A SENSITIVE ASSAY FOR MEASURING α-GAL EPITOPE EXPRESSION ON CELLS BY A MONOCLONAL ANTI-GAL ANTIBODY1


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Abstract

Background.The assessment of α-gal epitope(Galα1-3Galβ1-4GlcNAc-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 withα-gal-bovine serum albumin as a solid phase antigen. The extent ofα-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 forα-gal epitopes. Using this antibody in an ELISA inhibition assay with cells at a wide range of concentrations enables the detection of at least 5×104and up to more than 5×107α-gal epitopes per cell. This assay can be used also for the detection of α-gal epitopes on membranes from tissue homogenates, and thus it enables the determination of the extent of decrease in α-gal epitope expression in animals that are genetically manipulated to alter their carbohydrate make-up.

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