An improved phage-display panning method to produce an HM-1 killer toxin anti-idiotypic antibody

BMC Biotechnol. 2009 Dec 14;9:99. doi: 10.1186/1472-6750-9-99.

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.

Conclusion: 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Antibodies, Anti-Idiotypic / chemistry*
  • Antibodies, Fungal / chemistry
  • Antibodies, Immobilized / chemistry
  • Antibodies, Monoclonal / chemistry
  • Antibody Affinity
  • Antifungal Agents / chemistry*
  • Catalytic Domain
  • Cloning, Molecular
  • DNA Fingerprinting
  • Enzyme-Linked Immunosorbent Assay
  • Killer Factors, Yeast / immunology*
  • Molecular Sequence Data
  • Peptide Library*
  • Recombinant Proteins / biosynthesis
  • Sequence Alignment
  • Sequence Analysis, DNA
  • Single-Chain Antibodies / chemistry*
  • Williopsis / chemistry

Substances

  • Antibodies, Anti-Idiotypic
  • Antibodies, Fungal
  • Antibodies, Immobilized
  • Antibodies, Monoclonal
  • Antifungal Agents
  • Killer Factors, Yeast
  • Peptide Library
  • Recombinant Proteins
  • Single-Chain Antibodies