胶体金标免疫固相膜检测动物性食品中氟甲喹  

Colloidal Gold Labeled Solid Phase Membrane-Based Immunoassay to Detect Flumequine in Animal Foods

作　　者：刘卫华[1] 于文龙[1] 杨茜 王向红[1] LIU Wei-hua;YU Wen-long;YANG Xi;WANG Xiang-hong(College of Food Science and Technology,Hebei Agricultural University,Baoding 071000,Hebei,China)

机构地区：[1]河北农业大学食品科技学院

出　　处：《食品研究与开发》2020年第5期177-183,共7页Food Research and Development

基　　金：“十三五”国家重点研发计划(2016YFD0401101)

摘　　要：氟甲喹(flumequine,FLU)是动物专用的抗生素,滥用或超标使用会造成动物性食品中的残留问题,对人类健康造成危害。以活泼酯法制备氟甲喹完全抗原,免疫新西兰大耳白兔获得抗血清,通过间接竞争酶联免疫法(enzyme linked immunosorbent assay,ELISA)测定抗血清的效价和亲和性,效价为1∶12 800,纯化后抗体的IC50达到0.055 ng/mL。采用柠檬酸三钠还原法制备胶体金,并用于标记FLU多克隆抗体制备免疫金。以硝酸纤维素膜为固相载体,包被上包被原和酶标二抗作为检测线和控制线,优化检测条件:封闭液为5%脱脂乳、酶标二抗稀释40倍、包被量1.0μg、免疫金稀释度1∶5(体积比)、金标抗体与待测溶液混合比例1∶5(体积比)。在最优条件下,制成FLU胶体金标记免疫固相膜,最低检出限达40μg/L。建立的胶体金标记固相膜免疫检测方法适于大量样品的快速定性筛查。Flumequine(FLU),an animal-specific antibiotic,was abused or over standard used and resulted in the residual problem in animal foods,which caused great harm to human health. Complete antigen of FLU was prepared by active ester method and was used to immunize New Zealand white rabbits to obtain antiserum. The titer and affinity of the antiserum were determined by indirect competitive enzyme linked immunosorbent assay(ELISA). The titer was 1 ∶ 12800 and IC50 was 0.055 ng/mL after purification. Colloidal gold was prepared by trisodium citrate reduction method and immune gold was prepared by FLU antibody being labeled with colloidal gold. Nitro-cellulose membrane strip which used as solid carrier was separately coated with coating antigen(as test line)and enzyme-labeled secondary antibody(as control line). The optimum conditions were as follows:blocking solution was 5 % skimmed milk,enzyme-labeled antibody dilution times was 40 times,coating amount was 1.0 μg,immunogold dilution was 1 ∶ 5(volume ratio),the mixing ratio of gold-labeled antibody and the solution to be tested was 1 ∶ 5(volume ratio). Under the optimum conditions,the FLU colloidal gold labeled immunochromatographic strip was prepared with a minimum detection limit of 40 μg/L. The established solid phase membrane-based immunoassay was suitable for rapid qualitative screening.

关 键 词：氟甲喹(flumequine FLU) 多克隆抗体 固相膜免疫分析 胶体金 兽药残留 

分 类 号：O652[理学—分析化学] TS251.7[理学—化学]