CN102680684A - Detection method for specific whole blood thymus (T) cells of tuberculosis antigens - Google Patents
Detection method for specific whole blood thymus (T) cells of tuberculosis antigens Download PDFInfo
- Publication number
- CN102680684A CN102680684A CN2012101836473A CN201210183647A CN102680684A CN 102680684 A CN102680684 A CN 102680684A CN 2012101836473 A CN2012101836473 A CN 2012101836473A CN 201210183647 A CN201210183647 A CN 201210183647A CN 102680684 A CN102680684 A CN 102680684A
- Authority
- CN
- China
- Prior art keywords
- antigen
- mhc
- specific
- tuberculosis
- whole blood
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of mycobacterium tuberculosis detection, in particular to a detection method for specific whole blood T cells of tuberculosis antigens. Mycobacterium tuberculosis specific epitope peptide, a major histocompatibility complex (MHC) donor and a subject periphery are subjected to whole blood incubation, and signals of a composite formed by MHC donor-mycobacterium tuberculosisepitope-specific T cells are detected directly. According to the detection method, early, fast and specific detection on tuberculosis patients and infected persons can be achieved, the whole detection can be finished in 2 to 3 hours, the requirements for operation staff and operation environment are low and particularly manual and automated operation under an aseptic condition is not required.
Description
[technical field]
The present invention relates to tubercle bacillus detection technique field, specifically a kind of whole blood T cell detection method of tuberculosis antigen specific.
[background technology]
Tuberculosis is that to infect what caused by Much's bacillus (Macobacteriumtubercuolosis) be main chronic infectious disease with the infection in respiratory system; Be still so far that single pathogen causes the communicable disease that death toll is maximum in the global range; Being public health and the social concern that the whole world is paid close attention to, also is that China is one of principal disease of emphasis control.About 10% can develop into active tuberculosis behind the m tuberculosis infection, and major part becomes symptomless infection person.Even initial tuberculosis infection is by after the successful control, still not breed or the dormant state of low propagation level is present in the host, this Infection Status is called the tuberculosis latent infection to some bacteriums, no clinical symptoms.Symptomless infection person (immune system hypoplasia, decline or immunosupress etc.) when immunocompetence is low then is prone to develop into active tuberculosis, is the people at highest risk among stealthy the infected.Therefore, can be in early days, detected activity property tuberculosis and the latent tuberculosis sufferer that infects (latent tuberculosis infection LTBI) exactly, with the effect that greatly affects global tuberculosis prophylaxis control.Therefore, seek quick, easy, effective, cheap detecting method is of practical significance very much.The method that auxiliary tuberculosis commonly used clinically detects; Tuberculin skin test (tuberculin skin test; TST) be exactly the method for utilizing the cellular immune function that detects m tuberculosis infection; Required expense is less, and is simple, and the history that is used for detection lungy is above 100 years.But; The applied Much's bacillus purified proteins of TST derivant (purified protein derivative of tuberculin) PPD is more than 200 kind of mixture of ingredients in the mycobacterium nutrient solution; This protein mixture is that Much's bacillus, Mycobacterium bovis BCG and most environment mycobacterium are common, thereby lack of specific.When the sensitivity specificity of the associated antibodies of direct detection Much's bacillus pathogen, Much's bacillus still can not satisfy requiring that tuberculosis detects, scientists want that the way through indirect detects the infection of Much's bacillus.The immune response that the tuberculosis mycomycete infects mainly is the cell immune response by the T cell mediated; Can produce the specificity sensitized T lymphocyte behind the organism infection Much's bacillus; Utilize the principle of this T lymphocyte, check individuality whether to infect Much's bacillus in external activation energy generation series of effects process.
Over nearly 100 years, be based on the check and analysis of IFN-γ in one of of paramount importance progress of tuberculosis context of detection.The principle of this method be when specific sensitized T lymphocyte once more exposure phase with antigen the time just be activated and secretion of gamma-IFN, detect through the amount that detects IFN-γ.1991; Wood etc. use enzyme linked immunosorbent assay (ELISA) or enzyme linked immunological spotting method (Enzyme-linked immunospot assay the earliest; ELISPOT), thereby 13091 oxen on farm are checked that the susceptibility of detection is 76.8% ~ 93.6% in the amount of vitro detection T lymphocyte specific secretion of gamma-IFN; Specificity is 96.2% ~ 98.1%, and is highly sensitive in TST65.6%.But this method still has following two aspects not enough: the one, and sense cycle is long, needs two day time, and the 2nd, complex operation, very high to operating personnel and operating environment requirement, be inappropriate for basic unit and promote.
[summary of the invention]
The present invention be directed to the difficulty and the weak point of above-mentioned detection method, propose a kind of whole blood T cell detection method of tuberculosis antigen specific.
To achieve these goals; The present invention designs a kind of whole blood T cell detection method of tuberculosis antigen specific; It is characterized in that the tubercle bacillus differential epitope peptide; MHC donor and person under inspection's periphery whole blood is hatched, and directly detects the signal of the compound of MHC donor-antigen of mycobacterium tuberculosis epi-position-specific T-cells formation.
This application process may further comprise the steps,
A. described tubercle bacillus differential epitope peptide is carried out mark, and hatch preparation mark epitope peptide-MHC donor compound with the MHC donor; Gather person under inspection's peripheric venous blood sample;
B. the compound of using step (a) to obtain stimulates the T lymphocyte in the peripheric venous blood, the 37 ℃ of static cultivation of degree incubator 0.5-16h,
C. collect and cultivate the T cell,
D. detect the signal of the compound of MHC donor-antigen of mycobacterium tuberculosis epi-position-specific T-cells formation.
This specific antigen epi-position has the Much's bacillus specificity, comprises and is not limited to the contained epi-position of one or more tubercle bacillus differential antigens.
The bacteria culture in described negre antigen source only limits to the different types of Much's bacillus and the bacterial isolates of being derived by these types.
Described tubercle bacillus differential antigen is that native antigen, recombinant antigen or peptide and other possibly be used to discern the material of Much's bacillus specific t-cell receptor.
Described negre antigen is antigen itself or antigen conjugate or derivant.
Described MHC donor comprises and is not limited to the cell of the natural MHC of containing, artificial constructed MHC monomer, MHC dimer, the MHC tetramer.
Described compound input comprises and is not limited to and can be used for the label that antigen specific T CR identification detects, the mark of CD3 through epitope peptide label, TCR label and other.
The present invention can realize tuberculosis patient and early stage, quick, the special detection of the infected, can accomplish in whole detection 2-3 hour, and is lower to operating personnel and operating environment requirement, particularly need not be in manual operation under the gnotobasis and automation mechanized operation.
The whole blood TCR of the tuberculosis antigen specific that the present invention proposes detects has following advantage: the one, and the time that detection needs is short, and is comparatively quick, needs 2-4 hour altogether.And tubercle bacillus cultivation detection takes time the 1-2 month, and the TST detection needs 3 days.The ELISPOT method needs 2 days.Because in a single day tubercle bacillus infects human body, at first will be discerned by the immune system of body, activate body fluid and cellullar immunologic response, therefore individual month of time of origin 1-2 after infection utilizes our cellular immunology detection method can make detection.The X-ray sheet detects only just can make detection after tangible pathological change appears in the infected lung, this needs long time usually.The antigen-antibody detection method also only detects under disease symptoms is in the situation of active stage, but mycobacterium extensively exists in the environment, the common antigen of itself and tubercle bacillus, but the testing result of interference experiment.The 2nd, special detection because what adopt is the antigen of tubercle bacillus specific, only with the mycobacterium tuberculosis infection human body after the immunological memory T cell that produces be identified.The 3rd, susceptibility is high, detects with TST and compares, and the susceptibility that the TCR of tuberculosis antigen specific detects is 97.2%, and specificity has good detection coincidence rate (k=0.70) up to 92.3%.Therefore, the present invention proposes the whole blood TCR detectable and the application process thereof of tuberculosis antigen specific, to control lungy with eliminate significant.
[embodiment]
Below in conjunction with specific embodiment, further illustrate the present invention.Should understand these embodiment and only be used to that the present invention is described rather than be used to limit scope of the present invention, this patent relates to epitope peptide and comprises and be not limited to epitope peptide in the following table.Unreceipted concrete implementation method in the following example, normally the instructions according to the kit of laboratory technique routine or market public offering carries out.
Behind the m tuberculosis infection human body, at first can be activated the T cellullar immunologic response of body, the effect of performance resisting tuberculosis infection by the immune system recognition of human body.Part T cell transformation is an immunological memory cell, after meeting with the negre antigen epitope peptide once more, is discerned by specificity TCR.If by the infected and patient's T cell recognition, but not mycobacterium tuberculosis infection person or non-tuberculosis patient T cell can not be identified the antigen that therefore adopts tubercle bacillus differential external, thereby realize the purpose of detection.
It is as shown in the table to relate to the epitope peptide combination among the embodiment:
| Numbering | Sequence |
| 1 | EISTNIRQAGVQYSR |
| 2 | LAQEAGNFERISGDLKTQID |
| 3 | MTEQQWNFAGIEAAASAIQG |
| 4 | ISEAGQAMASTEGNVTGMF |
| 5 | PSALLADHPDRIRWN |
| 6 | KPGQPESEL |
| 7 | LLHGSQGIRLHAPLP |
| 8 | PTREDQALIYRLSGD |
Embodiment 1
Fluidic cell with fluorescence labeling Much's bacillus specificity epitope peptide-MHC tetramer compound detects
Get the 1-6 epitope peptide, hatch mark with the FITC luciferin, epitope peptide-MHC tetramer compound is for use to hatch the preparation fluorescence labeling with the MHC tetramer again.
Gather the fresh anticoagulant heparin venous blood 3ml of the positive healthy population of tens of routine TST feminine genders and TST.Respectively get the 1ml whole blood, with the lymphocyte separation medium isolated lymphocytes, collect lymphocyte, place 24 orifice plates, 1 hole adds fluorescently-labeled Much's bacillus specificity epitope peptide-MHC tetramer compound and hatches 30min jointly.Other two holes add Much's bacillus epitope peptide (5 μ g/ml) or physiological saline respectively for contrast, behind the mixing 10min, hatch 2h for 37 ℃.200 μ L cell suspending liquids are collected in every hole, and flow cytometer detects.
Embodiment 2
Micro-image with fluorescence labeling Much's bacillus specificity epitope peptide-MHC dimer compound detects
Get the 3-8 epitope peptide, hatch mark with the FITC luciferin, epitope peptide-MHC dimer compound is for use to hatch the preparation fluorescence labeling with the MHC dimer again.
Gather the fresh anticoagulant heparin venous blood 3ml of the positive healthy population of tens of routine TST feminine genders and TST.Respectively get the 1ml whole blood, with the lymphocyte separation medium isolated lymphocytes, collect lymphocyte, place 24 orifice plates, 1 hole adds fluorescently-labeled Much's bacillus specificity epitope peptide-MHC dimer compound and hatches 30min jointly.Other two holes add Much's bacillus epitope peptide (5 μ g/ml) or physiological saline respectively for contrast, behind the mixing 10min, hatch 0.5h for 37 ℃.Directly bottom, every hole cell is carried out fluorescent microscopic imaging with inverted microscope, the cell with fluorescence is counted.。
Embodiment 3
ELIASA with fluorescence labeling Much's bacillus specificity epitope peptide-MHC tetramer compound detects
Get the 1-8 epitope peptide, hatch mark with the FITC luciferin, epitope peptide-MHC tetramer compound is for use to hatch the preparation fluorescence labeling with the MHC tetramer again.
Gather the fresh anticoagulant heparin venous blood 3ml of the positive healthy population of tens of routine TST feminine genders and TST.Respectively get the 1ml whole blood, with the lymphocyte separation medium isolated lymphocytes, collect lymphocyte, place 96 orifice plates that are coated with anti-T-cell antibody, 1 hole adds fluorescence labeling epitope peptide-MHC tetramer compound and hatches 30min jointly.Other two holes add Much's bacillus epitope peptide (5 μ g/ml) or physiological saline respectively for contrast, behind the mixing 10min, hatch 30min for 37 ℃.Relaxing the cleaning back carries out quantitatively with each hole fluorescent value of fluorescence microplate reader mensuration.
Embodiment 4
ELIASA with horseradish peroxidase-labeled Much's bacillus specificity epitope peptide-MHC tetramer compound detects
Get the 1-8 epitope peptide, hatch mark with the horseradish peroxidase of activation, epitope peptide-MHC tetramer compound is for use to hatch the preparation horseradish peroxidase-labeled with the MHC tetramer again.
Gather the fresh anticoagulant heparin venous blood 3ml of the positive healthy population of tens of routine TST feminine genders and TST.Respectively get the 1ml whole blood; With the lymphocyte separation medium isolated lymphocytes; Collect lymphocyte, place 96 orifice plates that are coated with anti-T-cell antibody, Much's bacillus specificity epitope peptide-MHC tetramer compound that 1 hole adds horseradish peroxidase-labeled is hatched jointly.Other two holes add Much's bacillus epitope peptide (5 μ g/ml) or physiological saline respectively for contrast, behind the mixing 10min, hatch 1h for 37 ℃.Clean according to the ELISA measurement operation, each hole adds tmb substrate reaction 30min, and each hole is measured each hole light absorption value and carried out quantitatively after adding reaction response liquid.
Embodiment 5
ELIASA with alkali phosphatase enzyme mark Much's bacillus specificity epitope peptide-MHC tetramer compound detects
Get the 2-8 epitope peptide, hatch mark with the alkaline phosphatase of activation, epitope peptide-MHC tetramer compound is for use to hatch the preparation alkali phosphatase enzyme mark with the MHC tetramer again.
Gather the fresh anticoagulant heparin venous blood 3ml of the positive healthy population of tens of routine TST feminine genders and TST.Respectively get the 1ml whole blood, with the lymphocyte separation medium isolated lymphocytes, collect lymphocyte, place 96 orifice plates that are coated with anti-T-cell antibody, Much's bacillus specificity epitope peptide-MHC tetramer compound that 1 hole adds alkali phosphatase enzyme mark is hatched jointly.Other two holes add Much's bacillus epitope peptide (5 μ g/ml) or physiological saline respectively for contrast, behind the mixing 10min, hatch 1h for 37 ℃.Clean according to the ELISA measurement operation, each hole adds para-nitro-pheneye phosphate substrate reactions 30min, and each hole is measured each hole light absorption value and carried out quantitatively after adding reaction response liquid.
The ELIASA of embodiment 6 usefulness Much's bacillus specificity epitope peptides and the anti-MHC antibody of mark detects
Gather the fresh anticoagulant heparin venous blood 3ml of the positive healthy population of tens of routine TST feminine genders and TST.Respectively get the 1ml whole blood, with the lymphocyte separation medium isolated lymphocytes, collect lymphocyte, place 96 orifice plates that are coated with anti-T-cell antibody, 1 hole adds gets 1-8 epitope peptide mixed liquor and hatches jointly.Other two holes add epitope peptide mixed liquor (5 μ g/ml) or physiological saline respectively for contrast, behind the mixing 10min, hatch 1h for 37 ℃.Clean according to the ELISA measurement operation, each hole adds fluorescently-labeled MHC antibody incubation 30min, after the cleaning, measures each hole fluorescent value with ELIASA and carries out quantitatively.
Those skilled in the art can make various changes and technology modification to the present invention, and these equivalent form of values fall within claims institute restricted portion of the application equally.
Claims (8)
1. the whole blood T cell detection method of a tuberculosis antigen specific; It is characterized in that the tubercle bacillus differential epitope peptide; MHC donor and person under inspection's periphery whole blood is hatched, and directly detects the signal of the compound of MHC donor-antigen of mycobacterium tuberculosis epi-position-specific T-cells formation.
2. the whole blood T cell detection method of tuberculosis antigen specific as claimed in claim 1 is characterized in that this application process may further comprise the steps,
A. described tubercle bacillus differential epitope peptide is carried out mark, and hatch preparation mark epitope peptide-MHC donor compound with the MHC donor; Gather person under inspection's peripheric venous blood sample;
B. the compound of using step (a) to obtain stimulates the T lymphocyte in the peripheric venous blood, the 37 ℃ of static cultivation of degree incubator 0.5-16h,
C. collect and cultivate the T cell,
D. detect the signal of the compound of MHC donor-antigen of mycobacterium tuberculosis epi-position-specific T-cells formation.
3. according to claim 1 or claim 2 the whole blood T cell detection method of tuberculosis antigen specific is characterized in that this specific antigen epi-position has the Much's bacillus specificity, comprises and is not limited to the contained epi-position of one or more tubercle bacillus differential antigens.
4. according to claim 1 or claim 2 the whole blood T cell detection method of tuberculosis antigen specific is characterized in that the bacteria culture in described negre antigen source only limits to the different types of Much's bacillus and the bacterial isolates of being derived by these types.
5. according to claim 1 or claim 2 the whole blood T cell detection method of tuberculosis antigen specific is characterized in that described tubercle bacillus differential antigen is that native antigen, recombinant antigen or peptide and other possibly be used to discern the material of Much's bacillus specific t-cell receptor.
6. according to claim 1 or claim 2 the whole blood T cell detection method of tuberculosis antigen specific is characterized in that described negre antigen is antigen itself or antigen conjugate or derivant.
7. according to claim 1 or claim 2 the whole blood T cell detection method of tuberculosis antigen specific is characterized in that described MHC donor comprises and be not limited to the cell of the natural MHC of containing, artificial constructed MHC monomer, MHC dimer, the MHC tetramer.
8. according to claim 1 or claim 2 the whole blood T cell detection method of tuberculosis antigen specific is characterized in that described compound input comprises and is not limited to and can be used for the label that antigen specific T CR identification detects, the mark of CD3 through epitope peptide label, TCR label and other.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210183647.3A CN102680684B (en) | 2012-06-06 | 2012-06-06 | Detection method for specific whole blood thymus (T) cells of tuberculosis antigens |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210183647.3A CN102680684B (en) | 2012-06-06 | 2012-06-06 | Detection method for specific whole blood thymus (T) cells of tuberculosis antigens |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102680684A true CN102680684A (en) | 2012-09-19 |
| CN102680684B CN102680684B (en) | 2014-12-10 |
Family
ID=46812928
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210183647.3A Active CN102680684B (en) | 2012-06-06 | 2012-06-06 | Detection method for specific whole blood thymus (T) cells of tuberculosis antigens |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102680684B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH04505401A (en) * | 1990-02-14 | 1992-09-24 | アンステイテユ・ナシオナル・ドウ・ラ・サンテ・エ・ドウ・ラ・ルシエルシユ・メデイカル | Monoclonal antibody that recognizes peptides associated with major histocompatibility antigen |
| WO2006082391A1 (en) * | 2005-02-01 | 2006-08-10 | Queen Mary & Westfield College | Method for the detection of activated t-cells using antigenic peptide- loaded microsomes |
| CN101638430A (en) * | 2009-07-10 | 2010-02-03 | 郑州大学 | Anti-tuberculosis CTL epitope peptide |
| CN102243233A (en) * | 2010-05-12 | 2011-11-16 | 张舒林 | Immunological detection method and kit for detecting mycobacterium tuberculosis |
| CN102305855A (en) * | 2011-05-19 | 2012-01-04 | 中山大学 | Reagent and method for detecting Mycobacterium tuberculosis infection in vitro |
-
2012
- 2012-06-06 CN CN201210183647.3A patent/CN102680684B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH04505401A (en) * | 1990-02-14 | 1992-09-24 | アンステイテユ・ナシオナル・ドウ・ラ・サンテ・エ・ドウ・ラ・ルシエルシユ・メデイカル | Monoclonal antibody that recognizes peptides associated with major histocompatibility antigen |
| WO2006082391A1 (en) * | 2005-02-01 | 2006-08-10 | Queen Mary & Westfield College | Method for the detection of activated t-cells using antigenic peptide- loaded microsomes |
| CN101638430A (en) * | 2009-07-10 | 2010-02-03 | 郑州大学 | Anti-tuberculosis CTL epitope peptide |
| CN102243233A (en) * | 2010-05-12 | 2011-11-16 | 张舒林 | Immunological detection method and kit for detecting mycobacterium tuberculosis |
| CN102305855A (en) * | 2011-05-19 | 2012-01-04 | 中山大学 | Reagent and method for detecting Mycobacterium tuberculosis infection in vitro |
Non-Patent Citations (1)
| Title |
|---|
| 徐苗等: "结核病免疫研究进展", 《国外医学.内科学分册》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102680684B (en) | 2014-12-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Cossarizza et al. | SARS‐CoV‐2, the virus that causes COVID‐19: cytometry and the new challenge for global health | |
| CN102004155B (en) | Kit and method for detecting mycobacterium tuberculosis infection and application | |
| CN102175875B (en) | Detection kit for diagnosing tuberculosis | |
| CN104628833B (en) | A kind of tuberculosis infection cellular immunization detectable antigens composition and application thereof | |
| CN101221173A (en) | ELISpot tuberculosis infection diagnostic reagent kit and its application | |
| CN104020297B (en) | Test kit for m tuberculosis infection detection and clinical therapeutic efficacy monitoring and application thereof | |
| CN103604933A (en) | Kit for detecting active tuberculosis based on antigen-specific TNF-alpha-ELISA (enzyme linked immunosorbent assay) and application thereof | |
| Ndikuyeze et al. | Immunogenicity and safety of measles vaccine in III african children | |
| Gupta et al. | Prevalence and trends of transfusion transmitted infections in a regional blood transfusion centre | |
| US6630316B1 (en) | Method for measurement of lymphocyte function | |
| Zavaglio et al. | Robust and persistent B-and T-cell responses after COVID-19 in immunocompetent and solid organ transplant recipient patients | |
| Brunner et al. | More sensitive methods for detection of antibody to Mycoplasma pneumoniae | |
| CN103063836B (en) | Detect the reagent of mycobacterial infections, method and kit | |
| CN101493454A (en) | Tuberculosis antigen specific whole blood IFN-gamma diagnosis kit, method for producing the same and method for using same | |
| CN106053783A (en) | Quick time-resolved fluorescence immunoassay kit for detection of T cells infected with tuberculosis and detection method of kit | |
| Wang et al. | The source of Mycobacterium tuberculosis-specific IFN-γ production in peripheral blood mononuclear cells of TB patients | |
| CN106939035A (en) | A kind of mycobacterium tuberculosis T cell antigen epitope polypeptide and its application | |
| CN101492497A (en) | Expression purification process for tubercle bacillus differential recombinant protein and uses thereof | |
| CN106405107B (en) | Antigen of mycobacterium tuberculosis albumen Rv2941 and its t cell epitope peptide application | |
| CN106198971A (en) | The application of antigen of mycobacterium tuberculosis albumen Rv2351c | |
| CN102898513A (en) | ELISpot optical neuromyelitis diagnostic kit and application thereof | |
| CN102680684B (en) | Detection method for specific whole blood thymus (T) cells of tuberculosis antigens | |
| CN104628834B (en) | A kind of tuberculosis infection T cell immunodetection antigen and application thereof | |
| CN102062774B (en) | Kit for detecting tuberculosis treatment effect | |
| US9678071B2 (en) | Detecting latent tuberculosis infections |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |