Cloning antifungal single chain fragment variable antibodies by phage display and competitive panning elution.
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.
Krishnaswamy
Senthilkumar
S
Department of Biochemistry, Niigata University of Pharmacy and Applied Life Sciences, Japan.
Kabir
M Enamul
ME
Miyamoto
Masahiko
M
Furuichi
Yasuhiro
Y
Komiyama
Tadazumi
T
D000595:Amino Acid Sequence; D000818:Animals; D000888:Antibodies, Anti-Idiotypic; D000908:Antibodies, Fungal / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000915:Antibody Affinity; D000935:Antifungal Agents / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D001667:Binding, Competitive; D002175:Candida / Q000276:immunology*; D002462:Cell Membrane / Q000276:immunology / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007135:Immunoglobulin Variable Region; D020128:Inhibitory Concentration 50; D055646:Killer Factors, Yeast / Q000276:immunology; D051379:Mice; D018716:Molecular Mimicry / Q000276:immunology; D008969:Molecular Sequence Data; D019151:Peptide Library; D012441:Saccharomyces cerevisiae / Q000276:immunology; D016415:Sequence Alignment; D020349:Surface Plasmon Resonance; D055320:Williopsis / Q000378:metabolism
D000888:Antibodies, Anti-Idiotypic; D000908:Antibodies, Fungal; D000935:Antifungal Agents; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D055646:Killer Factors, Yeast; D019151:Peptide Library