title: [Demonstration of β-1,2 mannan structures expressed on the cell wall of Candida albicans yeast form but not on the hyphal form by using monoclonal antibodies].
abstract: Candida albicans is a polymorphic fungus that may be observed as both commensal and opportunistic pathogen in humans. As one of the major components of Candida cell wall structure, mannan plays an important role in the fungus-host cell interaction and in virulence. The ability to switch from yeast to hypha form of microorganism is crutial in the development of C.albicans infections. Hyphal form has different antigenic properties compared to yeast form and structural changes occur in the yeast cell wall during transition from yeast to hypha form. Although there are several factors associated with this transition process, sufficient information is not available. The aim of this study was to investigate the change of configuration in mannan structure found in C.albicans cell wall by using monoclonal antibodies. C.albicans (NIHA 207) serotype A strains were used as test strains throughout the study, together with Salmonella choleraesuis 211 and Salmonella infantis as controls with similar cell wall structures to that of C.albicans. Cultures were maintained on YPD-agar medium by incubating at 28°C for yeast forms, and on YPD-broth medium in a shaking incubator at 37°C for 3-4 hours for the growth of hyphal forms. Cells were harvested in the exponential phase, and after being washed, the mannan content from C.albicans were extracted from pellet by heating in 20 mM sodium citrate buffer for 90 minutes at 125°C. Hybridoma technique was used for the production of monoclonal antibodies. After immunizing the Balb/C mice with antigen, the splenocytes were harvested and fusion was performed between spleen cells and F0 myeloma cells. The clones grown in HAT medium were screened for the presence of antibody producing hybrid cells by ELISA method. The antibody isotypes were determined by using a commercial kit (Pierce Biotechnology, ABD). The culture supernatants which contained monoclonal antibodies were collected and purified according to the ammonium sulphate method. Sandwich ELISA and immunofluorescence (IF) methods have been used to detect the experimental reactions. In our study, highly specific class IgM murine monoclonal antibodies (mAb-2B7) against C.albicans yeast cell wall were obtained from clone 2B7. These antibodies cross-reacted with S.choleraesuis 211 and S.infantis bacteria sharing similar cell wall structure of C.albicans. The existence of mannan β-1,2 bonds on the surface of C.albicans yeast form was confirmed with a commercial monoclonal antibody (mAb-ACMK-1; Matriks Biotek(®), Turkey) specific for those bonds. Besides, mAb-ACMK-1 interacted with C.albicans yeast form and gave intense fluorescence (high positive reaction) in IF method, but no fluorescence (negative) was detected with hyphal form. This data, obtained for the first time with this study, indicates that the mannan β-1,2 bonds are either found infrequently or none in the fungal hyphal wall. Although both monoclonal antibodies recognize the mannan antigen, mAb-2B7 reacted with S.choleraesuis 211, while mAb-ACMK-1 did not, due to the difference of epitope specificity. In conclusion, monoclonal antibodies may facilitate the characterization of antigenic structures of Candida, which will lead for the identification of new determinants that may increase the sensitivity and specificity of commercial tests used for mannan detection in serum.
authors.forename: Cevahir
authors.initials: C
authors.lastname: Aydın
authors.affiliation: Matriks Biotechnology Industry and Trade Ltd. Co., Gazi Teknopark, Ankara, Turkey. halukataoglu@matriksbiotek.com.
authors.forename: Haluk
authors.initials: H
authors.lastname: Ataoğlu
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000946:Antigens, Fungal / Q000097:blood / Q000737:chemistry / Q000276:immunology; D002176:Candida albicans / Q000737:chemistry* / Q000254:growth & development / Q000276:immunology / Q000472:pathogenicity; D002177:Candidiasis / Q000382:microbiology*; D002473:Cell Wall / Q000737:chemistry; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D025301:Hyphae / Q000737:chemistry / Q000254:growth & development / Q000276:immunology; D007075:Immunoglobulin M / Q000276:immunology; D008351:Mannans / Q000097:blood / Q000737:chemistry* / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D012475:Salmonella / Q000276:immunology; D012680:Sensitivity and Specificity; D014774:Virulence
chemical_list: D000911:Antibodies, Monoclonal; D000946:Antigens, Fungal; D007075:Immunoglobulin M; D008351:Mannans