重组人内抑素单克隆抗体的制备及初步鉴定

目的制备抗重组人内抑素(ES)单克隆抗体(McAb)并进行鉴定。方法利用重组人ES为抗原,免疫Balb/c小鼠,应用杂交瘤技术将免疫小鼠的脾细胞与同源小鼠骨髓瘤细胞(SP2/0)进行融合,间接ELISA法对细胞培养上清进行筛检,对能稳定分泌抗重组人ES的细胞株,进行扩大培养。体内诱生法制备腹水并对其初步鉴定。结果获得3B4、1D10、1G5、2D10,4株能稳定分泌抗重组人ESMcAb的杂交瘤细胞株,并对3B4和1D10两株给予鉴定,其免疫亚型为IgG2a、IgG3。腹水ELISA效价为10-6、10-5。通过蛋白免疫印迹法和免疫组化证明抗体有特异性和敏感性。结论成功制备分泌抗重组人ES的杂交瘤细胞株,为研究人ES的表达及相关领域奠定了基础。

Preparation and characterization of monoclonal antibody against recombinant human endostatin

Objective To prepare mouse anti-recombinant human endostatin monoclonal antibodies and identify it.Methods Recombinant human ES was used as antigen to immune BALB/c mouse,and hybridoma technique to fuse immunized spell cells of the mouse with the source mouse myeloma cells(SP2/0),Indirect ELISA method was used to screen supernatant of positive wells.The extensive development could stabilize the secretive cells of anti-recombinant ES.The ascites developed by injecting the hybridoma cells into abdomen cavity of the BALB/c mouse and then identification of antibodies was performed.Results We have successfully obtained four hybridoma cell lines 3B4,1D10,1G5 and 2D10 and then identified 3B4 and 1D10.The subtypes of the monoclonal antibodies were IgG2a and IgG3.The titer of ascites was 10-6 and 10-5.Western blotting and immunohistochemistry showed these antibodies have speciality and sensitivity.Conclusion Succeeded in preparing hybridoma cell strains of anti-recombinant human endostatin monoclonal antibodies,which lay the foundation of study human's ES expression level and related realm.