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A kind of monoclonal antibody that recognizes bevacizumab and application thereof

A monoclonal antibody and bevacizumab technology, applied in the biological field, can solve the problems of unable to reflect drug content, unable to detect free drugs at the same time, unable to detect bevacizumab, etc., and achieve huge market value, The effect of broad application prospects

Active Publication Date: 2018-12-18
DRAGONBOAT BIOPHARMACEUTICAL (SHANGHAI) CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the commonly used detection methods in China mainly use antigen VEGF-coated plate detection, which cannot detect bevacizumab that has been combined with VEGF, and cannot simultaneously detect free drugs and drugs that have been bound to ligands. Therefore, it cannot be used in clinical testing. Reflects the total drug content in biological samples

Method used

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  • A kind of monoclonal antibody that recognizes bevacizumab and application thereof
  • A kind of monoclonal antibody that recognizes bevacizumab and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] The preparation process of embodiment 1 monoclonal antibody 5A9

[0050] The main steps of this example include: animal immunization, cell fusion, screening of hybridoma cells, cloning of hybridoma cells and preparation of monoclonal antibodies, etc.

[0051] (1) Animal immunization: 6-week-old BALB / C female mice were used, and the antigen was commercialized bevacizumab Avastin. Conventional immunization method was adopted, and the immunization route was subcutaneous injection, with 50 μg Avastin and Freund's complete adjuvant Mix equal volumes, and inject subcutaneously at 3 to 4 points on the back of the mouse; after two weeks, perform two immunizations with the same dose, with Freund's incomplete adjuvant; two weeks later, perform three immunizations, with Freund's incomplete adjuvant; Boost immunization two weeks later with Freund's incomplete adjuvant; take splenocytes for fusion after 3 days;

[0052] (2) Cell fusion: prepared on the third day after the last immu...

Embodiment 2

[0082] Example 2 Evaluating the Specificity of Monoclonal Antibody 5A9 Binding to Bevacizumab

[0083] Experimental procedure

[0084] (1) Coating: Dilute Avastin and the control antibody Erbitux to 5 μg / mL with coating buffer, respectively add to different 96-well plates, 100 μL / well, overnight at 2-8°C;

[0085] (2) Plate washing: wash the plate 3 times with washing solution, 300 μL / well;

[0086] (3) Blocking: 200 μL / well 0.1% BSA solution, room temperature for 2.5 hours;

[0087] (4) Plate washing: wash the plate 3 times with washing solution, 300 μL / well;

[0088] (5) Dilute samples: Dilute antibody 5A9 with buffer, and then serially dilute 2 times to obtain 12 concentration samples, in order: 400ng / mL, 200ng / mL, 100ng / mL, 50ng / mL, 25ng / mL, 12.5ng / mL, 6.25ng / mL, 3.12ng / mL, 1.56ng / mL, 0.78ng / mL, 0.39ng / mL and 0.19ng / mL;

[0089] (6) Transfer the diluted sample to a 96-well plate, 100 μL / well;

[0090] (7) Incubation: overnight incubation at 2-8°C;

[0091] (8) Plate...

Embodiment 3

[0099] Example 3 Evaluation of Neutralizing Activity of Monoclonal Antibody 5A9

[0100] Experimental procedure

[0101] (1) Coating plate: Dilute the recombinant human VEGF antigen to 50ng / mL with coating solution, add it to the ELISA plate, 100 μL per well, and incubate overnight at 4°C;

[0102] (2) Plate washing: on the second day, take out the plate, empty the liquid, wash three times with 300 μL washing solution and pat dry the residual liquid;

[0103] (3) Blocking: add 200 μL of blocking solution to each well and incubate at room temperature for 2 h;

[0104] (4) Plate washing: empty the liquid, wash three times with 300 μL washing liquid and pat dry the residual liquid;

[0105] (5) Dilution and loading of samples: Dilute the antibody Avastin to 6 μg / mL with buffer, and then serially dilute to 2 times to obtain 12 concentrations: 3000ng / mL, 1500ng / mL, 750ng / mL, 375ng / mL, 187.5ng / mL, 93.8ng / mL, 46.88ng / mL, 23.4ng / mL, 11.72ng / mL, 5.86ng / mL, 2.93ng / mL, 1.96ng / Ml, dil...

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Abstract

The invention provides a monoclonal antibody for recognizing bevacizumab and application of the monoclonal antibody. A light chain CDR1 (Complementarity-Determining Region 1) of the monoclonal antibody has an amino acid sequence of SEQ ID NO.3 as shown in the specification, a light chain CDR2 has an amino acid sequence of SEQ ID NO.4 as shown in the specification, a light chain CDR3 has an amino acid sequence of SEQ ID NO.5 as shown in the specification, a heavy chain CDR1 of the monoclonal antibody has an amino acid sequence of SEQ ID NO.6 as shown in the specification, a heavy chain CDR2 hasan amino acid sequence of SEQ ID NO.7 as shown in the specification, and a heavy chain CDR3 has an amino acid sequence of SEQ ID NO.8 as shown in the specification. A great number of complex experiments screen and verify that the monoclonal antibody can be specifically combined with bevacizumab, meanwhile combination of the bevacizumab with VEGF (Vascular Endothelial Growth Factor) is not affected, the metabolic process of the bevacizumab can be monitored and evaluated, and wide application prospects and great market values can be achieved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a monoclonal antibody recognizing bevacizumab and its application. Background technique [0002] In recent years, antibody drugs have been widely used in the clinical treatment of tumors, transplant rejection, infectious diseases, autoimmune diseases and other diseases due to their advantages of safety, effectiveness and high specificity. The drug metabolism of antibody drugs is an important detection index in preclinical and clinical trial research. [0003] Bevacizumab is a recombinant humanized IgG1 monoclonal antibody that specifically targets vascular endothelial growth factor (VEGF). It can specifically bind to VEGF and block VEGF and VEGF receptors. The combination and signal transduction of 1 and 2 can inhibit the biological activity of VEGF, block angiogenesis, and thus fail to provide oxygen and other nutrients needed for tumor tissue growth, and play an anticancer role. B...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/22C12N5/20G01N33/68
CPCC07K16/22C07K2317/55C07K2317/565G01N33/6854G01N2333/485
Inventor 邵喆刘聪刘钰山黄应峰
Owner DRAGONBOAT BIOPHARMACEUTICAL (SHANGHAI) CO LTD