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目的:制备抗人Dysbindin-1特异性单克隆抗体,建立DAS-ELISA检测体系,并初步应用于肝癌血清的检测。方法:采用合成免疫原免疫BALB/c小鼠,通过杂交瘤技术制备抗Dysbindin-1单克隆抗体,用HRP标记单克隆抗体,ELISA和SDS-PAGE电泳法检测抗体的亚类、滴度;采用DAS-ELISA技术制备Dysbindin-1检测试剂盒,检测正常、肝硬化及肝癌患者各30例血清并比较其差异。结果:细胞融合后获得了4株稳定产生抗Dysbindin-1单克隆抗体的杂交瘤细胞株(1C6A11、1E8H3、2D1C11、5B6D4),抗体亚类分别为IgG2b,IgG2b,IgG1和IgG2a,杂交瘤细胞诱生的腹水抗体效价达106以上,通过抗体配对实验筛选确定2D1C11作为包被抗体,1E8H3作为酶标抗体。该ELISA方法线性范围为62.5-1000ng/mL,检测限为62.5ng/mL;应用该方法检测显示正常组与肝硬化组及肝癌组间差异显著(P<0.001),Dysbindin-1诊断肝癌的敏感性和特异性分别为90%和93.3%。结论:Dysbindin-1可以成为新的候选的肝癌血清标志物,抗Dysbindin-1多肽双抗夹心ELISA体系可以初步应用于肝癌的早期诊断。
OBJECTIVE: To prepare anti-Dysbindin-1 specific monoclonal antibody and to establish a DAS-ELISA assay system for the detection of liver cancer serum. Methods: The BALB / c mice were immunized with the synthetic immunogen and the monoclonal antibodies against Dysbindin-1 were prepared by hybridoma technique. The anti-Dysbindin-1 monoclonal antibodies were prepared by HRP. The subclasses and titer of antibodies were detected by ELISA and SDS-PAGE electrophoresis. DAS-ELISA technique was used to prepare Dysbindin-1 test kit to detect the serum of 30 patients with normal, cirrhosis and liver cancer and to compare the differences. RESULTS: Four hybridoma cell lines stably producing monoclonal antibodies against Dysbindin-1 (1C6A11, 1E8H3, 2D1C11, 5B6D4) were obtained after cell fusion. The antibody subtypes were IgG2b, IgG2b, IgG1 and IgG2a, Health ascites antibody titer of 106 or more, through the antibody paired experiment screening to determine 2D1C11 as coating antibody, 1E8H3 as enzyme-labeled antibody. The linear range of this ELISA was 62.5-1000 ng / mL with a detection limit of 62.5 ng / mL. The detection of this method showed significant difference (P <0.001) between normal group and cirrhosis and hepatocellular carcinoma, and the sensitivity of Dysbindin-1 to diagnose hepatocellular carcinoma The specificity and specificity were 90% and 93.3% respectively. Conclusion: Dysbindin-1 can be a new candidate serum marker of liver cancer. Anti-Dysbindin-1 polypeptide double-antibody sandwich ELISA system can be initially applied in the early diagnosis of liver cancer.