Differential Immunoassay
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.
1 - 48 . (canceled)
49 . A method of detecting the presence of a first analyte in a sample, said method comprising:
contacting the sample with a conjugate comprising an epitope of the second analyte and a binding portion that recognizes a first epitope of the first analyte to form a labeled complex comprising the first analyte and the conjugate; and
detecting the presence of the labeled complex, wherein when a second analyte is present in the sample, the formation of the labeled complex is inhibited and wherein the presence of the labeled complex indicates the presence of the first analyte in the sample.
50 . The method of claim 49 , wherein said contacting comprises:
contacting the sample with
a) the conjugate, and
b) a mobile, labeled first binding partner that recognizes an epitope of the second analyte to form a reaction mixture; and
contacting the reaction mixture with an immobilized second binding partner that recognizes a second epitope of the first analyte to form a labeled complex,
wherein said labeled complex comprises the immobilized third binding partner, the first analyte, the conjugate, and the mobile, labeled second binding partner, and wherein said first analyte and second analyte are distinct from one another, said binding portion does not recognize the second analyte, and said first binding partner does not recognize the first analyte.
51 . The method of claim 50 , wherein the conjugate comprises a full-length second analyte.
52 . The method of claim 50 , wherein the binding portion and the first and second binding partners are antibodies.
53 . The method of claim 50 , wherein the first epitope and second epitope are distinct binding sites on the first analyte.
54 . The method of claim 50 , wherein the epitope of the second analyte and the binding portion are covalently linked.
55 . The method of claim 50 , wherein the conjugate is a single-chain polypeptide that comprises the epitope of the second analyte and the binding portion in operable form.
56 . The method of claim 55 , wherein the conjugate comprises the amino acid sequence of SEQ ID NO: 1.
57 . The method of claim 50 , wherein the conjugate further comprises a binding interface between the epitope of the second analyte and the binding portion, said binding interface comprising biotin and streptavidin respectively linked to:
the epitope of the second analyte and the binding portion, or
the binding portion and the epitope of the second analyte.
58 . The method of claim 50 , wherein the first analyte is a cardiac analyte.
59 . The method of claim 58 , wherein the cardiac analyte is myoglobin and the second analyte is released from a non-cardiac source.
60 . The method of claim 59 , wherein the second analyte is carbonic anhydrase III
61 . The method of claim 50 , comprising contacting the sample first with the mobile, labeled first binding partner then the conjugate.
62 . A method of diagnosing a condition in a subject, said method comprising:
contacting a sample from the subject with a conjugate comprising an epitope of a second analyte and a binding portion that recognizes a first epitope of a first analyte to form a labeled complex comprising the first analyte and the conjugate; and
detecting the presence of the labeled complex, wherein when the second analyte is present in the sample, the formation of the labeled complex is inhibited and wherein the presence of the labeled complex is indicative of the condition in the subject.
63 . The method of claim 62 , wherein the condition being diagnosed is a heart attack, the first analyte is myoglobin, and the second analyte is carbonic anhydrase III.
64 . The method of claim 62 , wherein the condition being diagnosed is a heart attack, the first analyte is fatty acid binding protein, and the second analyte is carbonic anhydrase III.
65 . The method of claim 62 , wherein the condition being diagnosed is risk for atherosclerotic disease, and the first analyte is total cholesterol and the second analyte is high-density lipoprotein.
66 . A method of detecting analyte from a first source, said method comprising:
contacting a sample with a conjugate comprising an epitope of a marker from a second source and a binding portion that recognizes a first epitope of the analyte to form a labeled complex comprising the analyte and the conjugate; and
detecting the presence of the labeled complex, wherein the first source and second source are distinct from one another, and when the marker is present in the sample, the formation of the labeled complex is inhibited, and wherein the presence of the labeled complex is indicative of the presence of analyte from the first source.
67 . The method of claim 66 , wherein the marker is selected to be a biomolecule that is released from the second source in proportion to the level of analyte released from the second source.
68 . The method of claim 66 , wherein the analyte is selected to be a biomolecule that can originate from the first source and the second source.
69 . The method of claim 66 , wherein the first source is cardiac tissue and the second source is muscle tissue.