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A sensitive assay for measuring alpha-Gal epitope expression on cells by a monoclonal anti-Gal antibody
U Galili1, D C LaTemple, M Z Radic
1Department of Microbiology and Immunology, MCP-Hahnemann School of Medicine, Allegheny University of the Health Sciences, Philadelphia, Pennsylvania 19129, USA.
Background:
The assessment of a-gal epitope (Galalpha1-3Galbeta1-4GlcNAc-R) expression on various cells and tissues is important for the prediction of anti-Gal-mediated immune rejection of xenografts. This study describes an enzyme-linked immunosorbent assay (ELISA inhibition assay) developed for this purpose, which uses the monoclonal anti-Gal antibody M86.
Methods:
Cells at various concentrations were incubated overnight with M86 at 1/100 dilution. The cells and bound antibody were removed, and the residual antibody in the supernatant was measured in an ELISA assay with a-gal-bovine serum albumin as a solid phase antigen. The extent of a-gal epitope expression on cells correlates with the subsequent inhibition of M86 binding in ELISA. The inhibition binding curves at various cell concentrations were compared with those of a standard cell line with a known number of epitopes per cell.
Results And Conclusions:
The mouse IgM M86 monoclonal antibody was highly specific for a-gal epitopes. Using this antibody in an ELISA inhibition assay with cells at a wide range of concentrations enables the detection of at least 5 x 10(4) and up to more than 5 x 10(7) a-gal epitopes per cell. This assay can be used also for the detection of a-gal epitopes on membranes from tissue homogenates, and thus it enables the determination of the extent of decrease in a-gal epitope expression in animals that are genetically manipulated to alter their carbohydrate make-up.