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建立稳定分泌抗人Y盒结合蛋白1单克隆抗体(anti-YB-1 mAb)的杂交瘤细胞株,鉴定其表位与免疫学应用。将重组YB-1蛋白免疫BALB/c小鼠,取脾细胞与Sp2/0骨髓瘤细胞融合。经ELISA法筛选鉴定、定株后采用腹水诱生法制备anti-YB-1 mAb;Protein G亲和层析法纯化mAb,ELISA法测定mAb效价、亚型及相对亲和力。采用抗原表位预测法鉴定anti-YB-1 mAb识别表位所在区域。Western blot和免疫组化鉴定mAb识别内源性YB-1的特异性。经筛选鉴定获得2株稳定分泌anti-YB-1 mAb的杂交瘤细胞(1-D9,3-E8);腹水抗体效价均≥1×10-6,亚型均为IgGl;1-D9和3-E8单抗识别表位分别位于(134-160aa)与(266-303aa)肽段。Western blot、免疫组化结果证实anti-YB-1 mAb能特异性识别内源性YB-1。该研究为YB-1免疫学定性、定量检测方法的建立、肿瘤靶向抗体治疗及进一步探讨YB-1的生物学功能奠定了基础。
To establish a hybridoma cell line which can stably secrete anti-YB-1 monoclonal antibody (mAb) and identify its epitope and immunological application. BALB / c mice were immunized with recombinant YB-1 protein and splenocytes were fused with Sp2 / 0 myeloma cells. Anti-YB-1 mAb was prepared by ascitic fluid induction method after immobilized strain. The mAb was purified by Protein G affinity chromatography. The titer, subtype and relative affinity of mAb were determined by ELISA. The anti-YB-1 mAb recognition epitope was identified by antigen epitope prediction. Western blot and immunohistochemistry were used to identify the specificity of mAb to recognize endogenous YB-1. Two hybridoma cells secreting anti-YB-1 mAb (1-D9,3-E8) were obtained by screening and identification. The antibody titer of ascites was ≥1 × 10-6, The epitopes of 3-E8 mAb were located at (134-160aa) and (266-303aa) peptides, respectively. Western blot, immunohistochemistry results confirmed that anti-YB-1 mAb can specifically recognize endogenous YB-1. This study laid the foundation for the qualitative and quantitative immunodeficiency test of YB-1, the establishment of quantitative detection methods, the treatment of tumor targeted antibodies and the further exploration of the biological function of YB-1.