抗人双特异性蛋白磷酸化酶Dusp23单克隆抗体的制备及鉴定

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K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。

Preparation and Identification of the Monoclonal Antibody against Human Dual-Specificity Protein Phosphatase 23

Objective To prepare the human dual-specificity protein phosphatase 23 （dusp23） hybridoma cell lines using classical hybridoma technique and to perform purification and identification of the monoclonal antibodies （mAbs） against dusp23 antigen. Methods BALB/c mice were immunized with purified recombinant dusp23 protein. After immunization, hybridoma were produced by fusing the immune spleen cells with the myeloma cells （Sp2/0）. mAbs against dusp23 antigen were screened by indirect enzyme linked immunosorbent assay （ELISA）. The immunoglobulin subtypes and the titer of the mAb against dusp23 antigen were identified and measured by Western blot analysis and immunohistochemistry, respectively. Results Two hybridoma cell lines stably, namely N012 and N013, secreting mAbs （IgG1, K） against dusp23 antigen were obtained. The antibody titers in the hybridoma culture supernatant were 1：5 120 and 1：2 560 for N012 and N013, respectively. The titers in the ascites fluid were 1：25 600 （N012） and 1：12 800 （N013）. Western blot analysis and immunohistoehemistry showed that the two antibodies can specifically bind with dusp23 antigen derived from human euearyotie cells or tissues. Conclusion Two specific mAbs against dusp23 antigen were successfully produced. These antibodies can be used for the identification of dusp23 protein, and may be used for the study of the biological properties of dusp23 protein.