醋酸甲羟孕酮单克隆抗体制备及ELISA检测方法的建立

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

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McAb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∶1×10^7,属于IgG1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、莱克多巴胺和己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。

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 MeAb against MPA was prepared and identified with the routine methods; then an indirect competitive inhibition ELISA（eiELISA） 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 （5131MPA1p[-3]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 IgG1 subclass, the iELISA titer of anti-MPA McAb was 1 ： 1× 10^7, and the IC50 value was 5 ng/mL; the limit of detection of this eiELISA 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, Ractapomine 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. Key words.