Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-02-23
2003-07-22
Saunders, David (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007950, C436S506000, C436S508000, C436S515000, C436S518000
Reexamination Certificate
active
06596501
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the field of diagnosing autoimmune disease. More particularly, it concerns the use of specific antigens to detect the presence of SR protein-specific antibodies in an individual suspected of having autoimmune disease, wherein the presence of such antibodies is indicative of said individual suffering from autoimmune disease.
2. Description of Related Art
Autoimmune diseases are known to afflict a significant portion of the population. Some of the more common autoimmune diseases include, among others, scleroderma, systemic lupus erythrematosus and mixed connective tissue disease. These diseases are characterized by the presence of a multitude of autoreactive antibodies that arise spontaneously. To date, high levels of circulating autoantibodies to DNA are the best evidence of these maladies.
In the blood of systemic lupus erythematosus (SLE) patients, there typically are found antibodies directed against one or more components of cell nuclei. Certain manifestations of SLE seem to be associated with the presence of different anti-nuclear antibodies and genetic markers, raising the possibility that SLE may be a family of diseases (Mills, 1994). The more common type of lupus erythematosus, discoid lupus erythematosus (DLE), affects exposed areas of the skin. The more serious and fatal form, systemic lupus erythematosus (SLE), affects many systems of the body, including the joints and the kidneys.
SLE is highly variable in its clinical presentation and affects individuals with an intensity that varies over time. Among the organs targeted in this disease are skin, joints kidneys, nervous system serosal surfaces and blood. These manifestations do not appear synchronously over time. Months or years can pass before a definitive diagnosis of a multisystem disease is ascertained.
Regardless of the clinical manifestations of the disease, patients with lupus almost invariably express anti-nuclear antibodies (Mohan, et al., 1993; Mills, 1994; Swaak, et al., 1986, ter Borg, et al., 1990). Such antibodies also are produced in most other rheumatic diseases (Tan at al., 1989), are produced transiently in viral infections and are present, usually in low titers, in about two percent of the normal population. Of the many anti-nuclear antibodies produced, two are considered diagnostic for SLE, namely, anti-double stranded DNA antibodies (anti-dsDNA, Casals, et al., 1964; Tan et al., 1966) and antibodies to Sm (anti-Sm; Reichlin, 1994). Anti-dsDNA antibodies bind sites on the helical backbone of the native DNA. The anti-Sm bind to proteins on an RNA-protein complex termed snRNP (small nuclear ribonucleoprotein complex; Tan, 1989). Further, in addition to these two antibodies, patients with SLE produce a variety of other autoantibodies which, although not disease specific, are characteristic of the disease.
Although anti-dsDNA and anti-Sm are disease-specific, they do not occur in all patients. Thus, despite the presence of such antibodies, a clear diagnostic assay for individuals with SLE is not available due to the heterogeneous nature of SLE and other autoimmune diseases, requiring the diagnosis to be based on an array of different criteria. Indeed, in order to be classified as lupus, an individual must show at least 4 out of 14 criteria selected from such wide ranging characteristics as malar rash, discoid rash, photosensitivity, oral ulceration, arthritis, serositis, renal defects, neurological disorder (seizures, psychoses), hematological disorder (hemolytic anemia, leukopenia, lymphopenia, thrombocytopenia), immunological disorders (positive LE cell preparation, anti-DNA, anti-Sm, false-positive for syphilis) and the presence of anti-nuclear antibody. These characteristics are not exclusive to lupus but may manifest in other connective tissue disease (Tan, 1982). Furthermore, these criteria are limited as definitive manifestations of lupus. For example, those characterized by low serum complement and/or vasiculitis are not characterizable by these criteria because they lack diagnostic specificity.
Autoimmune diseases typically cause a great deal of discomfort and pain in the patient. Clearly, there is a need for a rapid, distinctive and definitive assay that will be diagnostic for SLE and other autoimmune diseases. This rapid diagnosis would aid the clinician in properly prescribing an effective therapeutic regimen to alleviate the pain and symptoms associated with the disease.
SUMMARY OF THE INVENTION
Thus, in order to facilitate a diagnosis, the present invention provides a method of diagnosing an autoimmune disease in a mammal comprising the steps of obtaining an antibody-containing sample; contacting the sample with a composition comprising an SR antigen; and detecting the presence of an SR antigen/anti-SR antibody complex; wherein the presence of an SR antigen/anti-SR antibody complex is diagnostic for an autoimmune disease.
In particularly preferred embodiments, the autoimmune disease is a systemic autoimmune disease. In more particular embodiments, the systemic autoimmune disease may be selected from the group consisting of systemic lupus erythematosus (SLE), progressive systemic scleroderma, mixed connective tissue disease and antiphospholipid syndrome.
In preferred embodiments, the sample tested may be blood, plasma, serum or any other tissue sample employed in the diagnostic assays. In certain embodiments, the determining comprises the use of a technique selected from the group consisting of ELISA, RIA, immunoprecipitation and Western blotting. In those preferred embodiments, in which ELISA is used the ELISA may be a sandwich ELISA.
In preferred embodiments, the sandwich ELISA comprises the steps of providing a preparation comprising an SR antigen bound to a support; contacting the preparation with the sample whereby an SR antigen/anti-SR antibody complex is formed; and contacting the complex with a detection agent.
It is contemplated that the detection agent may be an anti-Fe antibody that binds the anti-SR antibody. In particularly preferred embodiments, the antibody is labeled with a label selected from the group consisting of a radiolabel, an enzyme, biotin, a dye, a fluorescent tag label, a hapten and a luminescent label. In certain embodiments, the fluorescent tag may be selected from the group consisting of fluorescein, rhodamine, luciferase and green fluorescent protein. In other embodiments, the dye may be selected from the group consisting of phycoerythrin, phycocyanin, allophycocyanin, texas red and o-phthaldehyde. The enzyme may be alkaline phosphatase, or horseradish peroxidase. In defined aspects the support may be any solid support that is routinely used in the art, for example a microtiter plate, a polystyrene bead, test tube or dipstick. In particular embodiments, the SR antigen may be bound to the solid support using an anti-SR antibody.
Also contemplated herein is a kit comprising an SR protein preparation, and a suitable container means therefor. The kit may further comprise a first antibody preparation that binds to an anti-SR antibody and a suitable container means therefor. In alternative and equally preferred embodiments, the kit may further comprise a second antibody preparation that binds to an antigenic determinant on an SR antigen, wherein the second antibody composition is immunoreactive against an epitope distinct from the epitope for the anti-SR antibody composition, and a suitable container means therefor. In certain detailed aspects, the second antibody preparation comprises a detectable label. In other aspects, the SR protein preparation is attached to a support. In particular embodiments, the support is a microtiter plate, polystyrene bead, test tube or dipstick. In particularly preferred embodiments, the anti-SR antibody-binding antibody preparation comprises a detectable label. In defined embodiments, the detectable label is a radiolabel, an enzyme, biotin, a dye, a fluorescent tag label, a hapten and a luminescent label. The enzyme may be alkaline pho
Fred Hutchinson Cancer Research Center
Saunders David
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