Proximity assays using chemical ligation and hapten transfer

A method of detecting a first target located proximally to a second target in a sample, the method comprising:

• labelling the first target with a first conjugate comprising a cleavable bridge component, the cleavable bridge component comprises a cleavage site, a detectable moiety, and a first chemical ligation group,
• labelling the second target with a second conjugate comprising a non-cleavable bridge component, the non-cleavable bridge component comprises a second chemical ligation group,
• activating the first chemical ligation group or the second chemical ligation group so a covalent bond can form where the first target and the second target are proximal;
• cleaving the detectable moiety from the first target site;
• washing the sample to remove unbound detectable moiety;
• and
• detecting the detectable moiety visible.

A method of in situ detection in a sample of a target protein having a post-translational modification, said method comprising:

• binding a first modified binding molecule to the target protein to form a first complex, the first modified binding molecule comprises a cleavable bridge component, the cleavable bridge component comprises a cleavage site, a detectable moiety, and a first chemical ligation group, the cleavage site is more proximal to the first modified binding molecule 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;
• binding a second modified binding molecule to the post-translational modification to form a second complex, the second modified binding molecule comprises a non-cleavable bridge component, the non-cleavable bridge component comprises a second chemical ligation group, the second chemical ligation group is at a terminus of the non-cleavable bridge component, the second chemical ligation group is stable under physiological conditions;
• 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 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.