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目的制备呋喃妥因代谢物AHD单克隆抗体,并进行鉴定。方法利用对羧基苯甲醛合成1-氨基乙内酰胺脲(AHD)的衍生物1-氨基乙内酰脲-4-羧苯基肟(4-CPAHD),通过活性脂法将4-CPAHD与牛血清白蛋白(BSA)和卵清蛋白(OVA)偶联,获得免疫抗原4-CPAHD-BSA和包被抗原4-CPAHD-OVA,采用紫外分光光度法检测偶联是否成功。将人工合成抗原4-CPAHD-BSA免疫BALB/c小鼠,通过杂交瘤技术将免疫小鼠脾脏细胞与Sp2/0骨髓瘤细胞融合,筛选能稳定产生抗体的细胞株;通过间接ELISA法检测该细胞株分泌的单克隆抗体的效价、邻硝基-PAHD(2-NPAHD)对单克隆抗体的半数抑制浓度(IC50),并分析抗体的特异性。结果偶联后4-CPAHD-BSA的最大吸收峰有较明显的偏移,表明4-CPAHD与BSA偶联成功,平均偶联比为17.4∶1。制备的单克隆抗体的效价为1∶64 000,2-NPAHD对单克隆抗体的IC50为1.45μg/L,单克隆抗体与同类抗生素及其代谢物无交叉反应。结论制备的AHD单克隆抗体各项指标均较好,为呋喃妥因代谢物免疫检测试剂的研发奠定了基础。
Objective To prepare the monoclonal antibody of nitrofurantoin metabolites AHD and identify it. Methods 4-CPAHD, a derivative of 1-aminobutylaminamide (AHD), was synthesized by the reaction of 4-CPAHD with bovine (BSA) and ovalbumin (OVA) to obtain the immunogen antigen 4-CPAHD-BSA and coating antigen 4-CPAHD-OVA. The coupling assay was successful by UV spectrophotometry. The BALB / c mice were immunized with the synthetic antigen 4-CPAHD-BSA, and the spleen cells of the immunized mice were fused with Sp2 / 0 myeloma cells by the hybridoma technique to screen the cell strains which can stably produce the antibody; The titer of the monoclonal antibody secreted by the cell line, the half-maximal inhibitory concentration (IC50) of the o-nitro-PAHD (2-NPAHD) to the monoclonal antibody, and the specificity of the antibody were analyzed. Results The peak of 4-CPAHD-BSA showed a significant shift after coupling, indicating that 4-CPAHD was successfully coupled with BSA with the average coupling ratio of 17.4:1. Monoclonal antibody titer of 1: 64 000, 2-NPAHD monoclonal antibody IC50 of 1.45μg / L, monoclonal antibodies and similar antibiotics and their metabolites no cross-reaction. Conclusion All the indexes of AHD monoclonal antibody prepared are good, which lays the foundation for the research and development of nitrofurantoin metabolite immunoassay reagents.