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Anti-osteopontin OPN monoclonal antibody and application thereof

A monoclonal antibody and osteopontin technology, which is applied in the direction of antibodies, anti-animal/human immunoglobulins, anti-tumor drugs, etc., can solve the problem that there is no good standard for OPN detection, affecting OPN clinical detection, and antibody missed detection And other issues

Inactive Publication Date: 2010-11-24
CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing ELISA kits for detecting OPN on the market mainly include R&D, Assay Designs, IBL, etc. However, the antibodies in these kits are all prepared by immunizing animals with recombinant OPN, and OPN is a protein with multiple glycosylation and phosphorylation site proteins, so the structure and epitope of recombinant OPN and natural OPN are different to a certain extent, so the antibody obtained by immunizing with recombinant OPN may have missed detection when detecting natural OPN. Literature research shows that these When the kit was used in clinical research to detect serum OPN levels, there were differences in the detection values ​​of the same sample (BMC Cancer 2008, 8: 38 doi: 10.1186 / 1471-2407-8-38), which caused There is no good standard for the detection of OPN, which affects the application of OPN in clinical detection

Method used

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  • Anti-osteopontin OPN monoclonal antibody and application thereof
  • Anti-osteopontin OPN monoclonal antibody and application thereof
  • Anti-osteopontin OPN monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0151] Example 1: Purification of native OPN from human milk

[0152] The present inventors used an optimized two-step method to extract natural OPN from milk. The separation and purification method of the present invention provides a new method for obtaining natural OPN. After long-term exploration and experiments, the inventors optimized a two-step chromatography method to obtain OPN with a purity of up to 95% from milk, which not only shortened the separation steps and purification time, but also reduced the loss of OPN and activity loss. The specific steps are:

[0153] 1. Milk processing: Centrifuge 200ml of milk (from lactating women) at 1000rpm / 10min, remove the upper layer of milk fat, and reserve the whey for the next step of purification.

[0154] 2. The ion exchange method enriches OPN in milk and removes most of the foreign proteins.

[0155] Materials: whey (200ml); DEAE sepharose (GE, 20ml); GE company Akta Purify purifier.

[0156] Process: After equilibrati...

Embodiment 2

[0172] Example 2: Animal immunization

[0173]Select healthy BALB / c mice homologous to the myeloma cells used, and the age of the mice is 8-12 weeks, male or female. The antigen is the natural purified OPN protein prepared in Example 1. Antigen stock solution concentration: 1 mg / ml; Balb / c mouse immunization dose: 100 μg OPN per mouse. The injection method is multi-point intramuscular injection. Dilute with PBS or normal saline before use. Immunization schedule: three immunizations in weeks 0, 3 and 6. Three days before the fusion, take 100μg and dilute it with PBS to 0.5ml for intraperitoneal injection for memory stimulation. Three days after the last immunization, splenocytes were isolated for fusion. Immunization results are shown in Table 1 below.

[0174] Table 1: Antibody titers in mouse sera after the third immunization with OPN

[0175]

Embodiment 3

[0176] Example 3: Construction of hybridoma cell lines and preparation of monoclonal antibodies

[0177] 1. Culture of myeloma cell lines

[0178] The most important point in selecting tumor cells is homology with the B cells to be fused. If spleen cells are to be fused, various myeloma cell lines can be used, and we use SP2 / 0 cell line. The growth and fusion efficiency of the cell line is average. In addition, the cell line itself does not secrete any immunoglobulin heavy chain or light chain. The highest growth scale for cells is 9*10 5 / ml, the doubling time is usually 10-15h. For fusion cells, select cells in the logarithmic growth phase, cell shape and activity (the activity is greater than 95%). The myeloma cell lines were first adapted to culture medium containing 8-azaguanine before fusion, and the day before cell fusion, fresh medium was used for 2*10 5 / ml, the next day is generally logarithmic growth phase cells.

[0179] 2. Culture of feeder cells

[0180] U...

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Abstract

The invention relates to an anti-osteopontin OPN monoclonal antibody, an application thereof in preparing a medicament for treating diseases mediated by OPN, an application thereof in preparing a kit for detecting the OPN, a hybridoma cell strain generating the monoclonal antibody, and the kit containing the monoclonal antibody.

Description

technical field [0001] The present invention relates to anti-osteopontin OPN monoclonal antibody. Specifically, the present invention relates to a method for preparing natural osteopontin, using the produced natural osteopontin to prepare a hybridoma cell line producing anti-osteopontin monoclonal antibody, having the hybridoma cell line produce monoclonal antibody, and The use of the monoclonal antibody of the present invention in the preparation of medicines for treating diseases mediated by osteopontin and the preparation of kits for detecting osteopontin. Background technique [0002] Osteopontin (OPN) is an important functional protein in the extracellular matrix (ECM). The secreted glycosylated phosphoprotein has a relative molecular mass of about 40-75KDa. At first, it was believed that OPN was involved in bone resorption, which was related to the mineralization of bone tissue. Now it is found that OPN is widely distributed in human tissues and has multiple function...

Claims

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Application Information

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IPC IPC(8): C07K16/18C12N5/20C07K19/00G01N33/577A61K39/395A61K47/48A61P37/02A61P35/00C07K14/435C07K1/20C07K1/18A61K47/68
Inventor 孙兵曹刘丽田林季永镛朱静燕李炳南边超
Owner CENT FOR EXCELLENCE IN MOLECULAR CELL SCI CHINESE ACAD OF SCI
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