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Monoclonal antibody TBEF3 of tubercle bacillus-resistant ESAT-6 and application thereof

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, cumbersome ELISPOT detection steps, expensive reagents, 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 TBEF3 of tubercle bacillus-resistant ESAT-6 and application thereof
  • Monoclonal antibody TBEF3 of tubercle bacillus-resistant ESAT-6 and application thereof
  • Monoclonal antibody TBEF3 of tubercle bacillus-resistant ESAT-6 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1. Preparation method of monoclonal antibody against ESAT-6

[0029] (1) Immunization of mice: For the first immunization, emulsify the 14 peptides (sequence aa1-14MTEQQWNFAGIEAA) of cross-linked KLH ESAT-6 with Freund's complete adjuvant, and emulsify with incomplete adjuvant for the second and third times, each BALB / C mouse 0.2ml (containing ESAT-6 antigen 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 periton...

Embodiment 2

[0038] Embodiment 2. carry out the identification (qualitative) detection of ESAT-6 with this monoclonal antibody

[0039] The anti-ESAT-6 monoclonal antibody prepared by the present invention can be used for identification (qualitative detection), and identification method can be realized by following two methods:

[0040] 1. Western Blotting: The recombinant ESAT-6 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 ESAT-6 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 from the b...

Embodiment 3

[0056] Embodiment 3, carry out the quantitative detection of ESAT-6 molecule with this monoclonal antibody

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

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Abstract

The invention provides a monoclonal antibody TBEF3 of tubercle bacillus-resistant secretory antigen target protein in early stage. The monoclonal antibody TBEF3 is prepared by the following steps: selecting a specific antigen peptide mouse; collecting sensitized mouse B lymphocyte and mouse myeloma cell to fuse; judging positive clones by using an enzyme linked immunosorbent assayelisa and an immunoblotting testing method; establishing a hybridoma cell line TBEF3 for secreting monoclonal antibody of tubercle bacillus-resistant secretory ESAT-6 in early stage; carrying out multiplication culturing on the positive cells and injecting to congenic mouse enterocoelia for induction of generation of ascitic fluid containing antibody; and carrying out collection and antibody affinity purification to obtain the monoclonal antibody TBEF3 of tubercle bacillus-resistant secretory antigen target protein in early stage. The invention also provides an application of the monoclonal antibody in testing the secretory antigen target protein of mycobacterium tuberculosis in early stage.

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 early secretory antigen target protein (ESAT-6). Clone the hybridoma cell line of the antibody, induce ascites from the same strain of mice, prepare the anti-EAST-6 monoclonal antibody TBEF3, identify it as IgM, κ type, and then realize the antibody through affinity purification, electrophoresis, immunization and other technologies Applications. 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 emergence and accumulation of drug resistance, Mycobacterium tuberculosis is still a real threat to human beings in the 21st century. More seriously,...

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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