Use of antibody-surrogate antigen systems for detection of analytes

Abstract

Compositions, methods and kits for labeling proteins and uses in reporter systems for detecting, quantifying and/or characterizing analytes.

1 . A labeling molecule useful for specifically labeling a protein comprising: 
 (a) a target moiety;    (b) a label moiety;    (c) an activatable reactive moiety; and    (d) a cleavable tether linking the target moiety to the molecule;    wherein the target moiety, label moiety, and reactive moiety are connected to one another such that the target moiety can bind the protein and the reactive moiety can crosslink the labeling molecule to the protein.    
     
     
         2 . The labeling molecule of  claim 1  in which the protein is an antibody and the target moiety is an antigen comprising an epitope recognized by said antibody.  
     
     
         3 . The labeling molecule of  claim 2  which has the structure:  
         TM-(L) N -CT-(L) N -LM-(L) N -RM  
       wherein: 
 TM represents the target moiety;  
 CT represents the cleavable tether;  
 LM represents the label moiety;  
 RM represents the reactive moiety;  
 L represents an optional linker moiety; and  
 n is an integer from 0 to 5.  
 
     
     
         4 . The labeling molecule of  claim 3  in which the label moiety comprises a fluorescent moiety.  
     
     
         5 - 8 . (canceled)  
     
     
         9 . The labeling molecule of  claim 3  in which the reactive moiety is specifically activatable.  
     
     
         10 - 11 . (canceled)  
     
     
         12 . The labeling molecule of  claim 3  in which the reactive moiety is linked to the label moiety via a linker moiety.  
     
     
         13 . (canceled)  
     
     
         14 . The labeling molecule of  claim 3  in which the cleavable tether comprises a cleavage moiety linked to one or more linker moieties, said cleavage moiety comprising a linkage that can be cleaved by a chemical cleaving agent.  
     
     
         15 . (canceled)  
     
     
         16 . The labeling molecule of  claim 14  in which the cleavable tether has the structure:  
         (TE) N CM(TE) N    
       wherein: 
 CM represents the cleavage moiety;  
 TE represents an optional tether element; and  
 n is an integer from 0 to 5.  
 
     
     
         17 - 23 . (canceled)  
     
     
         24 . The labeling molecule of  claim 16  in which the cleavable moiety comprises the structure:  
       
         
           
           
               
               
           
         
       
       wherein: 
 R 1  and R 2  are hydrocarbons;  
 TE represents an optional tether element; and  
 n is an integer from 0 to 5.  
 
     
     
         25 . (canceled)  
     
     
         26 . A protein, capable of binding an analyte, that is specifically labeled in close proximity to the analyte binding site with one or more label moieties, said protein being substantially unlabeled at other positions.  
     
     
         27 . The protein of  claim 26  which is an antibody capable of binding an antigen comprising an epitope.  
     
     
         28 . The antibody of  claim 27  in which the protein is labeled with a fluorescent moiety.  
     
     
         29 - 32 . (canceled)  
     
     
         33 . The antibody of  claim 27  which is bound to a surrogate antigen comprising a quenching moiety.  
     
     
         34 . (canceled)  
     
     
         35 . The antibody of  claim 33  which is encapsulated.  
     
     
         36 . The antibody of  claim 33  which is encapsulated in a semi-permeable nanocapsule.  
     
     
         37 . The antibody of  claim 36  in which the nanocapsule is permeable to an analyte molecule and is impermeable to said antibody.  
     
     
         38 . (canceled)  
     
     
         39 . The antibody of  claim 36  in which the nanocapsule comprises a cross-linked water soluble vinyl polymer or copolymer.  
     
     
         40 . The antibody of  claim 36  in which the nanocapsule further comprises a cell membrane targeting moiety.  
     
     
         41 - 43 . (canceled)  
     
     
         44 . A method of making a labeled protein of interest comprising: 
 (a) contacting the protein with a labeling molecule comprising a target moiety, a label moiety, a reactive moiety, and a cleavable tether connected to the target moiety, wherein the target moiety, label moiety, and reactive moiety are connected to one another such that the target moiety can bind the protein, and the reactive moiety can link the molecule to the protein, under conditions such that the protein binds the target moiety;    (b) activating the reactive moiety so as to form a covalent linkage between the reactive moiety and the protein;    (c) cleaving the cleavable tether; and    (d) removing the cleaved target moiety from the labeled protein complex.    
     
     
         45 - 50 . (canceled)  
     
     
         51 . A method of detecting the presence or absence of a target analyte molecule in a sample, comprising contacting the sample with a surrogate analyte-protein complex, said complex comprising a reporter system capable of generating a detectable fluorescent signal upon dissociation of the complex that is at least 3-fold greater than the background signal, wherein the presence of the detectable fluorescent signal indicates that the target analyte is present in the sample.  
     
     
         51 - 62 . (canceled)