摘要
用多元酸酐与混合酸酐相结合的方法合成了莱克多巴胺（Rac）抗原，免疫动物获得特异性抗体，并以蛋白A柱纯化得到IgG抗体。琼脂糖凝胶（Sepharose4B）经溴化氰（CNBr）活化后与IgG抗体偶联，制备莱克多巴胺免疫吸附剂。据此建立了尿液中莱克多巴胺的免疫亲和柱净化／液相色谱-荧光法（HPLC-FL）测定的分析方法。免疫制备特异抗体50％抑制浓度（IC50）为5μg／L。Sepharose4B经CNBr活化后与2mg抗体的偶联率达87．4％。1mL吸附剂的柱容量为67．57ng。尿液中莱克多巴胺的回收率为76％～90％。
A polyclonal antibody-based immunoaffinity column （IAC） was developed as a cleanup method for the detection of ractopamine （Rac）. Anti-rac specific antibody was gotten with immunized New Zealand rabbit. The IgG was isolated from antiserum with HiTrap Protein AHP column （PrA） and SephadexG25 cartridge. The lmmunosorbents were prepared by coupling anti-Rac IgG with sepharose 4B aetived by cyanogens bromide. Rac was cleaned up with the prepared immunoaffinity col- umn and detected by high-performance liquid chromatography with fluorescent detection （HPLC - FL）. The titer of anti-Rac antibody was more than 6 400 by indirect ELISA and the IC50 was 5 μg/L by indirect competitive ELISA method. The coupling efficiency of 2 mg antibody with aetived Sepharose 4B was 87.4% and the column capacity for 1 mL immunosorbents was 67.57 ng. The optimal clean-up condition for immunoaffinity column was sequentially rinsed with 5 mL phosphate buffer solu- tion, 5 mL 10% methanol and eluted with 5 mL 95% ethanol （pH =4. 0）. The recoveries of fortified samples at levels of 1, 5, 15, 30, 50 ng Rac in 1 mL blank pig urine were in the range of 76%- 90%. The immunoaffinity column could keep stable for 3 months in PBS containing 0. 02% NaN3 at 4 ℃.
出处
《分析测试学报》
CAS
CSCD
北大核心
2010年第8期812-816,共5页
Journal of Instrumental Analysis
基金
农业部948项目资助(2003-Q08)
作者简介
第一作者：王迪（1980-），女，黑龙江望奎人，助理研究员，博士
通讯作者：杨曙明，Tel：010—82106561，E—mail：smyang@foss．com．cn
