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VEGFR-2 resistant monoclonal antibody with human and mouse cross reaction as well as preparation method and application thereof

A VEGFR-2, monoclonal antibody technology, applied in applications, antibodies, anti-animal/human immunoglobulin, etc., can solve the problem of large market demand for biological reagents

Inactive Publication Date: 2010-02-10
NANJING MEDICAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At the same time, the market demand for biological reagents for medical scientific research and clinical diagnosis is large. According to the current domestic and foreign markets and existing literature reports, there is no monoclonal antibody with human-mouse cross-reactivity and high specificity against VEGFR-2. Research using various immunological methods

Method used

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  • VEGFR-2 resistant monoclonal antibody with human and mouse cross reaction as well as preparation method and application thereof
  • VEGFR-2 resistant monoclonal antibody with human and mouse cross reaction as well as preparation method and application thereof
  • VEGFR-2 resistant monoclonal antibody with human and mouse cross reaction as well as preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0069] Example 1, Preparation and Screening of Anti-VEGFR-2 Monoclonal Antibody

[0070] Application of hybridoma technology to prepare anti-VEGFR-2 monoclonal antibody, the specific steps are as follows:

[0071] 1. Preparation of mouse immunogen and antigen for detection:

[0072] 从GenBank获取VEGFR-2基因的核苷酸片段序列(accession No.NM002253)(nucleotides975-1283)gatgtggttctgagtccgtctcatggaattgaactatctgttggagaaaagcttgtcttaaattgtacagcaagaactgaactaaatgtggggattgacttcaactgggaatacccttcttcgaagcatcagcataagaaacttgtaaaccgagacctaaaaacccagtctgggagtgagatgaagaaatttttgagcaccttaactatagatggtgtaacccggagtgaccaaggattgtacacctgtgcagcatccagtgggctgatgaccaagaagaacagcacatttgtcagggtccatgaaaaa,设计并合成上下游引物:F:5-cgggatccgatgtggttctgagtccgtctcatgg-3;R:5-cggaattctcatttttcatgaactctaacaaaggtg-3。 The total RNA was extracted from normal human umbilical endothelial vein cells, the cDNA was reverse transcribed, and the VEGFR-2 gene was amplified by PCR using the cDNA as a template and the above primer conditions.

[0073] Th...

Embodiment 2

[0090] Example 2 Identification and Application of Functional Activity of Anti-VEGFR-2 Monoclonal Antibody A8H1

[0091] 1. ELISA method for functional identification and application of A8H1

[0092] Dilute commercially available recombinant VEGFR-2 protein (purchased from American R&D Company) to 2 μg / ml with coating solution, add 100 μl to each well to coat a 96-well ELISA plate, overnight at 4°C; PBST (PBS containing 0.5% Tween20) 5% Skim milk-washing buffer blocking, incubate at 37°C for 2h; after washing with PBST 5 times, add 100μl A8H1 (2μg / ml initial concentration, 11 concentration gradient dilutions) to each well overnight at 4°C; dilute at 1:5000 Goat anti-human secondary antibody (Santa Cruz Company) 100 μl / well was added to the well, and incubated at 37°C for 1 hour; peroxidase substrate chromogenic solution was 100 μl / well, and the reaction was terminated with 2M sulfuric acid after 15 minutes at room temperature, and detected on the machine Colorimetry adopts du...

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Abstract

The invention relates to a VEGFR-2 resistant monoclonal antibody with human and mouse cross reaction as well as a preparation method and an application thereof. The antibody is generated by the secretion of a hybridoma cell strain 8H1, has human and mouse cross reaction and recognizes three forms of a natural antigen VEGFR-2 in a high specificity way. The VEGFR-2 resistant monoclonal antibody A8H1with human and mouse cross reaction is generated by the secretion of the hybridoma cell strain 8H1 of a VEGFR-2 resistant monoclonal antibody. The preparation method comprises the following steps: preparing a mouse immunogen and an antigen for detection; preparing the VEGFR-2 resistant hybridoma cell strain and adopting His-VEGFR-2 recombinant protein as the immunogen to carry out immunity inoculation on hypodermic injection at the belly of a pure line BALB / c mouse; screening a VEGFR-2 positive monoclonal antibody and screening the monoclonal antibody with positive VEGFR-2 and negative normalhuman serum by an ELISA method; authenticating the subtype of the monoclonal antibody; producing the monoclonal antibody in the BALB / c mouse and purifying; and carrying out function authentication and application on A8H1 by methods of ELISA, and the like.

Description

technical field [0001] The present invention relates to an anti-VEGFR-2 monoclonal antibody with human-mouse cross-reactivity and its preparation and application. The antibody is produced by the secretion of hybridoma cell line 8H1, has human-mouse cross-reactivity, and highly specifically recognizes the natural antigen VEGFR Three forms of -2. Background technique [0002] Vascular endothelial growth factors (vascular endothelial growth factors, VEGFs) family play an important regulatory role in embryonic development and postnatal vascular development, mainly involved in the formation of new blood vessels and lymphatic vessels. Members of the VEGF family play a role after combining with three tyrosine kinase receptors, vascular endothelial growth factor receptors (VEGFR1, VEGFR2, VEGFR3). VEGFR mediates a series of cell behaviors including cell migration, survival, proliferation, etc. It also has special functions in mediating angiogenesis, such as regulating tissue edema ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/06C12P21/08G01N33/577A61K39/395A61P9/00A61P35/00C12N5/20
Inventor 冯振卿黄剑飞朱进谭洋唐奇刘新建管晓虹
Owner NANJING MEDICAL UNIV
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