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A method for rapid determination of cgrp/cgrp receptor antibody drug biological activity

A technology of biological activity and antibody drugs, which is applied in biochemical equipment and methods, microbiological measurement/testing, microbiology, etc., can solve problems such as cell contamination, and achieve the effects of good specificity, short experimental period, and easy operation

Active Publication Date: 2022-05-17
NAT INST FOR FOOD & DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no method for measuring the biological activity of CGRP / CGRPR antibody in China. The present invention adopts the bioassay method of transgenic cells to measure the biological activity of CGRP / CGRPR antibody. The result can be obtained within 4 hours, the experimental period is short, and the operation is simple. , and avoid the problem of objective factors such as the possibility of cell contamination caused by long-term incubation, and provide a biological activity determination method for CGRP / CGRPR antibody drugs for the first time

Method used

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  • A method for rapid determination of cgrp/cgrp receptor antibody drug biological activity
  • A method for rapid determination of cgrp/cgrp receptor antibody drug biological activity
  • A method for rapid determination of cgrp/cgrp receptor antibody drug biological activity

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1 Screening of SK-N-MC cell lines stably expressing CRE-luciferase

[0065] 1. Experimental materials

[0066] SK-N-MC cells were derived from ATCC; Plv-CRE-Luc-PGK-blasticidin plasmid was purchased from Wuhan Jinkairui Bioengineering Co., Ltd.; CGRP was purchased from Qiangyao Biotechnology Co., Ltd.; CGRP antibody was purchased from Suzhou Junmeng Biomedical Technology Co., Ltd. Co., Ltd. (corresponding to Junshi Biology in the description of the invention); the Bright-Glo luciferase kit was purchased from Promega.

[0067] 2. Lentiviral transduction

[0068] The SK-N-MC cells were transduced by the lentivirus packaging method, and after incubation for 48 hours, the medium was changed and the cell culture medium containing 5 μg / mL Blasticidin was added for pressurized selection. After the cell density and viability were recovered, the limited dilution method was used to screen monoclonal clones in a 96-well plate at a density of 0.4-0.6 cells / well. During c...

Embodiment 2

[0075] The optimization of embodiment 2 detection method

[0076] 1. Optimization of CGRP concentration, incubation time, and different cell densities

[0077] Dilute the CGRP concentration to a higher concentration of 100ng / mL (final concentration), 1:3 dilution, 11 concentration points, and determine CGRP according to the four-parameter curve fitted by the measured chemiluminescence value under different cell densities and incubation times The range of action, as well as the more appropriate incubation time and cell density.

[0078] Experimental method: SK-N-MC-CRE-Luc cells were divided into 5×10 3 -4×10 4 pcs / well into 96-well white plate, 37°C, 5% CO 2 Overnight culture; CGRP initial concentration 100ng / mL (final concentration), 1:3 dilution 11 concentration gradients, a total of 12 concentration points (including the concentration point 0ng / mL), 37 ° C, 5% CO 2 to cultivate. Add Bright-Glo at 2h, 4h, 6h, and 22h respectively, detect the chemiluminescence value, and...

Embodiment 3

[0088] Verification of embodiment 3 detection method

[0089] The method of the present invention is aimed at the biological activity of anti-CGRP / CGRPR antibody, therefore, the verification of its specificity has adopted the monoclonal antibody drug of different targets: CGRP monoclonal antibody (CGRP monoclonal antibody, target is CGRP) , Denosumab (target RANKL), trastuzumab (target HER2), infliximab (target TNF-α) and velbutuximab (Brentuximab , the target is CD30). Under the same experimental conditions, use the SK-N-MC-CRE-Luc reporter gene bioassay system described in the present invention to measure the activity of the above-mentioned monoclonal antibody drugs with different targets, so as to be specific to the detection method described in the present invention. sex is verified.

[0090] Experimental method: SK-N-MC-CRE-Luc cells were divided into 1×10 4 pcs / well into 96-well white plate, 37°C, 5% CO 2 Cultivate overnight; add 5 ng / mL (final concentration) of CGRP...

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Abstract

The invention discloses a method for rapidly measuring the biological activity of a CGRP / CGRP receptor antibody drug. The method includes constructing effector cells stably expressing the CRE reporter gene, stimulating and activating the expression of the reporter gene with CGRP, and blocking the CGRP signal pathway with a CGRP / CGRP receptor antibody drug, and fitting a four-parameter curve according to the measured reporter gene signal value. Biological activity of antibodies. The present invention establishes for the first time a fast and accurate quantitative detection method for the determination of the biological activity of the CGRP / CGRP receptor antibody drug, and the method can accurately, simply and rapidly detect the biological activity of the CGRP / CGRP receptor antibody drug .

Description

technical field [0001] The invention belongs to the field of biomedicine detection, and in particular relates to a method for quickly measuring the biological activity of CGRP / CGRP receptor antibody drugs. Background technique [0002] Migraine is the third most common disease and the sixth most disabling disease in the world. It is a common chronic neurovascular disease characterized by recurrent severe headaches, mostly hemilateral. It is estimated that migraineurs total more than 1 billion people worldwide, approximately 90% are episodic migraines (EM), characterized by up to 14 migraine days per month; the remaining 10% are chronic migraines (CM), characterized by the occurrence of headaches on at least 15 days per month, of which 8 or more days are migraines, and the patient's condition lasts for more than 3 months. Currently, there are no medications that can cure migraines. The World Health Organization (WHO) has listed migraine as one of the top 10 most disabling d...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N5/10C12Q1/02
CPCC12N5/0693G01N33/5058C12N2510/00
Inventor 王军志王兰于传飞付志浩郭潇黄璟刘春雨段茂芹郭莎
Owner NAT INST FOR FOOD & DRUG CONTROL
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