莫米松糠酸酯高特异性抗体制备及酶联免疫分析方法

Preparation of high specificity antibody against mometasone furoate and enzyme-linked immunosorbent assay method

莫米松糠酸酯(mometasone furoate,MF)是一种常见的糖皮质激素。为快速、便捷地监控MF的滥用情况,制备了MF单克隆抗体并建立了其竞争酶联免疫检测方法。将氨氧乙酸分别连接至MF和莫米松二糠酸酯(mometasone difuroate,MDF),合成了免疫半抗原和包被半抗原。免疫半抗原以活泼脂法连接钥孔血蓝蛋白获得全抗原,通过免疫小鼠、细胞融合及筛选获得能够稳定分泌抗MF单克隆抗体的细胞株,制备并纯化了抗体。该抗体的主要识别区域为MF的五元环区域,因此不和常见的64种糖皮质激素发生交叉反应。建立了基于异源包被的MF间接竞争酶联免疫检测法,其半抑制浓度(IC_(50))为1.2 ng/mL,对肌肉和肝脏的最低检出限分别为0.52μg/kg和0.59μg/kg。灵敏度相比于同源包被提升了20倍。使用60%甲醇水溶液提取动物组织的添加回收率为70%~82%。该方法能有效应用于动物组织中MF的快速筛查。

Mometasone furoate(MF)is a common glucocorticoid.A monoclonal antibody against MF was prepared,and a competitive enzyme-linked immunosorbent assay for MF was developed to quickly and conveniently monitor the abuse of MF.Aminooxyacetic acid was connected to MF and mometasone difuroate(MDF),respectively,to synthesize the immune hapten and the coating hapten.The immune hapten was linked to keyhole limpet hemocyanin using the active ester method to obtain the whole antigen.A cell line capable of stably secreting monoclonal antibodies against MF was obtained through immunizing mice,cell fusion,and screening.Furthermore,this antibody was prepared and purified.The main recognition region of the antibody was the five-membered ring region of MF,thus it did not cross-react with the common 64 glucocorticoids.An indirect competitive enzyme linked immunosorbent assay based on heterologous coating was established.The IC_(50) was 1.2 ng/mL.The minimum detection limit for meat and liver were 0.52μg/kg and 0.59μg/kg,respectively.The sensitivity was 20 times higher than that of the homologous coating.The recovery rate of animal tissues extracted with a 60%methanol aqueous solution was between 70%and 82%.This method can be effectively applied to the rapid screening of MF in animal tissues.