PMC

Western blotting of C. albicans antigens by using scFv5 and scFv12. C. albicans filaments were extracted with Zymolyase, and extracts were separated by SDS-PAGE (4 to 20% polyacrylamide), transferred to nitrocellulose filters, and probed with scFv. A polydisperse, high-molecular-weight band was detected with each scFv clone (5, scFv5; 12, scFv12). The position of the 205-kDa molecular size marker is indicated.

IFA detection of scFv5 and scFv12 binding to C. albicans in clinical samples. Paired panels are immunofluorescent and bright-field photomicrographs of the same microscopic fields. Clinical specimens were collected from infants with thrush (oral mucosa) or urinary candidiasis (urine) and spread immediately on microscope slides. Binding of scFv5 and scFv12 was detected by IFA. Bars, 10 μm.

Comparison of the deduced amino acid sequences of scFv5 and scFv12. The cloning strategy yields a VL-linker-VH orientation of the scFv. The framework (FR) and CDRs as defined by Kabat et al. () are indicated. The position and sequence of the linker between the VL and VH regions are also shown. A dot indicates amino acid identity at a given position in scFv12 to the corresponding residue in scFv5.

IFA detection of binding of scFv5 and scFv12 to Candida species. Paired panels are immunofluorescent and bright-field photomicrographs of the same microscopic fields. (A) scFv λ2-18 binding to C. albicans, showing the highest intensity for binding to the blastoconidia as previously described (). (B, D, and F) scFv5 binding to C. albicans (B), C. dubliniensis (D), and C. rugosa (F). C. albicans and C. dubliniensis stained intensely, while C. rugosa was negative. (C and E) scFv12 binding to C. albicans (C) and C. dubliniensis (E). scFv12 stained only C. albicans. Arrows indicate blastoconidia that did not stain. Bars, 10 μm.

IFA detection of binding of phage clones to C. albicans. Representative fields are shown. Paired panels are immunofluorescent and bright-field photomicrographs of the same microscopic fields of C. albicans strain 3153A stained via IFA with representative M13 phage clones displaying scFv. Numbers identify the clone from the original 16. Clone 1 did not bind and was included as a negative control. Arrows point to blastoconidia that were not stained by phage IFA. Bars, 10 μm.

IFA detection of scFv5 and scFv12 binding to C. albicans after proteinase K treatment. Paired panels are immunofluorescent and bright-field photomicrographs of the same microscopic fields. C. albicans filaments were subjected to proteinase K digestion, and the presence of antigen was detected by IFA. Mock-treated cells were treated with buffer alone. scFv λ2-18 has previously been shown to recognize a carbohydrate epitope () and was included as a control. Proteinase K eliminated binding by scFv5 and scFv12. Bars, 15 μm.