Analyte Detection

Abstract

The present disclosure provides methods and/or kits for detecting an analyte in a sample. Some embodiments provide a method for detecting a non-nucleic acid analyte in a sample using a solid substrate comprising a bound immobilisation agent and an antibody capture agent and a detectable agent, which can bind to the analyte. The antibody capture agent comprises, at a plurality of sites, a ligand for the immobilisation agent. A complex between the analyte, the antibody capture agent and a detectable agent is formed and immobilised on the solid substrate by binding between the immobilisation agent and the ligand. In some embodiments, the ligand and the immobilisation agent are a binding pair comprising a peptide tag and an anti-peptide tag antibody.

What is claimed is: 
     
         1 . A method for detecting at least one analyte, comprising:
 i) forming at least one analyte complex, the at least one analyte complex comprising the at least one analyte bound to at least one tagged antibody capture agent and separately bound to at least one detectable agent, a first tagged antibody capture agent of the at least one tagged antibody capture agent comprising a plurality of covalently conjugated peptide tags;   ii) binding at least one anti-peptide antibody that is immobilized on at least one bead to at least one of the plurality of covalently conjugated peptide tags, to form at least one bead-bound complex; and   iii) detecting the at least one bead-bound complex.   
     
     
         2 . The method of  claim 1 , wherein the at least one bead-bound complex comprises a luminescent compound. 
     
     
         3 . The method of  claim 1 , wherein the at least one anti-peptide antibody is covalently attached to the at least one bead. 
     
     
         4 . The method of  claim 1 , wherein the method comprises no washing step. 
     
     
         5 . The method of  claim 1 , wherein the at least one bead is a magnetic bead. 
     
     
         6 . The method of  claim 1 , wherein the at least one detectable agent comprises at least one luminescent compound. 
     
     
         7 . The method of  claim 6 , wherein the at least one luminescent compound comprises one or more of luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, oxalate ester, a catalytic protein, luciferin, luciferase, and/or aequorin. 
     
     
         8 . The method of  claim 1 , wherein the at least one analyte is provided in a cell lysate or derivative of a cell lysate. 
     
     
         9 . The method of  claim 1 , wherein the at least one analyte comprises a phosphoprotein. 
     
     
         10 . The method of  claim 1 , wherein the at least one of the plurality of covalently conjugated peptide tags comprises the amino acid sequence DYKDDDDK (SEQ ID NO: 1). 
     
     
         11 . The method of  claim 1 , wherein the at least one anti-peptide antibody comprises a monoclonal antibody. 
     
     
         12 . The method of  claim 1 , wherein the at least one detectable agent comprises a monoclonal antibody. 
     
     
         13 . The method of  claim 1 , wherein a first analyte of the at least one analyte is present in the sample at a concentration of less than 10 ng/ml. 
     
     
         14 . A method for detecting an analyte, comprising:
 i) forming an analyte complex, said analyte complex comprising the analyte bound to a tagged antibody capture agent and separately bound to a detectable agent, said tagged antibody capture agent comprising a plurality of covalently conjugated peptide tags;   ii) binding an anti-peptide antibody that is immobilized on a first bead to at least one of the plurality of covalently conjugated peptide tags; and   iii) detecting the analyte by detection of the detectable agent.   
     
     
         15 . The method of  claim 14 , wherein the tagged antibody capture agent is bound to a first epitope of the analyte and the detectable agent is bound to a second epitope of the analyte. 
     
     
         16 . The method of  claim 14 , wherein the detectable agent comprises a second bead. 
     
     
         17 . The method of  claim 16 , wherein the second bead comprises a quencher. 
     
     
         18 . The method of  claim 16 , wherein the second bead comprises a fluorophore. 
     
     
         19 . The method of  claim 16 , wherein the second bead comprises a FRET donor. 
     
     
         20 . The method of  claim 16 , wherein the second bead comprises a FRET acceptor.