Purification of a Candida albicans germ tube specific antigen

Marot-Leblond, A

doi:10.1016/0928-8244(95)00058-f

Cited by 6 publications

(10 citation statements)

References 6 publications

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“…Many Candida filament antigens characterized to date are extractable under reducing conditions and with Zymolyase (29,40,43,50). The cognate antigens of scFv5 and scFv12 were not extractable with 2-mercaptoethanol and SDS despite multiple attempts.…”

Section: Discussionmentioning

confidence: 99%

Differentiation of Candida albicans and Candida dubliniensis by Using Recombinant Human Antibody Single-Chain Variable Fragments Specific for Hyphae

Bliss¹,

Sullivan²,

Malone³

et al. 2003

J Clin Microbiol

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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.

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Section: Discussionmentioning

confidence: 99%

Differentiation of Candida albicans and Candida dubliniensis by Using Recombinant Human Antibody Single-Chain Variable Fragments Specific for Hyphae

Bliss¹,

Sullivan²,

Malone³

et al. 2003

J Clin Microbiol

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“…These antibodies were most frequently directed toward carbohydrates carried by cell wall proteins. Among these antibodies, three MAbs have been demonstrated by indirect immunofluorescence assay (IFA) to only recognize C. albicans germ tubes and hyphae (Ollert & Calderone, 1990; Marot‐Leblond et al , 1993, 1995, 2000; Ponton et al , 1993). Molecular genetic approaches have identified genes such as ALS3, HWP1 and HYR1 that encode hypha‐specific surface proteins that may contribute to differences in cell wall structure and functions (Bailey et al , 1996; Hoyer et al , 1998; Staab & Sundstrom, 1998).…”

Section: Introductionmentioning

confidence: 99%

“…In previous work (Marot‐Leblond et al , 1993, 1995), a C. albicans germ tube‐specific antigen (3D9 antigen) was identified by the use of an MAb (MAb 3D9.3). By indirect immunofluorescence, MAb 3D9.3 was specific for the surface of C. albicans germ tubes and hyphae, and was not able to label the cell wall of any other Candida species (Marot‐Leblond et al , 1993).…”

Section: Introductionmentioning

confidence: 99%

Recognition ofCandida albicansAls3 by the germ tube-specific monoclonal antibody 3D9.3

Beucher¹,

Marot-Leblond²,

Billaud-Nail³

et al. 2009

FEMS Immunol Med Microbiol

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Monoclonal antibody 3D9.3 (MAb 3D9.3) reacts with the surface of Candida albicans germ tubes and recognizes a protein epitope. We used a two-step chromatography procedure to purify and identify the antigen (3D9) from C. albicans strain 66396 germ tubes. MAb 3D9.3 recognized two intense protein bands at 140 and 180 kDa. A comparative analysis between theoretical and experimental mass spectrum peaks showed that both bands corresponded to Als3. This conclusion was supported by lack of reactivity between MAb 3D9.3 and an als3Δ/als3Δ mutant strain, and the fact that an immunoglobulin preparation enriched for Als3 specificity recognized the purified 3D9 antigen. PCR demonstrated that C. albicans strain 66396 has two different-sized ALS3 alleles that correspond to the two purified protein bands. Strain- and species-specificity of the 3D9 epitope were studied with various C. albicans strains and Candida species, such as closely related C. dubliniensis. The 3D9 epitope was detected only in C. albicans, demonstrating the utility of MAb 3D9.3 for differentiation between C. albicans and C. dubliniensis. Adhesion assays demonstrated that MAb 3D9.3 blocks adhesion of C. albicans germ tubes to human buccal epithelial cells and vascular endothelial cells.

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“…These methods are based on extracting two distinct types of antigens, from the cell wall (mannoproteins) and from the cytosol. Major cell wall C. albicans antigens are glyceraldehyde 3-phosphate dehydrogenase (23), the proteins of Mw from 110 to 170 kDa (24), and the mannoproteins of Mw 260 kDa (25) and 58 kDa (26). Major cytosol C. albicans antigens are enolase Mw 47 kDa (27) and the proteins of Mw 52 kDa, from 64 to 67 kDa and 70 kDa (15,28).…”

Section: Discussionmentioning

confidence: 99%

Preparation of combined extract of cell wall and cytosol antigens of Candida albicans for immunoblot analysis

Dojnov¹,

Božić²,

Bulajić

et al. 2007

Clinical Laboratory Analysis

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Immunoblot analysis is not in wide use for diagnosis of invasive candidiasis, mostly because the procedure is not standardized and hence not reliable. This work describes a standardized method for C. albicans antigen extract preparation and immunochemical detection. The major improvement of the method is the preparation of combined antigen extract -- consisting of the cell wall and cytosol antigens. The fungal cells and lysis buffer were mixed at a 1:3 ratio and disintegrated by ultrasound for six cycles of one minute each. After centrifugation, cytosol antigens were obtained in the supernatant and cell wall antigens were in the precipitate. Precipitate was dissolved in lysis buffer with 2% sodium dodecyl sulfate (SDS) and boiled at 100 degrees C for 2 min. After centrifugation, the supernatant was combined with the previous one, so the extract of the combined antigens was obtained in the mixture. With those combined antigen extracts and with sera of three different groups of patients, immunoblot analysis showed sensitivity of 90.2%, specificity of 84.4%, and accuracy of 88.0%.

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Purification of a Candida albicans germ tube specific antigen

Cited by 6 publications

References 6 publications

Differentiation of Candida albicans and Candida dubliniensis by Using Recombinant Human Antibody Single-Chain Variable Fragments Specific for Hyphae

Differentiation of Candida albicans and Candida dubliniensis by Using Recombinant Human Antibody Single-Chain Variable Fragments Specific for Hyphae

Recognition ofCandida albicansAls3 by the germ tube-specific monoclonal antibody 3D9.3

Preparation of combined extract of cell wall and cytosol antigens of Candida albicans for immunoblot analysis

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