呋喃它酮代谢物AMOZ单克隆抗体的制备及免疫学检测方法的研究

发布时间：2018-03-13 19:13

本文选题：AMOZ　切入点：单克隆抗体　出处：《暨南大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的：用自制的抗AMOZ单克隆抗体建立检测组织样品中AMOZ残留的间接竞争ELISA和胶体金免疫层析法。方法：AMOZ与对醛基苯甲酸(4-CBA)反应,衍生成半抗原CP-AMOZ,采用活化酯法将CPAMOZ与载体蛋白牛血清白蛋白(BSA)、卵清蛋白(OVA)分别偶联制备完全抗原,高效液相色谱质谱法(LC-MS)、SDS-PAGE、紫外光谱扫描鉴定抗原合成效果。通过免疫BALB/c小鼠、杂交瘤制备技术制备抗CPAMOZ的单克隆抗体,并对其效价、亚类、特异性等方面进行鉴定,建立检测组织样品中AMOZ残留的间接竞争ELISA和胶体金免疫层析检测方法。结果：1.成功合成AMOZ的衍生物CPAMOZ及完全抗原CPAMOZ-BSA及CPAMOZ-OVA。2.成功获得稳定分泌抗CPAMOZ单克隆抗体的杂交瘤细胞株,该细胞株分泌的抗体效价高,属于IgGl类抗体,与CPAMOZ特异性结合,与载体蛋白BSA、OVA及硝基呋喃类代谢物及其衍生物交叉反应均小于1%。3.建立了检测AMOZ的间接竞争ELISA法,ICso为0.86μg/L,最低检测限(LOD)为0.07μg/L,组织样品的样品回收率为81.48%-85.24%；胶体金免疫层析法的最低检测限为5μg/L。结论：1.成功制备了呋喃它酮代谢物AMOZ的完全抗原及单克隆抗体,该抗体灵敏度高,特异性好。2.成功建立了能用于检测组织样品中AMOZ残留的间接竞争ELISA法和快速、灵敏、稳定的胶体金免疫层析法。
[Abstract]:Aim: to establish indirect competitive ELISA and colloidal gold immunochromatographic assay for the detection of AMOZ residues in tissue samples with self-made anti-AMOZ monoclonal antibody. Methods CPAMOZ was conjugated with bovine serum albumin (BSA) and ovalbumin (ovalbumin) to prepare complete antigen by the reaction of: Amoz with 4-CBA), and then the hapten CP-AMOZ was derived into CP-AMOZ by activation ester method, which was coupled with bovine serum albumin (BSA) and ovalbumin (ovalbumin) respectively to prepare complete antigen. High performance liquid chromatography-mass spectrometry (HPLC / MS) / SDS-PAGE and UV scanning were used to identify the effect of antigen synthesis. Monoclonal antibodies against CPAMOZ were prepared by immunizing BALB/c mice and hybridoma preparation technique, and their titers, subclasses and specificity were identified. To establish indirect competitive ELISA and colloidal gold immunochromatography for the detection of AMOZ residues in tissue samples. Results 1. CPAMOZ, a derivative of AMOZ, and its complete antigen CPAMOZ-BSA and CPAMOZ-OVA.2.The hybridoma cell lines secreting monoclonal antibodies against CPAMOZ were successfully obtained. The hybridoma cells secreted high titers of antibodies, which belonged to IgGl class antibodies and specifically bound to CPAMOZ. The cross reaction with the carrier protein BSA-OVA, nitrofuran metabolites and their derivatives was less than 1.3.The indirect competitive ELISA method for the detection of AMOZ was established. The ELISA method was 0.86 渭 g / L, the lowest detection limit was 0.07 渭 g / L, and the sample recovery of tissue samples was 81.48 and 85.24.The colloidal gold immune layer was established. The detection limit of the method is 5 渭 g / L. Conclusion the complete antigen and monoclonal antibody of furantadone metabolite AMOZ were successfully prepared. The antibody was sensitive and specific. 2. An indirect competitive ELISA method was successfully established for the detection of AMOZ residues in tissue samples. Stable colloidal gold immunochromatography.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

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