Platelet immunoglobulin bead suspension and flow cytometry

#The present invention is directed to immunoassay methods and apparatus, and more particularly concerns an immunobead-flow cytometry method, apparatus, assay, device, system, kit, and the like for detecting and quantifying platelets, antigens, antibodies and the like.

#Typically, autoimmune testing for Systemic Lupus Erythematosus (SLE), Systemic Rheumatic Disease, rheumatoid arthritis, Sjogren's Syndrome, Progressive Systemic Sclerosis (PSS), Subacute Erythematosus, congenital complete heart block, neonatal complete heart block, neonatal lupus dermatitis, Polymyositis, Human Immunodeficiency Virus (HIV), Acquired Immunodeficiency Syndrome (AIDS), as well as other diseases has involved the use of extractable nuclear antigens (ENA) and immunological assays including hemagglutination, counter immunoelectrophoresis (CIE), immunodiffusion, Enzyme Linked Immunosorbent Assay (ELISA), and the like. For example, the Ro(SS-A) antigen having one major band at 60 kD by SDS gel electrophoresis (silver stain) has been purified through the use of immobilized human anti-Ro(SS-A) immunoglobulins. La(SS-B) antigen has two major bands, one at 40 kD and the other at 23 kD (a degradation product) by SDS gel electrophoresis (silver stain) and has been purified through the use of immobilized human anti-La(SS-B) immunoglobulin. Smith (Sm) antigen has two major bands in the 10 and 14 kD region by SDS gel electrophoresis (silver stain) has been purified through the use of immobilized human anti-Sm (Smith) immunoglobulins. Smith (Sm/RNP) complex antigen has five bands, one each at 70, 40, 24, 12 and 10 kD, respectively, by SDS gel electrophoresis (silver stain) and has been purified through the use of immobilized human anti-RNP immunoglobulin. Scl-70 antigen has one major band at 68 kD by SDS gel electrophoresis (silver stain) and has been purified through the use of immobilized human anti-Scl-70 immunoglobulins. Jo-1 antigen has one major band at 50 kD by SDS gel electrophoresis (silver stain) and has been purified through the use of immobilized human anti-Jo-1 immunoglobulins. dsDNA double-stranded (native) deoxyribonucleic acid, ssDNA single-stranded DNA, whole Histones, Histone subclasses (distinct molecular fractions) tissue extracts, human antibodies, animal tissue acetone powders, sera and immunoglobulin fractions, second antibodies, anti-whole sera, whole antisera to animal proteins and to human proteins have been used in enzyme immunoassay (ELISA) for detecting or evaluating systemic rheumatic diseases. Thus, other antigens may be added to this invention as combined with or separate from the existing 5-bead immunoasay. Immunovision, Inc. of Springdale, Ark. has developed a number of enzyme-linked immunoassays, ouchterlony immunoprecipitation assays, and Western blot assays for detecting human antibody to particular nuclear antigens (ENA).

#The presence of human autoantibodies to nuclear antigens, for examples antibodies against RNP/Sm, Sm, SS-A, SS-B, dsDNA and Scl-70 antigens, in combination with IFA, have been diagnostic when evaluating patients with Systemic Lupus Erythematosus (SLE). Positivity may indicate more progressive disease states or simply rheumatoid arthritis. Currently, enzyme linked immunosorbent assay (ELISA) has been the assay of choice to detect these antibodies. Antibodies to Smith (Sm) antigen have been shown to occur in twenty-five to thirty percent of patients with Systemic Lupus Erythematosus. Antibodies to Sm are less commonly found in patients with other rheumatic diseases. Antibodies to ribosomal nuclear protein (nRNP) have been found in patients with Systemic Lupus Erythematosus. They are also found in sera from patients with rheumatoid arthritis, Sjogren's Syndrome (SS), Progressive Systemic Sclerosis (PSS), and Mixed Connective Tissue Disease (MCTD). Twenty to thirty percent of the patients with antibodies to Scl-70 antigen have Progressive Systemic Sclerosis. Antibodies to Scl-70 are rarely found in patients with other systemic rheumatic diseases. Antibodies to Ro (SS-A) antigen are found in half of Systemic Lupus Erythematosus patients, most patients with Sjogren's Syndrome or Subacute Lupus Erythematosus and nearly all mothers of infants with congenital complete heart block or Neonatal Lupus Dermatitis. Antibodies to the La (SS-B) antigen usually occur in twenty to thirty percent of Sjogren's Syndrome patients and with five to ten percent of Systemic Lupus Erythematosus patients. Antibodies to Jo-1 antigen are usually found in patients with polymyositis. Antibodies to Ribosomal P antigens are found to occur in five to ten percent of Systemic Lupus Erythematosus patients and ninety percent of those patients will demonstrate signs of lupus psychosis. Antibodies to mitochondrial antigens are found in all primary biliary cirrhosis patients. Antibodies to histone antigens (H1, H2A, H2B, H3, H4) are found in ninety-five to one-hundred percent of drug-induced Lupus Erythematosus, fifteen to twenty percent rheumatoid arthritis, and thirty percent of all patients with Systemic Lupus Erythematosus. Antibodies to cytoplasmic components of neutrophil granulocytes are present in the serum of patients with acute Wegener's granulomatosis and microscopic polyarteritis. Myeloperoxidase and proteinase 3 are the two major antigens present.

#FIG. 1 is a schematic representation of an exemplary immunological structure of the bead-antigen-antibody indicator complex,

#FIG. 2 is a schematic illustration of the flow cytometer histogram of forward angle light scatter (size) versus fluorescence on a positive control sample in a multiple bead system,

#FIG. 3 is a schematic representation of a flow cytometer histogram of a negative control in a multiple bead system,

#In accordance with an exemplary embodiment of the present invention, antigen coated latex surfaces, anti-nuclear antibodies, fluorescenated antibodies against such anti-nuclear antibodies, and flow cytometry are combined to provide multiparameter devices for the detection of a plurality of antigens in a single tube.

#One basic principle of the present invention is to conjugate antigens or antibodies to the exterior of latex microspheres (beads) of different sizes. The coated microspheres are used to detect the appropriate specific antibodies or antigens simultaneously in one tube, with or without washing between sample incubation and indicator antibody phases. The ability to detect multiple analytes in one reaction tube eliminates the variability often seen in results arising from separate assays. Procedurally, latex beads are coated with specific antigens or antibodies. These beads vary in size and may also contain fluorescent dyes e.g. FITC, PE, etc. One or more of these precoated beads are then incubated with the sample (serum, body fluid) solution. If an antibody-antigen complex has been formed, a 2° indicator fluorochrome labelled antibody will bind to the appropriate bead (FIG. 1).

#A no wash procedure is optional and has been utilized in most serum assays. However, as control substrates, washing may be necessary. The control beads are centrifuged, washed, and analyzed using forward angle light scatter to discriminate the different sized beads, each bound to a different antigen or antibody, and analyzed to detect fluorescence with a flow cytometer. The solution containing beads is passed through a series of tubes until it reaches the optical quartz cell of the flow cytometer. Because of the laminar flow of sheath fluid, single particle analysis is achieved. The signal is converted from analog to a digital display representing the size of the spheres and fluorescence of each (FIG. 2). Controls are used to adjust for the fluorescence background created by electronic and particle noise (FIG. 3). A forward scatter (size) adjustment of the multiple sized bead antigen or antibody complexes is necessary in order to semi-quantitate or quantitate the relative concentration of antigen or antibody on the bead surface through single screens visual distribution As seen in FIG. 3, a fluorescent threshold (x-axis) is established below which fluorescence values are considered negative. Upon addition of a “positive” sample, (containing appropriate antibody or antigen) the reaction between the fluorochrome labelled indicator antibody and antigen or antibody bead complex, amplifies the fluorescence signals detected by the flow cytometer (FIG. 2). Thus, the definition of “positivity” in this system is relative to the negative control (background) and can easily be interpreted.