Sets of generalized target-binding e-tag probes

Abstract

Probe sets for the multiplexed detection of the binding of, or interaction between, one or more ligands and target antiligands are provided. Detection involves the release of identifying tags as a consequence of target recognition. The probe sets include electrophoretic tag probes or e-tag probes, comprising a detection region and a mobility-defining region called the mobility modifier, both linked to a target-binding moiety. Target antiligands are contacted with a set of e-tag probes and the contacted antiligands are treated with a selected cleaving agent resulting in a mixture of e-tag reporters and uncleaved and/or partially cleaved e-tag probes. The mixture is exposed to a capture agent effective to bind to uncleaved or partially cleaved e-tag probes, followed by electrophoretic separation. In a multiplexed assay, different released e-tag reporters may be separated and detected providing for target identification.

1 - 20 . (Canceled)  
     
     
         21 . A probe set for detecting the presence or absence of one or more target compounds, the probe set comprising a plurality of electrophoretic probes selected from the group defined by the formula: 
       [(D, M)-L] k -T 
       wherein: 
 T is a target-binding moiety specific for a target compound;  
 k is an integer in the range of from 1 to 20;  
 L is a cleavable linkage;  
 D is a detection group;  
 M is a mobility modifier consisting of from 1 to 500 atoms selected from the group consisting of carbon, hydrogen, oxygen, sulfur, nitrogen, phosphorus, and boron; and wherein, upon cleavage of L an eTag reporter comprising a detection group, D, and a mobility modifier, M, is produced with a distinct charge/mass ratio so that eTag reporters from different electrophoretic probes form distinct peaks upon electrophoretic separation.  
 
     
     
         22 . (New) The probe set of  claim 21  wherein said plurality is in the range of from 5 to 100, and wherein M is a mobility modifier consisting of from 1 to 300 atoms selected from the group consisting of carbon, hydrogen, oxygen, phosphorus, nitrogen, sulfur, and boron.  
     
     
         23 . The probe set of  claim 22  wherein said cleavable linkage is cleavable by oxidation and is selected from the group consisting of olefins, thioethers, sulfoxides, and selenium analogs of thioethers or sulfoxides.  
     
     
         24 . The probe set of  claim 23  wherein said cleavable linkage is cleavable by singlet oxygen.  
     
     
         25 . The probe set of  claim 21  wherein said cleavable linkage is a silyl group that is cleaved with fluoride, oxidation, acid, bromine, or chlorine; an o-nitrobenzyl group that is cleaved with light; a catechol that is cleaved with cerium salts; an olefin that is cleaved with ozone, permanganate, or osmium tetroxide; a sulfide that is cleaved with singlet oxygen or enzyme catalyzed oxidative cleavage with hydrogen peroxide; a furan that is cleaved with oxygen or bromine in methanol; a tertiary alcohol that is cleaved with acid; a ketal or an acetal that are cleaved with acid; or an α-and β-substituted ether or ester that is cleaved with base, the α-and β-substituents being electron-withdrawing groups.  
     
     
         26 . The probe set of  claim 21  wherein said cleavable linkage is cleaved by singlet oxygen, hydrogen peroxide, NADH, and hydrogen radicals.  
     
     
         27 . The probe set of  claim 21  wherein said cleavable linkage is a disulfide bond.  
     
     
         28 . The probe set of claims  21 ,  22 ,  23 ,  24 ,  25 ,  26 , or  27  wherein said detection group is a fluorophore, a chromophore, or an electrochemical label.  
     
     
         29 . The probe set of  claim 28  wherein said target-binding moiety is a monoclonal antibody or a polyclonal antibody; and wherein k is in the range of from 1 to 3.  
     
     
         30 . The probe set of  claim 29  wherein said charge/mass ratio is in the range of from −0.001 to 0.5, and wherein said detection group is a fluorescein.