title: Altered specificity of single-chain antibody fragments bound to pandemic H1N1-2009 influenza virus after conversion of the phage-bound to the soluble form.
abstract: BACKGROUND
  In 2009, a novel influenza A/H1N1 virus (H1N1pdm) quickly spread worldwide and co-circulated with then-existing seasonal H1N1 virus (sH1N1). Distinguishing between these 2 viruses was necessary to better characterize the epidemiological properties of the emergent virus, including transmission patterns, pathogenesis, and anti-influenza drug resistance. This situation prompted us to develop a point-of-care virus differentiation system before entering the 2009-2010 influenza season. Aiming to establish H1N1pdm-specific detection tools rapidly, we employed phage display libraries to select H1N1pdm-specific single-chain variable fragments (scFvs).
  
  
  RESULTS
  Human single-fold scFv libraries (Tomlinson I + J) underwent selection for the ability to bind H1N1pdm virus particles. Three rounds of panning brought 1152 phage-bound scFvs, of which 58 clones reacted with H1N1pdm specifically or preferentially over sH1N1 in an enzyme-linked immunosorbent assay (ELISA). After conversion of the scFvs to soluble form, 7 clones demonstrating high/stable expression were finally obtained. However, all the soluble scFvs except No. 29 were found to have lost their specificity/preference for H1N1pdm in ELISA. The specificity/preference of No. 29 was also confirmed by immunofluorescence assay and immunoprecipitation, and the viral nucleoprotein was identified by ELISA as its target protein. The change in specificity associated with scFv conversion from phage-bound to soluble form could be due to loss of phage scaffold pIII protein, which likely provides structural support for the scFv antigen-binding site. It is also possible that the similar antigenic properties of H1N1pdm and sH1N1 led to the observed alterations in scFv specificity.
  
  
  CONCLUSIONS
  Using a phage display library, we obtained 7 soluble scFv clones reactive against H1N1pdm; however, only 1 showed specificity/preference toward H1N1pdm. Our results confirmed that using phage display libraries was highly advantageous for the rapid development of molecules to detect target antigens. However, our results also indicated that this strategy might not have been effective for selecting H1N1pdm-specific antibodies during the 2009 pandemic, where the co-circulating sH1N1 virus shared similar antigenic properties. This suggests that it might be advisable to use a synthetic scFv phage display library by strategically considering the characteristics of target antigens and the potential situations.
authors.affiliation: Department of Veterinary Science, National Institute of Infectious Diseases, Shinjuku, Tokyo, Japan. ykaku@nih.go.jp
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Kaku
authors.forename: Akira
authors.initials: A
authors.lastname: Noguchi
authors.forename: Akiko
authors.initials: A
authors.lastname: Okutani
authors.forename: Satoshi
authors.initials: S
authors.lastname: Inoue
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Tanabayashi
authors.forename: Yoshie
authors.initials: Y
authors.lastname: Yamamoto
authors.forename: Akitoyo
authors.initials: A
authors.lastname: Hotta
authors.forename: Michio
authors.initials: M
authors.lastname: Suzuki
authors.forename: Naoko
authors.initials: N
authors.lastname: Sugiura
authors.forename: Akio
authors.initials: A
authors.lastname: Yamada
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism; D000918:Antibody Specificity; D000956:Antigens, Viral / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D001435:Bacteriophages / Q000235:genetics / Q000276:immunology; D001665:Binding Sites; D002999:Clone Cells; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D007251:Influenza, Human / Q000175:diagnosis* / Q000453:epidemiology* / Q000821:virology; D007564:Japan / Q000453:epidemiology; D058873:Pandemics; D019151:Peptide Library; D019095:Point-of-Care Systems / Q000458:organization & administration; D011485:Protein Binding; D057127:Single-Chain Antibodies / Q000276:immunology* / Q000378:metabolism; D012995:Solubility
chemical_list: D000911:Antibodies, Monoclonal; D000956:Antigens, Viral; D019151:Peptide Library; D057127:Single-Chain Antibodies