Methods for characterizing molecular interactions

A qualitative analysis and specific technology, applied in analytical materials, chemical method analysis, instruments, etc., can solve problems such as slow apparent dissociation rate constants

Inactive Publication Date: 2009-02-11
FORTEBIO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Antibody rebinding on the sensing surface may result in an unusually slow apparent dissociation rate constant

Method used

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  • Methods for characterizing molecular interactions
  • Methods for characterizing molecular interactions
  • Methods for characterizing molecular interactions

Examples

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example 1

[0042] Example 1: Overview of methods for qualitative analysis of antibody-antigen responses.

[0043] A scheme for practicing the method of the present invention to qualitatively analyze antigen-antibody responses is disclosed in FIG. 1 . In Step 1, samples are prepared as follows: affinity-tagged (e.g., biotinylated) antigen ( L * ) forms an immune complex with the antibody (R) whose dissociation rate constant is to be estimated. The labeled antigen and antibody concentrations are preferably selected such that at equilibrium substantially all of the labeled antigen is bound by the antibody. In step 2, a large excess of unlabeled antigen (L) is added to elicit a reaction that competes with the biotin-antigen for the antibody binding site. In this format, since the biotin-antigen has been previously bound by the antibody, the immune complex must dissociate in order to bind the unlabeled antigen. After dissociation, a molar excess of unlabeled antigen preferentially binds...

example 2

[0044] Example 2: Qualitative analysis of anti-FITC / FITC responses using glass fiber biolayer interferometry sensors.

[0045] figure 2 The step 3 dissociation results for the anti-fluorescein / fluorescein-dextran binding pair are shown (schematically in Figure 1). In this example, a glass fiber biolayer interferometer sensor was used to perform a solid phase binding assay. Sensors and methods of making and using them are described in detail in commonly owned US Patent Application Publication No. 20050254062, the entirety of which is hereby incorporated by reference for all purposes. In this example, the fibers were derivatized with streptavidin conjugated to a biotin label present on the FITC-dextran ligand. The use of this type of sensor in the practice of the method may provide unexpected advantages. In the early stages of developing a therapeutic antibody, the amount of antibody available may be limited. The accuracy of off-rate estimates is improved by performing rep...

example 3

[0091] Example 3: Implementation of the inventive method on a biomolecular interaction analyzer device

[0092] 1. Sample preparation was performed in a manner similar to that described in Example 2, except that samples were prepared in larger batches (eg, 10-20 mL). Each analysis consumes an aliquot (eg, approximately 1-2 mL). Additionally, use a solution control to correct for any refractive index changes due to protein concentration. For this working example, the ideal solution control consisted of analyte plus antibody that was not biotinylated to give a similar total protein concentration to the sample.

[0093] 2. Using a streptavidin Bacow chip (Sensor Chip SA, Bacow Registry No. BR-1000-32). Alternatively, streptavidin can be immobilized by standard protocols using an amine-reactive CM5 chip (Bacow Accession No. BR-1006-68).

[0094] 3. For each time point, a new Bacow chip is used. Each chip contained four flow cells that could be used to analyze three samples a...

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Abstract

Methods are provided for measuring rate constants for high affinity molecular interactions using an assay format for determining dissociation rates in liquid phase. The invention uses a biosensor that at selected time intervals is contacted with a sample solution to estimate the ratio of bound vs. free ligand. Dissociation rate constants determined according to the methods of the invention more closely mimic in vivo binding constants and avoid diffusional barrier artifacts that accompany measurements performed using solid phase devices. The methods of the invention provide further advantage by not requiring continuous measurements be made on a biosensor instrument thus leaving it available to process other samples. The methods permit accurate determination of dissociation rates of reactions for which dissociation slowly occurs over intervals of hours to days or more.

Description

technical field [0001] The present invention relates to methods and compositions that can be used to qualitatively analyze high-affinity molecular reactions. Background technique [0002] The goal of many biotechnology companies producing therapeutic monoclonal antibodies is to develop antibodies that bind biological targets with high affinity. Most therapeutic antibodies have affinity constants in the nanomolar range and much research has been devoted to increasing the affinity to approach the picomolar range. Instruments capable of binding reaction kinetics or real-time measurements (e.g., etalon fiber-based systems available from Forté Bio and surface plasmon resonance-based instruments available from Biacore) are available in Monitoring bimolecular interactions is advantageous because it can be used to determine rate constants for both the association and dissociation phases of binding, and thus offers advantages over equilibrium-based binding measurements. Therefore, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N31/00G01N33/00G01N33/53
CPCG01N33/53G01N33/543G01N33/536G01N31/00G01N33/00
Inventor 罗伯特·朱克克丽斯塔·利娅·威特贝蒂娜·海德克尔
Owner FORTEBIO
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