Affiliations: [a] Department of Bioscience and Biotechnology, Malek ashtar University of Technology, Tehran, Iran | [b] The Lister Laboratory of Microbiology, Tehran, Iran | [c] Student of Medical Biotechnology, Pasteur Institute of Iran, Tehran, Iran
Correspondence:
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Corresponding author: Jalil Fallah Mehrabadi, The Lister Laboratory of Microbiology, Tehran, Iran. Tel.: +98 21 88849707; E-mail:[email protected]
Abstract: BACKGROUND: Human monoclonal antibodies are important molecules in clinical
research. Current Limitations of mAb technologies namely instability of
immortalized B-cell line and probability of forming unusual VH-VL pairs in
phage-display method led to mAbs technology based on single plasma cell
called ``SYMPLEX''. OBJECTIVE: In this method, cognate VH and VL fragments generated from
individual antibody genes exactly the same as natural ones. METHODS: PBMCs of whole blood of an immunized candidate was used as a
resource of rearranged Ab genes. Then flow-cytometric screening was
performed to isolate VH and VL from PBMCs. Various VH and VLκ were
amplified by six pairs of primers. Overlap Extension PCR was accomplished to
link VH and Vκ regions. ScFv inserted into T-vector and its sequence
was determined and eventually analyzed by using blast analysis tools. RESULTS: Electrophoresis results indicated that VH and VL fragments were
separately amplified by PCR with a length of about 400bp and linked through
OE-PCR. Hence, ScFv, which was approximately 800bp in size, was constructed
then sequencing and BLASTn results of the ScFv fragment consequently proved
the accuracy. CONCLUSION: Results showed 88% similarity to available sequences in
mentioned databank. ScFv was ultimately inserted into expression vector for
producing recombinant human anti-tetanus mAb.
