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

中国生物工程杂志

2012, Vol. 32

Issue (6): 13-19

研究报告

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

李朴¹, 史静², 成凤¹, 梁勤东¹, 匡文斌¹, 王秦¹, 董晋豫¹, 涂植光¹

1. 重庆医科大学临床检验诊断学教育部重点实验室重庆 400016;
2. 重庆医科大学附属第一医院检验科重庆 400016

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

LI Pu¹, SHI Jing², CHENG Fen¹, LIANG Qin-dong¹, KUANG Wen-bin¹, WANG Qin¹, DONG Jin-yu¹, TU Zhi-guang¹

1. Key Laboratory of Laboratory Medical Diagnostics, Ministry of Education, Chongqing Medical University, Chongqing 400016, China;
2. Department of Laboratory, The First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China

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摘要： 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 IgG_l;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。

关键词： YB-1; 单克隆抗体; 抗原表位; 免疫学检测

Abstract: 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 IgG_l. 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.

Key words: YB-1 Monoclonal antibodies Epitope Immunoassay

收稿日期: 2011-12-15 出版日期: 2012-06-25

ZTFLH:

Q819

基金资助: 国家自然科学基金资助项目(30872417)

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引用本文:

李朴, 史静, 成凤, 梁勤东, 匡文斌, 王秦, 董晋豫, 涂植光. 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[J]. 中国生物工程杂志, 2012, 32(6): 13-19.

LI Pu, SHI Jing, CHENG Fen, LIANG Qin-dong, KUANG Wen-bin, WANG Qin, DONG Jin-yu, TU Zhi-guang. Preparation, Characterization and Application of Monoclonal Antibodies Against Human Y box-binding Protein 1. China Biotechnology, 2012, 32(6): 13-19.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2012/V32/I6/13

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