呋喃它酮代谢物单链抗体制备和酶联免疫分析方法  

Preparation of Single-chain Variable Fragment Antibody against Furaltadone Metabolite and Development of Indirect Competitive Enzyme-linked Immunosorbent Assay for Its Detection in Shrimp Samples

作　　者：杨武英[1] 王弘[2] 洪艳平[1] 王丹[1] 徐振林[2] 沈玉栋[2] 柯法钧 陈薪竹 孙远明[2] YANG Wuying;WANG Hong;HONG Yanping;WANG Dan;XU Zhenlin;SHEN Yudong;KE Fajun;CHEN Xinzhu;SUN Yuanming(Key Laboratory of Agricultural Products Processing and Quality Control of Nanchang City,School of Food Science and Engineering,Jiangxi Agricultural University,Nanchang 330045,China;Key Laboratory of Food Quality and Safety of Guangdong Province,College of Food Science,South China Agricultural University,Guangzhou 510642,China)

机构地区：[1]南昌市农产品加工与质量控制重点实验室,江西农业大学食品科学与工程学院,江西南昌330045 [2]广东省食品质量安全重点实验室,华南农业大学食品学院,广东广州510642

出　　处：《食品科学》2020年第24期251-258,共8页Food Science

基　　金：国家自然科学基金地区科学基金项目(31660486)。

摘　　要：从呋喃它酮代谢物5-吗啉甲基-3-氨基-2-恶唑烷基酮(5-morpholinomethyl-3-amino-2-oxazolidinone,AMOZ)衍生物单克隆抗体杂交瘤细胞中克隆抗体重链可变区和轻链可变区基因,通过(G1y4Ser)3连接肽采用重叠延伸聚合酶链式反应技术构建单链抗体基因,pET-22b(+)表达载体进行表达,Ni亲和柱进行纯化,制备得到抗AMOZ衍生物单链抗体,采用棋盘滴定实验优化包被原包被浓度和单链抗体稀释度,建立基于此单链抗体的AMOZ残留间接竞争酶联免疫分析方法并对方法进行评价。结果表明:最佳包被抗原质量浓度和单链抗体稀释度分别为0.0625μg/mL和20倍;建立的间接竞争酶联免疫分析方法半抑制浓度为8.65μg/L,线性范围为2.90~53.28μg/L,检测限为1.48μg/L;除与原药呋喃它酮有交叉反应外,此单链抗体与其他硝基呋喃类抗生素及其代谢物交叉反应率均小于0.1%,虾肉样品添加回收率为74.3%~86.6%;用高效液相色谱-串联质谱法对免疫测定结果进行确证实验,两种方法相关性良好(R^2=0.9992),说明建立的间接竞争酶联免疫分析方法可用于虾类等水产品中AMOZ残留的快速筛查。In this study,the genes of heavy chain and light chain variable fragment were cloned from hybridoma cells secreting a monoclonal antibody against a derivative of the furaltadone metabolite 5-morpholinomethyl-3-amino-2-oxazolidinone(AMOZ).The gene of single-chain variable fragment(scFv)antibody against this derivative was constructed by splicing overlap extension-polymerase chain reaction(SOE-PCR)through the ligating peptide(G1y4Ser)3,inserted into the expression vector pET-22b(+)and expressed in E.coli BL21(DE3).The expressed product was purified by Ni affinity column chromatography.The concentration of coating antigen and the dilution factor of scFv antibody were optimized by chessboard titration to establish and evaluate an indirect competitive enzyme-linked immunosorbent assay(icELISA)based on this scFv antibody for AMOZ residues.The results showed that the optimal concentration of coating antigen and the dilution factor of scFv antibody were 0.0625μg/mL and 20,respectively.The IC50 value,linear range and limit of detection(LOD)of the established icELISA method were 8.65μg/L,2.90–53.28μg/L and 1.48μg/L,respectively.In addition to the cross-reactivity with the original drug furantadone,the cross-reactivity rates of the scFv antibody with other nitrofuran antibiotics and their metabolites were lower than 0.1%.The average recoveries of AMOZ from the spiked shrimp samples ranged from 74.3%to 86.6%.Good correlation(R^2=0.9992)was obtained between the results of icELISA and high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS).The icELISA method established in this study is suitable for the rapid screening of AMOZ residues in shrimp samples and other aquatic products.

关 键 词：呋喃它酮 呋喃它酮代谢物 单链抗体 间接竞争酶联免疫分析 

分 类 号：TS207.3[轻工技术与工程—食品科学]