Chinese Journal of Tissue Engineering Research ›› 2012, Vol. 16 ›› Issue (46): 8703-8708.doi: 10.3969/j.issn.2095-4344.2012.46.031

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Preparation and purification of a great amount of monoclonal antibodies against the c-terminal of very low density lipoprotein receptor

Yang Pu1, 2, Li Qi2, 3, Liu Zhi-guo2, 3, Tian Jun2, Zong Yi-qiang2, Qu Shen2   

  1. 1Department of Biomedical Engineering, School of Biological Science and Engineering, South China University of Technology, Guangzhou 510006, Guangdong Province, China
    2Department of Biochemistry and Molecular Biology, Basic Medical School, Tongji Medical College, Huangzhong University of Science and Technology, Wuhan 430030, Hubei Province, China
    3School of Biotechnology and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan 430023, Hubei Province, China
  • Received:2012-04-05 Revised:2012-04-24 Online:2012-11-11 Published:2013-03-16
  • Contact: Qu Shen, Professor, Department of Biochemistry and Molecular Biology, Basic Medical School, Tongji Medical College, Huangzhong University of Science and Technology, Wuhan 430030, Hubei Province, China qushen@mails.tjmu.edu.cn
  • About author:Yang Pu☆, Ph.D., Department of Biomedical Engineering, School of Biological Science and Engineering, South China University of Technology, Guangzhou 510006, Guangdong Province, China; Department of Biochemistry and Molecular Biology, Basic Medical School, Tongji Medical College, Huangzhong University of Science and Technology, Wuhan 430030, Hubei Province, China yangpu254@yahoo.com.cn

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Abstract:

BACKGROUND: Very low density lipoprotein receptor is regarded as a Swiss Army Knife receptor, and many researchers have studied this receptor. But there are only several kinds of commercial antibodies and the price is on the high side.
OBJECTIVE: To get the monoclonal antibody against the c-terminal of very low density lipoprotein receptor for the future study.
METHODS: In order to obtain the very low density lipoprotein receptor, hybridoma IgG 6A6 cells related with very low density lipoprotein receptor intracellular domain was injected into peritoneal of Balb/c mice. After 2 weeks, ascites was collected, and the target protein was preliminary purified by salting-out method (n-octanoic acid-saturated ammonium sulfate precipitation method) and further purified by affinity chromatography, and the monoclonal antibody related with very low density lipoprotein receptor intracellular domain was obtained. The relative molecular mass and purity were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the activity was detected by enzyme-linked immunosorbent assay.
RESULTS AND CONCLUSION: Western Blotting results showed that the antibody from the ascites directly can bind the very low density lipoprotein receptor protein, but the background was not clear. The purified antibody from the ascites through salting-out method (n-octanoic acid-saturated ammonium sulfate precipitation method) and then by protein A affinity chromatography can bind specifically with the very low density lipoprotein receptor. The pure and activity monoclonal antibodies can be obtained through purification by the salting-out method and Protein A Agarose

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