title: New avenues for phage-display library to produce a Cryptococcus-specific anti-idiotypic antibody of HM-1 killer toxin.
abstract: Existing antifungal drugs are notable for their inability to act rapidly, as well as their toxicity and limited spectrum. The identification of fungal-specific genes and virulence factors would provide targets for new and influential drugs. The display of repertories of antibody fragments on the surface of filamentous phage offers a new way to produce immunoreagents as defined specificities. Here we report the selection of Cryptococcus-specific targets by using phage-display panning from a cDNA library, where bactericidal antibodies have been developed against conserved surface-exposed antigens. A single-chain variable fragment (scFv) phage library was constructed from splenocyte of an immunized mouse by idiotypic vaccination with HM-1 killer toxin (HM-1) neutralizing monoclonal antibody (nmAb-KT) that was used for selection against Cryptococcus neoformans membrane fraction (CnMF). Key elements were the selection against antigen (nmAb-KT and CnMF) and the release of bound phages using competitive panning elution with CnMF at neutral pH condition. Isolated scFvs react specifically with C. neoformans and some other pathogenic and non-pathogenic fungal strain's cell wall receptors by exerting strong antifungal activity in vitro. A high affinity clone, designated M1 was selected for detailed characterization and tested anti-cryptococcal activity with IC(50) values at 5.33 × 10(-7) to 5.56 × 10(-7) M against C. neoformans. The method described here is a new technique for the isolation of cell membrane specific immunoreactive phages in the form of scFv using CnMF that contained cell membrane associated proteins.
authors.affiliation: Department of Biochemistry, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, 265-1 Higashijima, Niigata 956-8603, Japan.
authors.forename: M Mamunur
authors.initials: MM
authors.lastname: Rahman
authors.forename: M Enamul
authors.initials: ME
authors.lastname: Kabir
authors.forename: Senthilkumar
authors.initials: S
authors.lastname: Krishnaswamy
authors.forename: Masahiko
authors.initials: M
authors.lastname: Miyamoto
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Furuichi
authors.forename: Tadazumi
authors.initials: T
authors.lastname: Komiyama
mesh_terms: D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000276:immunology* / Q000378:metabolism*; D015153:Blotting, Western; D003454:Cryptococcus / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D055646:Killer Factors, Yeast / Q000276:immunology*; D051379:Mice; D019151:Peptide Library
chemical_list: D000888:Antibodies, Anti-Idiotypic; D055646:Killer Factors, Yeast; D019151:Peptide Library