Immunoenzymometric assays for simultaneous quantitation and distribution of erythrocyte antigen

We describe an adaptation and modification of the enzyme-labeled immunosorbent assay (ELISA), which yields information on the quantity and distribution of surface antigens on erythrocytes. The distribution assay, named "ELADA" (for Enzyme-Linked Antigen Distribution Assay), involves use of an alkaline phosphatase-immunoglobulin conjugate with alpha-naphthol phosphate and a diazonium salt as substrate. The insoluble reaction product forms a complex on the cell surface at the location of the antigen. Antigens in aliquots from the same enzyme-immunoglobulin conjugate are quantified with the soluble substrate p-nitrophenyl phosphate. One can thus establish whether the distribution of erythrocyte antigens is random by examining the ELADA-stained cells with a scanning electron microscope. Combining the quantitative ELISA with measurements of cell numbers makes it possible to estimate the number of antibody molecules-and thus the number of antigenic sites per cell. By using various modified antisera, substituted naphthol phosphates with higher avidities, and reaction conditions designed to optimize the kinetic reaction, we have been able to resolve the enzyme product deposited on the antigenic site to within 20 nm. It is not always possible to decide whether each deposit represents a cluster of antigenic determinants or a single determinant; however, if the number of antibody sites is known from ELISA quantitation, information about the number of antibody sites per deposit can be ascertained.