摘要
为制备洛美沙星(LMF)多克隆抗体,采用碳二亚胺(EDC)法,将洛美沙星(LMF)与BSA/OVA进行偶联制备了人工免疫抗原LMF-BSA和包被抗原LMF-OVA。用紫外分光光度法对人工抗原LMF-BSA和LMF-OVA进行鉴定,判断人工抗原合成成功。用LMF-BSA免疫小鼠后,制备多克隆抗体,用方阵滴定法检测包被原在ELISA试验中的最佳工作浓度。选用6种封闭液,进行间接ELISA试验,以P/N大小为标准,选出最佳封闭液,使用建立的ELISA方法检测多抗血清的效价和灵敏度。紫外分光光度法测得LMF-BSA和LMF-OVA相对于BSA和OVA的最大吸收峰均发生偏移,表明人工抗原合成成功。方阵滴定法确定包被原最佳工作浓度为2μg/mL,最佳封闭液为5%猪阴性血清。3只小鼠多抗血清效价和敏感性均较好,多抗血清效价达到1∶12800,半数抑制浓度(IC50)为23.69 ng/mL。表明洛美沙星多克隆抗体制备成功,为洛美沙星单克隆抗体的制备和ELISA检测试剂盒研制等后续试验奠定基础。
In order to prepare polyclonal antibody of lomefloxacin(LMF).The artificial immune antigen LMF-BSA,and coated antigen LMF-OVA were prepared by coupling lomefloxacin(LMF)with BSA/OVA by carbodiimide method.The artificial antigen LMF-BSA and LMF-OVA were identified by UV spectrophotometry to judge the success of synthetic antigen.The polyclonal antibody was prepared by immunizing mice with LMF-BSA,and the optimum working concentration of coating was detected by phalanx titration in ELISA test;six kinds of sealants were tested by indirect ELISA.The best sealants were selected according to the P/N size.Titer and sensitivity of polyclonal antibody serum were detected using established ELISA method.The results showed that the LMF-BSA and LMF-OVA were offset relative to the maximum absorption peaks of BSA and OVA,by UV spectrophotometry.By square array titration,the optimum working concentration of coating was 2μg/mL,and the best sealing solution was 5% pig negative serum.The titer and sensitivity of polyclonal antibody serum were good in mice No.3,the titer of polyclonal antibody serum reached 1∶12800,half inhibitory concentration(IC50)23.69 ng/mL.The successful preparation of polyclonal antibody of lomefloxacin lays a foundation for the preparation of monoclonal antibody of lomefloxacin and the development of ELISA detection kit.
作者
杨树宇
王文哲
吴玉苹
吴世秀
陈俊杰
刘长忠
姜金庆
Yang Shuyu;Wang Wenzhe;Wu Yuping;Wu Shixiu;Chen Junjie;Liu Changzhong;Jiang Jinqing(College of Animal Science and Veterinary Medicine,Henan Institute of Science and Technology,Xinxiang 453003,China;Xinxiang University,Xinxiang 453003,China)
出处
《畜牧兽医科学（电子版）》
2021年第6期1-6,共6页
Graziery Veterinary Sciences：Electronic Version
基金
中原千人计划-中原科技创新领军人才项目(204200510010)
河南省高校科技创新团队(20IRTSTHN025)
河南省重点科技攻关项目(202102110090)
新乡市酵守康饲料有限公司横向项目(1344010016)资助。
关键词
洛美沙星
人工抗原
ELISA
多克隆抗体
鉴定
lomefloxacin
artificial antigen
ELISA
polyclonal antibody
identification
