抗人和肽素单克隆抗体的研制

Preparation of monoclonal antibodies against human copeptin

目的制备抗人和肽素单克隆抗体并完成鉴定。方法合成人和肽素C端及N端氨基酸片段并耦联钥孔戚血蓝蛋白(KLH)作为抗原,分别免疫BALB/c小鼠,利用经典杂交瘤技术筛选出分泌抗人和肽素抗体的单克隆细胞株,动物体内诱生法收集腹水,葡萄球菌蛋白A亲和层析柱纯化腹水中的单克隆抗体(单抗),完成抗体亚类鉴定,用间接ELISA鉴定其活性,初步应用于免疫细胞化学检测。结果获得了3株特异性抗人和肽素单抗2h7、5b10及5e7,均属于IgG1亚类,2h7针对的抗原表位在和肽素C端,而5b10及5e7针对的抗原表位在和肽素N端。间接ELISA检测证实3株单抗均特异性识别人和肽素,其中2h7和5b10可应用于免疫细胞化学分析。结论成功制备出抗人和肽素单抗,能特异识别和肽素C端和N端,可应用于细胞免疫化学检测,为人和肽素的进一步研究提供依据。

Objective To prepare and identify the monoelonal antibodies against human copeptin. Methods The peptides from human C-terminal and N-terminal copeptin were synthesized, and coupled with keyhole limpet hemocyanin（KLH） to be col-KLH and eo2-KLH antigens. Then, BALB/c mice were immunized with the two antigens, respectively. The monoclonal cell strains secreting monoclonal antibodies against col-KLH and co2-KLH were screened with classic hybridoma technology. Then, ascite production in vivo was induced and the monoelonal antibodies in ascite were purified with staphylococcal protein A affinity chromatography. Next, the subtypes and activity of monoelonal antibodies were identified with the isotyping ELISA kit. Last, the monoelonal antibodies were applied for the detection of immunoeytoehemistry. Results Three hybridoma colonies, such as 2h7, 5b10 and 5e7, were obtained. All of the three monoelonal antibodies secreted by them belonged to IgG1 isotype. The monoclonal antibody from the 2h7 colony recognized the epitope on C-terminal copeptin, while the antibodies from the 5b10 and 5e7 colonies recognized the epitopes on N-terminal eopeptin. As shown by indirect ELISA, the three monoclonal antibodies specifically reacted with human eopeptin, and the antibodies from the 2h7 and 5b10 colonies could react with copeptin expressed in human lung adenoearcinoma SPCA-1 cells as shown by immunocytochemistry. Conclusion The monoclonal antibodies against human copeptin are obtained successfully, which may specifically recognize the C-terminal and N-terminal copeptin and be applied for the immunocytochemistry detection, and lay the foundation for the further study of human copeptin application.