Y盒结合蛋白1单克隆抗体的研制、表位测定及其免疫学应用

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。

Preparation,Characterization and Application of Monoclonal Antibodies Against Human Y box-binding Protein 1

The purpose is to prepare and characterize the monoclonal antibodies(mAbs) of anti-human Y-box binding protein 1(anti-YB-1) and identify its application of immunoassay.Hybridoma cells were generated by fusing the Sp2/0 myeloma cells with spleen cel1s,which were obtained from BALB/c mice immunized by recombinant YB-1 protein.Indirect ELISA was used to screen positive clones,and to detect titers,subtypes,and relative affinity of the mAbs.Protein G affinity chromatography system was applied to prepare highly-purified mAbs.B-cell epitope prediction method was applied to assay the corresponding epitopes of mAbs.Western blot and immunohistochemisty were adopted to identify immunoassay application of the mAbs.The results showed that two positive hybridoma-cell clones were obtained(1-D9,3-E8).The titers of mAbs were ≥ 1∶1×106,and the subtype of the two mAbs were IgGl.The recognized epitope of the two mAbs might harbor in 134-160aa and 266-303aa of the YB-1 protein,respectively.The results of Western blot and immunohistochemisty showed that mAbs(1-D9) could specifically react with endogenous and recombinant YB-1 protein.All these results indicate that two kinds of anti-YB-1 mAbs which could recognize different epitope of the YB-1 protein are successfully prepared;it would provide foundations for quantitative and qualitative detection of YB-1,diagnostic immunoassay,anti-cancer targeted-therapy,and investigation of YB-1 function.