The isolation and characterization of human natural alpha Gal-specific IgG antibodies applicable to the detection of alpha Gal-glycosphingolipids

The Ga1 alpha 1-3Ga1 beta (alpha Ga1) hapten is xenogeneic for humans; natural anti-alpha Ga1 antibodies are present in human serum. To study the possible abnormal expression of the alpha Ga1 in humans and the pathophysiological role of antibodies, the method of affinity purification of human anti-alpha Ga1 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 alpha Ga1-restricted specificity. The IC50 value for alpha Ga1-PAA was equal to 4 x 10(-8) M. In a competitive assay, the Ga1 alpha 1-3(Fuc alpha 1-2)Ga1 beta-PAA (trisaccharide of blood group B) was found to be one hundred times less active inhibitor than alpha Ga1-PAA. The multivalent alpha Ga1-PAA was 1100 times more potent an inhibitor than the monovalent spacered alpha Ga1-saccharide. The antibodies did not show any reactivity to the negatively charged antigens (DNA, human tumor-derived mucins). At a concentration of 2 mu g/mL, the antibodies agglutinated rabbit erythrocytes but not hare erythrocytes. The high reactivity of antibodies to the alpha Ga1-glycosphingolipids of rabbit erythrocytes and the pig kidney was shown by a modified sensitive method of thin-layer chromatography with immunodetection.