PH-gradient SPR-based binding assay
AssigneeHoffmann-La Roche Inc.
InventorsTilman Schlothauer, Christian Spick
Life of this patent
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Abstract
Herein is reported a method for determining the dissociation rate constant kd of an antibody from its antigen at the dissociation pH-value, wherein the method comprises the steps of immobilizing at a first pH-value the antibody on a solid phase to which the antigen of the antibody has been conjugated; applying a pH-gradient from the first pH-value to the dissociation pH-value and thereafter maintaining the pH-value at said dissociation pH-value; and recording the binding signal during the maintaining of the pH-value and calculating therefrom the dissociation rate constant kd of the antibody from its antigen at the dissociation pH-value.
Claims7
A surface plasmon resonance method for determining a dissociation rate constant kd of an antibody from its antigen at the dissociation pH-value, wherein the method comprises the following steps:
- a) immobilizing at a first pH-value the antibody on a solid phase using an anti-Fc capture antibody as a capture reagent,
- b) incubating the immobilized antibody with its antigen to form a captured antibody-antigen complex,
- c) applying a pH-gradient from the first pH-value to a second pH-value to the solid phase to determine the dissociation pH-value of the antigen from the antibody and thereafter maintaining the pH-value at said dissociation pH-value, and
- d) recording the binding signal during the maintaining of the pH-value and calculating therefrom the dissociation rate constant kd of the antibody from its antigen at the dissociation pH-value;
- wherein the first pH-value is about 7.4 and the second pH-value is between and including pH 4.0 to pH 6.5.
Dependencies are inferred from the claim text.