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Antibody capable of efficiently and rapidly binding VEGF and application thereof

An antibody and antibody fragment technology, applied in the direction of anti-growth factor immunoglobulin, anti-animal/human immunoglobulin, instruments, etc., can solve the problem of insignificant early screening and achieve the effect of inhibiting immune function

Active Publication Date: 2019-08-23
浙江众意生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Other tumor markers are mostly produced in stage III and stage IV tumors, which are of little significance for early screening

Method used

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  • Antibody capable of efficiently and rapidly binding VEGF and application thereof
  • Antibody capable of efficiently and rapidly binding VEGF and application thereof
  • Antibody capable of efficiently and rapidly binding VEGF and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] To obtain high-titer VEGF antibody, the specific method is:

[0043] Step 1, mice immunization, using 6 to 8 weeks old female BALB / c mice as experimental animals (purchased from Shanghai Lingchang Biotechnology Co., Ltd.), VEGF-A antigen protein (purchased from Guangzhou Ruibo Biotechnology Co., Ltd. ).

[0044]Step 2. Dissolve the antigenic protein in normal saline, mix 50 μg of human with an equal volume of complete Freund’s adjuvant for the first immunization, and inject mice at 1 ml / mouse, subcutaneously in the abdomen at multiple points.

[0045] Step 3. Booster immunization was carried out after 2 weeks. For booster immunization, 25 μg of human VEGF protein was fully mixed with incomplete Freund’s adjuvant to form an emulsion, and injected into the abdominal cavity of mice at an injection volume of 0.5 ml / mouse, and boosted immunization 3 times.

[0046] Step 4. Three days after the last immunization, the venous blood of the mice was taken and the serum was separ...

Embodiment 2

[0051] Coat human VEGF (purchased from Guangzhou Ruiboao Biotechnology Co., Ltd.) on a 96-well high-adsorption enzyme plate with carbonate buffer solution, the coating volume is 100 μ L per well, and the buffer solution is washed 3 times; The blocking buffer was blocked and incubated at room temperature for 1 hour. The blocking volume was 250 μL / well. After the incubation was completed, the buffer was washed 3 times, and 100 μL of supernatant samples (A1-A85) and positive serum were added to wells 1-90 respectively. (control, CK1-5), incubate at room temperature for 1 hour, wash buffer 3 times after incubation, add 100 μL anti-mouse IgG antibody diluted in 1% BSA buffer at a ratio of 1 / 10000 to each well, the anti-mouse IgG antibody Mouse IgG antibody was labeled with horseradish peroxidase and incubated at room temperature for 1 hour. After incubation, the buffer was washed 3 times, and 100 μL of colorimetric substrate 3,3',5,5'-tetramethylbenzidine was added to each well. Af...

Embodiment 3

[0053] In Example 2, a total of three clones GX-81, NX-06, and XY-57 with strong antigen-binding activity were obtained, and the screened clones with both strong antigen-binding activity and antigen neutralization activity were subjected to nucleotide Sequence determination, briefly, the first strand of cDNA was synthesized after extracting the cellular mRNA, the first strand of cDNA generated by reverse transcription was used in the subsequent PCR reaction, the target band obtained by PCR amplification was cloned into the pGEM-T vector, and picked Single clones were subjected to DNA sequencing. Sequencing was performed by Nanjing GenScript Biotechnology Co., Ltd.

[0054] The antibody light chain variable region and the antibody heavy chain variable region are obtained by PCR amplification, and the complementarity determining region sequence can be obtained after excluding the framework region sequence:

[0055] The amino acid sequence of the three complementarity determinin...

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PUM

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Abstract

The invention provides an antibody capable of efficiently and rapidly binding a VEGF and application thereof. The antibody comprises CDR-L1 of the amino acid sequence shown as SEQ ID NO:07, and / or CDR-L2 of the amino acid sequence shown as SEQ ID NO:08, and / or CDR-L3 of the amino acid sequence shown as SEQ ID NO:09. L3, and / or CDR-H1 of the amino acid sequence shown as SEQ ID NO:10, and / or CDR-H2of the amino acid sequence of SEQ ID NO:11, and / or CDR-H3 of the amino acid sequence shown as SEQ ID NO:12.

Description

technical field [0001] The invention relates to the field of monoclonal antibodies, in particular to an antibody that binds VEGF efficiently and rapidly and uses thereof. Background technique [0002] Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (Vascular permeability factor, VPF), is a highly specific factor that promotes the growth of vascular endothelial cells. Matrix degeneration, vascular endothelial cell migration, proliferation and angiogenesis. [0003] There are many subtypes of the vascular endothelial growth factor family, including VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E and placental growth factor (PGF), and VEGF usually refers to VEGF-A. VEGF-A can promote the formation of new blood vessels and increase the permeability of blood vessels, VEGF-B plays a role in non-neovascular tumors, VEGF-C and VEGF-D play a role in the formation of new blood vessels and new lymphatic vessels in cancer tissues VEGF-E is also a potential...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/22G01N33/577G01N33/574
CPCC07K16/22C07K2317/24C07K2317/565G01N33/574G01N33/57407G01N33/57411G01N33/57438G01N33/57446G01N33/57449G01N33/577G01N2800/16
Inventor 吕鹏辉
Owner 浙江众意生物科技有限公司
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