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目的制备抗日本血吸虫重组信号蛋白14-3-3单克隆抗体,并鉴定抗体性质。方法以重组表达纯化的日本血吸虫信号蛋白14-3-3为抗原免疫BALB/c小鼠,采用杂交瘤技术制备单克隆抗体,采用酶联免疫吸附试验及免疫印迹法对获得的单克隆抗体进行类和亚类、效价、浓度、纯度、亲和常数、特异性和检测灵敏度的测定与鉴定。结果共获得6株针对日本血吸虫信号蛋白14-3-3的单克隆抗体,分别为5G9、3F1、3F7、5C6、5D1和1G6,抗体类型分别为IgG1、IgG1、IgG2a、IgG2b、IgG1和IgG1。对其中的单克隆抗体5G9分析显示,制备的腹水抗体效价为1∶1.28×105,亲和层析纯化后的浓度为5.3 mg/ml,纯度>95%,亲和常数为5.1×107mol/L。免疫印迹试验结果表明,该单克隆抗体可与体外重组表达纯化的日本血吸虫14-3-3蛋白特异性结合,同时也能与日本血吸虫可溶性虫卵抗原、成虫排泄分泌抗原和成虫抗原特异性反应,而与大肠埃希菌、健康人血清和华支睾吸虫抗原均无明显交叉反应。HRP标记的单克隆抗体5G9检测14-3-3蛋白的灵敏度为31.25 ng/ml。结论本研究获得了6株能稳定分泌抗日本血吸虫14-3-3蛋白的单克隆抗体杂交瘤细胞株,为建立新的日本血吸虫活动性感染检测方法奠定了良好的基础。
Objective To prepare anti-Japanese Schistosoma japonicum recombinant protein 14-3-3 monoclonal antibody and identify its properties. Methods BALB / c mice were immunized with the purified recombinant protein of Schistosoma japonicum signaling protein 14-3-3 and the monoclonal antibodies were prepared by hybridoma technique. The obtained monoclonal antibodies were tested by enzyme - linked immunosorbent assay and immunoblotting Class and subclass, titer, concentration, purity, affinity constant, specificity and detection sensitivity of the determination and identification. Results Six monoclonal antibodies against Schistosoma japonicum signaling protein 14-3-3 were obtained, which were 5G9, 3F1, 3F7, 5C6, 5D1 and 1G6 respectively. The antibody types were IgG1, IgG1, IgG2a, IgG2b, IgG1 and IgG1. The analysis of monoclonal antibody 5G9 showed that the prepared ascites antibody titer was 1: 1.28 × 105, the affinity chromatography purified concentration was 5.3 mg / ml, the purity was> 95% and the affinity constant was 5.1 × 107mol / L. The results of Western blotting showed that the monoclonal antibody could specifically bind to the purified recombinant protein of Schistosoma japonicum 14-3-3 in vitro and could also react with the soluble antigen of wormworm, the secreted antigen of adult worm and the antigen of adult worm , But no significant cross-reaction with Escherichia coli, healthy human serum and Clonorchis sinensis antigen. The sensitivity of HRP-labeled monoclonal antibody 5G9 to detect 14-3-3 protein was 31.25 ng / ml. Conclusion Six monoclonal antibody hybridoma cell lines that can stably secrete 14-3-3 protein against Schistosoma japonicum were obtained in this study, which laid a good foundation for establishing a new detection method of active infection of Schistosoma japonicum.