基于异源包被的 曲安奈德竞争酶联免疫检测方法

为监控曲安奈德的滥用情况,本研究建立了曲安奈德竞争酶联免疫检测方法。将曲安奈德(Triamcinolone acetonide,TCA)半抗原通过活泼酯法与钥孔戚血蓝蛋白(keyhole limpet hemocyanin,KLH)偶联获得曲安奈德完整抗原TCA-KLH;通过免疫小鼠、细胞融合及筛选获得一株能够稳定分泌抗曲安奈德的单克隆抗体细胞株,制备并纯化了腹水抗体。将TCA和地塞米松(Dexamethasone,DEX)半抗原通过活泼酯法分别与牛血清白蛋白(bovine serum albumin,BSA)偶联作为包被原,建立曲安奈德同源及异源的间接竞争酶联免疫分析方法(indirect competitive enzyme linked immunosorbent assay,ic-ELISA)。在同源包被情况下,曲安奈德灵敏度为5.4 ng/mL,而DEX异源包下灵敏度为0.53 ng/mL。未观察该方法到对哈西奈德、布地奈德、安西奈德以外的其他糖皮质激素的交叉反应。在添加回收实验中,使用20%甲醇水溶液提取动物组织的添加回收率在75%~95%之间。该方法能有效应用于动物组织中曲安奈德的快速筛查。

Detection of Triamcinolone Acetonide Indirect Competitive ELISA Based on Heterology Coating Strategy

An indirect competitive ELISA for triamcinolone acetonide(TCA)was developed to monitor TCA abuse. To obtain the TCA complete antigen,TCA hapten and keyhole limpet hemocyanin(KLH)were coupled by using N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride(EDC)and N-Hydroxysuccinimide(NHS). The homologous detection antigen TCA-bovine serum albumin(TCA-BSA)and heterologous detection antigen Dexamethasone-BSA(DEX-BSA)were synthesized in the same way. After immunization and cell fusion,one strain of hybridoma cell line that could steadily secrete anti-TCA monoclonal antibody was prepared and its antibody(ascites)was prepared and purified. By using the antibody and two different detection antigens,both homologous and heterologous strategy indirect competitive enzyme linked immunosorbent assay(ic-ELISA)standard curve for detection of TCA was built,recoveries of TCA added to liquid milk was detected by the heterologous strategy. The results showed that the IC₅₀ of homologous strategy ic-ELISA was 5.4 ng/mL,the IC₅₀ of heterologous strategy ic-ELISA was 0.53 ng/mL.The method did not cross reacted with other glucocorticoids except halcinonide,budesonide and amcinonide. The recovery rates of TCA added to animal tissues ranged from 75% to 90% using 20% methanol/water. The method can be applied in TCA fast screening in animal tissue.