A sensitive assay for measuring alpha-Gal epitope expression on cells by a monoclonal anti-Gal antibody.
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.


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.
Galili
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Department of Microbiology and Immunology, MCP-Hahnemann School of Medicine, Allegheny University of the Health Sciences, Philadelphia, Pennsylvania 19129, USA.
LaTemple
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Radic
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MZ
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D002240:Carbohydrate Sequence; D002417:Cattle; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006084:Graft Rejection / Q000175:diagnosis / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D011237:Predictive Value of Tests; D011817:Rabbits; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine; D014183:Transplantation, Heterologous / Q000276:immunology; D014312:Trisaccharides / Q000032:analysis* / Q000096:biosynthesis
D000911:Antibodies, Monoclonal; D014312:Trisaccharides; C076569:alpha-galactosyl epitope; D012710:Serum Albumin, Bovine