Abstract: Immunoassay methods and apparatus are provided which utilize flow cytometry, coated latex microspheres, and fluorochrome labeled antibodies, to simultaneously detect the presence and amount of several antigens or antibodies in a sample. The use of microspheres, beads, or other particles as solid supports for antigen-antibody reactions in order to detect antigens or antibodies in serum and other body fluids is particularly attractive when linked to flow cytometry. Flow cytometers have the capacity to detect particle size differences and are highly sensitive fluorescence detectors. It is practical to use beads of several different sizes, colors or shapes, each bead coated with a different protein or antibody, for the simultaneous detection of multiple analytes in a sample.

Abstract: A method of making a no wash bead based assay comprises preparing a first reagent comprising a buffer, and preparing a second reagent comprising a protein. Beads of preselected size and having a coefficient of variation less than 5% are prepared, including washing the beads in the buffer to form a bead-buffer matrix and reducing the surfactancy of the beads to an effective amount. Thereafter, an antigen for detecting the presence of a target species is added to the bead-buffer matrix such that the antigen attaches to the beads to form a bead-antigen mixture. The surfactancy of the beads facilitates attachment of the antigen thereto. Buffer is added to the bead-antigen mixture and thereafter the mixture is incubated. The second reagent is added to the bead-antigen mixture to reduce or eliminate non-specific binding sites.

Abstract: Immunoassay methods and apparatus are provided which utilize flow cytometry, coated latex microspheres, and labelled antibodies, to simultaneously detect the presence and amount of several antigens or antibodies in a sample. Microspheres can be sized by forward angle light scatter (FALS) or electronic volume. By combining FALS and fluorescence, it is practical to use beads of several different sizes, colors or shapes, each bead coated with a different protein or antibody, for the simultaneous detection of multiple analytes in a sample. Available auto-sampling systems make it even more appealing in this regard. In accordance with one embodiment, highly purified RNP. Sm, SS-A, SS-B and Scl-70 antigens are bound to 4, 5, 6, 7 and 10 .mu.m latex beads, respectively and stabilized for extended shelflife. Diluted patient serum is placed into test tubes containing a mixture of the five antigen coated beads and incubated. If an antibody is present for a specific antigen, it will bind to that specific bead.