The isolation and characterization of human natural alphaGal-specific IgG antibodies applicable to the detection of alphaGal-glycosphingolipids.
The Galalpha1-3Galbeta (alphaGal) hapten is xenogeneic for humans; natural anti-alphaGal antibodies are present in human serum. To study the possible abnormal expression of the alphaGal in humans and the pathophysiological role of antibodies, the method of affinity purification of human anti-alphaGal IgG was developed. The specificity of antibodies was evaluated using polyacrylamide (PAA)-based glycoconjugates in direct and competitive enzyme-linked immunosorbent assays (ELISA). The purified antibodies exhibited alphaGal-restricted specificity. The IC50 value for alphaGal-PAA was equal to 4 x 10(-8) M. In a competitive assay, the Galalpha1-3(Fucalpha1-2)Galbeta-PAA (trisaccharide of blood group B) was found to be one hundred times less active inhibitor than alphaGal-PAA. The multivalent alphaGal-PAA was 1100 times more potent an inhibitor than the monovalent spacered alphaGal-saccharide. The antibodies did not show any reactivity to the negatively charged antigens (DNA, human tumor-derived mucins). At a concentration of 2 microg/mL, the antibodies agglutinated rabbit erythrocytes but not hare erythrocytes. The high reactivity of antibodies to the alphaGal-glycosphingolipids of rabbit erythrocytes and the pig kidney was shown by a modified sensitive method of thin-layer chromatography with immunodetection.
Smorodin
E P
EP
Department of Oncology & Immunology, National Institute for Health Development, Hiiu 42, 11619, Tallinn, Estonia. evgeni.smorodin@tai.ee
Kurtenkov
O A
OA
Shevchuk
I N
IN
Tanner
R H
RH
D000818:Animals; D000918:Antibody Specificity; D004187:Disaccharides / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D004912:Erythrocytes / Q000378:metabolism; D006001:Glycoconjugates / Q000276:immunology; D006028:Glycosphingolipids / Q000097:blood* / Q000276:immunology; D006241:Haptens / Q000276:immunology*; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology*; D007668:Kidney / Q000378:metabolism; D009077:Mucins / Q000276:immunology; D011817:Rabbits; D013552:Swine; D014312:Trisaccharides / Q000276:immunology*
D004187:Disaccharides; D006001:Glycoconjugates; D006028:Glycosphingolipids; D006241:Haptens; D007074:Immunoglobulin G; D009077:Mucins; D014312:Trisaccharides; C076569:alpha-galactosyl epitope; C055075:galactosyl-(1-3)galactose