醋酸甲羟孕酮间接竞争ELISA检测方法建立

Indirect competitive enzyme-linked immunosorbent assay for detection of medroxyprogesterone acetate residues

目的建立以单抗为基础的醋酸甲羟孕酮间接竞争ELISA检测方法。方法将羧甲基羟胺盐酸盐和醋酸甲羟孕酮（MPA）反应生成MPA-3-（O-羧甲基）肟（MPS）,MPS与牛血清白蛋白（BSA）偶联生成免疫原,采用淋巴细胞杂交瘤技术制备MPA单克隆抗体（MPA-McAb）,建立水产品中MPA残留的间接竞争ELISA（ciELISA）检测方法。结果经飞行质谱法测得MPS-BSA分子量为76 045.656 3,MPS与BSA偶联比为25.17;MPA-McAb最佳稀释倍数为1∶400 00;包被抗原最佳质量浓度为1μg/mL;标准曲线呈线性,相关系数R2=0.983 7,IC50=3.630 ng/mL;最低检测限为0.133 ng/mL;批内和批间变异系数分别为3.125%和3.557%;黄鳝鱼肉样的平均添加回收率为80.46%~101.00%;检测方法与甲羟孕酮、甲地孕酮的交叉反应（CR%）分别为0.26%,10.67%,与其他抗生素药物无交叉反应。结论醋酸甲羟孕酮间接竞争ELISA检测方法可用于水产品中醋酸甲羟孕酮残留量检测。

Objective To establish an indirect competitive enzyme-linked immunosorbent assay for the detection of medroxyprogesterone acetate residue.Methods Medroxyprogesterone acetate（MPA） was activated by carboxymethyl hydroxylamine hemi-HCL to form MPA-3-10-carboxymethyl oximel.The hapten was coupled to bovine serum albumin（BSA） with diisopropylcarbodiimide method to synthesize artificial immunogen.The monoclonal antibody（McAb） against MPA was prepared and identified with routine methods.Indirect competitive enzyme-linked immunosorbent assay（ciELISA） based on McAb was developed for the detection and estimation of medroxyprogesterone acetate residue in aquatic products.Results The artificial antigen MPS-BSA was confirmed by MALDI-TOF and its molecular weight was 76 045.656 3.The couple ratio of MPS-BSA was 25.71.The optimal concertration of the coating antigen（MPS-OVA） and that of McAb against MPA was 1ug/ml and 1：40 000,respectively.Linear detection of ciELISA covered a range from 0.2ng/mL to 200ng/mL（R2 = 0.9707） with a sensitivity of 0.133ng/mL.The intra-assay and inter-assay coefficients of variation were 3.125% and 3.557%,respectively.And the average fortified recovery of the method was 80.5%-101.00%.ELISA had 0.26%,10.67% of cross-reactivity with megestrol and medroxyprogesterone acetate,respectively.There was no cross-reactivity with other antibiotics.Conclusion The indirect competitive enzyme-linked immunosorbent assay was established,which could be used to detect residues of medroxyprogesterone acetate in aquatic products.