Proximity assays using chemical ligation and hapten transfer

• US 10,620,217 B2
• Filed: 05/23/2017
• Issued: 04/14/2020
• Est. Priority Date: 11/25/2014
• Status: Active Grant

• Current Assignee: Ventana Medical Systems Incorporated (Roche Holding AG)
• Original Assignee: Ventana Medical Systems Incorporated (Roche Holding AG)
• Inventors: Bieniarz, Christopher, Hong, Rui
• Primary Examiner(s): Haq, Shafiqul
• Application Number:
  US15/603,079
• Publication Number:
  US 20170254813A1
• Time in Patent Office: 1,057 Days
• CPC Class Codes: G01N 2440/00 Post-translational modifications [PTMs] in chemical analysis of biological material
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/54353 with ligand attached to the carrier via a chemical coupling agent coatings G01N33/54393
• CPC Class Codes: G01N 33/6845 Methods of identifying protein-protein interactions in protein mixtures

Abstract

Methods for in situ detecting proximity of two targets of interest featuring an antibody conjugated with a cleavable bridge component having a detectable moiety and an antibody conjugated with a non-cleavable bridge component. The bridge components each have a chemical ligation group adapted to form a covalent bond under particular conditions and when the targets are in close proximity. Following covalent bond formation, the cleavable bridge component can be cleaved from the antibody, effectively transferring the detectable moiety to the non-cleavable bridge component. Detection of the detectable moiety is indicative of the targets being in close proximity. The methods are compatible with both chromogenic and fluorogenic detection systems. The methods may be used to perform assays wherein one or more than one proximity event is detected on the same slide.

Claims

1. A method of determining that a first target and a second target in a sample are proximal, the method comprising the sequential steps of:

binding a first modified binding molecule to the first target to form a first complex, wherein, the first modified binding molecule comprises a cleavable bridge component and a first specific binding moiety capable of binding to the first target, the cleavable bridge component comprises a cleavable linker having a cleavage site, a detectable moiety, and a first chemical ligation group, wherein the cleavable bridge component is bonded to the first specific binding moiety through said cleavable linker and wherein the cleavage site is more proximal to the first specific binding moiety than is the detectable moiety and the first chemical ligation group, the first chemical ligation group is at a terminus of the cleavable bridge component, the first chemical ligation group is stable under physiological conditions, and wherein the first chemical ligation group comprises a first member of a pair of reactive functional groups capable of specifically reacting with a second member of a pair of reactive functional group to form a covalent bond;

binding a second modified binding molecule to the second target to form a second complex, wherein the second modified binding molecule comprises a non-cleavable bridge component and a second specific binding moiety capable of binding to the second target, the non-cleavable bridge component comprises a non-cleavable linker and a second chemical ligation group, the second chemical ligation group is at a terminus of the non-cleavable bridge component, wherein the non-cleavable bridge component is bonded to the second specific binding moiety through said non-cleavable linker and wherein the second chemical ligation group is stable under physiological conditions, and wherein the second chemical ligation group comprises said second member of a pair of reactive functional groups;

initiating a chemical reacting to covalently linking the first chemical ligation group to the second chemical ligation group to form a covalently bonded unit;

cleaving the cleavage site of the cleavable bridge component such that the covalently bonded unit is bound to the second modified binding molecule and not to the first modified binding molecule;

removing cleavable bridge components that are not part of a covalently bonded unit; and

making the detectable moiety visible, and detection of visible signal indicates that the first target and the second target are proximal.

2. The method of claim 1, wherein the first target and the second target are proximal in that they are less than about 40 nm apart.

3. The method of claim 1, wherein the method is performed using an automated staining instrument.

4. The method of claim 1, wherein the first modified binding molecule comprises a first antibody and the second modified binding molecule comprises a second antibody.

5. The method of claim 1, wherein the first modified binding molecule comprises a first primary antibody and a first secondary antibody, and the second modified binding molecule comprises a second primary antibody and a second secondary antibody, wherein the first secondary antibody specifically binds the first primary antibody and not the second primary antibody, and the second secondary antibody specific binds the second primary antibody and not the first primary antibody, wherein the cleavable bridge component is bound to the first secondary antibody and the non-cleavable bridge component is bound to the second secondary antibody.

6. The method of claim 1, wherein the detectable moiety comprises hapten, a peptide tag, or an oligonucleotide.

7. A method of in situ detection in a sample of a target protein having a post-translational modification comprising the sequential steps of:

binding a first modified binding molecule to the target protein to form a first complex, wherein the first modified binding molecule comprises a cleavable bridge component and a first specific binding moiety capable of binding to the target protein, the cleavable bridge component comprises a cleavage site, a detectable moiety, and a first chemical ligation group, wherein the cleavable bridge component is bonded to the first specific binding moiety through said cleavable linker and the cleavage site is more proximal to the first specific binding moiety than is the detectable moiety and the first chemical ligation group, the first chemical ligation group is at a terminus of the cleavable bridge component, the first chemical ligation group is stable under physiological conditions, and wherein the first chemical ligation group comprises a first member of a pair of reactive functional groups capable of specifically reacting with a second member of a pair of reactive functional group to form a covalent bond;

binding a second modified binding molecule to the post-translational modification of the target protein to form a second complex, wherein the second modified binding molecule comprises a non-cleavable bridge component and a second specific binding moiety capable of binding to the post-translational modification of the target protein, the non-cleavable bridge component comprises a non-cleavable linker and a second chemical ligation group, the second chemical ligation group is at a terminus of the non-cleavable bridge component, wherein the non-cleavable bridge component is bonded to the second specific binding moiety through said non-cleavable linker and wherein the second chemical ligation group is stable under physiological conditions, and wherein the second chemical ligation group comprises said second member of a pair of reactive functional groups;

initiating a chemical reaction to covalently linking the first chemical ligation group to the second chemical ligation group to form a covalently bonded unit;

cleaving the cleavage site of the cleavable bridge component such that the covalently bonded unit is bound to the second modified binding molecule and not to the first modified binding molecule;

removing cleavable bridge components that are not part of a covalently bonded unit; and

making the detectable moiety visible, wherein the visibility of the detectable moiety indicates the presence of the target protein with the post-translational modification.