The enzyme-linked immunospot (ELISPOT) assay for detection of single antibody-secreting cells has become the best alternative method to the conventional plaque-forming cell (PFC) assays. Among its several advantages are better antigen stability and specificity as well as fewer limitations in the diversity of antigens that can be used in the assay. In addition, the ELISPOT assay can be used to detect two antigenically different secreted antibodies simultaneously by two-color analysis and offers the unique possibility of quantifying the number of antibody molecules secreted per cell. Finally, the assay can be used to detect single antibody-secreting cells in tissues that usually confront the immunologist with difficulties, e.g., gut lamina propria from humans or mice. This unit presents the ELISPOT assay in three steps: coating of antigen to a solid phase, incubation of antibody-producing cells in appropriate dilution, and detection of the antigen-antibody complex formed at the site of the active antibody-secreting cell. The assay can be performed using polystyrene plates or nitrocellulose membrane in microtiter plates or using nitrocellulose membrane in a blotting manifold.
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