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Method for Determination of Binding Constants by Equilibrium Shifts

A technology of binding constants and dissociation constants, applied in measuring devices, biological tests, instruments, etc., can solve problems such as uncertain interactions

Active Publication Date: 2020-06-30
3B药品有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of this method is that the interaction between substances exhibiting very high or low affinity for the membrane and the target cannot be determined

Method used

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  • Method for Determination of Binding Constants by Equilibrium Shifts
  • Method for Determination of Binding Constants by Equilibrium Shifts
  • Method for Determination of Binding Constants by Equilibrium Shifts

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] A) Experimental design

[0067] A particularly preferred embodiment is a microtiter plate used in the above method, in which 5 columns have 7 chambers, so that there are 25 samples with immobilized target and 10 samples without immobilized target as a control . Human serum albumin (HSA) was used as the immobilized target (Target 1). HSA was immobilized on commercial agarose beads (Mini-Leak beads medium, Kem-En-Tec Nordic A / S) according to a standard method, and human plasma was used as a soluble target. The substance to be tested is a peptide. Five concentrations of immobilized HSA were used for each of the five plasma concentrations. For this, the chambers of the microtiter plate are filled as follows:

[0068] Column I of the microtiter plate (use rows 1 to 7):

[0069]

[0070]

[0071] Column II of the microtiter plate (use rows 1 to 7):

[0072]

[0073]

[0074] Column III of the microtiter plate (use rows 1 to 7):

[0075]

[0076] Column I...

Embodiment 2

[0097] A) Experimental design

[0098] A particularly preferred embodiment is a microtiter plate used in the above method, in which 5 columns have 7 chambers, so that there are 25 samples with immobilized target and 10 samples without immobilized target as a control . Human serum albumin (HSA) was used as the immobilized target (Target 1). HSA was immobilized on commercial agarose beads (Mini-Leak beads medium, Kem-En-Tec Nordic A / S) according to several standard methods and human plasma was used as soluble target. The substance to be tested is liraglutide. Five concentrations of immobilized HSA were used for each of the five plasma concentrations. For this, the chambers of the microtiter plate are filled as follows:

[0099] Column I of the microtiter plate (use rows 1 to 7):

[0100]

[0101] Column II of the microtiter plate (use rows 1 to 7):

[0102]

[0103]

[0104] Column III of the microtiter plate (use rows 1 to 7):

[0105]

[0106]

[0107] Co...

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Abstract

The invention relates to a method for determining the binding constant between a substance or a mixture of substances and an object of interest, and a kit for carrying out the method of the invention. The basic functional principle of the method of the present invention is to determine the substance binding constant of the target substance by changing the binding balance. In this case, the concentrations of the two targets, immobilized and soluble, are varied, and the affinity to the target is determined by changing the binding equilibrium in the respective medium. Herein, at least four samples are used, wherein according to the present invention, first and second samples of substances comprising different amounts of the immobilized target and the same amount of the dissolved target are incubated, and the The first and second samples of the same amount of immobilized target were incubated with the third and fourth samples of the same amount of soluble target, wherein the third and fourth sample containers contained the same amount of soluble target The mass is different from that of the soluble target substance in the first and second sample containers, and the sample containers contain the same volume of liquid phase during incubation, consisting of buffer solution, dissolved target substance and substance sample.

Description

technical field [0001] The invention relates to a method for determining the binding constant between a substance or substance mixture and a target according to claim 1 and to a kit according to claim 16 . Background technique [0002] The determination of interactions and binding constants between substances or substance mixtures and target substances plays an important role in the pharmaceutical industry, especially in drug research and development. Both fields deal with the interaction between substances or mixtures of substances and living things. Here, the binding properties of the individual substances must be quantified with binding parameters and binding constants. [0003] A method for determining the binding constant is known from DE 102010018 965 A1. In the method, the protein is immobilized on a plurality of solids, eg, contacted with an aqueous solution of the substance to be tested, and incubated for a period of time sufficient for the dissolved substance to ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/543G01N33/92
CPCG01N33/54306G01N33/54313G01N33/92G01N33/48G01N33/50
Inventor 亨涅克·波瑞斯
Owner 3B药品有限责任公司