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Monoclonal antibody TBCD6 resistant to mycobacterium tuberculosis CFP-10 and application

A monoclonal antibody and Mycobacterium tuberculosis technology, applied in anti-bacterial immunoglobulin, biochemical equipment and methods, instruments, etc., can solve the problems of difficult promotion, expensive reagents, cumbersome ELISPOT detection steps, etc.

Inactive Publication Date: 2011-03-16
ZHEJIANG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, ELISPOT detection steps are cumbersome, require special equipment and expensive reagents, and are difficult to promote in most low-income countries and regions with a high burden of tuberculosis

Method used

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  • Monoclonal antibody TBCD6 resistant to mycobacterium tuberculosis CFP-10 and application
  • Monoclonal antibody TBCD6 resistant to mycobacterium tuberculosis CFP-10 and application
  • Monoclonal antibody TBCD6 resistant to mycobacterium tuberculosis CFP-10 and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1. Preparation method of monoclonal antibody against CFP-10

[0029] (1) Immunization of mice: For the first immunization, emulsify the 14 peptide (sequence aa23-36GDLKTQIDQVESTA) of cross-linked KLH CFP-10 with Freund's complete adjuvant, and emulsify with incomplete adjuvant for the second and third times, each BALB / C mouse 0.2ml (containing CFP-10 antigenic peptide 200μg), multi-point intradermal injection on the back, once every two weeks. A booster immunization was given 6 weeks later, and cell fusion was carried out 3 days later.

[0030] (2) Cultivation of mouse myeloma cell SP2 / 0: the SP2 / 0 myeloma cell line from BALB / C mice was cultured and subcultured in 10% FBS-DMEM medium, in 5% CO 2 Cultured in a humidified 37°C incubator. Subculture the day before fusion to ensure that the cells enter the logarithmic growth phase at the time of fusion.

[0031] (3) Cell fusion: use BALB / C mouse peritoneal macrophages as feeder cells, inoculate BALB / C mouse perito...

Embodiment 2

[0038] Embodiment 2. Carry out the identification (qualitative) detection of CFP-10 with this monoclonal antibody

[0039] The anti-CFP-10 monoclonal antibody prepared by the present invention can be used for identification (qualitative detection), and the identification method can be realized by the following two methods:

[0040] 1. Western Blotting: The recombinant CFP-10 and ESAT-6 / CFP-10 fusion protein and Mycobacterium tuberculosis whole protein lysate were detected by Western Blotting with this monoclonal antibody, and the target band appeared at the corresponding position, indicating that the detection to the expression of CFP-10 protein. Specific steps:

[0041] (1) SDS polyacrylamide gel electrophoresis: For the method, refer to F. Osper et al., "Refined Molecular Biology Experiment Guide" (Science Press, 1998). Using 15% separating gel and 5% stacking gel, the electrophoresis condition is a voltage of 150V, and the bromophenol blue dye band is about 1.5 cm away fr...

Embodiment 3

[0056] Embodiment 3, carry out quantitative detection of CFP-10 molecule with this monoclonal antibody

[0057] The anti-CFP-10 monoclonal antibody prepared by the present invention can be used to quantitatively detect recombinant CFP-10 and ESAT-6 / CFP-10 fusion protein, Mycobacterium tuberculosis culture supernatant, and various clinical body fluid samples in tuberculosis specific antigen CFP -10 level, the detection method can be realized by the following two methods:

[0058] 1. Indirect ELISA method:

[0059]① Dilute the test sample appropriately in the coating solution of 0.01M pH9.6 carbonate buffer solution, and at the same time dilute the recombinant CFP-10 and ESAT-6 / CFP-10 fusion protein in appropriate serial concentrations in the coating solution, as Quantitative standard control. Add 100ul of the above-mentioned samples to be tested and standard products of serial concentrations to the corresponding microplate wells, incubate at room temperature for 2h or coat ov...

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Abstract

The invention provides a monoclonal antibody TBCD6 resistant to mycobacterium tuberculosis culture filter protein (CFP) 10. A preparation method of the monoclonal antibody is characterized by immunizing the mice with the specific antigenic peptide, collecting sensitized B lymphocyte of the mice to undergo fusion with the myeloma cells of the mice, judging positive clones by utilizing enzyme-linked immunosorbent assay (ELISA) and western blotting, building a hybridoma cell system TBCD6 which secretes the monoclonal antibody, amplifying, culturing and injecting the positive cells to the peritoneal cavities of the mice of the same strain, using the mice to induce ascites containing the antibody and obtaining the monoclonal antibody through collection and affinity purification of the antibody. The invention provides application of the monoclonal antibody to detection and quantitative analysis of the mycobacterium tuberculosis CFP 10.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to the preparation and application of a monoclonal antibody against mycobacterium tuberculosis culture filtrate protein 10 (CFP-10), and uses cell engineering and antibody engineering techniques to obtain and secrete a monoclonal antibody against CFP-10 The hybridoma cell line of the same strain was used to induce ascites from the same strain of mice to prepare the anti-CFP-10 monoclonal antibody TBCD6, which was identified as IgM and κ type, and then the application of the antibody was realized through affinity purification, electrophoresis, immunization and other technologies . Background technique [0002] Early diagnosis of tuberculosis is an important problem that has been plaguing the field of medical diagnosis. In the 20th century, due to the development and wide application of antibiotic research, humans successfully controlled tuberculosis. However, due to the continuous emerge...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/12C12N5/20G01N33/577G01N33/569C12R1/91
Inventor 姚航平卢洪洲
Owner ZHEJIANG UNIV
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