Method of determining thermodynamic and kinetic parameters from measured off rates

摘要

A method for measuring a property of a binding interaction between a capture agent and a binding partner for the capture agent is provided. In certain embodiments, this method comprises: a) contacting a population of particles that are linked to a capture agent with a substrate comprising a binding partner to produce capture agent/binding partner complexes, wherein the population of particles comprises first particles that are bound to a single molecule of the capture agent and second particles that are bound to two molecules of the capture agent; b) applying a force to the bound support, wherein the force is in a direction that separates the particles from the support; and c) separately measuring the forces required to disassociate the first particles and the second particles from their respective complexes.

主权项

1. A method for measuring a property of a binding interaction between a capture agent and a binding partner for said capture agent, comprising:
a) contacting a population of particles that are linked to a capture agent with a substrate comprising a binding partner to produce capture agent/binding partner complexes, wherein said population of particles comprises first particles that are bound to a single molecule of said capture agent and second particles that are bound to two molecules of said capture agent; b) applying a force to the particles, wherein said force is in a direction that separates said particles from said substrate; c) separately measuring:
(i) the force required to disassociate the first particles from their respective complexes and(ii) the force required to dissociate the second particles from their respective complexes; d) calculating the off rates of said first particles and said second particles as a function of the applied force; e) calculating the off rate for said first particles in the absence of applied force from the dependence of off rates on the applied force; and f) calculating the on rates for binding said first particles and said second particles, using the respective off rates calculated in d) and e).