A fluorescence immunoassay for soluble antigens employing flow cytometric detection

A “sandwich” fluorescence immunoassay is described which does not require the physical separation of bound from free label. Antibody coated microspheres, sample and fluorescent antibody are reacted together as in a conventional ‘sandwich’ immunoassay except that separation and washing steps are omitted. After the reaction is completed, the suspension is introduced directly into a flow cytometer equipped with a laser light source and both fluorescent and scattered light detection capabilities. By gating fluorescence light accumulation on scattered light pulses, particle associated fluorescence may be selectively measured. The system was evaluated in a model immunoassay for human immunoglobulin (hIgG), employing anti-hIgG coated microspheres (1–5 μm and 40–50 μm polyacrylamide beads and 30–40 μm dextran beads), fluorescein-labeled rabbit anti-hIgG and a Spectrum III flow cytometer. Sensitivities of 10 ng/ml and intra-assay precisions of 2–10% were achieved in a serum matrix. The approach potentially provides a general non-separation immunoassay format for quantitatively measuring both small and large molecular weight soluble antigens, as well as cell surface antigens.