A double substrate immunoassay has been developed that allows simultaneous quantitation and localization of red blood cell surface antigens. The assay can be used to quantitate surface antigens, or the titer of antisera specific to cell-surface antigens, on the red blood cell. The assay has been adapted to the study of both D antigen and erythrocyte membrane (EM) antigen, which were found to be uniformly dispersed over the surface of the cell. The number of EM antigens per cell appeared to be approximately twenty times that of the D antigen. For typing antisera, this assay makes it feasible to express antibodies in absolute units (microgram antibody) rather than conventional dilution titers, and thus offers a simpler means of rapidly comparing antisera.
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