A flow cytometric immunoassay for beta2-microglobulin in whole blood

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 beta2M (beta2-microglobulin) in whole blood, utilizing an antibody that discriminates soluble from cellular beta2M. A 4 microm 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 beta2M. This antibody appears to recognize an epitope on beta2M 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 beta2M (present in roughly equal amounts in normal blood). The various parameters required for a flow cytometric immunoassay were optimized. The 4 microm 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 beta2M concentrations with a single blood dilution.