Direct and Indirect Cell-Based Enzyme-Linked Immunosorbent Assay

Abstract

The direct cell-based enzyme-linked immunosorbent assay (ELISA) has developed into a popular alternative immunoassay for the rapid detection of expressed cell-surface antigens or receptors. It is used to determine cell surface antigen expression profiles using existing reporter-labeled antibodies. The target specificity of newly developed antibodies can be determined by the indirect approach, which screens hybridoma supernatants for antibody reactivity against the target antigen of interest. The cell-based ELISA acts as a surrogate for the identification of immunohistochemistry-reactive (IHC) antibodies and aids in hybridoma-derived antibody identification, which is used for further ELISA development. Briefly, cells are adsorbed onto the microtiter plate surface and then fixed. Next, either hybridoma supernatant or reporter-labeled antibody is added and allowed to bind, followed by a washing step to remove unbound antibodies. For the indirect approach—during the screening of hybridoma supernatant—a reporter-labeled species-specific secondary antibody is added. For either approach, the next step is the addition of substrate. Substrate hydrolysis is proportional to the level of cell-surface antigen expression and can be measured using a microplate reader. Cell-based ELISAs have detection sensitivities comparable to quantitative analyses by flow cytometry and can be developed in multiplex format using different reporters for the analysis of cell populations.