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  Differential immunoassayRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Antigen-antibody Binding, Specific Binding Protein Assay Or Specific Ligand-receptor Binding Assay, Involving Avidin-biotin BindingThe Patent Description & Claims data below is from USPTO Patent Application 20050260689. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATIONS [0001] Priority under 35 U.S.C. .sctn.119(e) is claimed to Provisional Applications Ser. Nos. 60/227,536, filed Aug. 24, 2000, and 60/292,497, filed May 21, 2001, both of which are incorporated by reference herein in their entireties. BACKGROUND OF THE INVENTION [0002] Innumerable qualitative and quantitative tests are available for detecting the presence or level of particular substances in a sample. Sources of such samples range from industrial environments such as mines, wastewater processing, food quality, soil testing, among many others. In the medical field, tests for substances in bodily fluids are well known, and are aids to prognostication, diagnosis, and monitoring the progression and treatment of various conditions and diseases. In many cases, multiple tests are performed on a sample and a health care professional then makes a presumptive diagnosis based on the various levels of particular analytes in the sample, among other information gained, for example, from examining a patient. [0003] In certain circumstances, particular in the emergency room and ambulance call, time is of the essence in arriving at a diagnosis and initiating appropriate therapy to intervene in the morbidity and mortality of a rapidly deteriorating condition. One such example is diagnosis of a heart attack in an individual with chest pain or recent onset. Multiple diagnoses may be attributable to chest pain, yet diagnosis based on electrocardiogram or levels of cardiac markers released into the circulation are needed for a confirmatory diagnosis and initiation of a course of therapy, which would be unwise in a patient not having a heart attack. Thus, the need for rapid and accurate, early diagnostic tests is apparent for such emergency conditions. [0004] Although such early tests are available, even such tests are not without flaws. For example, diagnosis of a heat attack within six hours of the onset of chest pain is difficult to perform with a single test. While the cardiac marker troponin I has been recently adopted as a single and highly accurate indicator, it is not detectable until after about six hours, leaving a large window where early initiation of treatment would be highly desirable but dangerous without an accurate diagnosis. Another cardiac marker, myoglobin, is released into the circulation earlier than troponin I, but is not specific for cardiac tissue, as skeletal muscle damage also releases myoglobin into the circulation. Additional tests may be performed together with myoglobin to attempt to identify its origin, in order to improve the accuracy of an early diagnosis. [0005] The foregoing example of heart attack is merely one example of a myriad of diagnoses, which if to be carried out with a high degree of accuracy, need additional, corroborative tests. Although the combination of multiple assays performed simultaneously increases diagnostic precision, it is undesirable in that it also increases the complexity of the testing, the coordination of the timing of the separate test procedures and availability of the information, and the amount of information that must be processed manually or otherwise, often under emergency conditions. it is towards the simplification of multiple analyte diagnostic tests to provide a single readout reliably indicative of a particular diagnosis that the present invention is directed. BRIEF DESCRIPTION OF THE INVENTION [0006] In its broadest aspect, the present invention provides an assay useful to determine the relative levels at which different analytes are present in a sample. In the present assay, the extent of the readout is related to the level of the first analyte and that of the second analyte. Thus, in one embodiment, the readout provides either a ratio of the level of the first analyte to the second, or the difference in levels between the first analyte and the second analyte. [0007] The assay of the invention is useful for situations in which a ratio or difference between the levels of the first and second analytes is diagnostically useful, and a single readout that takes the two values into consideration in generating a single differential value can be as informative and directive of further action as would be obtaining the individual values and mentally evaluating or arithmetically calculating the difference or ratio, and then acting upon the result. The method of the invention simplifies decision making by internally integrating the results of at least two individual analyte levels. [0008] By way of non-limiting example, the first analyte and second analytes may be markers useful for determining the health status of an individual, wherein the ratio or difference among the markers is diagnostically informative. In a particular embodiment, elevated levels of the first analyte may be indicative of a life-threatening medical event only if the level of the second analyte is not elevated. In another embodiment, the second analyte also being elevated is indicative of an event. In a third and preferred embodiment, an analyte may originate from different bodily sources and the origin is diagnostically useful; the assay of the invention is useful for identifying the source of elevated levels of the first analyte. In this embodiment, the format of the assays of the invention takes advantage of the co-release of another analyte from the bodily source other than the intended source (referred to herein as a non-target-source marker) whose level is effectively subtracted from that of the total level of desired analyte to provide in a single test, a readout specific to the analyte source. [0009] In one broad aspect, the single assay for a preselected analyte is indicated as the level of the first analyte reduced proportionally by the level of a second analyte present in the sample. A reading is obtained only if the first analyte is present, and the detected level of the first analyte is reduced as the level of the second analyte increases. [0010] In the present assay, the relative presence or level of first and second analytes in a given sample is revealed by utilizing a labeling reagent for one of the two analytes that labels that analyte through a reaction that is inhibited by the other analyte. More particularly, in the present assay, the readout is dependent on binding, to one of the analytes selectively, of a labeling reagent complex the formation of which is inhibited by any second analyte present in the sample. Thus, labeling of the analyte targeted by the labeling reagent proceeds in the absence of the second analyte, and a reading is obtained. No reading is obtained when the first analyte is absent from the sample. When both analytes are present in the sample, the labeling reaction proceeds but in a manner that is competitively inhibited by the second analyte. Thus, the relative levels at which the first and second analytes are present in the sample is reflected by the extent to which the first analyte is labeled, and this is ultimately reflected in the readout obtained following performance of the assay. [0011] Thus, in one of its aspects, the present invention provides a method useful to assay a sample to detect the presence or relative levels therein of first and second analytes, the method comprising the step of bringing the sample into contact with a labeling reagent means adapted to form a labeling complex that binds to and thereby labels the first analyte, wherein formation of the labeling complex is inhibited by second analyte present in the sample. [0012] In a preferred aspect of the invention, the labeling reagent means comprises two components: a labeled binding partner for the second analyte, and a conjugate formed by coupling of a second analyte itself and a binding partner for the first analyte. When second analyte is absent, the first analyte is thus labeled by formation of complexes between the first analyte and the first analyte binding partner, and the second analyte and the labeled second analyte binding partner. When present in the sample, however, the second analyte becomes a competitor for binding to the labeled second analyte binding partner, and thereby inhibits binding of that labeled second analyte binding partner to the conjugate, thus reducing labeling of the first analyte. [0013] In a particular embodiment, a method is provided for identifying in a sample the presence or level of a first analyte above the level of a second analyte comprising the steps of [0014] (a) forming a reaction mixture by contacting the sample with reagent means for labeling the first analyte, the labeling reagent means comprising a mobile, labeled binding partner to the second analyte, and a conjugate between the second analyte and a binding partner to the first analyte; [0015] (b) contacting the reaction mixture with an immobilized binding partner to the first analyte; [0016] wherein the extent of formation of a complex comprising the mobile, labeled binding partner to the second analyte, the conjugate between the second analyte and the binding partner to the first analyte, the first analyte, and the immobilized binding partner to the first analyte, is indicative of the presence or level of the first analyte in the sample reduced by the level of the second analyte in the sample. Desirably, the reaction is staged by first bringing the sample into contact with the mobile, labeled binding partner to the second analyte to allow any second analyte in the specimen to become bound thereto, and then presenting the conjugate before finally contacting the resultant mixture with immobilized binding partner to the first analyte. In this way, the inhibitory effect of sample-borne second analyte is maximized, by allow it to react first with the labeled second analyte binding partner before allowing the conjugate to compete therewith for binding. [0017] By way of non-limiting example, the aforementioned binding partners may be antibodies. The label may be colloidal gold. The sample may be, by way of non-limiting example, a bodily fluid, wastewater, a foodstuff; preferably, it is a bodily fluid such as whole blood, serum, plasma, or urine. By way of example, the first analyte may be a cardiac marker, such as myoglobin, and the second analyte may be a different analyte co-released from a non-cardiac source along with the first analyte, such as carbonic anhydrase III which is released from damaged skeletal muscle along with myoglobin. For determining the level of myoglobin originating from the heart, the mobile, labeled binding partner to the second analyte may be a gold-labeled monoclonal anti-carbonic anhydrase III antibody, the conjugate between the second analyte and a binding partner to the first analyte may be a conjugate between carbonic anhydrase III and an anti-myoglobin monoclonal antibody, and the immobilized binding partner to the first analyte may be an anti-myoglobin monoclonal antibody. [0018] The conjugate between the second analyte and a binding partner to the first analyte may be a covalent conjugate between the members, such as is achievable using a homobifunctional or heterobifunctional cross-linking agent or carbodiimide, or it may comprise a single-chain polypeptide on which reside both the second analyte, or an epitope thereof, and a binding partner, or binding portion thereof, to the first analyte, such that each member retains its desired activities within the conjugate or single-chain polypeptide. For example, the conjugate between an antibody to myoglobin and carbonic anhydrase III may include a single-chain polypeptide comprising carbonic anhydrase III and the immunoglobulin heavy chain, which when assembled into the functioning antibody, provides binding sites for myoglobin and a carbonic anhydrase III portion to which the labeled anti-carbonic anhydrase III antibody may bind. The analyte portion of any of the conjugates herein may be the full-length analyte or a fragment bearing the epitope recognized by the corresponding binding partner. The foregoing example may be used to diagnose a heart attack by indicating an elevated level of myoglobin exists over that which may derived from a non-cardiac source. In this case, the level of cardiac and skeletal (i.e., total) myoglobin detected in the assay is reduced by the amount of carbonic anhydrase III present in the sample, the latter equivalent to the level of skeletal muscle-derived myoglobin. [0019] In a second embodiment, a homogeneous assay similar to that above is provided which employs slightly different reagents, but applies the same principles. In this embodiment, a further binding interface is incorporated into the labeling reaction. Particularly, the conjugate between the first analyte binding partner and the second analyte instead introduces a further biotin/streptavidin interaction, and the conjugate thus is represented by two reagents; one in which biotin is conjugated with either the first analyte antibody or the second analyte, and another in which streptavidin or a biotin-binding component thereof is conjugated with the other of the first analyte antibody or the second analyte. In an embodiment, the conjugate reagents are a first conjugate between first analyte antibody and biotin, and a second conjugate between streptavidin and the second analyte. [0020] Thus, a method is provided for identifying in a sample the presence or level of a first analyte above a second analyte, the method comprising conducting an assay following the steps of [0021] (a) forming a reaction mixture by contacting the sample with Continue reading... 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