醋酸甲羟孕酮单克隆抗体制备及ELISA检测方法的建立

建立饲料和食品中醋酸甲羟孕酮(MPA)残留快速、灵敏的检测方法.分别以碳二亚胺法、混合酸酐法合成MPA的人工抗原MPA-BSA、MPA-OVA为免疫原和包被原,采用淋巴细胞杂交瘤技术制备抗MPA单克隆抗体(McAb),在Protein G Sepharose 4 Fast Flow纯化MeAb基础上通过对ELISA反应条件的优化建立了MPA检测的间接竞争ELISA方法,并对MPA标品添加试样进行了初步应用.结果获得1株能稳定分泌抗MPA McAb的杂交瘤细胞株M68F9H9,分泌McAb腹水效价为1∶1×107,属于IgGl亚类,MPA半数抑制浓度(IC50)为5.0 ng/mL,具有良好特异性;以纯化McAb为基础建立的间接竞争ELISA,MPA最低检测限为0.16 ng/mL,线性检测范围为0.1～80 ng/mL;除与甲羟孕酮、甲地孕酮、盐酸克伦特罗、莱克多巴胺和已烯雌酚交叉反应率分别为100%、25%、<0.01%、<0.01%和<0.01%;MPA添加试样应用检测结果与进口试剂盒检测结果相符.

The Study on Monoclonal Antibody and ELISA Method for the Medroxyprogesterone Acetate(MPA) Residue Detection

To develop a rapid and sensitive method based the monoclonal antibody(McAb) against for the Medroxyprogesterone Acetate(MPA)residues detection in food. The artificial antigen MPA-BSA and MPA-OVA were synthesized by using diisopropylcarbodiimide method and mixed anhydrides method,respectively ;and the McAb against MPA was prepared and identified with the routine methods;then an indirect competitive inhibition ELISA (ciELISA) method for the quantitative determination of MPA was developed based on the anti-MPA McAb purified with the Protein G Sepharose 4 Fast Flow-affinity chromatography,and was compared with the commercial MPA EIA Kit (5131MPAlp3]08.05) in the primary application test of spike samples with different concentration MPA.One hybridoma cell line named M68F9H9 was obtained,the McAb against MPA belongs to IgGI subclass,the iELISA titer of anti-MPA McAb was 1:1×10⁷,and the ICso value was 5 ng/mL; the limit of detection of this ciELISA method established in this study was 0.16 ng/mL;the linear detection range was from 0.1 ng/mL to 80 ng/mL,and the cross-reactivity of Medroxyprogesterone, megestrol acetate (MA),Clenbuterol,Ractapo mine and DES were 100%,25%,<0.01%,<0.01% and<0.01% respectively;the application detection results of spike samples with the ciELISA are concordance with the method of MPA EIA Kit imported.