Glycosphingolipid antigens from Leishmania (L.) amazonensis amastigotes. Binding of antiglycosphingolipid monoclonal antibodies in vitro and in vivo

Specific glycosphingolipid antigens of Leishmania (L.) amazonensis amastigotes reactive with the monoclonal antibodies (MoAbs) ST-3, ST-4 and ST-5 were isolated, and their structure was partially elucidated by negative ion fast atom bombardment mass spectrometry. The glycan moieties of five antigens presented linear sequences of hexoses and N-acetylhexosamines ranging from four to six sugar residues, and the ceramide moieties were found to be composed by a sphingosine d18:1 and fatty acids 24:1 or 16:0. Affinities of the three monoclonal antibodies to amastigote glycosphingolipid antigens were also analyzed by ELISA. MoAb ST-3 reacted equally well with all glycosphingolipid antigens tested, whereas ST-4 and ST-5 presented higher affinities to glycosphingolipids with longer carbohydrate chains, with five or more sugar units (slow migrating bands on HPTLC). Macrophages isolated from footpad lesions of BALB/c mice infected with Leishmania (L.) amazonensis were incubated with MoAb ST-3 and, by indirect immunofluorescence, labeling was only detected on the parasite, whereas no fluorescence was observed on the surface of the infected macrophages, indicating that these glycosphingolipid antigens are not acquired from the host cell but synthesized by the amastigote. Intravenous administration of 125I-labeled ST-3 antibody to infected BALB/c mice showed that MoAb ST-3 accumulated significantly in the footpad lesions in comparison to blood and other tissues.

Immunochemical characterization of carbohydrate antigens from fungi, protozoa and mammals by monoclonal antibodies directed to glycan epitopes

Cell surface carbohydrates constitute the major antigenic determinants of fungi and protozoa. Glycoconjugates also represent a large variety of antigens or markers present in mammals such as histo-blood groups ABO, differentiation and heterophile antigens, among others. This article focuses on the general properties of glycoconjugate antigens and production and characterization of the anti-carbohydrate monoclonal antibodies (MoAbs). It describes the specificity and some properties of monoclonal antibodies directed against carbohydrate epitopes present in tumor-associated glycoproteins, in clycosaminoglycans of higher eukaryotes and in glycolipid antigens of protozoa and fungi. The epitopes recognized by the anti-carbohydrate MoAbs range from one sugar unit up to ten sugar units. Although most anti-carbohydrate MoAbs are directed predominantly toward terminal sugar residues, a few MoAbs are also reactive with internal sugar residues. The fine structure of the carbohydrate epitopes has been chemically defined by [H]NMR, GC/MS of alditol acetates of partially permethylated compounds, FAB/MS, degradation with exoglycosidases and inhibition with different methyl-glycosides and oligosaccharides.