title: Screening and evaluation of human single-chain fragment variable antibody against hepatitis B virus surface antigen.
abstract: BACKGROUND
  Phage display technology has become a vital tool in studies aimed at identifying molecules binding to a specific target. It enables the rapid generation and selection of high affinity, fully human antibody product candidates to essentially any disease target appropriate for antibody therapy. In this study, we prepared the recombinant single-chain fragment variable (ScFv) antibody to hepatitis B virus surface antigen (HBsAg) by the phage display technology for obtaining a virus-targeting mediator.
  
  
  METHODS
  mRNA was isolated from B-lymphocytes from a healthy volunteer and converted into cDNA. The fragment variables of heavy and light chain were amplified separately and assembled into ScFv DNA with a specially constructed DNA linker by polymerase chain reaction. The ScFv DNA was ligated into the phagmid vector pCANTAB5E and the ligated sample was transformed into competent E.coli TG1. The transformed cells were infected with M13K07 helper phage to form a human recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by bacterial colony count and restriction analysis. After two rounds of panning with HBsAg, the phage clones displaying ScFv of the antibody were selected by enzyme-linked immunosorbant assay (ELISA) from the enriched phage clones. The antigen binding affinity of the positive clone was detected by competition ELISA. HB2151 E.coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the anti-HBsAg ScFv. ELISA assay was used to detect the antigen binding affinity of the soluble anti-HBsAg ScFv. Finally, the relative molecular mass of soluble anti-HBsAg ScFv was measured by SDS-PAGE.
  
  
  RESULTS
  The variable heavy (VH) and variable light (VL) and ScFv DNAs were about 340 bp, 320 bp and 750 bp, respectively. The volume of the library was up to 2 x 10(6) and 8 of 10 random clones were recombinants. Two phage clones could strongly compete with the original HBsAb for binding to HBsAg. Within 2 strong positive phage clones, the soluble anti-HBsAg ScFv from one clone was found to have the binding activity with HBsAg. SDS-PAGE showed that the relative molecular weight of soluble anti-HBsAg ScFv was 32 kDa.
  
  
  CONCLUSIONS
  The anti-HBsAg ScFv successfully produced by phage antibody technology may be useful for broadening the scope of application of the antibody.
authors.affiliation: Department of Biochemistry and Molecular Biology, Shanxi Medical University, Taiyuan 030001,China.
authors.forename: Jian-Lin
authors.initials: JL
authors.lastname: Zhang
authors.forename: Jian-Jin
authors.initials: JJ
authors.lastname: Gou
authors.forename: Zi-Yan
authors.initials: ZY
authors.lastname: Zhang
authors.forename: Yi-Xin
authors.initials: YX
authors.lastname: Jing
authors.forename: Lin
authors.initials: L
authors.lastname: Zhang
authors.forename: Rui
authors.initials: R
authors.lastname: Guo
authors.forename: Ping
authors.initials: P
authors.lastname: Yan
authors.forename: Niu-Liang
authors.initials: NL
authors.lastname: Cheng
authors.forename: Bo
authors.initials: B
authors.lastname: Niu
authors.forename: Jun
authors.initials: J
authors.lastname: Xie
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism*; D000915:Antibody Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D005069:Evaluation Studies as Topic; D006514:Hepatitis B Surface Antigens / Q000276:immunology*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000276:immunology* / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000378:metabolism*; D008403:Mass Screening / Q000379:methods; D019151:Peptide Library; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012494:Sampling Studies; D012680:Sensitivity and Specificity
chemical_list: D000911:Antibodies, Monoclonal; D006514:Hepatitis B Surface Antigens; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D019151:Peptide Library