Candida Albicans Serotype Research Articles

Monoclonal antibodies against Candida tropicalis mannan: antigen detection by enzyme immunoassay and immunofluorescence

Three strains of mice were immunized with Candida tropicalis cell walls, and antibodies against mannan were detected by indirect enzyme immunoassay (EIA) in 3 of 9 BALB/c mice, 4 of 11 C57BL/6 mice, and 4 of 8 CFW mice. Responding mice produced immunoglobulin M (IgM), but IgG was not detected in their sera. Fusion of the high-responder BALB/c mouse with a plasmacytoma cell line resulted in 41 clones secreting antimannan monoclonal antibodies (MAbs). Four clones selected for propagation included one IgM and one IgG MAb that reacted with mannans of Candida albicans serotypes A and B and of C. tropicalis and two IgM MAbs specific for an epitope only in the mannans of C. albicans serotype A and C. tropicalis. One of the IgM MAbs, CB6, was an effective substitute for rabbit antibodies in the double-antibody sandwich EIA to detect antigenemia produced in rabbits infected with C. albicans A or C. tropicalis. It could function either as the peroxidase-conjugated indicator antibody or as the capture antibody. Two MAbs, CB6 (C. tropicalis and C. albicans A specific) and AC3 (C. tropicalis and C. albicans A and B specific), functioned in place of polyclonal antisera in the serotyping of C. albicans by immunofluorescence. There was 95.8% agreement in the results of serotyping using MAbs as reagents compared with rabbit antisera. Competitive inhibition in EIA between CB6 and monospecific antisera against C. albicans factors 1, 4, and 6 indicated that CB6 binds to an epitope which is probably factor 6. Serologic similarity between factor 4 and the binding site of MAb AC3 was also determined.

Production and characterization of agglutinating monoclonal antibodies against predominant antigenic factors for Candida albicans.

Two clones, CA4-2 and CA5-4, which produced agglutinating monoclonal immunoglobulin M (IgM) antibodies (MAbs) against mannan antigens of Candida albicans serotype A, were established. The specificity of each MAb was determined by slide agglutination tests for cross-reactivity patterns against the homologous and six other strains of Candida and a strain of Torulopsis: C. albicans serotype B, C. tropicalis, C. guilliermondii, C. krusei, C. parapsilosis, C. pseudotropicalis, and Torulopsis glabrata. The MAb produced by CA4-2 reacted with the homologous, C. tropicalis, and T. glabrata strains, whereas the MAb produced by CA5-4 reacted with the homologous, C. albicans serotype B, and C. tropicalis strains. These results are consistent with results obtained by comparative experiments with several strains of each serotype or species. Specificity of these two MAbs by agglutination was also consistent with the cross-reactivity patterns demonstrated by indirect immunofluorescence staining. The competitive binding experiments by immunofluorescence staining with two MAbs and polyclonal factor sera (PAb factors) 5 and 6 suggested that the MAb from clone CA4-2 did not completely correspond to PAb factor 6 and that the MAb from CA5-4 was distinct from PAb factor 5 in its manner of binding to determinants (the latter was designated 5b), Cross-reactivity patterns, however, furnished evidence that these two MAbs could replace the known PAb factors 6 and 5, respectively, as reagents for aid in the identification of the strains of C. albicans and their serotypes.

Antigenemia in rabbits infected withCandida albicansserotype B: detection by enzyme immunoassay and preliminary characterization of the antigen

Peroxidase-labeled IgG against Candida albicans serotype A cell walls failed to detect antigenemia in rabbits infected with C. albicans type B. Therefore type B antisera were produced in rabbits immunized with either type B cell walls; whole blastoconidia heated and killed at 60 degrees C (HK); a sublethal intravenous dosage of 10(6) colony forming units (c.f.u.); or a normally lethal dose of 10(7) c.f.u. injected into subcutaneously implanted plastic chambers. The four antisera were conjugated to peroxidase for a double antibody sandwich enzyme immunoassay. Sixteen rabbits, immunosuppressed with cortisone, received 10(7) type B blastoconidia intravenously. On day 4 their kidneys contained between 3.4 X 10(8) and 1.4 X 10(11) c.f.u. Antigenemia was detected only with the conjugated anti-HK-IgG. A mean concentration of 246 ng ml-1 of circulating antigen was detected only when serum-antigen complexes were dissociated by boiling in EDTA. The properties of the circulating antigen were as follows: relative molecular weight between 50 000 and 100 000 da; stable to boiling and proteinases, labile to sodium periodate oxidation, and bound by concanavalin A. These properties are consistent with a polysaccharide or glycoprotein, probably the mannoprotein of the cell wall. Antigenic specificity was probed further using IgG produced against heat-killed C. albicans serotype A. Antigenemia resulting from infection with serotype B was detected in 5 of 5 rabbits tested with the anti-HK-serotype B IgG but only with 3 of 5 tested with the anti-HK-serotype A IgG. In addition, the concentrations detected with serotype B reagents were 31 times higher.

Production and partial characterization of anti- Candida monoclonal antibodies

Spleen cells of Biozzi HR mice immunized with formolized, lyophilized Candida albicans serotype A cells were fused with P3-X63-Ag8.653 mouse myeloma cells. Twenty-one monoclonal antibodies (mAb) selected by an indirect ELISA technique were produced and partially characterized. All mAb reacted with a C. albicans cell wall extract. Five of the mAb were directed against C. albicans serotype A, but not against serotype B. These mAb also recognized C. tropicalis. The 16 other mAb cross-reacted with several yeast species. The immunoreactivity profiles of 5 representative anti-Candida mAb were confirmed in most cases by inhibition studies.

A novel antibody overlay technique for two-dimensional immunoelectrophoresis

A new antibody overlay technique for 2-dimensional immunoelectrophoresis (2-D-IEP) is described. After the first dimension electrophoresis of the antigen, the desired amount of antiserum is applied to the initial agarose layer and then evenly distributed over a defined surface of the gel with a PVC film. This modification of the conventional 2-D-IEP procedure makes it possible to perform tandem 2-D-IEPs comparing 2 antisera on the same gel plate, rocket IEPs where several antisera are compared by electrophoresis on the same gel plate, and 2-D-IEPs with an intermediate antiserum, avoiding the need to pour an intermediate gel. With this technique, 77 antigens have been demonstrated in a Candida albicans serotype A somatic antigen preparation. This is the first description of such a large number of immunoprecipitates on the same immunoplate.

Relative effectiveness of various yeasts, Candida spp. and Torulopsis glabrata, for inducing palatal infection in the Wistar rat

Candida albicans serotypes A and B, Candida tropicalis and Torulopsis gtabrata were tested for their ability to produce palatal candidosis in the rat. C. albicans serotype A consistently produced palatal candidosis whereas serotype B did so in only two of five animals tested, indicating a difference in pathogenicity of the two serotypes. C. tropicalis and T. glabrata did not induce changes under the conditions tested and their colonization of palatal mucosa in commensal form was dependent on the presence of an acrylic plate.

Interactions of serotypes A and B ofCandida albicansin mice

Candida albicans serotypes A and B were mixed in different proportions and administered orally to white mice receiving a protein-free diet. Recoveries of the serotype from the stomach and rectosigmoid showed that the relative proportion of serotype A was consistently higher than in the inoculating suspension. No modification of serotype was observed in mice given only one serotype, and there was no change in proportions of serotypes coincubated in vitro. These observations indicate that host factors must be involved in selective colonization by the serotypes in vivo.

Structural analysis of mannans from Candida albicans serotypes A and B and from Torulopsis glabrata by methylation gas chromatography mass spectrometry and exo-alpha-mannanase.

Mannans of Candida albicans serotypes A and B and of Torulopsis glabrata were subjected to methylation analysis and the resulting monosaccharides were converted to the peracetylaldononitrile derivatives and subsequently analyzed by gas chromatography mass spectrometry. Serotype A differed from B in having more 1 leads to 2 linked mannosyl residues (46.2 vs 37.1 mol%) indicating longer oligomannosyl sidechains; mannosyl branch points linked through C-1, C-3 and C-6 were detected for the first time in C. albicans mannans and were more abundant in serotype B (9.2 vs 5.3 mol%). T. glabrata mannan differed from that of C. albicans in having virtually no (1.6 mol%) unsubstituted sugars in the linear backbone and less 1 leads to 2 linked mannosyl residues (33.1 mol%) interpreted as shorter oligomannosyl sidechains. Model building of a repeating unit for the outer chain region of these mannans was prevented by the large amount of nonreducing terminal mannosyl residues, 23.0 mol% for C. albicans A, that probably arise from the inner core region. These mannans were exposed to exo-alpha-mannanase and the percentage of digestion, measured as reducing sugar released, was 33.5% for C. albicans B; 27.4% for T. glabrata, and C. albicans A was refractory (5.4% digested).

Cell wall glycopeptides of Candida albicans serotypes A and B

Extraction of isolated cell walls of Candida albicans serotype A and B by anhydrous ethylenediamine yielded water-soluble and -insoluble glycopeptides. The water-soluble glycopeptides were separated into mannan peptides and glucan peptides by chromatography on Sephadex G-75. The ratio of proteins to carbohydrates of both types of water-soluble glycopeptides is higher in serotype A than in serotype B. In mannan peptides of both serotypes 12 amino acids were determined, with relatively large proportions of threonine and glutamic acid. The mannan peptides of serotype A exhibit greater proportions of aromatic amino acids, tyrosine and phenylalanine. In glucan peptides of both serotypes 10 amino acids were estimated, with relatively high contents of aspartic and glutamic acids. The alkali-induced β-elimination of mannan peptides with 0.1 M NaOH gave evidence for the presence of O- glycosidic linkages between disaccharides consisting of d-mannopyranose units and threonine. The O- glycosidic type of linkage was not estimated in water-soluble glucan peptides. Periodate oxidation of the mannan component of mannan peptides showed that the mannan is a highly branched polysaccharide with higher average chain length in serotype A than in serotype B. The glucan part of the insoluble glucan peptides possesses predominantly the 1 → 3 linkages configuration.

Studies on the antigenic activities of yeasts. VII. The reaction between concanavalin A and the mannans isolated from five species of yeasts including two serotypes of Candida and Saccharomyces cerevisiae.

ABSTRACTFive antigenic mannans isolated from the cells of Candida albicans serotype A, C. albicans serotype B, C. tropicalis, C. stellatoidea and Saccharomyces cerevisiae were examined for their reactivities against concanavalin A, the size of the combining site has been estimated to be relatively small, up to 4 hexopyranosyl residues. In the quantitative precipitation reaction, all mannan‐concanavalin A systems afforded nearly the same amounts of nitrogen or mannan precipitated, and the ratios of precipitation‐inhibition with α1→2 linked manno‐oligosaccharides, from biose to tetraose, were also equal regardless of the structural differences of these mannans. Furthermore, in agar‐gel double diffusion analysis, all the systems gave a corresponding precipitation arc which completely fused with the adjacent ones. These behaviors of mannan‐concanavalin A systems resemble those of antigen‐antibody systems consisting of the same mannans and anti‐C. albicans serotype B serum. It also provided evidence that the previous interpretation on the lesser serologic specificity of this serum compared to that of anti‐C. albicans serotype A was due to the smaller size of combining sites for antibodies of the former than of the latter serum.

Isolation and properties of mannans from Alternaria kikuchiana Tanaka and Alternaria zinniae.

Japanese Journal of MicrobiologyVolume 15, Issue 4 p. 373-376 NoteFree Access Isolation and Properties of Mannans from Alternaria kikuchiana Tanaka and Alternaria zinniae Ichiro Azuma, Ichiro Azuma The Third Department of Internal Medicine, School of Medicine, and Institute of Scientific and Industrial Research, Osaka University, OsakaSearch for more papers by this authorShigeru Negoro, Shigeru Negoro The Third Department of Internal Medicine, School of Medicine, and Institute of Scientific and Industrial Research, Osaka University, OsakaSearch for more papers by this authorFuminori Kanetsuna, Fuminori Kanetsuna The Third Department of Internal Medicine, School of Medicine, and Institute of Scientific and Industrial Research, Osaka University, OsakaSearch for more papers by this authorYuichi Yamamura, Yuichi Yamamura The Third Department of Internal Medicine, School of Medicine, and Institute of Scientific and Industrial Research, Osaka University, OsakaSearch for more papers by this authorHideki Miyaji, Hideki Miyaji The Third Department of Internal Medicine, School of Medicine, and Institute of Scientific and Industrial Research, Osaka University, OsakaSearch for more papers by this authorAkira Misaki, Akira Misaki The Third Department of Internal Medicine, School of Medicine, and Institute of Scientific and Industrial Research, Osaka University, OsakaSearch for more papers by this author Ichiro Azuma, Ichiro Azuma The Third Department of Internal Medicine, School of Medicine, and Institute of Scientific and Industrial Research, Osaka University, OsakaSearch for more papers by this authorShigeru Negoro, Shigeru Negoro The Third Department of Internal Medicine, School of Medicine, and Institute of Scientific and Industrial Research, Osaka University, OsakaSearch for more papers by this authorFuminori Kanetsuna, Fuminori Kanetsuna The Third Department of Internal Medicine, School of Medicine, and Institute of Scientific and Industrial Research, Osaka University, OsakaSearch for more papers by this authorYuichi Yamamura, Yuichi Yamamura The Third Department of Internal Medicine, School of Medicine, and Institute of Scientific and Industrial Research, Osaka University, OsakaSearch for more papers by this authorHideki Miyaji, Hideki Miyaji The Third Department of Internal Medicine, School of Medicine, and Institute of Scientific and Industrial Research, Osaka University, OsakaSearch for more papers by this authorAkira Misaki, Akira Misaki The Third Department of Internal Medicine, School of Medicine, and Institute of Scientific and Industrial Research, Osaka University, OsakaSearch for more papers by this author First published: April 1971 https://doi.org/10.1111/j.1348-0421.1971.tb00594.xCitations: 2 From the Center for Microbiology, Instituto Venezolano de Investigaciones Cientificas, Apartado 1827 Caracas, Venezuela. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL REFERENCES 1Azuma, I., Ajisaka, M., and Yamamura, Y. 1970. Polysaccharides of Mycobacterium bovis Ushi 10, Mycobacterium smegmatis, Mycobacterium phlei, and atypical Mycobacterium P 1. Infec. Immun. 2: 347– 349. 2Azuma, I., Kimura, H., Hirao, F., Tsubura, E., and Yamamura, Y. 1969. Biochemical and immunological studies on Aspergillus. II. Immunological properties of protein and polysaccharide fractions obtained from Aspergillus fumigatus. Mycopathol. Mycol. Appl. 37: 289– 303. 3Ballou, C. E. 1970. 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Studies on the antigenic activities of yeasts. VI. Analysis of the antigenic determinants of the mannan of Candida albicans serotype B-792.

ABSTRACTIn order to provide further information about the immunochemical differences between two mannans of Candida albicans serotype A and serotype B, quantitative precipitation‐inhibition tests of anti‐C. albicans serotype B serum were carried out in the present study. Oligosaccharides were prepared by acetolysis of a homologous mannan, and a1→2 linked di‐, tri‐, tetra‐, penta‐ and hexasaccharide were separated in chromatographically homogeneous states. The latter two oligomers contained a small amount of an isomer containing a1→2 and a1→3 linkages. In the precipitation‐inhibition tests of anti‐C. albicans serotype B serum with its homologous mannan and heterologous mannan of C. albicans serotype A, the inhibitory power of the oligomers was of the following order; hexa‐> penta‐> tetra‐> tri‐> disaccharide, and the amounts for 50%‐inhibition of the former 4 oligomers were 0.02–0.03, 0.05–0.07, 0.1–0.2 and 0.3–0.4 μmoles respectively, whereas disaccharide was very poor inhibitor. The lower oligomers, a1→2 linked tri‐ and tetrasaccharide, showed considerably strong inhibitory activities. The results obtained in the present study confirmed that the antigenic determinants of the mannan of C. albicans serotype B is the hexasaccharide moiety corresponding to the longest branched chains of mannan, and moreover, the a1→2 linked tri‐ and tetrasaccharide moieties play an important factor in dominating immunochemical specificity.