Multiplexed particle-based flow cytometric assays.

Several methods have been developed to quantify soluble analytes in biological fluids and tissue culture samples, including bioassays, ELISA, RPA and PCR. However, each of these techniques possesses one or more significant limitations; ELISA will only measure one analyte as a time; PCR does not detect native protein. The recent development of particle-based flow cytometric assays has raised hopes that many of these limitations can be overcome. The technology utilizes microspheres as the solid support for a conventional immunoassay, affinity assay or DNA hybridization assay which are subsequently analyzed on a flow cytometer. Several multiplexed bead systems are currently marketed by different vendors. We have used the Luminex FlowMetrix system which consists of 64 different bead sets manufactured with uniform, distinct proportions of red and orange fluorescent dyes (detected by FL2/FL3 on a FACScan). Each bead set forms the basis of an individual assay using a green fluorescent reporter dye (FL1). This system facilitates the development of multiplexed assays that simultaneously measure many different analytes in a small sample volume. They can also be developed into rapid, 'no wash' assays that can be completed in <2 h. This review traces the historical association between microspheres and flow cytometry, the development and use of particle-based flow cytometric assays, how they compare with current assays and potential future developments of this very exciting technology.