检测牛IL-4双抗体夹心ELISA方法的建立  

Development of a double-antibody sandwich ELISA for detection of BolL-4

作　　者：朱月姝 文志发 孔文君 徐正中 焦新安 陈祥 ZHU Yueshu;WEN Zhifa;KONG Wenjun;XU Zhengzhong;JIAO Xin'an;CHEN Xiang(Jiangsu Key Laboratory of Zoonosis/Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou University,Yangzhou 225009,China;Key Laboratory of Prevention and Control of Biological Hazard Factors(Animal Origin)for Agrifood Safety and Quality,Ministry of Agriculture and Rural Af fairs,Yangzhou University,Yangzhou 225009,China)

机构地区：[1]扬州大学江苏省人兽共患病学重点实验室/江苏省动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009 [2]扬州大学农业农村部农产品质量安全生物性危害因子(动物源)控制重点实验室,江苏扬州225009

出　　处：《扬州大学学报（农业与生命科学版）》2023年第3期65-71,共7页Journal of Yangzhou University：Agricultural and Life Science Edition

基　　金：国家重点研发计划项目(2021YFD1800403);江苏省重点研发计划项目(BE2021331);江苏省农业科技自主创新资金项目[CX(21)1004];江苏高等学校学科创新引智计划项目(D18007);江苏高校优势学科建设工程项目(PAPD)。

摘　　要：为利用牛白细胞介素-4(BoIL-4)的特异性单克隆抗体(MAb)建立检测BoIL-4的双抗体夹心ELISA方法,以多种ELISA方法对7株抗BoIL-4的单抗进一步筛选,得到反应最佳的2株单抗8B7和4A10,以A9表达的重组BoIL-4为阻断剂的阻断ELISA试验中,表明这2株单抗也可识别所构建的细胞系A9表达的重组BoIL-4。以单抗8B7作为包被抗体,以标记了HRP的单抗4A10作为检测抗体建立的双抗体夹心ELISA方法可检测大肠埃希菌、毕赤酵母和Flp-In-293细胞系3种表达系统表达的重组BoIL-4,最低检测限分别为0.25、8和2 ng·mL^(-1),且对A9细胞系上清中表达的BoIL-4检测效果和特异性良好。该研究建立的BoIL-4双抗体夹心ELISA方法为深入研究BoIL-4、开发BoIL-4 ELISA检测试剂盒奠定了基础。To establish a double antibody sandwich ELISA method for the detection of BoIL-4 using the specific monoclonal antibody(MAb)of bovine interleukin-4(BoIL-4).In this study,seven MAbs against BoIL-4 were further screened by multiple ELISA methods,and two MAbs 8B7 and 4A10 with the best response were obtained.The seven anti-BoIL-4 MAbs were further screened by various ELISA methods,and the two MAbs with the best reaction were obtained,8B7 and 4A10.The blocking ELISA experiment using the recombinant BoIL-4 expressed by A9 as the blocking agent showed that these two MAbs could also recognize the recombinant BolL-4 expressed by the cell line A9 constructed in our laboratory.The double antibody sandwich ELISA method established with MAb 8B7 as the encapsulated antibody and MAb 4A1o labeled with HRP as the detection antibody could detect recombinant BoIL-4 expressed in three expression systems of Escherichia coli,Pichia pastoris and Flp-In-293 cell line,with the minimum detection limit of 0.25,8 and 2 ng·mL^(-1),respectively,and the detection effect and specificity of BolL-4 expressed in the supernatant of A9 cell line were good.The BoIL-4 double antibody sandwich ELISA method established in this study lays the foundation for the indepth study of BoIL-4 and the development of BoIL-4 ELISA test kits.

关 键 词：检测牛白细胞介素-4 单克隆抗体 抗体夹心ELISA 

分 类 号：S858.23[农业科学—临床兽医学]