title: An improved phage-display panning method to produce an HM-1 killer toxin anti-idiotypic antibody.
abstract: BACKGROUND
  Phage-display panning is an integral part of biomedical research. Regular panning methods are sometimes complicated by inefficient detachment of the captured phages from the antigen-coated solid supports, which prompted us to modify. Here, we produce an efficient antigen-specific single chain fragment variable (scFv) antibody by using a target-related molecule that favored selection of recombinant antibodies.
  
  
  RESULTS
  To produce more selective and specific anti-idiotypic scFv-antibodies from a cDNA library, constructed from HM-1 killer toxin (HM-1)-neutralizing monoclonal antibodies (nmAb-KT), the method was modified by using an elution buffer supplemented with HM-1 that shares structural and functional similarities with the active site of the scFv antibody. Competitive binding of HM-1 to nmAb-KT allowed easy and quick dissociation of scFv-displayed phages from immobilized nmAb-KT to select specific anti-idiotypic scFv antibodies of HM-1. After modified panning, 80% clones (40/50) showed several times higher binding affinity to nmAb-KT than regular panning. The major populations (48%) of these clones (scFv K1) were genotypically same and had strong cytocidal activity against Saccharomyces and Candida species. The scFv K1 (K(d) value = 4.62 x 10(-8) M) had strong reactivity toward nmAb-KT, like HM-1 (K(d) value = 6.74 x 10(-9) M) as judged by SPR analysis.
  
  
  CONCLUSIONS
  The scFv antibodies generated after modified subtractive panning appear to have superior binding properties and cytocidal activity than regular panning. A simple modification of the elution condition in the phage-display panning protocol makes a large difference in determining success. Our method offers an attractive platform to discover potential therapeutic candidates.
authors.affiliation: Department of Biochemistry, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, Higashijima, Niigata, Japan. enamul.kabir_2000@yahoo.com
authors.forename: M Enamul
authors.initials: ME
authors.lastname: Kabir
authors.forename: Senthilkumar
authors.initials: S
authors.lastname: Krishnaswamy
authors.forename: Masahiko
authors.initials: M
authors.lastname: Miyamoto
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Furuichi
authors.forename: Tadazumi
authors.initials: T
authors.lastname: Komiyama
mesh_terms: D000595:Amino Acid Sequence; D000888:Antibodies, Anti-Idiotypic / Q000737:chemistry*; D000908:Antibodies, Fungal / Q000737:chemistry; D055910:Antibodies, Immobilized / Q000737:chemistry; D000911:Antibodies, Monoclonal / Q000737:chemistry; D000915:Antibody Affinity; D000935:Antifungal Agents / Q000737:chemistry*; D020134:Catalytic Domain; D003001:Cloning, Molecular; D016172:DNA Fingerprinting; D004797:Enzyme-Linked Immunosorbent Assay; D055646:Killer Factors, Yeast / Q000276:immunology*; D008969:Molecular Sequence Data; D019151:Peptide Library; D011994:Recombinant Proteins / Q000096:biosynthesis; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D057127:Single-Chain Antibodies / Q000737:chemistry*; D055320:Williopsis / Q000737:chemistry
chemical_list: D000888:Antibodies, Anti-Idiotypic; D000908:Antibodies, Fungal; D055910:Antibodies, Immobilized; D000911:Antibodies, Monoclonal; D000935:Antifungal Agents; D055646:Killer Factors, Yeast; D019151:Peptide Library; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies