A simple and sensitive ELISA to detect immune (gamma) interferon induced I-A on a macrophage line

A convenient and sensitive cell surface ELISA is described to detect immune (gamma) interferon induced I-A determinants on a murine macrophage line, P388D1. The ELISA is performed on monolayers of P388D1 cells grown in 96-well culture plates for 2 days in the presence of recombinant gamma IFN or supernatants from cultures of antigen-activated T cells. After glutaraldehyde fixation, the cultured cells are overlayed with fetal calf serum to block nonspecific antibody binding during the assay. A double sandwich technique employing a monoclonal anti-I-Ad (MKD6) antibody followed by peroxidase-conjugated goat anti-mouse gamma chain antibody is then used to detect the presence of I-A on the cell monolayers. Detection of I-A induced by both r gamma IFN and the supernatant from an antigen-stimulated T cell line was highly reproducible and sensitive using this method. Furthermore, the assay is easily performed and many sample supernatants can be rapidly screened for this activity. The assay is used by this laboratory to detect the antigen-stimulated production of gamma IFN by T cell clones and hybridomas.