Compositions and Methods to Expedite Antibody-Based Exchange Imaging

Abstract

A method for exchange imaging of at least two targets in a sample includes (a) incubating a sample with at least two or more target-recognizing antibodies, each bound to a corresponding monovalent tight antibody binder-docketing moiety (MTAB-DM) reagent capable of binding monovalently to the target-recognizing antibodies, (b) applying at least two imager moieties corresponding to the MTAB-DM, either in series, in batches, or in parallel, and (d) imaging the at least two imager moieties either in series, in batches, or in parallel.

1 - 10 . (canceled) 
     
     
         11 . A composition comprising:
 a. a docking moiety covalently bound to a monovalent tight antibody binder (MTAB), the MTAB being bound to a target-recognizing antibody; and   b. an intermediate moiety having a first domain and a second domain, wherein the first domain is capable of specifically binding to the docking moiety and wherein the second domain is not capable of specifically binding to the docking moiety.   
     
     
         12 . The composition of  claim 11 , wherein the MTAB is Protein A, Protein G, Protein A/G, Protein L, or a monovalent antibody fragment. 
     
     
         13 . A method of making reagents for exchange imaging comprising:
 a. providing a monovalent tight antibody binder (MTAB);   b. conjugating the MTAB to a docking moiety to form a monovalent tight antibody binder-docking moiety (MTAB-DM), the MTAB being bound to a target-recognizing antibody;   c. providing a plurality of intermediate moieties, each having a first domain capable of specifically binding to the docking moiety and a second domain that is not capable of specifically binding to the docking moiety;   d. combining the plurality of intermediate moieties with the MTAB-DM.   
     
     
         14 . The method of  claim 13 , wherein the MTAB is Protein A, Protein G, Protein A/G, Protein L, or a monovalent antibody fragment. 
     
     
         15 . The method of  claim 13 , wherein the plurality of intermediate moieties is combined with the MTAB-DM in a batch reaction. 
     
     
         16 . The method of  claim 13 , wherein the plurality of intermediate moieties is combined with the MTAB-DM separately. 
     
     
         17 - 25 . (canceled) 
     
     
         26 . The composition of  claim 11 , wherein the docking moiety comprises a single-stranded nucleic acid. 
     
     
         27 . The method of  claim 13 , wherein the docking moiety comprises a single-stranded nucleic acid. 
     
     
         28 . The method of  claim 13 , wherein the intermediate moiety comprises a single-stranded nucleic acid. 
     
     
         29 . The composition of  claim 11 , where the MTAB is monovalently bound to a target-recognizing antibody. 
     
     
         30 . The method of  claim 13 , where the MTAB is monovalently bound to a target-recognizing antibody. 
     
     
         31 . The composition of  claim 11 , wherein the MTAB-DM is capable of binding at least two different target-recognizing antibodies with an affinity of from about 1 fM to 1 nM. 
     
     
         32 . The method of  claim 13 , wherein the MTAB-DM is capable of binding at least two different target-recognizing antibodies with an affinity of from about 1 fM to 1 nM.