The Galα1-3Galβ (αGal) hapten is xenogeneic for humans; natural anti-αGal antibodies are present in human serum. To study the possible abnormal expression of the αGal in humans and the pathophysiological role of antibodies, the method of affinity purification of human anti-αGal 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 αGal-restricted specificity. The IC 50 value for αGal-PAA was equal to 4 × 10 −8 M. In a competitive assay, the Galα1-3(Fucα1-2)Galβ-PAA (trisaccharide of blood group B) was found to be one hundred times less active inhibitor than αGal-PAA. The multivalent αGal-PAA was 1100 times more potent an inhibitor than the monovalent spacered αGal-saccharide. The antibodies did not show any reactivity to the negatively charged antigens (DNA, human tumor-derived mucins). At a concentration of 2 µg/mL, the antibodies agglutinated rabbit erythrocytes but not hare erythrocytes. The high reactivity of antibodies to the αGal-glycosphingolipids of rabbit erythrocytes and the pig kidney was shown by a modified sensitive method of thin-layer chromatography with immunodetection.