2001
DOI: 10.1074/jbc.m102711200
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Structure of a Neutral Glycosphingolipid Recognized by Human Antibodies in Polyagglutinable Erythrocytes from the Rare NOR Phenotype

Abstract: NOR is a rare inheritable polyagglutination phenomenon that has been described in two families. Our recent studies on these erythrocytes showed they contained at least two unique neutral glycosphingolipids, and based on their reactivity with Griffonia simplicifolia IB4 (GSL-IB4) isolectin (Kusnierz-Alejska, G., Duk, M., Storry, J. R., Reid, M. E., Wiecek, B., Seyfried, H., and Lisowska, E. (1999) Transfusion 39, 32-38), both oligosaccharide chains terminated with an ␣-galactose residue. The reactivity with GSL… Show more

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Cited by 26 publications
(35 citation statements)
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“…The dried plates were immersed in 0.05% polyisobutylmethacrylate (Aldrich, Steinheim, Germany) in hexane for 1 min, dried, sprayed with TBS (0.05 M Tris buffer, 0.15 M NaCl (pH 7.4)), and blocked in 5% BSA for 1 h. For antibody assays, the plates were successively overlain with 1) mouse monoclonal or rabbit polyclonal antibody diluted in TBS/1% BSA (TBS-BSA) for 1-1.5 h; 2) biotinylated goat anti-mouse Ig antibody or goat anti-rabbit Ig antibody conjugated with alkaline phosphatase (Dako, Glostrup, Denmark), diluted 1:5000 with TBS-BSA; 3) ExtrAvidin-alkaline phosphatase conjugate (Sigma) diluted 1:1000 with TBS/ BSA/0.2% Tween20 for 1 h; and 4) the substrate solution (nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate, Sigma). Other details were as described previously (3,4). Each HPTLC experiment was repeated three times.…”
Section: Methodsmentioning
confidence: 99%
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“…The dried plates were immersed in 0.05% polyisobutylmethacrylate (Aldrich, Steinheim, Germany) in hexane for 1 min, dried, sprayed with TBS (0.05 M Tris buffer, 0.15 M NaCl (pH 7.4)), and blocked in 5% BSA for 1 h. For antibody assays, the plates were successively overlain with 1) mouse monoclonal or rabbit polyclonal antibody diluted in TBS/1% BSA (TBS-BSA) for 1-1.5 h; 2) biotinylated goat anti-mouse Ig antibody or goat anti-rabbit Ig antibody conjugated with alkaline phosphatase (Dako, Glostrup, Denmark), diluted 1:5000 with TBS-BSA; 3) ExtrAvidin-alkaline phosphatase conjugate (Sigma) diluted 1:1000 with TBS/ BSA/0.2% Tween20 for 1 h; and 4) the substrate solution (nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate, Sigma). Other details were as described previously (3,4). Each HPTLC experiment was repeated three times.…”
Section: Methodsmentioning
confidence: 99%
“…Finally, washed cells were resuspended (5 ϫ 10 5 cells per 0.75 ml) and analyzed as described above. Extraction and Purification of Glycosphingolipids-The isolation and fractionation of glycolipids and the orcinol staining were performed using the standard procedures applied previously to erythrocytes (2,3). Briefly, glycolipids were extracted from 2102Ep cells with chloroform/methanol, and the neutral glycolipids were separated from the phospholipids and gangliosides, purified in peracetylated form, de-O-acetylated, and desalted.…”
Section: Methodsmentioning
confidence: 99%
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