Differentiation of Candida albicans and Candida dubliniensis by using recombinant human antibody single-chain variable fragments specific for hyphae.
To identify antigens specific for the filamentous form of Candida albicans, a combinatorial phage display library expressing human immunoglobulin heavy and light chain variable regions was used to select phage clones capable of binding to the surfaces of viable C. albicans filaments. Eight distinct phage clones that bound specifically to filament surface antigens not expressed on blastoconidia were identified. Single-chain antibody variable fragments (scFv) derived from two of these phage clones (scFv5 and scFv12) were characterized in detail. Filament-specific antigen expression was detected by an indirect immunofluorescence assay. ScFv5 reacted with C. dubliniensis filaments, while scFv12 did not. Neither scFv reacted with C. glabrata, C. parapsilosis, C. rugosa, C. tropicalis, or Saccharomyces cerevisiae grown under conditions that stimulated filament formation in C. albicans and C. dubliniensis. Epitope detection by the two scFv was sensitive to proteinase K treatment but not to periodate treatment, indicating that the cognate epitopes were composed of protein. The antigens reactive with scFv5 and scFv12 were extractable from the cell surface with Zymolyase, but not with sodium dodecyl sulfate (SDS) and 2-mercaptoethanol, and migrated as polydisperse, high-molecular-weight bands on SDS-polyacrylamide gel electrophoresis gels. The epitopes were detected on clinical specimens obtained from infants with thrush and urinary candidiasis without passage of the organisms on laboratory media, confirming epitope expression in human infection. The availability of a monoclonal immunologic reagent that recognizes filaments from both C. albicans and C. dubliniensis and another specific only to C. albicans adds to the repertoire of potential diagnostic reagents for differentiation between these closely related species.
Bliss
Joseph M
JM
Department of Pediatrics. Department of Microbiology and Immunology. Center for Oral Biology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.
Sullivan
Mark A
MA
Malone
Jane
J
Haidaris
Constantine G
CG
D000946:Antigens, Fungal / Q000032:analysis; D015153:Blotting, Western; D002176:Candida albicans / Q000145:classification* / Q000276:immunology / Q000302:isolation & purification; D006801:Humans; D025301:Hyphae / Q000276:immunology*; D007128:Immunoglobulin Fragments / Q000276:immunology; D010446:Peptide Fragments; D011994:Recombinant Proteins
D000946:Antigens, Fungal; D007128:Immunoglobulin Fragments; D010446:Peptide Fragments; D011994:Recombinant Proteins; C055966:immunoglobulin Fv