Quantitation and characterization of anti-Galα1-3Gal antibodies in sera of 200 healthy persons

The natural occurrence of complement-activating anti-Gal alpha l-3Gal antibodies (anti-Gal) in human serum is considered as a major obstacle to xenotransplantation. In this study we determined anti-Gal in sera of 200 healthy volunteers (100 male, 100 female) of different age groups using an IgG- and IgM-isotype specific ELISA. In addition, we used a direct hemagglutination test involving rabbit erythrocytes (E-R), which are known for abundant surface expression of the Gal alpha l-3Gal antigen. The measured arbitrary ELISA units (U) for anti-Gal ranged from 5 to 204 U (77 +/- 47; mean +/- 1 SD) for IgM, and from 1 to 162 U(73 +/- 32) for IgG. Anti-ER hemagglutination titers were between 1: 65536 and 1: 64 (mean 1: 703), with 75% of all serum samples being in the range of 1: 8192-1: 512. Specificity of the tests was determined by immunoabsorption of anti-Gal on Sepharose-coupled synthetic Gal alpha l-3Gal antigen, which reduced ELISA as well as agglutination titers by 90% or more. Overall, inter-individual differences of both anti-Gal ELISA values and E-R agglutination titers exceeded age-, gender-, or ABO-type related changes. A tendency was found to higher anti-Gal IgM values in women than in men (P < 0.005 by Student's t-test), whereas the gender-difference for anti-Gal IgG or ER titers was not significant. Application of the anti-Gal ELISA as well as the E-R agglutination assay for pre-transplant screening of potential pig xenograft recipients is discussed.