Mapping of Candida albicans oligomannosidic epitopes by using monoclonal antibodies

Abstract

Six monoclonal antibodies (MAbs) from various laboratory sources (EB-CA1, EB-CA2, H5, AF1, C6, and 5B2), reacting with the polysaccharidic moieties of Candida albicans mannoproteins, were used for epitope mapping by an enzyme-linked immunosorbent assay (ELISA) with neoglycolipids and by Western blotting (immunoblotting) of a C. albicans germ tube extract. The ELISA involved neoglycolipids constructed from three families of oligomannosides released by sequential depolymerization of C. albicans phosphopeptidomannan by acid hydrolysis (NGLH), 1-elimination (NGLO), and acetolysis (NGLA). All of the MAbs exhibited low reactivities against NGLO. MAbs EB-CA1, EB-CA2, and H5 reacted mainly against NGLA, and MAbs C6 and AF1 recognized mainly NGLH, whereas MAb 5B2 reacted with both families of neoantigens. When this method was compared with Western blotting, strong reactivity to NGLA was associated with the presence of epitopes shared by high-molecular-weight mannoproteins, whereas strong reactivity to NGLH was associated with a * Corresponding author.

Nature of Candida albicans-derived carbohydrate antigen recognized by a monoclonal antibody in patient sera and distribution over Candida species

“…The Candida mannan has long been recognized for its antigenic properties [2^4]. We have con¢rmed the reactivity of MAb EB-CA1 with NGLs constructed with mannan acid stable oligomannosides [14], and shown that within this pool of oligomannose residues, the length of the oligomannoside chain was a limiting factor for recognition. A minimal number of ¢ve mannose residues was required for binding.…”

“…The sequential depolymerization of the mannan has been previously described [14]. The ¢rst step consisted of a mild acid hydrolysis: 500 mg mannan was dissolved in 10 mM HCl and heated for 30 min at 100³C.…”

“…Yeast cells were obtained on SDA after 24 h at 37³C. Cytoplasmic and cell wall components were prepared according to a method of alkali extraction in reducing conditions (AERC) previously described [14]. Electrophoresis of these extracts (100 mg ml 3I proteins) was performed for 5 h at a constant current of 30 mA on 7^20% gradient polyacrylamide gel slabs.…”

“…the`Pastorex Candida' used for detection of mannanemia in patient sera [11]. We further used a method which consisted in coating ELISA plates with neoglycolipids (NGLs) constructed from mannan released oligomannosides [13,14]. Binding experiments and NMR analysis have shown the MabEBCA1 minimal epitope to consist in an K-linked mannopentaose of the C. albicans mannan acid stable domain.…”

Existence of novel β‐1,2 linkage‐containing side chain in the mannan of Candida lusitaniae, antigenically related to Candida albicans serotype A

“…We have reported the presence of three kinds of b-1,2 linkage-containing side chains in the mannans from Candida species [11][12][13][14][15][16][17]. Because the b-1,2-linked mannose unit is present among the pathogenic fungi only in the mannan of the genus Candida and the b-1,2 linkage-containing side chains behave as strong antigens, many workers [18][19][20][21][22][23][24][25] have produced monoclonal antibodies to b-1,2-linked mannose units to protect against candidiasis, for the serodiagnosis of candidiasis, or for the identification of mechanisms of Candida infection. Furthermore, studies have indicated the b-1,2-linked mannose units participate in the adherence of Candida cells to mammalian cells as the first step in infection [26][27][28].…”