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Reagent for detecting specific immune response of mycobacterium tuberculosis and use thereof

A technology for mycobacterium tuberculosis and immune response, applied in the field of biomedical testing, can solve problems such as false positives, unsatisfactory results, and poor detection value, and achieve the effects of simple operation, easy promotion, and improved detection sensitivity and specificity

Active Publication Date: 2014-05-21
SHANGHAI JIAOTONG UNIV SCHOOL OF MEDICINE
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0004] However, existing reports indicate that genes such as ESAT-6, CFP-10, and TB7.7 are mainly expressed in the early infection stage of Mycobacterium tuberculosis, and that M. kansasii, M. szulgai, M. marinum or M.gordonae, etc. also express ESAT-6, CFP-10 or TB7.7, so when patients are infected with such bacteria, there will be false positive results
Moreover, existing literature has shown that there are relatively serious non-tuberculous mycobacteria infections in the Chinese population, so the effect of T-spot.TB and QuantiFERON (QFT)-IT in the diagnosis and application of tuberculosis in my country is not ideal, which is manifested in the sensitivity of its detection and specificity between 80% and 90%, and the detection value of these two kits after the clinical prognosis of tuberculosis is poor

Method used

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  • Reagent for detecting specific immune response of mycobacterium tuberculosis and use thereof
  • Reagent for detecting specific immune response of mycobacterium tuberculosis and use thereof
  • Reagent for detecting specific immune response of mycobacterium tuberculosis and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Preparation of Mycobacterium tuberculosis Rv0733 polypeptide and antigen.

[0054] 1. The experimental steps for the preparation of the Rv0733 fusion protein (antigen) are as follows:

[0055] 1. The Rv0733 gene was cloned from the DNA of Mycobacterium tuberculosis by PCR. After sequencing, the gene fragment was inserted into the E. coli expression plasmid pET28a to construct the pET28a-Rv0733 prokaryotic expression plasmid.

[0056] 2. The pET28a-Rv0733 prokaryotic expression plasmid was transformed into E.coli BL21 (DE3) to construct expression engineering bacteria.

[0057] 3. Induce the expression with 0.4mM IPTG at 30℃, 220rpm for 3h.

[0058] 4. Lysate the bacteria by ultrasonic and collect the supernatant protein solution after centrifugation.

[0059] 5. Purify the Rv0733 fusion protein with Ni-NTA magnetic beads.

[0060] 6. The concentration of fusion protein was detected by BCA (Pierce, 23227) method, and stored in -80 ℃ refrigerator.

[0061] ...

Embodiment 2

[0082] Example 2: ELISPOT detection of the number of specific gamma interferon-releasing cells stimulated by Rv0733 and its antigenic peptides

[0083] The experimental steps are as follows:

[0084] 1. Coat 96-well PVDF plate (Millipore MSIPS4510) for ELISPOT with IFN-γ antibody, overnight at 4°C.

[0085] 2. Wash 3 times with 200 μl per well of PBS or RPMI1640 and block with RPMI1640 containing 10% FBS for 1 hour at 37°C.

[0086] 3. About 5 ml of heparin sodium, sodium citrate or dipotassium EDTA anticoagulant tubes are collected from the periphery of tuberculosis patients or normal controls.

[0087] 4. Use Ficoll density gradient centrifugation, 1000×g, 18-22℃, centrifuge for 22min. Peripheral blood mononuclear cells (PBMC) were isolated.

[0088] 5. PBMCs were washed twice with 5-10ml PBS or RPMI1640 and resuspended in RPMI1640 containing 10% FBS, counted, and adjusted to a final concentration of 2.5×10 6 cells / ml.

[0089] 6. Add 100 μl of cell suspension to each w...

Embodiment 3

[0097] Example 3: Detection of Rv0733-specific antibody levels in peripheral blood of tuberculosis patients and normal control populations by ELISA.

[0098] The experimental steps are as follows:

[0099] 1. Coat the ELISA 96-well plate (Corning9018) with 1 μg / ml Rv0733 fusion protein, overnight at 4°C.

[0100] 2. Discard the supernatant, wash 5 times with 300 μl of PBS containing 0.05% Tween-20 per well, and pat dry each time on absorbent paper.

[0101] 3. Block with PBS containing 1% gelatin at 37°C for 1 hour.

[0102] 4. Wash 5 times with 300 μl of PBS (PBST) containing 0.05% Tween-20 per well. Add 100 μl of peripheral blood plasma from tuberculosis patients or normal persons diluted 1:100 to each well, and the dilution solution is PBST containing 1% gelatin. Incubate for 1 hour at 37°C.

[0103] 5. Wash 6 times with 300 μl of PBS containing 0.05% Tween-20 per well. Add 100 μl of HRP-labeled anti-human IgG antibody diluted with 1% gelatin in PBST to each well, and ...

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Abstract

The invention discloses a reagent for detecting specific immune response of mycobacterium tuberculosis and use thereof. The invention discloses an immune positive reagent Rv0733 of mycobacterium tuberculosis by a gamma interferon release assay and a serum enzyme-linked immune method. The reagent comprises antigen or polypeptide and analogues thereof for detecting the specific cellular immunity of a tuberculosis sufferer and humoral immune response level. After the mice are immunized by using the reagent disclosed by the invention, the mice can generate specific cell factors such as gamma interferon and the like, and an antibody. T cells of tuberculosis patients or normal people are stimulated by using the reagent in vitro, the quantity of gamma interferon release cells is detected, or the content of gamma interferon in supernatant is cultivated, the tuberculosis patients can be distinguished from the normal people, the detection sensitivity and specificity of the tuberculosis sufferer can be improved by detecting the specific antibody level of resisting the reagent in the peripheral serum, and the clinical prognosis conditions of the tuberculosis patients can be disclosed by utilizing an Rv0733 specific immune response contrast experiment in the process of tracking treatment of the tuberculosis sufferer.

Description

technical field [0001] The invention belongs to the field of biomedical testing, and in particular relates to a reagent for detecting the specific immune response of Mycobacterium tuberculosis and use thereof. Background technique [0002] Tuberculosis is one of the three major infectious diseases in the world. In 2011, WHO estimated that the total number of tuberculosis cases in China was 1.4 million, accounting for 11.67% of the world's 12 million total cases, second only to India's 3.1 million (25.83%), ranking first in the world. It ranks second in the world, with 912,000 new cases and 47,000 deaths. Tuberculosis has caused a huge economic and social burden in our country. [0003] With the deepening of research, the clinical diagnosis methods of tuberculosis have been greatly developed, but there are still some deficiencies in the specificity and sensitivity of diagnosis, which need to be improved. Immunological detection method is one of the important auxiliary metho...

Claims

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Application Information

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IPC IPC(8): C07K19/00G01N33/569
Inventor 肖洋炯王颖沈浩王慧宇田兆峰赖允鑫
Owner SHANGHAI JIAOTONG UNIV SCHOOL OF MEDICINE
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