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Method for testing bioactivities of GLP-1 receptor agonist

A GLP-1, receptor agonist technology, applied in biochemical equipment and methods, microbial determination/inspection, color/spectral property measurement, etc., can solve the problems of high technical strength, long time, instability, etc

Active Publication Date: 2010-08-11
SHANGHAI HUAYI BIO LAB CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing experimental methods, whether cAMP or cell assays with chemiluminescent substances as target detection substances, are only for the determination of intermediate products, which are easy to degrade and unstable, and there are many kinds of intermediate products. way
[0009] Furthermore, the cells required for the chemiluminescent assay need to be specially constructed so that they can secrete the chemiluminescent substances that can be detected, which requires a lot of cost, high technical strength and long time

Method used

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  • Method for testing bioactivities of GLP-1 receptor agonist
  • Method for testing bioactivities of GLP-1 receptor agonist
  • Method for testing bioactivities of GLP-1 receptor agonist

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Determination of Biological Activity of Recombinant Human Glucagon Peptide-1(7-36)(GLP-1(7-36)) by This Method

[0031] Reagents and materials (1) DMEM high-glucose medium Take 1 bag of DMEM high-glucose medium powder (specification 1L), add water to dissolve and dilute to 1000ml, wherein the final concentration of sodium pyruvate is 1mmol / L, and the final concentration of HEPES is 0.5%. , plus penicillin 10 5 IU and streptomycin 10 5 IU, 3.7g of sodium bicarbonate, dissolved, mixed evenly, sterilized and filtered, stored at 4°C.

[0032] (2) Complete medium Take 10ml of fetal bovine serum, add 90ml of DMEM high-glucose medium, and store at 4°C.

[0033] (3) Assay medium (50 mM glucose) 88 ml of DMEM high-sugar medium, 2 ml of 10% BSA, 10 ml of 250 mM glucose solution.

[0034] (4) 10% BSA Take 10g of BSA, add 100ml of water to dissolve and dilute to 100ml, sterilize and filter, and store at -20°C.

[0035] (5) Take 8.0 g of sodium chloride, 0.20 g of potassium chlo...

Embodiment 2

[0064] This method was used to determine the biological activity of Exendin-4.

[0065] The experimental materials and methods are basically the same as in Example 1.

[0066] Preparation of the test solution: take the Exendin-4 test product and dilute it to 50ug / ml with the assay medium.

[0067] In a 1.5ml centrifuge tube, make 2-fold serial dilutions.

[0068] See the experimental results figure 2 ,Data are as follows:

[0069] Standard:

[0070]

[0071] testing sample:

[0072]

[0073] Placebo:

[0074]

[0075] Such as figure 2 As shown, the sample and standard curves are parallel and linear, according to ED 50 , the titer of the sample can be determined after the titer of the standard substance is determined, which proves the feasibility of the method.

Embodiment 3

[0077] Comparison of the results of measuring the biological activity of the standard GLP-1 (7-36) with the assay medium under different glucose concentration conditions.

[0078] Experimental materials, methods are basically the same as in Example 1 (without test sample). The glucose concentrations of the assay medium were configured to be 17.5 mM, 25 mM, 50 mM and 75 mM, respectively. See the experimental results image 3 , 4 , 5 and 6. Depend on image 3 , 4 The comparison of , 5 and 6 shows that increasing the concentration of glucose is conducive to the secretion of insulin by cells, and the amount of insulin secreted by different concentrations of standard products shows an obvious dose-effect relationship, and the comparison with placebo tends to be more obvious.

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Abstract

The invention relates to a method for testing bioactivities, in particular to a method for testing the bioactivities of a GLP-1 receptor agonist. Based on the function of the GLP-1 receptor agonist which can promote mouse insulinoma cells (Min-6) to secrete the insulin, the method comprises the steps of: using insulin quantitatively secreted by a mouse / rat insulin kit to improve the precision of determination through a series of condition optimization processes, and testing the absorbency at the wavelength of 450 nm and the reference wavelength of 590 nm to obtain the effect curve that the GLP-1 receptor agonist induces the Min-6 cells to secrete the insulin; and finally testing the bioactivities of the GLP-1 receptor agonist on the basis of the effect curve.

Description

technical field [0001] The invention relates to a biological activity assay method, more specifically to a biological activity assay method of a GLP-1 receptor agonist. Background technique [0002] Diabetes has become a major disease that threatens human life and health, and GLP-1 receptor agonists occupy an increasingly important position in diabetes drug research [Drug Review. 2009, 24(1): 32-33; Drug Review. 2008, 5(11):504-505]; GLP-1 receptor agonists mainly include glucagon-like peptide-1 (GLP-1), insulin-secreting peptide (Exendin-4), human GLP-1 analogues For Liraglutide, the determination of its biological activity has become a major difficulty because there is no international standard for activity determination [Chinese Journal of Biological Products. 2004, 17(1): 29-31]. [0003] The biological function of GLP-1 receptor agonists is mainly to specifically interact with the GLP-1 receptor of islet cells, causing changes in the corresponding signaling pathways an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/02G01N21/31
Inventor 蔡永青陈霞梁成罡李克坚
Owner SHANGHAI HUAYI BIO LAB CO LTD
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