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Method for analyzing binding rate of recombinant human insulin and analogue or conjugate thereof with insulin receptor

A technology for recombining human insulin and insulin receptors, which is applied in analytical materials, biological material analysis, and measuring devices, can solve problems such as cumbersome steps, and achieve the effects of simplified experimental steps, high accuracy, and good safety

Active Publication Date: 2019-04-26
CHONGQING PEG BIO BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method is practical, has high accuracy, and has no radioactive hazards, and solves the problem of radiation and cumbersome steps in the existing methods

Method used

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  • Method for analyzing binding rate of recombinant human insulin and analogue or conjugate thereof with insulin receptor
  • Method for analyzing binding rate of recombinant human insulin and analogue or conjugate thereof with insulin receptor
  • Method for analyzing binding rate of recombinant human insulin and analogue or conjugate thereof with insulin receptor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Enzyme-linked immunoassay (ELISA method) to determine the binding rate of recombinant human insulin receptor

[0045] The recombinant human insulin receptor plasmid was constructed by Shanghai Jikai Gene Technology Co., Ltd. The HEK-293 cells used were donated by the School of Chemistry and Biological Engineering, Chongqing University of Technology, and the HEK-293-IR-B cells were transfected and screened by our company and purchased by Biotin From Shanghai Dongren Chemical Technology Co., Ltd., Insulin-biotin is labeled by our company, recombinant human insulin standard product (from China Food and Drug Control Institute, each labeled content is 20mg / tube 28.3IU / mg), recombinant human insulin is made by Company preparation. The method for determining the binding rate of recombinant human insulin receptor includes the following steps:

[0046] (1) After cell transfection, culture HEK-293-IR-B cells in the logarithmic growth phase with complete medium. When the cel...

Embodiment 2

[0062] Example 2 Enzyme-linked immunoassay (ELISA) method to determine insulin receptor binding rate and 125 Comparison of I-labeled Insulin Radiometric Determination of Recombinant Human Insulin Receptor Binding Rate

[0063] At the same time, two methods are used to determine the binding rate of recombinant human insulin receptor, one is enzyme-linked immunoassay (ELISA), the other is 125 I-labeled insulin radiation method.

[0064] Enzyme-linked immunoassay (ELISA) method to determine the binding rate of insulin receptor: using the method of Example 1 to determine the binding rate of recombinant human insulin receptor (see figure 2 )

[0065] 125 I-labeled insulin radiation method to determine the binding rate of recombinant human insulin receptor:

[0066] ①After the neck of the adult Wistar rat was dissected and the blood was taken, the liver was immediately dissected and the liver was removed, the connective tissue was removed, cut into pieces, rinsed with ice-cold saline until ...

Embodiment 3

[0074] Example 3 Enzyme-linked immunoassay (ELISA) method to determine the receptor binding rate of recombinant human insulin, insulin aspart, insulin lispro, insulin glargine, insulin detemir, PEG and modified conjugate (PEG-Lispro)

[0075] The enzyme-linked immunoassay (ELISA) method of Example 1 was used to determine insulin aspart (from Novo Nordisk Novo Nordisk 300 units / 3ml / piece), and insulin lispro (from Eli Lilly and Company Euler 300 units) / 3ml / piece), insulin degludec (from Novo Nordisk, trade name: Tresiba 300 units / 3ml / piece), recombinant human insulin (from China Institute for Food and Drug Control, each with a labeled content of 20mg / 28.3IU / mg), insulin glargine (from Sanofi-Aventis Pharmaceuticals, 300 units / 3ml / piece), insulin detemir (from Novo Nordisk, Nope, 300 units / 3ml / piece) ), PEG-Lispro (according to patent EP2476430A1, self-made by the company) insulin receptor binding rate, after 5 repeated experiments, using ORIGIN software to perform four-parameter...

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Abstract

The invention discloses a method for analyzing the binding rate of recombinant human insulin and an analogue or conjugate thereof with an insulin receptor. The method comprises the steps that the insulin receptor is transfected to an HEK-29 cell firstly; an enzyme label plate is wrapped with the antibody of the insulin receptor, the insulin receptor is combined with the insulin receptor, the humaninsulin and the analogue or conjugate thereof and a biotin labeled insulin are competitively bound with the insulin receptor, an avidin is bound with the biotin, and the receptor binding rate is determined through HRP developing. The method has the advantages that the variation of the binding rate of insulin receptors can be accurately measured through the principals of antigen and antibody binding, ligand and receptor binding and the capability of binding avidin of the biotin, and the demands of being simple and convenient in operation steps, pollution free, low in cost high in accuracy andthe like of a measuring method of biological product receptor binding experiments are met.

Description

Technical field [0001] The present invention relates to the technical field of in vitro pharmacodynamic experimental detection of insulin receptor binding rate, in particular to the technical field of in vitro pharmacodynamic experimental detection of recombinant human insulin analog or conjugate receptor binding rate, including rapid-acting insulins such as insulin aspart (Insulin aspart), Insulin Lispro, Insulin Glulisine, long-acting insulins such as Insulin glargine, Insulin Detemir, Insulin Degludec and modified conjugates such as Methods for determining the binding rate of PEGylated insulin (such as PEG-Lispro, PEG-Insulin) and PEG-Insulin (PEG conjugated des B30-insulin) to the human insulin receptor. Background technique [0002] Insulin is a double-chain (A, B chain) protein hormone composed of 51 amino acids, with a molecular weight of 5374 and an isoelectric point of 5.35. The A chain contains 21 amino acids and the B chain has 30 amino acids. The two peptide chains a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543
CPCG01N33/54306G01N2333/62
Inventor 何跃杨黎曹宇刘日勇范开
Owner CHONGQING PEG BIO BIOTECH CO LTD
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