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VEGF (vascular endothelial growth factor) monoclonal antibody and fracture healing evaluation antibody chip with same

A technology of antibody chip and monoclonal antibody, which is applied in the direction of anti-animal/human immunoglobulin, combinatorial chemistry, organic compound library, etc., can solve the problems of high titer, and achieve accurate qualitative detection, specificity improvement, and convenient operation Effect

Inactive Publication Date: 2013-01-09
李彬
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, high titer and high specificity anti-human VEGF monoclonal antibody and fracture healing evaluation antibody chip have not been reported yet.

Method used

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  • VEGF (vascular endothelial growth factor) monoclonal antibody and fracture healing evaluation antibody chip with same
  • VEGF (vascular endothelial growth factor) monoclonal antibody and fracture healing evaluation antibody chip with same
  • VEGF (vascular endothelial growth factor) monoclonal antibody and fracture healing evaluation antibody chip with same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1: Preparation of the monoclonal antibody of the present invention

[0049] The preparation method of antibody comprises the following process steps:

[0050] (1) Cell fusion: Sp2 / 0 myeloma cells with high activity were mixed with splenocyte suspension sensitized with self-designed and synthesized VEGF polypeptide (sequence IFQEYPDEIE YIFKPS) at a ratio of 1:10-100, and polyethylene Diol (produced by Sigma, USA) makes the cells fuse with each other, and the culture medium is added dropwise to the mixed cell suspension of the two cells, and the cells are cultured with HAT selective medium (produced by Haiclone, USA);

[0051] (2) Screen hybridoma cells: when the cells to be fused are cultured to the 5th to 10th day, absorb the culture supernatant of the clonal cell clusters in the wells of the 96-well culture plate, and use the enzyme-linked immunosorbent assay method to detect the antibody content, and limit dilution Three times of subcloning screening were ...

Embodiment 2

[0072] Embodiment 2: the preparation method of antibody chip

[0073] Increase following processing steps on the basis of embodiment 1:

[0074] Step (5) Label complement C3 antibody with CY3 fluorescein.

[0075] Step (6) Design of standard spotting matrix: Repeat 2-5 points for each antibody, select 6-18 different antibody concentration gradients, 6-18 matrices, and use SmartArrayerTM microarray chip spotting system for spotting .

[0076] Step (7): Design of sample spotting matrix: 8 kinds of antibodies, two spots for each antibody, 16 spots in total, using spotting solution as negative control, and anti-complement C3 antibody as positive control, each The matrix totals 20 points. A total of 24 lattices per chip. The SmartArrayerTM microarray chip spotting system was used for spotting.

[0077] Step (8) After spotting the samples, vacuumize overnight at 30-40°C for drying; after drying, vacuumize and plastic seal, and store at 4-8°C for later use.

[0078] Sample para...

Embodiment 3

[0080] Embodiment 3: Detection of various indicators of the monoclonal antibody described in the present invention

[0081] The monoclonal antibody secreted by the VEGF hybridoma cell line screened in Example 1 is subjected to sequence analysis, and its heavy chain variable region is as follows:

[0082] QVQLV QSGAE VKKPG ASVKV SCKAS GGTFS SYAIS WVRQA PGQGL EWMGG FDPED GETIY AQKFQ GRVTM TEDTS TDTAY MELSS LRSED TAVYY CATGR SMVRG VIIPF NGMDV WGQGT TVTVS SY (shown in SEQ ID No.1)

[0083] Its light chain variable region is as follows:

[0084] DIR MTQSP SSLSA SVGDR VTITC RASQSISSYL NWYQQ KPGKA PKLLI YAASS LQSGV PSRFS GSGSG TDFTL TISSL QPEDFATYYC QQSYS TPLTF GGGTK VEIKP (shown in SEQ ID No.2)

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Abstract

The invention relates to the technical field of bioengineering and discloses a VEGF (vascular endothelial growth factor) monoclonal antibody and a fracture healing evaluation antibody chip with the same. The amino acid sequence of a heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO.1, and the amino acid sequence of a light chain variable region of the monoclonal antibody is shown as SEQ ID NO.2. Homogeneity and biological activity unicity of the monoclonal antibody enables antigen antibody reaction results to be convenient for quality control and beneficial to standardization and normalization. The chip with the monoclonal antibody can be directly applied to clinical therapeutic evaluation on fracture healing, solves the technical problems of time and labor consumption, poor result repeatability and the like of the prior art, has extremely high application value in the field of clinical treatment and has wide application prospect.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a VEGF monoclonal antibody and a fracture healing evaluation antibody chip comprising the monoclonal antibody. Background technique [0002] Angiogenesis plays an important role in the fracture healing process. New blood vessel formation may be related to many factors, such as fibroblast growth factor, bone morphogenetic protein, transforming growth factor. Platelet-derived growth factor, prostaglandin E, vascular endothelial cell growth factor, tumor necrosis factor, insulin growth factor Ⅰ, etc. [0003] Vascular endothelial growth factor (VEGF) is particularly important for endothelial cells to promote their proliferation and angiogenesis, and increase vascular permeability. VEGF binds to receptors on vascular endothelial cells, and has a powerful and specific effect of promoting vascular endothelial cell proliferation and angiogenesis. It is the final channel to promote angioge...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/22G01N33/577C40B40/10
Inventor 李彬
Owner 李彬
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