信刚
杨增岐
LIU Gang;YIN Zheng;LI Chen-lu;XU Hai-ling;WANG Jing-jing;ZHANG Qi;XU Xin-gang;YANG Zeng-qi(College of Veterinary Medicine,Northwest A&F University,Yangling,Shaanxi,712100,China)
出处
《动物医学进展》
北大核心
2021年第7期1-6,共6页
Progress In Veterinary Medicine
基金
2018年陕西省农业专项资金支持科技创新驱动项目(K3360218035)
国家绒毛用羊产业技术体系项目(CARS-39-14)。
关键词
伪结核棒状杆菌
间接ELISA
抗体检测
Corynebacterium pseudotuberculosis
indect ELISA
antibody detection
