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pH-gradient SPR-based binding assay

A gradient and conjugation technology that can be used in measurement devices, instruments, scientific instruments, etc., and can solve problems such as neutralizing antigens

Pending Publication Date: 2021-08-06
F HOFFMANN LA ROCHE & CO AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the stoichiometric neutralization of one antibody to one antigen (one bivalent antibody to two antigens), it is not possible to completely neutralize the antigen with a smaller amount of antibody than the antigen

Method used

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  • pH-gradient SPR-based binding assay
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  • pH-gradient SPR-based binding assay

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0194] Equipment and Reagents

[0195] All SPR experiments were performed on a Xantec SR7500DC with sensor chip Xantec SCR CMD500L

example 1

[0197] chip preparation

[0198] In order to immobilize the antigen, two kinds of flow cells (Fc) were prepared, Fc1 was used as a blank control, and Fc2 was used as a specific target binding interaction surface. In order to immobilize the antigen on the flow cell II, its surface has been activated by EDC / NHS for amine coupling of dimeric antigen constructs. Antigen dimers have been prepared in 10 mM acetate buffer, pH 4.5, at a concentration of 30 μg / ml. Inject this solution over Fc2 at a flow rate of 20 μl / min for 10 min

[0199] run pH gradient

[0200] To create the gradient, two PBS-P+ buffers were prepared. The pH of buffer A was set at 7.4. Buffer B was set to pH 4.9.

[0201] Antibody samples at a concentration of 100 nM were injected into buffer A at a flow rate of 100 μl / min for 90 s. After equilibrating for 210 seconds, the gradient was initiated from 100% buffer A to 100% buffer B in 2400 seconds. At the end of the run, the surface was regenerated with 1...

example 2

[0204] chip preparation

[0205] To immobilize anti-human Fc capture antibody ( GE BR-1008-39), two flow cells were prepared, Fc1 was used as a blank control, and Fc2 was used as a specific target binding interaction surface. To immobilize the capture antibody on the flow cell II, the surface has been activated with EDC / NHS for amine conjugation. It has been prepared in 10 mM acetate buffer, pH 4.5, at a concentration of 30 μg / ml. This solution was injected over Fc2 for 10 min at a flow rate of 20 μl / min.

[0206] Run pH Gradient After Antibody

[0207] To create the gradient, two PBS-P+ buffers were prepared. The pH of buffer A was set at 7.4. Buffer B was set to pH 4.9. Inject the antibody in question at a concentration of 100 nM into buffer A at a flow rate of 100 μl / min for 90 s. In the second step, different antigen preparations (dimer, wild type and monomer) at a concentration of 100 nM were injected into buffer A for 90 s. After equilibrating for 210 se...

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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 the dissociation pH-value; and recording a 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.

Description

technical field [0001] The present invention belongs to the field of functional assays. Here we report a novel SPR-based binding assay for measuring pH-dependent interactions of antibodies. Background technique [0002] SPR (Surface Plasmon Resonance) is a biosensor-based technology to measure real-time protein-protein interactions. The SPR technique has become a standard tool in biopharmaceutical research and development [1-5], and is commonly used to determine the binding constants of macromolecular interactions. The ability to determine the association and dissociation kinetics of molecular interactions provides detailed insights into the mechanism of complex formation [6]. This information is becoming an important part of the selection and optimization process for mAbs and other biopharmaceutical products [7-10]. Furthermore, SPR techniques allow determination of, for example, the binding activity (binding ability) of an antibody to a target. [0003] The antigen-neu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N33/557
CPCG01N33/543G01N33/557
Inventor T·施洛特豪尔C·斯皮克
Owner F HOFFMANN LA ROCHE & CO AG