Particle concentration fluorescence immunoassay (PCFIA): a new, rapid immunoassay technique with high sensitivity

A new solid-phase fluorescence immunoassay technique is described and is exemplified by the detection of murine monoclonal antibodies to human IgG in hybridoma culture supernatants and the detection of murine IgG. The assay is performed in a specially designed 96-well plate. For antibody detection, antigen bound to submicron polystyrene particles is bound to its specific antibody, which is in turn reacted with fluorescein-labeled affinity-purified goat anti-mouse IgG. The reaction is complete in 10 min at ambient temperature. The solid phase is separated from the reaction mixture by filtration, washed and the total particle-bound fluorescence is determined by front-surface fluorimetry. The sensitivity of the technique for antibody detection is equivalent to enzyme-linked immunoabsorbent assay and 2-4 ng/ml for murine IgG detection. It is readily amenable to automation.