Volume 5, Issue 17 (3-2015)
NCMBJ 2015, 5(17): 1-7 |
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Rohaninej H, Norouzi J, Fallah Mehrabadi J, Mahdavi M. Creating a human ScFv antibody library based on T-Vector against tetanus toxin. NCMBJ 2015; 5 (17) :1-7
URL: http://ncmbjpiau.ir/article-1-502-en.html
URL: http://ncmbjpiau.ir/article-1-502-en.html
Pasteur Institute of Iran , jalil.fallah@gmail.com
Abstract: (11881 Views)
Aims and Background: Nowadays, monoclonal antibodies (mAbs) became powerful therapeutic and diagnostic tools. Due to showing minimum immunogenic reaction and their properties, human mAbs are important. The purpose of this study was to construct an immune antibody library from a vaccinated donor against tetanus toxin.
Materials and Methods: A whole blood sample was taken from the donor who was vaccinated against tetanus toxoid. PBMC were isolated by using ficol. After RNA extraction, all variation of VH and VL regions by RT-PCR reactions were amplified and linked as a ScFv antibody. The amplicons were inserted in T-vector and transformed to E. coli DH5α strain, followed by an ELISA test. Plasmids were also extracted and sequenced.
Results: cDNA quality was confirmed by using HPRT primers. To confirm PCR, insertion and transformation, gel electrophoresis and restriction enzymes digestion were applied. Positive clones were selected based on growth on LB agar which is the blue/white selection method. After plasmid extraction and DNA sequencing, the sequences were aligned using igBLAST at NCBI. The result was shown to have admissible similarity among antibody gene library nucleotide sequences and the antibody genes were deposited in this database. ELISA confirmed this data too.
Conclusion: In this study, the immune human antibody library was constructed and confirmed using DNA sequencing and sequences alignment in NCBI database. ELISA test confirms antibody specifically. The next step is to screen the library to find an antibody specifically for tetanus toxin.
Materials and Methods: A whole blood sample was taken from the donor who was vaccinated against tetanus toxoid. PBMC were isolated by using ficol. After RNA extraction, all variation of VH and VL regions by RT-PCR reactions were amplified and linked as a ScFv antibody. The amplicons were inserted in T-vector and transformed to E. coli DH5α strain, followed by an ELISA test. Plasmids were also extracted and sequenced.
Results: cDNA quality was confirmed by using HPRT primers. To confirm PCR, insertion and transformation, gel electrophoresis and restriction enzymes digestion were applied. Positive clones were selected based on growth on LB agar which is the blue/white selection method. After plasmid extraction and DNA sequencing, the sequences were aligned using igBLAST at NCBI. The result was shown to have admissible similarity among antibody gene library nucleotide sequences and the antibody genes were deposited in this database. ELISA confirmed this data too.
Conclusion: In this study, the immune human antibody library was constructed and confirmed using DNA sequencing and sequences alignment in NCBI database. ELISA test confirms antibody specifically. The next step is to screen the library to find an antibody specifically for tetanus toxin.
Type of Study: Research Article |
Subject:
Immonology
Received: 2014/06/7 | Accepted: 2014/11/10 | Published: 2015/02/24
Received: 2014/06/7 | Accepted: 2014/11/10 | Published: 2015/02/24
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