大鼠血清中抗轮状病毒IgG抗体间接ELISA检测方法的建立及验证  

Development and validation of an indirect ELISA for IgG against rotavirus in sera of rats

作　　者：程满荣 徐葛林 李庆亮 杨露 李望华 孙营 彭瑾 CHENG Man-rong;XU Ge-lin;LI Qing-liang;YANG Lu;LI Wang-hua;SUN Ying;PENG Jin(Wuhan Institute of Biological Products Co.,Ltd.,Wuhan 430027,Hubei Province,China)

机构地区：[1]武汉生物制品研究所有限责任公司,湖北武汉430027 [2]国家联合疫苗技术研究中心,湖北武汉430027

出　　处：《中国生物制品学杂志》2020年第1期61-65,共5页Chinese Journal of Biologicals

基　　金：科技部863项目（2012AA02A401）;湖北省技术创新专项重大项目（2018ACA120）

摘　　要：目的建立大鼠血清中抗轮状病毒(rotavirus,RV)IgG抗体的间接ELISA检测方法,并进行验证。方法以兔抗RV多抗血清作为包被抗体,RV作为检测抗原,建立抗RV IgG抗体间接ELISA检测方法。棋盘滴定法优化包被抗体浓度(1∶1000~1∶1024000)和血清浓度(1∶20~1∶2560),直接ELISA法优化生物素标记的羊抗大鼠IgG工作浓度(1∶1000~1∶128000稀释)。采用优化的间接ELISA法检测39份RV抗体阴性大鼠血清样品,样品A450均值加上3倍标准差作为该检测条件的阴阳性判定界值。同时对该方法的专属性、检测限、耐用性进行验证。分别采用建立的方法及荧光灶形成单位(fluorescent focus-forming unit,FFU)方法检测96份临床前安全性评价试验样品,评价二者的符合率。结果建立的间接ELISA法的最适反应条件为:包被抗体稀释倍数为1∶8000,血清稀释倍数为1∶80,生物素标记的羊抗大鼠IgG工作浓度为1∶4000,阴阳性判定界值为0.105。方法检测限为0.031,且具有良好的专属性及耐用性。建立的方法与FFU方法检测结果的符合率达95.8%。结论成功建立了抗RV IgG抗体的间接ELISA检测方法,且方法准确可靠,为临床前安全性评价试验提供了一种简便的血清学诊断方法。Objective To develop and validate an indirect ELISA for IgG against rotavirus(RV)in sera of rats.Methods An indirect ELISA method for RV IgG was developed using rabbit antiserum against RV as coating antibody and RV as detection antigen.The working concentrations of coating antibody(1∶1000~1∶1024000)and serum samples(1∶20~1∶2560)were optimized by chessboard titration,while that of biotin-labeled goat anti-rat IgG antibody(1∶1000~1∶128000)by direct ELISA.Thirty-nine rat serum samples negative for RV were determined by the optimized indirect ELISA method,and the(mean±3 SD)of the A450 values of samples was served as the cutoff for judgment of negative and positive results.The method was validated for specificity,detection limit and robustness.A total of 96 samples for preclinical safety evaluation were determined by the developed method and fluorescent focus-forming unit(FFU)method respectively,of which the coincidence rate of determination results was calculated.Results The optional dilution of antibody for coating was 1∶8000,while that for serum sample was 1∶80,and that for biotin-labeled goat anti-rat IgG antibody was 1∶4000.The cutoff of A value for judgment of negative and positive results was 0.105.The developed method showed high specificity and good robustness,of with the limit of detection(LOD)was 0.031.The coincidence rate of determination results by the developed indirect ELISA and by traditional FFU test was 95.8%.Conclusion An indirect ELISA for RV IgG was successfully developed,which was accurate and reliable.It provided a simple serological diagnostic method for preclinical safety evaluation.

关 键 词：轮状病毒 IGG抗体 间接ELISA法 

分 类 号：R392-3[医药卫生—免疫学]