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

Abstract

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 Candida albicans 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 6.40 μM and 2.20 μM, respectively. By using surface plasmon resonance analysis, the scFv–C1 had a K d value of 3.09 × 10 −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.