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 . The labeling molecule of  claim 4  in which the fluorescent moiety comprises a xanthene dye. 
     
     
         6 . The labeling molecule of  claim 5  in which the xanthene dye is selected from a fluorescein dye and a rhodamine dye. 
     
     
         7 . The labeling molecule of  claim 3  in which the label moiety comprises a quenching moiety. 
     
     
         8 . The labeling molecule of  claim 7  in which the quenching moiety is capable of quenching the fluorescence of a fluorescent moiety. 
     
     
         9 . The labeling molecule of  claim 3  in which the reactive moiety is specifically activatable. 
     
     
         10 . The labeling molecule of  claim 9  in which the reactive moiety is photo-activatable. 
     
     
         11 . The labeling molecule of  claim 10  which the reactive moiety comprises a benzophenone. 
     
     
         12 . The labeling molecule of  claim 3  in which the reactive moiety is linked to the label moiety via a linker moiety. 
     
     
         13 . The labeling molecule of  claim 12  in which the linker moiety comprises one or more nitrogen atoms. 
     
     
         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 . The labeling molecule of  claim 14  in which the chemical cleaving agent is fluoride. 
     
     
         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 . The labeling molecule of  claim 16  in which tether elements are the same. 
     
     
         18 . The labeling molecule of  claim 16  in which tether elements are different. 
     
     
         19 . The labeling molecule of  claim 16  in which the tether elements comprise a substituted or unsubstituted, saturated or unsaturated hydrocarbon, having from 2 to 25 carbon atoms. 
     
     
         20 . The labeling molecule of  claim 16  in which the tether elements comprise a substituted or unsubstituted, saturated or unsaturated hydrocarbon, having from 2 to 10 carbon atoms. 
     
     
         21 . The labeling molecule of  claim 19  in which the tether elements are a linear, branched or cyclic, saturated or unsaturated alkyl. 
     
     
         22 . The labeling molecule of  claim 21  in which the tether elements is fully saturated n-alkyl. 
     
     
         23 . The labeling molecule of  claim 19  further comprising a substituted or unsubstituted heteroatom. 
     
     
         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 . The labeling molecule of  claim 1  in which the label moiety, reactive moiety, and target moiety, are linked to one another via a multivalent linker. 
     
     
         26 .- 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 . A method of  claim 44  in which the protein is an antibody and the target moiety is an antigen comprising an epitope that recognizes said antibody. 
     
     
         46 . The method of  claim 44  which further comprises isolating the labeled antibody from a cleavage product comprising the target moiety. 
     
     
         47 . The method of  claim 44  in which the label moiety comprises a fluorescent moiety. 
     
     
         48 . The method of  claim 47  further comprising contacting the antibody with a surrogate antigen comprising a quenching moiety. 
     
     
         49 . The method of  claim 44  in which the label moiety comprises a quenching moiety. 
     
     
         50 . The method of  claim 49  further comprising contacting the antibody with a surrogate antigen comprising a fluorescent moiety. 
     
     
         51 .- 62 . (canceled)