己烯雌酚完全抗原的合成及分析

Synthesis and analysis of complete antigen of diethylstilbestrol

以己烯雌酚(DES)?氯乙酸钠(CH2ClCOONa)和牛血清蛋白(BSA)等为主要原料,首先用氯乙酸钠修饰DES分子中的一个酚羟基从而合成单羧甲醚己烯雌酚(DES-MCME),然后以混合酸酐法将DES-MCME与载体蛋白(BSA?OVA)偶联形成完全抗原,并以紫外扫描分析完全抗原的偶联比率,再将得到的免疫原DES-BSA按免疫程序免疫新西兰白兔。结果表明己烯雌酚及其衍生物存在着顺反异构转化现象,极性有机溶剂的溶剂化作用促使顺反异构体的转化。高分辨质谱测得合成的DES-MCME相对分子质量为325。人工免疫原及包被原的最适结合比为13和10。采用间接酶联免疫测定,产生的抗血清的稀释度可以达到102400。从而建立了DES-MCME的制备方法,并通过高分辨质谱和ELISA检测方法验证了DES人工抗原的有效性,为研制DES残留免疫检测试剂盒奠定了基础。

Diethylstilbestrol monocarboxymethoxyflurane （DES-MCME） was synthesized by first retouching a phenolic hydroxyl group of DES molecule and using diethylstilbestrol, sodium monochloroacetate and bovine serum albumin as key raw materials. The conjugations of DES-MCME with proteins as a complete antigen were prepared by the mixed acid anhydride method and the conjugations were analyzed by ultraviolet scanning. According to the immune protocol, New Zealand white rabbits were immunized. The results of experimentation showed that isomerism was present in cis-and trans-forms of DES and its derivates, which was effected obviously by polar solvents. High resolution mass spectroscopy revealed that the molecular mass of DES-MCME was 325. The optimal conjugation ratios of immunogen and coating antigen were 13 and 10. The dilution rate of antiserum could reach 102400, through an indirect enzyme-linked immunosorbent assay （ ELISA ）. The preparation method of DES-MCME was developed. The effectiveness of the artificial antigen of DES was established by high resolution mass spectroscopy and ELISA methods. This could provide the basis for the DES ELISA kit.