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目的对6株抗日本血吸虫重组信号蛋白14-3-3单克隆抗体生物学特性进行鉴定,并探讨其在血吸虫病诊断中的应用价值。方法采用酶联免疫吸附试验及免疫印迹法对6株单克隆抗体的类和亚类、腹水效价、亲和常数、检测灵敏度和特异性进行测定与鉴定。建立检测日本血吸虫感染兔血清14-3-3蛋白的Dot-ELISA方法,对感染兔吡喹酮治疗前、后不同时间的配对血清中14-3-3蛋白含量的动态变化进行观察,并通过对一批血吸虫感染兔血清样本进行检测,以评价该方法的诊断价值。结果 6株抗日本血吸虫信号蛋白14-3-3单克隆抗体3F1、3F7、5C6、5D1、5G9、1G6的抗体亚类分别为IgG1、IgG2 a、IgG2b、IgG1、IgG1和IgG1;腹水单克隆抗体效价分别为1∶6.4×105、1∶8.0×105、1∶6.4×105、1∶3.2×105、1∶4.8×105和1∶2.0×104;亲和常数分别为8.82×108、4.93×108、1.56×108、5.12×108、1.41×108mol/L和2.30×107mol/L;Dot-ELISA方法检测14-3-3蛋白的灵敏度分别为1、10、100、10、10 ng和100 ng。免疫印迹试验结果表明,6株单克隆抗体均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中的天然14-3-3蛋白特异性反应,而与大肠埃希菌和华支睾吸虫抗原均无明显交叉反应。以单抗3F1和5D1为检测抗体建立的Dot-ELISA方法检测日本血吸虫感染兔治疗前、后不同时间点血清,发现随着感染时间的延长,兔血清中14-3-3蛋白含量逐渐增加,吡喹酮治疗后兔血清中14-3-3蛋白含量逐渐降低。采用此Dot-ELISA方法检测42份血吸虫感染兔血清,41份阳性,敏感性为97.6%;10份健康兔血清均为阴性,特异性为100%。结论 6株单克隆抗体均能有效识别天然的日本血吸虫信号蛋白14-3-3,初步证明以单抗3F1和5D1为检测抗体建立的Dot-ELISA方法具有一定的日本血吸虫活动性感染诊断及潜在的疗效考核价值。
Objective To identify the biological characteristics of six monoclonal antibodies against Schistosoma japonicum recombinant protein 14-3-3 and to explore its value in the diagnosis of schistosomiasis. Methods The types and subclasses of 6 monoclonal antibodies, ascites titer, affinity constant, detection sensitivity and specificity were determined and identified by enzyme-linked immunosorbent assay and Western blotting. To establish a Dot-ELISA method for the detection of 14-3-3 protein in rabbits infected with Schistosoma japonicum, the dynamic changes of 14-3-3 protein content in paired serums before and after infection with praziquantel in rabbits were observed and compared A number of sera infected rabbit serum samples were tested to evaluate the diagnostic value of the method. Results The antibody subtypes of 6 strains of 3F1, 3F7, 5C6, 5D1, 5G9 and 1G6 of 14-3-3 monoclonal antibodies against Schistosoma japonicum were IgG1, IgG2 a, IgG2b, IgG1, IgG1 and IgG1, respectively; ascites monoclonal antibody The titer was 1: 6.4 × 105, 1: 8.0 × 105, 1: 6.4 × 105, 1: 3.2 × 105, 1: 4.8 × 105 and 1: 2.0 × 104, respectively. The affinity constants were 8.82 × 108 and 4.93 × 108,1.56 × 108,5.12 × 108,1.41 × 108mol / L and 2.30 × 107mol / L respectively.The sensitivities of Dot-ELISA to detect 14-3-3 protein were 1, 10, 100, 10, 10, 10 ng and 100 ng. The results of Western blotting showed that all six monoclonal antibodies could specifically bind to the purified recombinant Schistosoma japonicum 14-3-3 protein in vitro and also could bind to the soluble antigen of Schistosoma japonicum, the secreted antigen of adult worms and the adult antigen Of the natural 14-3-3 protein-specific response, and Escherichia coli and Clonorchis sinensis antigen no significant cross-reaction. Dot-ELISA was used to detect the serum levels of 14-3-3 protein in rabbits infected with Schistosoma japonicum at different time points after Dot-ELISA with 3F1 and 5D1 as detection antibodies. After the praziquantel treatment rabbit serum 14-3-3 protein content decreased. Forty-two sera infected by Schistosoma japonicum were detected by this Dot-ELISA method, and 41 were positive with a sensitivity of 97.6%. The serum of 10 healthy rabbits was negative and the specificity was 100%. Conclusion All six monoclonal antibodies can effectively recognize the native Schistosoma japonicum signaling protein 14-3-3. The preliminary identification of Dot-ELISA using monoclonal antibodies 3F1 and 5D1 as detection antibodies has some diagnostic significance and potential for Schistosoma japonicum infection The efficacy of assessment value.