A flow cytometric immunoassay for β2-microglobulin in whole blood

Abstract Flow cytometers can discriminate a single particle type in an unwashed whole blood sample. Utilizing this capability, we devised a homogeneous bead-immobilized sandwich immunoassay for soluble β 2 M ( β 2 -microglobulin) in whole blood, utilizing an antibody that discriminates soluble from cellular β 2 M. A 4 μ m bead was chosen that fluoresces only in a FACScan™ flow cytometer's FL3 channel, thus allowing triggering on this bead to the exclusion of the many blood cell events. The bead was adsorbed with a capture antibody (clone A7801) which binds only to soluble and not to cellular β 2 M. This antibody appears to recognize an epitope on β 2 M which interfaces to the heavy chain of cellular Class I MHC molecules. The signal antibody (PE conjugate of clone L376, emitting in the FL2 channel) binds to both soluble and cellular β 2 M (present in roughly equal amounts in normal blood). The various parameters required for a flow cytometric immunoassay were optimized. The 4 μ m sized bead was adequately large to give a near full scale signal at saturation. The relative amounts of signal antibody and capture beads were balanced to give a low blank, minimal `hook effect', and reasonable event rate on the flow cytometer. The amount of blood added was selected to give a signal near the bottom of the immunassay range for normals with the higher range available for clinical samples. The assay requires no washing, minimizes blood handling, and has a working range (2.5 decades) that is compatible with the biological range of β 2 M concentrations with a single blood dilution.