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| 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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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 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.
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Received: 15 December 2011
Published: 25 June 2012
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Cite this article:
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.
URL:
https://manu60.magtech.com.cn/biotech/ OR https://manu60.magtech.com.cn/biotech/Y2012/V32/I6/13
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