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Method for measuring activity of anti-VEGF (Vascular Endothelial Growth Factor) antibody, and application thereof

An antibody activity and antibody technology, which is applied in the field of determination of anti-VEGF antibody activity, can solve the problems of long detection time, slow growth cycle, low sensitivity, etc.

Active Publication Date: 2018-11-30
BIO THERA SOLUTIONS LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since HUVEC is a primary adherent cell, the growth cycle is slow, and the whole experimental process takes 72 hours, and the detection time is long and the sensitivity is low.

Method used

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  • Method for measuring activity of anti-VEGF (Vascular Endothelial Growth Factor) antibody, and application thereof
  • Method for measuring activity of anti-VEGF (Vascular Endothelial Growth Factor) antibody, and application thereof
  • Method for measuring activity of anti-VEGF (Vascular Endothelial Growth Factor) antibody, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Construction of Jurkat cell lines stably expressing VEGFR-luciferase

[0055] 1.1 Experimental materials

[0056] The test consumables required for this detection system are as follows: NFAT-Jurkat cells containing the NFAT-luciferase reporter gene are obtained from Jurkat cells purchased from Promega, and after the NFAT-luciferase reporter gene is transferred, the stable cell line is screened. pcDNA3.1[G418] and pcDNA3.1[zeo] plasmids were obtained from General Biosystems (Anhui) Co., Ltd. The VEGFR-IL1R sequence was synthesized at General Biosystems (Anhui) Co., Ltd. The active recombinant human vascular endothelial growth factor-165 (VEGF-165) was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd. Bio-Glo luciferase kit was purchased from Promega.

[0057] 1.2 Plasmid transfection

[0058] Divided into two groups:

[0059] The first group: NFAT-Jurkat cells were co-transfected with pcDNA3.1[VEGFR1-IL1R3 / G418] and pcDNA3.1[VEGFR1-IL1R6 / zeo] pla...

Embodiment 2

[0067] Example 2 Detection method optimization

[0068] 2.1 Concentration optimization

[0069] According to the results of the VEGF-165 activation experiment in Example 1.3, VEGF-165 was set at three different concentrations of 200ng / mL, 100ng / mL, and 25ng / mL, and the inhibition experiment of VEGF monoclonal antibody was carried out respectively, and the VEGF-165 was determined according to the measured fluorescence value. The use concentration of 165.

[0070] Methods: Anti-CD3 antibody and anti-CD28 antibody were coated on 96-well white plate overnight at 4°C, and 100,000 VEGFR2-NFAT-Jurkat-1-2 cells were added to each well; the initial concentration of bevacizumab (purchased from Roche) was 125μg / mL, 1:3 gradient dilution of 10 concentration points, added to the above cell plate; VEGF-165 was diluted to 200ng / mL, 100ng / mL, 25ng / mL, respectively, transferred to the above cell plate; 37℃, 5 %CO2 incubator for 6 hours, add 50 μL of luciferase substrate to each well, and det...

Embodiment 3

[0080] Verification of embodiment 3 detection method

[0081] 3.1 Specificity Verification

[0082] This method is aimed at the biological activity of anti-VEGF antibody. Therefore, the verification of its specificity uses monoclonal antibody drugs with different targets: bevacizumab (Bevacizumab, the target is VEGF), tocilizumab ( Tocilizumab (target IL-6R), rituximab (target CD20), cetuximab (target EGFR). Under the same experimental conditions, use our VEGFR2-NFAT-Jurkat-1-2 reporter gene assay system to measure the activity of these four monoclonal antibodies.

[0083] Method: Anti-CD3 antibody and anti-CD28 antibody were coated on 96-well white plate overnight at 4°C, and VEGFR2-NFAT-Jurkat-1-2 cells were divided into 1×10 5 Add one per well into a 96-well white plate; add 100ng / mL VEGF-165; dilute the four monoclonal antibodies to an initial concentration of 125μg / mL, dilute 10 concentration gradients at 1:4, and transfer to the above-mentioned cell plate; 37°C, 5% CO...

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Abstract

The invention discloses a method for measuring the activity of an anti-VEGF (Vascular Endothelial Growth Factor) antibody. An effector cell which transfects a VEGF receptor, IL-1R3, IL-1R6 and a reporter gene is taken as a detection material, anti-CD3 and anti-CD28 antibodies activate Jurkat cells, a VEGF ligand is added and is combined with a VEGFR (Vascular Endothelial Growth Factor Receptor) onthe effector cell, so that IL-1R3 and IL-1R6 in the cell mutually act to activate a downstream NFAT (Nuclear Factor of Activated T) signal path, and therefore, the reporter gene can be expressed. After the anti-VEGF antibody is added, the anti-VEGF antibody can be competitively combined with the VEGF ligand so as to block the combination of the VEGF ligand and the receptor VEGFR thereof so as toreduce a downstream reporter gene expression quantity. The method has the advantages of convenient operation step, high detection sensitivity and good accuracy, and experiment time is greatly shortened.

Description

technical field [0001] The invention belongs to the technical field of drug activity detection methods, and in particular relates to the establishment and application of a method for measuring the activity of anti-VEGF antibodies. Background technique [0002] Tumor is an angiogenesis-dependent disease, and tumor growth is inseparable from blood vessels and blood supply. Tumor vascular targeting therapy has gradually developed into one of the main research directions in the field of tumors. Tumor tissue produces vascular endothelial growth factor (VEGF), which can promote vascular endothelial cell proliferation, Migration, lumen formation and microvascular permeability, and induce angiogenesis to maintain the continued growth of tumors. VEGF is the most important vascular growth factor found so far, and it is related to many physiological and pathological processes. Studies have shown that VEGF is overexpressed in various solid tumors, which makes VEGF a target for tumor ...

Claims

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Application Information

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IPC IPC(8): G01N33/68C12N15/85
CPCC12N15/85G01N33/6863G01N2333/475
Inventor 吴晓云杨少伟李胜峰俞金泉
Owner BIO THERA SOLUTIONS LTD
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