Proximity assay for in situ detection of targets
First Claim
1. A method for analyzing a sample to determine whether a first target is proximal to a second target, the method comprising:
- contacting a sample with a first conjugate comprising a biotin ligase and a first binding moiety specific for the first target;
contacting the sample with a second conjugate comprising a biotin ligase peptide substrate and a second binding moiety specific for the second target;
contacting the sample with biotin and ATP so that the biotin ligase labels the biotin ligase peptide substrate with biotin if the first target is proximal to the second target; and
detecting the biotin label;
wherein the first and second binding moieties are selected from an antibody, an antibody fragment, or an antibody derivative or an antibody mimetic.
1 Assignment
0 Petitions
Accused Products
Abstract
A proximity detection method is described that utilizes enzymatic biotinylation to detect targets in a sample, particularly formalin fixed paraffin embedded samples using automated staining platforms. One disclosed embodiment comprises contacting the sample with a first conjugate comprising a biotin ligase and a first specific binding moiety that binds proximally to the first target; contacting the sample with a second conjugate comprising a biotin ligase substrate and a second specific binding moiety that binds proximally to the second target; subjecting the sample to conditions that allow biotinylation of the biotin ligase substrate by the biotin ligase when the first target and the second target have a proximal arrangement; and detecting biotinylation of the biotin ligase substrate. The conditions that allow biotinylation of the substrate include addition of biotin and ATP. The method also may comprise contacting the sample with a streptavidin-enzyme conjugate. Signal amplification also can be used.
-
Citations
21 Claims
-
1. A method for analyzing a sample to determine whether a first target is proximal to a second target, the method comprising:
-
contacting a sample with a first conjugate comprising a biotin ligase and a first binding moiety specific for the first target; contacting the sample with a second conjugate comprising a biotin ligase peptide substrate and a second binding moiety specific for the second target; contacting the sample with biotin and ATP so that the biotin ligase labels the biotin ligase peptide substrate with biotin if the first target is proximal to the second target; and detecting the biotin label; wherein the first and second binding moieties are selected from an antibody, an antibody fragment, or an antibody derivative or an antibody mimetic. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
-
-
21. An automated method, comprising using an automated staining apparatus to perform one or more steps associated with a method of testing a formalin fixed paraffin embedded tissue sample for the presence of a first target and a second target in close proximity, the method comprising:
-
contacting the sample with a first set of reagents so that the first target is recognized by a first binding moiety specific for the first target and that results in the conjugation or deposition of a biotin ligase proximally to the first target; contacting the sample with a second set of reagents so that the second target is recognized by a second binding moiety specific for the second target and that results in the conjugation or deposition of a biotin ligase substrate proximally to the second target; contacting the sample with biotin and biotinylation reagents so that the biotin ligase biotinylates the biotin ligase substrate if the first target and the second target are in close proximity; and detecting the biotin; wherein the first and second binding moieties are selected from an antibody, an antibody fragment, an antibody derivative or an antibody mimetic.
-
Specification