title: Construction of human anti-tetanus single-chain variable fragment applying SYMPLEX technology.
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.
  
  
  CONCLUSIONS
  Results showed 88% similarity to available sequences in mentioned databank. ScFv was ultimately inserted into expression vector for producing recombinant human anti-tetanus mAb.
authors: Department of Bioscience and Biotechnology, Malek ashtar University of Technology, Tehran, Iran.
authors: Laya
authors: L
authors: Esmati
authors: The Lister Laboratory of Microbiology, Tehran, Iran.
authors: Jalil Fallah
authors: JF
authors: Mehrabadi
authors: Student of Medical Biotechnology, Pasteur Institute of Iran, Tehran, Iran.
authors: Masoumeh
authors: M
authors: Bazaz
authors: Department of Bioscience and Biotechnology, Malek ashtar University of Technology, Tehran, Iran.
authors: Hamideh Rouhani
authors: HR
authors: Nejad
mesh_terms: D000907:Antibodies, Bacterial / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D002469:Cell Separation; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007114:Immunization; D007963:Leukocytes, Mononuclear / Q000276:immunology*; D008958:Models, Molecular; D010950:Plasma Cells / Q000276:immunology*; D016133:Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA; D059010:Single-Cell Analysis / Q000379:methods; D057127:Single-Chain Antibodies / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D013742:Tetanus / Q000276:immunology / Q000382:microbiology / Q000517:prevention & control*
chemical_list: D000907:Antibodies, Bacterial; D017931:DNA Primers; D057127:Single-Chain Antibodies
keywords: Overlap-extension PCR; SYMPLEX technology; ScFv