A multiplicity of erythrocyte glycolipids of the neolacto series revealed by immuno-thin-layer chromatography with monoclonal anti-I and anti-i antibodies

Abstract

The thin-layer-chromatography immunostaining procedure was applied to human erythrocyte glycolipids using monoclonal anti-i and anti-I antibodies which are directed against epitopes on linear and branched carbohydrate chains of the neolacto (poly-N-acetyllactosamine) series. An examination of native and mild-acid-treated glycolipids from normal adult (I(adult) antigen type), neonatal (i(cord)), and I-antigen-deficient adult (i(adult)) erythrocytes enabled certain structural inferences to be made as follows: (a) cells of both I and i phenotypes contain a multiplicity of glycolipids of the neolacto series whose backbones consist of 8 or more sugar residues; (b) the octasaccharide backbones are predominantly linear in cells of i phenotype and branched in those of I type; and (c) more complex glycolipids having decasaccharide and larger backbones with both linear and branched sequences occur in erythrocytes of both phenotypes.

Carbohydrates as antigenic determinants of glycoproteins

“…We have observed that under the conditions used by Woodward et al (1985) the linear poly-N-acetyl-lactosamine determinant recognised by an anti-i antibody (Tho) and the branched poly-N-acetyllactosamine determinant recognised by anti-I (Step) that are expressed on meconium glycoproteins and sheep gastric mucins, respectively, are not destroyed, whereas SSEA-1, i (Den), on meconium, and I (Ma), on sheep gastric mucins, are destroyed (P. W. Tang & T. Feizi, unpublished work). Both the anti-i Tho and anti-I Step (Gardas, 1982;Uemura et al, 1983) can react with internal domains of poly-N-acetyllactosamine chains and do not require terminal galactose residues to be present. In contrast, anti-SSEA-1 has a strict requirement for a l-3-linked terminal fucose Hounsell et al, 1981) and anti-I Ma and anti-i Den require terminal galactose residues (Feizi et al, 1971;Tang et al, 1986).…”

Autoantibodies Against Ganglioside GM3 Represent a Portion of Anti‐Lymphocyte Antibodies in AIDS Patients

“…Immunostaining was performed using as antigen about 2^g of gangliosides obtained from AIDS lymphocytes. Gangliosides were separated by TLC, using HPTLC aluminium backed silica gel 60 (20x20) plates (Merck) [21]. Plates were soaked in a 0.2% solution of polyisobuthylmethacrylate in hexane for 90s, air dried and incubated in the blocking solution 3% bovine serum albumin (BSA) in 20 mmol Tris 0.5M NaCl, pH 7.5 (BSA-TBS) for lh at room temperature.…”

The reactivities of human erythrocyte autoantibodies anti-Pr2, anti-Gd, Fl and Sa with gangliosides in a chromatogram binding assay

“…lA and 1 B) which are known to be predominantly of the neolacto-series (Chien et al, 1978;Hakomori, 1981). The majority of immunoreactive glycolipid bands were clustered as two groups, the first migrating in the region of the GD1Ia standard and sialosylneolactohexaosylceramides and the second in the region of GDl b and GT1 b and slow migrating components corresponding to more complex gangliosides of the neolacto series (Feizi et al, 1978;Uemura et al, 1983). Some reactivity in the region-of GN13 was also present ( Fig.…”

“…Human brain gangliosides were prepared according to Ledeen -t -t cytes were used after storage at 4°C for 5 days and the iadult cells at -20°C in glycerol for 9 months according to Mollison (1979). From erythrocyte membranes (Dodge et al, 1963), glycolipids were extracted (Uemura et al, 1983) and evaporated to dryness. The dried residue was treated with 0.1 M-NaOH for 1 h at room temperature and dialysed against water, evaporated and finally dissolved in chloroform/methanol (1:…”

“…The binding of antibodies to glycolipids on t.l.c. plates was detected by overlaying with the human autoantibodies followed by I 251-labelled rabbit immunoglobulins to human ju or a chains and autoradiography as described previously (Magnani et al, 1981;Uemura et al, 1983).…”