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Isolation of Anti-Tumor Necrosis Factor-Alpha (TNF-?) scFvs Antibody from Phage Antibody Library
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J Abdolalizadeh   , J Majidi Zolbanin * , M Nouri , B Baradaran , A Barzegari , Y Omidi |
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Abstract: (9712 Views) |
ABSTRACT
BACKGROUND AND OBJECTIVE: Cytokine therapy is a fundamental step for the control of the most severe inflammatory diseases. Considering the importance of TNF-? in most of autoimmune and inflammatory diseases, it has been as an attractive target for many researchers and is an important candidate for cytokine therapy. Using monoclonal antibodies is a major approach in contrast to deleterious effects of TNF-?. Antibody phage display technology is a major technique for the preparation of monoclonal antibodies. Our goal in current study was production and preparation of monoclonal antibody against TNF-?.
METHODS: For Biopanning technique, we used ELISA plates for isolation of single chain variable fragments (scFv) from Tomlinson library. After coating of TNF-? protein in the wells, phage library was added. Specific phages were eluted using Trypsin after washing process and amplified in TG1 cells. Five rounds of panning were conducted for obtaining high specific phages. The evaluation of selected clones was done through monoclonal phage ELISA, PCR, RFLP and western blotting methods.
FINDINGS: Analysis of sequences using bioinformatic softwares showed that separated plasmids from TNF-? reactive clones contained insertions. Furthermore, most of variations are seen in three hot regions which can affect protein function.
CONCLUSION: The results of this study showed that forth generation of monoclonal antibodies against human TNF-? were successfully produced.
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| Keywords: Phage display, TNF-?, Monoclonal antibody, Single chain variable fragment. |
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Full-Text [PDF 1078 kb]
(2638 Downloads)
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Type of Study: Research |
Subject:
Biochemical
Accepted: 2014/06/11 | Published: 2014/06/11
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