基于猪附红细胞体膜蛋白的间接ELISA检测方法的建立及初步应用  

Establishment and preliminary application of an indirect ELISA detection method based on the Mycoplasma suis membrane protein

作　　者：徐颖[1] 王金琦 王淼 金鑫[1] 朴政霖 韩天龙 薛书江[1] 吕舟[4] XU Ying;WANG Jinqi;WANG Miao;JIN Xin;PIAO Zhenglin;HAN Tianlong;XUE Shujiang;LÜZhou(College of Agriculture,Yanbian University,Jilin Yanji 133002,China;Jilin Vocational and Technical College,Jilin Longjing 133400,China;Da’an Animal Husbandry Administration,Jilin Baicheng 131300,China;Jilin Agricultural Science and Technology University,Jilin Jilin 132019,China)

机构地区：[1]延边大学农学院,吉林延吉133002 [2]吉林职业技术学院,吉林龙井133400 [3]大安市畜牧业管理局,吉林白城131300 [4]吉林农业科技学院,吉林吉林132019

出　　处：《延边大学农学学报》2025年第1期59-64,共6页Journal of Agricultural Science Yanbian University

基　　金：吉林省自然科学基金项目(YDZJ202201ZYTS616);吉林省教育厅“十三五”科学技术项目(JJKH20191134KJ)。

摘　　要：为建立有效的猪附红细胞体病间接酶联免疫吸附(enzyme-linked immunosorbent assay,ELISA)检测方法,该试验利用猪附红细胞体水溶性膜蛋白为包被抗原,优化反应条件,建立了猪附红细胞体病间接ELISA检测方法。结果表明:提取的猪附红细胞体脂溶性膜蛋白的分子量主要集中在25~35Ku;猪附红细胞体水溶性膜蛋白的分子量主要集中在45~180Ku。建立的间接ELISA检测方法最佳反应条件:膜蛋白最佳包被浓度为0.15μg/μL;最佳封闭条件为5%脱脂乳37℃封闭2h;血清最佳稀释倍数为1∶320;酶标二抗最佳稀释倍数为1∶2000;TMB显色时间为37℃5min。特异性试验中,包被抗原与弓形虫、猪肺炎支原体,以及猪大肠杆菌阳性血清无交叉反应。重复性试验结果显示,批内变异系数为2.62%~8.89%,批间变异系数为5.49%~9.65%。对40份猪血清进行检测,建立的间接ELISA方法检出阳性样品19份,阳性率为47.5%。这说明建立的间接ELISA检测方法特异性强、敏感性高,将为猪附红细胞体病的临床诊断提供准确便捷的血清学检测手段。To create an accurate indirect enzyme-linked immunosorbent assay(ELISA)technique for detecting Mycoplasma suis,this experiment utilized water-soluble membrane protein of porcine eperythrozoon as coated antigen,an indirect ELISA method for detection of porcine eperythrozoon was established by optimizing reaction conditions.The findings demonstrated that the predominant distribution of the fat-soluble membrane protein of Mycoplasma suis’molecular mass is between 25~35 Ku,whereas the water-soluble membrane protein predominantly ranged between 45~180 Ku in terms of molecular weight.The most suitable reaction circumstances are as follows:membrane protein coating concentration is 0.15μg/μL;5%skim milk was sealed at 37℃for 2 h;dilution of serum was 1∶320;dilution of enzyme-labeled secondary antibody was 1∶2000;TMB color development time 37℃5 min.In the specificity test,the encapsulated antigens did not cross-react with Toxoplasma gondii,Mycoplasma pneumoniae,and swine Escherichia coli.The results of the repeatability test showed that the intra-batch variation coefficients ranged from 2.62%to 8.89%,and the inter-batch variation coefficients ranged from 5.49%to 9.65%.With a positive rate of 47.5%,the indirect ELISA approach was developed to identify 19 positive samples from 40 pig serum samples.The indirect ELISA detection method established in this experiment has strong specificity and high sensitivity,providing an accurate and convenient serological detection method for the clinical diagnosis of Mycoplasma suis.

关 键 词：猪附红细胞体 膜蛋白 临床检测 间接ELISA检测方法 抗体检测 

分 类 号：S858.28[农业科学—临床兽医学]