土霉素完全抗原的制备及ELISA检测方法的建立  

Preparation of OTC complete antigen and establishment of ELISA detection method

作　　者：曹金博 胡骁飞[1] 王耀[2] 李燕虹 曹夕丹[2] 邢云瑞 孙亚宁 邓瑞广[1] 张改平[1,3] CAO Jin-bo;HU Xiao-fei;WANG Yao;LI Yan-hong;CAO Xi-dan;XING Yun-rui;SUN Ya-ning;DENG Rui-guang;ZHANG Gai-ping(Key Laboratory of Animal Immunology,Henan Academy of Agricultural Sciences,Zhengzhou,Henan 450002,China;Food and Bioengineering College,Henan University of Science and Technology,Luoyang,Henan 471003,China;Henan Agricultural University,Zhengzhou,Henan 450002,China)

机构地区：[1]河南省农业科学院动物免疫学重点实验室,河南郑州450002 [2]河南科技大学食品与生物工程学院,河南洛阳471003 [3]河南农业大学,河南郑州450000

出　　处：《食品与机械》2019年第9期77-83,共7页Food and Machinery

基　　金：国家“十二五”科技支撑计划课题（编号：2014BAD13B05）;国家重点研发计划（编号：2018YFC1602903）;国家自然科学基金（编号：31702218）;河南省自然科学基金（编号：182300410038）;河南省科技攻关项目（编号：182102110445,182102310230）;河南省高等学校重点科研项目（编号：18B550001）;河南省动物免疫学重点实验室开放课题（编号：PKLAI20170603）

摘　　要：为制备土霉素(Oxytetracycline,OTC)完全抗原,获得免疫学特性良好的土霉素鼠源多抗血清并建立OTC免疫学检测方法,采用重氮化法在OTC分子上引入羧基,进而利用改进碳二亚胺法将其与载体蛋白偶联制备完全抗原,通过红外扫描、紫外扫描和凝胶电泳鉴定其偶联效果,然后将完全抗原免疫BLAB/c小鼠获得多抗血清(pAb)并对其免疫学特性进行鉴定,通过优化条件建立OTC间接竞争ELISA检测方法。结果表明,偶联效果良好,免疫的4只小鼠多抗血清效价均达到1 12 800;4只小鼠多抗血清均有抑制效果,其中2号小鼠产生的多抗血清效果最佳,基于该多抗血清建立的OTC间接竞争ELISA检测方法半数抑制浓度(IC 50)为32.92 ng/mL,检测限(LOD)为1.3 ng/mL,牛奶样品中添加回收率在84.70%~87.45%,与金霉素、四环素的交叉反应率分别为5.27%,4.10%,与其他竞争物交叉反应率<0.4%。该试验成功合成了OTC完全抗原,获得了免疫学特性良好的OTC多抗血清,并建立了OTC免疫学检测方法。To prepare complete antigen of oxytetracycline(OTC)and obtain mouse polyclonal antiserum with good immunological characteristics and establish immunological detection method of OTC.In this study,carboxyl groups were introduced into OTC molecules by diazotization,and then complete antigens were prepared by coupling with carrier proteins using an improved carbodiimine method.The coupling effect of complete antigen was identified by IR,UV and SDS-PAGE.Then the BLAB/c mice were immunized with complete antigens to obtain antiserum(pAb)and identify the antiserum immunological characteristics.After optimizing the reaction conditions of ELISA,indirect competitive ELISA method of OTC was established.The results showed that complete antigen has good coupling effect,and the titer of antiserum of all four mouse were higher than 1 12 800,indicating that the serum of all four mouse had inhibitory effect.The antiserum produced by mouse number 2 was the best.The sensitivity(IC 50)of the indirect competitive ELISA to assay OTC was 32.92 ng/mL,and the limit of detection(LOD)was 1.3 ng/mL.The addition recovery rate of milk samples was between 84.70%~87.45%.The cross-reaction rates with Chlortetracycline and Tetracyclines were 5.27%and 4.10%respectively,and the cross-reaction rates with other competitors were less than 0.4%.This study successfully synthesized OTC complete antigens and prepared OTC antiserum with good immunological properties,and initially established immunological detection method of it.

关 键 词：土霉素 完全抗原 多抗血清 ELISA 

分 类 号：S85[农业科学—兽医学]