Immunometric assay by flow cytometry using mixtures of two particle types of different affinity

An improved dynamic range in a particle based flow cytometric immunoassay for carcinoembryonic antigen (CEA) was obtained using a binary mixture of two distinguishable particle types, namely particles of 7 and 10 microns diameter that were distinguishable by their light scattering characteristics in the flow cytometer. The two particle types were coated with antibody of the same specificity but different affinity. The association constants were 3.2 x 10(10) and 3.3 x 10(9) for the antibodies on the 7 and 10 micron particles, respectively. A dilution series of CEA samples was incubated with aliquots of the particle mixture and secondary biotin-streptavidin-phycoerythrin-conjugated antibody directed against a different epitope on the CEA molecule. The fluorescence intensity of the two particle types was measured flow cytometrically, and a double standard curve plotted from the mean logarithmic fluorescence values. The precision profile derived from the standard curve demonstrated that an increase in the dynamic range of about 50% (from 2 to 3 log) was obtained by using a mixture of high and low affinity particles, compared to using the high affinity particles alone.