双交联法制备桔霉素-蛋白质偶联抗原及抗体

Preparation of CIT-protein Antigen and Antibody With The Bicoupling Method

以1,4-丁二醇二缩水甘油醚(双环氧试剂)为偶联剂,合成桔霉素-蛋白质偶联抗原CIT-BSA,经过HPLC分析、紫外扫描和红外光谱鉴定表明,偶联物成功制备,CIT/BSA的偶联比为8.16.通过免疫BALB/C小鼠,获得抗桔霉素多克隆抗体,经间接ELISA检测,效价达到1.1×105.间接竞争ELISA表明,桔霉素(CIT)的最低检测浓度为10μg/L,其线性范围为10～250μg/L,IC50为100μg/L.分析了不同方法制备的偶联抗原的免疫原性,实验表明,桔霉素抗原决定簇C7位置的羧基保留是获得针对桔霉素特异性抗体的必要条件.为快速检测桔霉素的酶联免疫检测技术的建立和检测试剂盒的研制提供技术依据.

To prepare Citrinin（CIT）-protein antigen, CIT was conjugated with bovine serum albumin （BSA） by 1,4-butanediol diglycidyl ether. HPLC, UV and IR absorption suggested that CIT was correlated with the carrier protein, and the molar ratio of CIT to BSA was 8.16. The polyclonal antibodies （PcAb） against CIT were produced in serum of immunized BALB/C mice with CIT-BSA, and the titer of antibody reached to 1.1 ×105 by indirect enzyme-linked immunoassay. The indirect competitive ELISA showed that the detection limit of CIT was 10 μg/L, with a good linearity ranging 10 ～250 μg/L, and IC50 was 100 μg/L. Immunogenicity of antigens prepared by different methods was analyzed. The result showed that the epitope was the carboxyl group at C7. This work would be helpful for establishing the technology and developing the kit to determine CIT-contaminated samples by ELISA.