Assays for detecting enzymatic activity

摘要

The present invention relates to method of detecting activity of a sample. Some methods involve detecting a neo-binding-site created by the activity such as detecting an activity of a protease by detecting a neo-binding-site created by cleavage of a substrate by the protease.

主权项

1. A method of detecting protease activity in a sample, comprising:
combining in a solution at least the sample and a substrate under conditions compatible for the protease activity,
wherein the substrate comprises an amino acid sequence selected from the group consisting of IEGRDYKDDDDKGSHHHHHH (SEQ ID NO:8), IDGRDYKDDDDK (SEQ ID NO:41), IDGRDYKDDDDKGS (SEQ ID NO:42), IDGRDYKDDDDKGSHHHHHH (SEQ ID NO:43), AEGRDYKDDDDK (SEQ ID NO:44), AEGRDYKDDDDKGS (SEQ ID NO:45), and AEGRDYKDDDDKGSHHHHHH (SEQ ID NO:46),wherein the amino acid sequence comprises a cleavage site for the protease,wherein cleavage at the cleavage site generates a neo-binding-site, andwherein the cleavage site is heterologous with respect to the neo-binding-site; previously, subsequently or concurrently adding to the solution a first binding molecule, wherein the first binding molecule preferentially binds the neo-binding-site after cleavage of the substrate by the protease activity as compared to binding to the neo-binding-site in the uncleaved substrate; and detecting the binding of the first binding molecule to a fragment of the cleaved substrate.