| | | 抗黄曲霉毒素M1抗体制备及检测方法建立
| | 免费毕业论文下载【关键词】 黄曲霉毒素M1；单克隆抗体；酶联免疫吸附试验
摘要： 目的免费毕业论文下载 制备针对黄曲霉毒素M1的免费毕业论文下载单克隆抗体并建立针对黄曲霉毒素M1的间接竞争酶联免疫吸附试验检测方法。方法 利用B细胞杂交瘤技术，建立能分泌抗黄曲霉毒素M1单克隆抗体的杂交瘤细胞株，制备抗黄曲霉毒素M1单克隆抗体，建立间接竞争酶联免疫吸附试验检测方法。结果 研制出1株能特异性分泌抗黄曲霉毒素M1单克隆抗体的杂交瘤细胞株，命名为2F2。该单克隆抗体的Ig亚类为IgG1，亲和常数为28×10-11mol/L。该抗体与黄曲霉毒素B1、黄曲霉毒素B2、黄曲霉毒素G1、黄曲霉毒素G2和黄曲霉毒素M2等结构类似物有微弱的交叉反应，具有较高的特异性。在此基础上建立了间接竞争酶联免疫吸附试验检测方法。该方法的最低检出浓度为007ng/ml，校正曲线的线性范围为002～2ng/ml，线性方程y=-04364x+02693(R2=09949)。方法的加标回收率为725%～1313%。结论 制备了具有高特异性和亲和力的抗黄曲霉毒素M1单克隆抗体，并建立了快速、灵敏的针对黄曲霉毒素M1的酶联免疫吸附试验检测方法。
关键词： 黄曲霉毒素M1；单克隆抗体；酶联免疫吸附试验
Development of monoclonal antibody against aflatoxin M1 and immunoassay for aflatoxin M1
Abstract： Objective To prepare monoclonal antibody against aflatoxin M1 and develop indirect competitive enzyme-linked immunosorbent assay for detection of aflatoxin M1.Methods Hybridoma cell line excreting monoclonal antibody against aflatoxin M1 was produced using B cell hybridoma technique and develop indirect competitive enzyme-linked immunosorbent assay for detection of aflatoxin M1.Results One hybridoma cell line excreting monoclonal antibodies against aflatoxin M1 coded 2F2 was obtained by fusing murine Sp2/0 cells with spleen cells from BALB/c mice immunized with AFM1-BSA conjugate.The monoclonal antibody produced by the hybridoma cell were tested for subtype and designated as IgG1 for 2F2.The affinity of IgG in purified ascites yielded by hybridoma cell was 28×10-11 mol/L.The monoclonal antibody obtained in the present study was specific to aflatoxin M1,because of lightly cross reactions among the monoclonal antibodies against aflatoxin M1 with the analogues of aflatoxin B1,aflatoxin B2,aflatoxin G1,aflatoxin G2 and aflatoxin M2 were found.The limit of detecting concentration of aflatoxin M1 was 0.07 ng/ml.The linear range of standard curve was 002～2ng/ml and the linear equation was y=-04364x+02693(R2=0.9949).The recovery of aflatoxin M1 was between 725%～1313%.Conclusion The monoclonal antibody obtained in the study was of relatively high specificity to aflatoxin M1,and a simple fast sensitive indirect competitive enzyme-linked immunosorbent assay for the detection of aflatoxin M1 was developed.
Key words： aflatoxin M1;monoclonal antibody;enzyme-linked immunosorbect assay
黄曲霉毒素M1(aflatoxin M1,AFM1)是动物摄入黄曲霉毒素B1(AFB1)后在体内经羟基化代谢的产物，存在于动物体内可食部分，如乳、肝、蛋类等，其中以乳最为常见，且乳与乳制品中的AFM1在生产和贮藏期间相对稳定，巴氏消毒难以破坏〔1〕。AFM1的急性毒性与AFB1大致相同，可引起实验动物发生肿瘤〔2〕，50μg/(kg・bw)的AFM1可致大鼠肝癌及结肠腺癌〔3〕，此外尚有AFM1引起牙原性肿瘤的报道〔4〕。鉴于AFM1对人类健康构成潜在的威胁，在我国食用乳与乳制品者又多为婴幼儿、老年人和体弱多病者，因此，必须对其含量进行监控，限制AFM1在牛奶或乳制品中的污染水平，达到保护消费者健康的目的。因此，建立精确、简便、快速、灵敏、回收率高、不需特殊设备和可在基层实验室开展的检测分析方法十分必要。本文旨在制备抗黄曲霉毒素M1单克隆抗体，并对抗体特性进行鉴定，为进一步研制具有我国自主知识产权的免疫学快速检测试剂盒提供技术支持。
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