醋酸甲羟孕酮(MPA)人工抗原的合成与单克隆抗体的制备  

Preparation of Artificial Antigen for Medroxyprogesterone Acetate(MPA) and McAb Against MPA

作　　者：杨泽晓[1] 张彦明[1] 林祥梅[2] 梅琳[2] 刘建[2] 韩雪清[2] 

机构地区：[1]西北农林科技大学动物医学院,杨凌712100 [2]中国检验检疫科学研究院,北京100029

出　　处：《上海交通大学学报（农业科学版）》2008年第2期119-123,共5页Journal of Shanghai Jiaotong University(Agricultural Science)

基　　金：国家质检总局科研项目(2006IK017)

摘　　要：为研制检测醋酸甲羟孕酮(MPA)的免疫学检测技术进行MPA单克隆抗体的制备与鉴定,研究分别采用碳二亚胺法、混合酸酐法将MPA偶联到牛血清白蛋白(BSA)和鸡卵清白蛋白(OVA)上制备免疫原(MPA-BSA)与包被原(MPA-OVA),通过BALB/c小鼠免疫、细胞融合、间接ELISA和阻断ELISA筛选、有限稀释法克隆亚克隆获得分泌MPA单克隆抗体的杂交瘤细胞株,并通过ELISA方法对其抗体效价、特异性、敏感性、亚类及稳定性进行鉴定。结果表明:成功合成2种人工抗原,MPA与BSA、OVA的偶联比分别为8:1、12:1;获得1株稳定分泌MPA单克隆抗体(McAb)的杂交瘤细胞株2G4H9,McAb腹水的间接ELISA效价为1:8×10~4,IgG1亚类,κ型轻链,腹水抗体IC_(50)为5.0 ng·mL^(-1),与甲地孕酮、盐酸克伦特罗、莱克多巴胺和己烯雌酚交叉反应率为12.5%、<0.01%、<0.01%和<0.01%。作者认为:MPA单克隆抗体特异性和敏感性良好,杂交瘤细胞株性能稳定,为进一步研究MPA相关免疫学分析技术奠定了基础。To prepare monoclonal antibody against medmxypmgestemne acetate（MPA） for the development of immunological analysis methods for MPA residues detection. The hapten MPA was coupled to bovine serum albumin（BSA） and ovalbulmin（OVA） by using diisopmpylcarbodiimide method and mixed anhydrides method to synthesize artificial immunogen and coating antigen, respectively, and the artificial antigen MPA-BSA, MPA-OVA were confirmed by UV spectrum and SDS-PAGE; then the monoclonal antibodies（McAb） against MPA was prepared by cell fusion and sub-cloning. The main characteristics of screened McAb against MPA were further identified by indirect ELISA and blocking ELISA. Two artificial immunogen MPA-BSA and coating antigen MPA-OVA were successfully synthesized, and the couple rates of MPA：BSA, MPA：OVA were 8：1, 12：1, respetively; one hybridoma cell line named 2G4H9 was obtained, the highest titers of the McAb against MPA determined by indirect ELISA was 1：8×10^4, it belongs to IgG1 subtype and κlight chain, the concentration of MPA that inhibits 50% of antigen-antibody binding was 5.0 ng.mL^-1, the cross-reactions rates of the prepared McAb with MA, Clenbuterol, Rac and DES were 12.5%, 〈0.01%, 〈0.01% and 〈0.01% respectively. The mcAb against MPA with excellent specificity and affinity, this study set basis for development of the immunological analysis methods of MPA residues.

关 键 词：醋酸甲羟孕酮 人工抗原 单克隆抗体 

分 类 号：S851[农业科学—预防兽医学] Q939.91[农业科学—兽医学]