Differential Immunoassay

Abstract

The invention provides assay methods and kits that in general measure the level of a first analyte in a sample reduced by the level of a second analyte present in the same sample. In one embodiment, where levels of a first analyte from a first source is desirably determined and first analyte in the sample released from a second source is accompanied by proportional co-release of a second analyte, the assay identifies the level of first analyte released only from the first source. For analytes within bodily fluids, the assay can differentiate between elevated levels of analyte specific to the particular physiological or pathological state and elevated levels not specific to the particular state, providing single tests with diagnostic utility.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]The present application is a divisional of U.S. patent application Ser. No. 10 / 673,781, filed Sep. 29, 2003; which is a divisional of U.S. patent application Ser. No. 09 / 938,270, filed Aug. 23, 2001, now issued as U.S. Pat. No. 6,673,562; which in turn claims the benefit of U.S. provisional application Ser. No. 60 / 292,497, filed May 21, 2001, and U.S. provisional application Ser. No. 60 / 227,536, filed Aug. 24, 2000 under 35 U.S.C. §119(e), the contents of which are incorporated herein by reference in their entirety.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,...

AI Technical Summary

Benefits of technology

[0008]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.

[0013]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.

[0017]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 allowing it to react first with the labeled second analyte binding partner before allowing the conjugate to compete therewith for binding.

[0031]In a preferred embodiment, the first or preselected analyte is an analyte originating from a target source whose level is desirably measured over the same analyte originating from a source other than the target source. A second analyte is a marker that is released from the non-target (other) source in proportion to the level of first analyte released from the non-target source. The assay of the invention subtracts or proportionally reduces, depending on binding partner affinities, from the total level of first analyte (from the target and non-target source) the level of the second analyte, which effectively subtracts the level of the first analyte derived from the non-target source from the readout value.

[0050]Of course, in the above methods, wherein two binding partners bind to the preselected analyte, each must be capable of recognizing a different binding site on the preselected analyte such that both binding partners can independently bind and permit the final labeled complex to form if the second analyte (marker) is not present at a level relatively greater than that of the first analyte. Moreover, the sensitivities and selectivities of the foregoing assays may be adjusted, for example, depending on the relative levels of the preselected analyte released from the target source, the level released from the non-target source, and the amount of co-release of the non-target-source marker relative to the release of the preselected analyte from the non-target source. The ratios of the components in the various reagents of the assays may be adjusted, and any reduced binding thereby compensated for in another reagent, as an example of the flexibility of the assay for various analytes.

Problems solved by technology

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.

Although such early tests are available, even such tests are not without flaws.

For example, diagnosis of a heart 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.

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.

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