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An antibody reactive with the galactosyl(α1-2)galactose [gal(α1-2)gal] epitope was characterized in human sera by enzyme-linked immunosorbent assay, red blood cell (RBC) and laminin absorption, and oligosaccharide inhibition. This antibody was found evenly distributed between the IgG and IgM classes and was present at high titers in the serum of all normal adults studied, but in 75% of children less than three years of age, it was observed at the lower limit of detection, and gradually increased to adult levels by the age of six. Although this antibody bound to gal(α1-3)gal-linked synthetic antigens, it did not bind to the same residues present in rabbit, rat, and guinea pig RBC or in murine laminin or nidogen. These latter results, plus the fact that antigen-antibody binding was strongly blocked by gal(α1-2)gal but not by methyl-α-galactopyranoside or melibiose, suggest that this antibody is indeed different from anti-gal(α1-3)gal antibody. Anti-gal(α1-2)gal antibody levels were significantly elevated in 66% of patients with chronic chagasic cardiomyopathy, but were not elevated in patients with different clinical forms of leishmaniasis, Trypanosoma rangeli-infected patients, or in patients with 15 other infectious and inflammatory diseases. Gal(α1-2)gal antibodies did not absorb to intact T. cruzi parasites, but absorbed strongly to trypomastigote and epimastigote sonicates, suggesting some masking of reactive epitopes. Since antibody binding is blocked by gal(α1-3)gal, previous results suggest that in chronic T. cruzi infection, at least three different antibody clones exist that react with gal(α1-3)gal epitopes: anti-gal(α1-3)gal IgG, anti-mannose [man](α 1-3)gal or anti-man(β1-3)gal IgM, and anti-gal(α1-2)gal IgM and IgG.