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目的制备高亲和力、高特异性的抗人双特异性磷酸化酶23(Dusp23)单克隆抗体,并对其生物学特性进行鉴定,为Dusp23功能的研究奠定基础。方法以纯化的原核表达Dusp23为免疫原,免疫BALB/c小鼠,待免疫小鼠血清效价满足融合需要,取其脾细胞与Sp2/0细胞融合,经多次筛选及克隆化建立可稳定分泌抗Dusp23单克隆抗体的杂交瘤细胞株,将细胞株打入BALB/c小鼠腹腔制备腹水,用ProteinG亲合层析柱纯化所得腹水获得纯化抗体,ELISA及Western-blot检测抗体的效价和亲和力,并用亚型鉴定试纸条鉴定单克隆抗体的亚型。利用纯化后的两株单克隆抗体对临床收集的肝癌组织标本进行免疫组化分析。结果筛选到2株(N012和N013)可以稳定分泌人双特异性磷酸化酶23(Dusp23)单克隆抗体的细胞株,并对其进行纯化和鉴定,两株杂交瘤细胞培养上清效价分别为1:5120和1:2560,腹水效价分别为1:25600和1:12800,以肝癌细胞HepG2和重组Dusp23蛋白为抗原,利用纯化后的两株抗体Western-blot鉴定结果均在预期位置出现阳性条带。两株杂交瘤细胞分泌抗体的亚型鉴定N012和N013均为IgG1型,轻链均为κ型。两株抗体分别与临床肝癌组织标本免疫组化结果均为阳性。结论成功制备两株能特异性识别Dusp23的单克隆抗体,为进一步研究Dusp23的生物学功能,揭示其与肿瘤发生发展之间的关系奠定了基础。
Objective To prepare monoclonal antibodies against human bispecific phosphorylase 23 (Dusp23) with high affinity and specificity, and to identify its biological characteristics, which laid the foundation for the study of Dusp23 function. Methods The purified prokaryotic expression of Dusp23 was used as immunogen to immunize BALB / c mice. After the serum titer of the immunized mice was satisfied, the spleen cells were fused with Sp2 / 0 cells. After multiple screening and cloning, The monoclonal antibody secreting Dusp23 hybridoma cell line, the cell line into BALB / c mice peritoneal preparation of ascites, with ProteinG affinity chromatography purification of the resulting ascites purified antibody, ELISA and Western-blot detection of antibody titer And affinity, and subtype identification test strips identified monoclonal antibody subtypes. Immunohistochemical analysis was performed on the clinically collected HCC samples using the two purified monoclonal antibodies. Results Two strains (N012 and N013) that could stably secrete monoclonal antibody against human bispecific phosphorylase 23 (Dusp23) were screened and purified. The titers of the two hybridoma cell culture supernatants were respectively 1: 5120 and 1: 2560 respectively. The ascites titer was 1: 25600 and 1: 12800, respectively. HepG2 and recombinant Dusp23 protein were used as the antigen. Western blot analysis of the two purified antibodies was performed at the expected position Positive band. Subtype Identification of Antibody Secreted by Two Hybridoma Cells Both N012 and N013 were of IgG1 type and both of the light chains were κ type. Two antibodies and clinical liver cancer tissue specimens were positive for immunohistochemistry results. Conclusion Two monoclonal antibodies that can specifically recognize Dusp23 were successfully prepared, which laid the foundation for further study on the biological function of Dusp23 and its relationship with tumorigenesis.