Cloning antifungal single chain fragment variable antibodies by phage display and competitive panning elution

Anal Biochem. 2009 Dec 1;395(1):16-24. doi: 10.1016/j.ab.2009.08.003. Epub 2009 Aug 7.


Phage display and two competitive panning elution conditions were used to isolate Candida-specific single chain fragment variable (scFv) antibodies. An scFv phage library constructed from splenic lymphocytes of mice immunized by idiotypic vaccination with an HM-1 killer toxin (HM-1)-neutralizing monoclonal antibody (nmAb-KT) was used for panning against Candidaalbicans membrane fraction (CaMF). Key steps were specific elution conditions to separately release the bound phages with original antigen HM-1+HM-1 peptide 6 and CaMF. The positive phages were screened by using enzyme-linked immunosorbent assay, and after nucleotide sequencing, clone expression, and purification, clone scFv-C1 was selected for detailed characterization. The scFv-C1 showed IC(50) values for cell growth against various Candida species and Saccharomyces cerevisiae as 2.40 to 6.40microM and 2.20microM, respectively. By using surface plasmon resonance analysis, the scFv-C1 had a K(d) value of 3.09x10(-11)M to nmAb-KT, indicating a 260-fold higher affinity than for HM-1. These results showed the generated scFv-C1 mimicking HM-1-binding affinity to nmAb-KT and in vitro antifungal activity. We believe that the effectiveness of the competitive panning elution method and antigen-specific recombinant scFv antibodies obtained in this study are excellent candidates for antimycotic drugs.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibodies, Anti-Idiotypic
  • Antibodies, Fungal / chemistry
  • Antibodies, Fungal / genetics*
  • Antibodies, Fungal / isolation & purification
  • Antibodies, Fungal / metabolism
  • Antibody Affinity
  • Antifungal Agents / chemistry
  • Antifungal Agents / isolation & purification
  • Antifungal Agents / metabolism
  • Binding, Competitive*
  • Candida / immunology*
  • Cell Membrane / immunology
  • Cell Membrane / metabolism
  • Cloning, Molecular / methods*
  • Humans
  • Immunoglobulin Fragments / chemistry
  • Immunoglobulin Fragments / genetics*
  • Immunoglobulin Fragments / isolation & purification
  • Immunoglobulin Fragments / metabolism
  • Immunoglobulin Variable Region
  • Inhibitory Concentration 50
  • Killer Factors, Yeast / immunology
  • Mice
  • Molecular Mimicry / immunology
  • Molecular Sequence Data
  • Peptide Library*
  • Saccharomyces cerevisiae / immunology
  • Sequence Alignment
  • Surface Plasmon Resonance
  • Williopsis / metabolism


  • Antibodies, Anti-Idiotypic
  • Antibodies, Fungal
  • Antifungal Agents
  • Immunoglobulin Fragments
  • Immunoglobulin Variable Region
  • Killer Factors, Yeast
  • Peptide Library