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Detection method for biological activity of liraglutide

A biologically active, liraglutide technology, applied in biological testing, chemiluminescence/bioluminescence, analysis by chemical reaction of materials, etc., can solve problems such as harm to the human body, and achieve good accuracy and repeatability , the effect of the simple method

Active Publication Date: 2014-06-25
HANGZHOU JIUYUAN GENE ENG +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since young hamster kidney cells expressing the human pancreatic GLP-1 receptor are difficult to obtain; and the scintillation proximity assay uses radioactive isotopes, which are harmful to humans, it is necessary to develop a simple, safe and reliable assay for the bioactivity of liraglutide method

Method used

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  • Detection method for biological activity of liraglutide
  • Detection method for biological activity of liraglutide
  • Detection method for biological activity of liraglutide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] (1) Take RIN-m5F cells in good growth state, spread them in white flat-bottomed 96-well cell culture plates, 7000 cells / well, and place them in a 5% carbon dioxide incubator at 37°C for static culture for three days.

[0048] (2) Weigh some liraglutide raw powder, dissolve it with 10mM disodium hydrogen phosphate solution (pH8.2), and heat it in a water bath at 80°C for 47 hours. Then weigh some liraglutide raw powder and dissolve it with 10mM disodium hydrogen phosphate solution (pH8.2). The heat-treated liraglutide solution, the untreated liraglutide solution and the original preparation of liraglutide were pre-diluted respectively to prepare a dilution solution with a concentration of 0.006 mg / ml. Then divide into two groups to prepare the concentration gradient, and set 7 concentration points. Group 1 was the original liraglutide preparation and untreated liraglutide solution. Group 2 was heat-treated liraglutide solution and untreated liraglutide solution. The d...

Embodiment 2

[0053] (1) Take RIN-m5F cells in a good growth state, spread them in a white flat-bottomed 96-well cell culture plate, 7000 cells / well, and place them in a 37°C, 5% carbon dioxide incubator for static culture for three days.

[0054] (2) Weigh some liraglutide test substance and reference substance, and dissolve them with 10mM disodium hydrogen phosphate solution (pH8.2). According to the weighing amount, the test sample was pre-diluted 156 times, the reference substance was pre-diluted 135 times, the same concentration gradient was prepared, the concentration was 0.006mg / ml, 8 concentration points were set, and the dilution times were 32 times, 64 times, 128 times Times, 256 times, 1280 times, 6400 times, 32000 times, 800,000 times.

[0055] (3) Aspirate the supernatant in the cell wells in step (1), add a concentration gradient of liraglutide to stimulate the cells, and place the cell plate at 37°C for 5 minutes.

[0056] (4) Use the cAMP-dependent protein kinase A activity...

Embodiment 3

[0059] Experimental procedure is identical with embodiment 2. Known that the activity of the test product is 39,500 U / ml, the biological activity of the test product was tested five times, and the test results are shown in Table 1. The recovery rate of the test results ranged from 70% to 120%, the average value was 102%, and the RSD was 20%, which indicated that the accuracy and repeatability of the method were good.

[0060] Table 1 Verification of accuracy and repeatability

[0061]

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Abstract

According to the invention, the change of the level of 3',5'-cyclic adenosine monophosphate (cAMP) is taken as a detection index and a cAMP-depending type protein kinase A active analysis method is used for determining the biological activity of liraglutide. Rat insulinoma cells RIN-m5F are cultivated and a diluted liraglutide comparison product solution and a diluted test solution are added; a kit is used for detecting the change of the level of the cAMP in stimulated cells; a super-sensitive chemiluminiscence detection module of a multifunctional enzyme linked immunosorbent assay is used for reading a relative chemiluminiscence unit (RLU) and statistical software is used for fitting experimental data; the biological activity of a product to be detected is calculated by a formula. The method can be used for simply, conveniently, rapidly and accurately detecting the change of the biological activity of the liraglutide; the accuracy and the repeatability accord with the requirements of a biological activity determination method of biological products.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for measuring the biological activity of liraglutide. Background of the invention [0002] Diabetes is an increasingly serious threat to human health worldwide. Glucagon-like peptide-1 (GLP-1) is a polypeptide hormone secreted by intestinal L cells, which has the functions of promoting insulin secretion and promoting the proliferation and differentiation of pancreatic β cells. It exerts its hypoglycemic effect by stimulating insulin secretion, inhibiting glucagon secretion, and inhibiting gastric emptying. Since its stimulation of insulin is glucose-dependent, it does not cause symptoms of hypoglycemia. [0003] Natural GLP-1 is not suitable for clinical application due to its short half-life (only 1-2 minutes). Liraglutide has 97% homology with GLP-1 and is clinically used to control blood sugar in adults with type 2 diabetes. The difference between its mole...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N21/76
CPCG01N21/76G01N33/68
Inventor 卢旻衎周亮曹阳戎亚雯吉鹏飞马国昌
Owner HANGZHOU JIUYUAN GENE ENG
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