Abstract
We have developed a sensitive enzyme immunoassay (EIA) that is useful for detecting antibodies directed against antigenic sites on the surface of mammalian cells. Approximately 100 antigen-bearing cells were trapped in the wells of 96-well Durapore membrane filter plates (Millititer GV plates, Millipore, Bedford, MA). Antibody binding was detected with alkaline phosphatase conjugated goat anti-mouse IgG + IgM. Conjugate alkaline phosphatase activity was detected with a fluorogenic substrate in the presence of levamisole, an inhibitor of endogenous cellular alkaline phosphatase using a Fluoroskan microtiter plate reader (Flow Laboratories). The method permits accurate and reproducible screening of hybridoma supernatants using a minimal number of antigen-bearing cells.
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