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

• Published in: Analytical biochemistry Vol. 395; no. 1; pp. 16 - 24
• Main Authors: Krishnaswamy, Senthilkumar, Kabir, M. Enamul, Miyamoto, Masahiko, Furuichi, Yasuhiro, Komiyama, Tadazumi
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.12.2009
• Subjects: 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; Candida albicans; Candida albicans membrane fraction; Cell Membrane - immunology; Cell Membrane - metabolism; Cloning, Molecular - methods; Competitive panning elution; High-affinity antibody; HM-1 killer toxin; HM-1-neutralizing monoclonal antibody; 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; Phage display; Saccharomyces cerevisiae; Saccharomyces cerevisiae - immunology; Sequence Alignment; Surface Plasmon Resonance; Williopsis - metabolism; High-affinity antibody; Phage display; Candida albicans membrane fraction; Competitive panning elution; HM-1-neutralizing monoclonal antibody; HM-1 killer toxin
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2009.08.003

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.