Particle concentration fluorescence immunoassay: a new immunoassay technique for quantification of human...

A new fluorescence immunoassay technique, particle concentration fluorescence immunoassay (PCFIA), has been developed for quantifying the human immunoglobulins (IgA, IgM, and IgG). In these "two-site sandwich assays," the capture antibody is immobilized on small polystyrene spheres and the tracer is fluorescein-labeled antibody. Polystyrene particles less than 1 microm in diameter make up the solid phase, to which goat anti-human antibody for each respective assay is attached. Serum specimens are diluted (5000-fold for IgA or IgM, 20 000-fold for IgG) placed on the 96-well Pandex assay plate; and mixed with the solid phase and tracer (fluorescein-labeled goat anti-human IgA, IgM, or IgG), which are added automatically by the Pandex Screen Machine. This instrument incubates the reaction mixture for 17 min at ambient temperature, separates the bound and free label by filtration, washes the solid phase, and determines the total particle-bound fluorescence by front-surface fluorimetry or epifluorescence, calculates results, and generates detailed reports. Ninety-six specimens may be analyzed in 29 min or 960 specimens in 136 min. Results by PCFIA for IgA, IgM, and IgG in serum correlated well with those by rate nephelometry.