绵羊肺炎支原体抗体ELISA检测方法的建立  

Development of an ELISA method for detection of antibody against Mycoplasma ovipneumoniae

作　　者：杜改梅[1] 孙玫 刘茂军[2] DU Gaimei;SUN Mei;LIU Maojun(Department of Animal Science and Technology,Jinling Technology Institution,Nanjing 210038,China;Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences/Key Laboratory of Veterinary Biological Engineering and Technology,Ministry of Agriculture,Nanjing 210014,China)

机构地区：[1]金陵科技学院动物科学与技术学院,江苏南京210038 [2]江苏省农业科学院兽医研究所/农业部兽用生物制品工程技术重点实验室,江苏南京210014

出　　处：《畜牧与兽医》2020年第6期107-111,共5页Animal Husbandry & Veterinary Medicine

基　　金：国家重点研发计划(2018YFD0502000);江苏高校“青蓝工程”;江苏省第五期“333工程”科研项目(BRA2019092)。

摘　　要：为了建立特异性好和敏感性高的绵羊肺支原体抗体ELISA检测方法,以绵羊肺炎支原体Y98标准菌株的全菌蛋白作为抗原,进行了反应体系和反应条件的优化筛选。结果显示:最佳蛋白抗原包被浓度为1.52μg/mL,最适封闭液及其稀释浓度为1%BSA,血清样品的稀释度为1/50,兔抗山羊IgG的最适稀释浓度为1/4000,一抗和二抗的最佳作用时间为37℃60 min,底物显色的最佳时间为10 min;通过与绵羊肺炎支原体抗体间接血凝试验(IHA)检测结果相比,60份羊血清样品中Y98 ELISA检出阳性为33份,阴性为20份,二者的检测符合率可达到88.33%,相对特异性达90.90%。提示:建立的绵羊肺炎支原体血清抗体检测方法具有较高的检测敏感性和特异性,为绵羊肺炎支原体的抗体检测及其感染的流行病学调查提供了一种快速简便的检测工具。To develop an indirect ELISA for detecting the antibody against Mycoplasma ovipneumoniae with high specificity and sensitivity,the Y98 standard strain was employed as a specific antigen for ELISA detection of antibodies in serum against M.ovipneumoniae.The reaction condition and the reaction system were optimized.The results showed that the coated concentration of Y98 was 1.52μg/mL.1%BSA showed the best blocking effect.The serum samples were diluted at 1/50.The optimal dilution of rabbit anti-goat IgG was 1/4000.The optimal incubation time for both the serum samples and the conjugate were 1 h at 37℃.The chromogenic substrate was incubated for 15 min in the dark.Compared with the IHA,the established Y98 ELISA detected 33 positive serum samples and 20 negative ones of the 60 serum samples.There was a rate of concordance of 88.33%and a relative specificity of 90.90%between the Y98 ELISA and the IHA assay for the same serum samples.The above results indicated that the Y98 ELISA was a sensitive and specific serological test for detecting antibodies against M.ovipneumoniae and for epidemiologic investigation.

关 键 词：绵羊支原体肺炎 抗体 间接ELISA 

分 类 号：S852.6[农业科学—基础兽医学]