半抗原直接包被的磺胺二甲氧嘧啶酶联免疫吸附分析法的建立  

Establishment of Hapten Coated Enzyme-Linked Immunosorbent Assay for Sulfadimethoxine

作　　者：张玉超 周洪波 刘良禹 朱思洁 刘旭东 ZHANG Yuchao;ZHOU Hongbo;LIU Liangyu;ZHU Sijie;LIU Xudong(Department of Brewing Engineering,Moutai Institute,Renhuai 564507,China;Department of Food Science and Engineering,Moutai Institute,Renhuai 564507,China;College of Food Science and Engineering,Ocean University of China,Qingdao 266071,China;Guizhou Specialty Food Development and Comprehensive Utilization Engineering Research Center,Renhuai 564507,China)

机构地区：[1]茅台学院酿酒工程系,贵州仁怀564507 [2]茅台学院食品科学与工程系,贵州仁怀564507 [3]中国海洋大学食品科学与工程学院,山东青岛266071 [4]贵州特色食品开发及综合利用工程研究中心,贵州仁怀564507

出　　处：《食品科技》2023年第3期303-309,共7页Food Science and Technology

基　　金：遵义市科学技术局茅台学院市校联合科技研发资金项目(遵市科合HZ字[2020]315号);教育部“春晖计划”国际合作项目(mych2019001);茅台学院科学研究项目(myk2019008);贵州省大学生创新创业训练计划项目(X202214625041)。

摘　　要：作为食品中抗生素残留的主要类型之一,传统的磺胺类抗生素(Sulfonamides,SAs)酶联免疫吸附分析法(Enzyme-linked immunosorbent assay,ELISA)常以多克隆抗体和完全抗原包被作为检测基础,可能会出现检测灵敏度低、标准化难等问题。为建立更优的SAs ELISA检测方法,以磺胺二甲氧嘧啶(Sulfadimethoxine,SDM)为研究对象,并将其作为半抗原与大分子蛋白质偶联制备人工完全抗原免疫BALB/c小鼠,获得抗SDM的单克隆抗体(Monoclonal antibody,MAb)。并采用酸性高锰酸钾羧化酶标板,将SDM直接包被在酶标板上后对ELISA反应体系条件进行优化,构建一种基于MAb的半抗原直接包被的SDM间接竞争ELISA(Indirect competitive ELISA,icELISA)检测方法。所制备的MAb特异性良好,交叉反应率低。所构建的icELISA检测法检测条件为:半抗原包被浓度为0.5μg/mL,抗体稀释比为256000,竞争反应体系温度为25℃,pH7.4,以1%BSA溶液作为封闭剂。该方法最低检测限IC_(10)为0.274 ng/mL,半数抑制率IC50为8.39 ng/mL,水和牛乳中加标回收率分别为97.28%~101.2%、98.34%~101.28%。与同等条件下建立的人工抗原包被的icELISA检测方法(IC_(10):0.579 ng/mL,IC50:22.5 ng/mL,水和牛乳中加标回收率分别为92.08%~105.22%、94.62%~103.76%)相比检测灵敏度有明显提高,并可用于实际样品中SDM含量的检测,具有良好的应用前景。As one of the main types of antibiotic residues in food,the traditional sulfonamides(SAs)antibiotic enzyme-linked immunosorbent assay(ELISA)is often based on polyclonal antibody and complete antigen coating.Problems such as low detection sensitivity and difficult standardization may occur.In order to establish a better SAs ELISA method,sulfadimethoxine(SDM)was used as hapten,coupled with macromolecular protein to prepare artificial complete antigen to immunize BALB/c mice,monoclonal antibody(MAb)against SDM was obtained.The ELISA reaction system conditions were optimized after SDM was coated directly on the ELISA plate with acid potassium permanganate carboxylase plate,and an indirect competitive ELISA(icELISA)method for SDM,which directly coated with hapten based on MAb was constructed.The prepared MAb has good specificity and low crossreaction rate.The detection conditions of the constructed icELISA assay were as follows:hapten coating concentration was 0.5μg/mL,antibody dilution ratio was 256000,competitive reaction system temperature was 25℃,pH7.4,1%BSA solution was used as blocking agent.The IC_(10) of the method was 0.274 ng/mL,the half inhibition rate IC50 was 8.39 ng/mL,and the recoveries of water and milk were 97.28%~101.2%and 98.34%~101.28%,respectively.Compared with the artificial antigen coated icELISA method established under the same conditions(IC_(10):0.579 ng/mL,IC50:22.5 ng/mL,the recoveries of water and milk were:92.08%~105.22%,94.62%~103.76%),the detection sensitivity was significantly improved,and can be used for the detection of SDM content in actual samples,which has a good application prospect.

关 键 词：磺胺二甲氧嘧啶 半抗原包被 人工完全抗原 单克隆抗体 酶联免疫吸附分析法 

分 类 号：TS207.3[轻工技术与工程—食品科学]