title: An improved semi-quantitative enzyme immunostaining procedure for glycosphingolipid antigens on high performance thin layer chromatograms.
abstract: An immunoassay is described which allows the detection of glycosphingolipid (GSL) antigens on high performance thin layer chromatograms (HPTLC). The method involves: (1) the separation of GSL on HPTLCs; (2) incubation with specific antibodies against carbohydrate structures of GSL, and (3) the detection of specifically bound antibodies with alkaline phosphatase-conjugated second antibodies and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) as substrate. Using a monoclonal rat IgG2c antibody against Forssman GSL, a BALB/c monoclonal antibody against asialo GM2, and polyclonal rabbit antibodies against asialo GM1, it was shown that as little as 3 ng GSL antigen could be detected in a procedure taking detected in a procedure taking only 4 h to perform. The assay should be useful for screening mono- and polyclonal antibodies with potential specificity for GSL antigens, for the detection and quantification of GSL-antigens in tissue extracts, and for defining the specificity of anti-GSL antibodies.
mesh_terms: D000469:Alkaline Phosphatase; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001923:Brain Chemistry; D002855:Chromatography, Thin Layer / Q000379:methods; D005677:G(M1) Ganglioside; D006028:Glycosphingolipids / Q000032:analysis* / Q000276:immunology; D006801:Humans; D007158:Immunologic Techniques; D008563:Membrane Lipids / Q000032:analysis / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D006028:Glycosphingolipids; D008563:Membrane Lipids; D005677:G(M1) Ganglioside; C018835:asialo GM1 ganglioside; D000469:Alkaline Phosphatase