CN111443208B - Composition for distinguishing active tuberculosis from latent tuberculosis - Google Patents
Composition for distinguishing active tuberculosis from latent tuberculosis Download PDFInfo
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Abstract
The invention relates to the fields of molecular biology and immunology, in particular to application of Rv1733c in preparing a kit for identifying active tuberculosis and latent tuberculosis infection. The invention uses a fluorescence immune spot method to detect the specific Th1 cell immune response caused by the stimulus of the mycobacterium tuberculosis latency related antigen Rv1733c and identify active tuberculosis and latency tuberculosis infection. Experiments show that the number of T cell spots of IL-2 generated after the stimulation of a novel latent infection related antigen Rv1733c has a certain value for identifying active tuberculosis and latent tuberculosis infection, and can improve the identification accuracy.
Description
Technical Field
The invention relates to the fields of molecular biology and immunology, in particular to application of Rv1733c in preparing a kit for identifying active tuberculosis and latent tuberculosis infection.
Background
Tuberculosis (TB) is a serious threat to human health. About 1/3 of the population worldwide is currently potentially infected with mycobacterium tuberculosis (Mycobacterium tuberculosis, mtb), of which 5% to 10% are likely to develop tuberculosis. When the immunity of the latent infected person is reduced, MTB in the body is activated, proliferated and disseminated, and finally the latent infected person develops into active tuberculosis.
Mycobacterium tuberculosis exists mainly in dormant bacteria state during latent infection. Mycobacterium tuberculosis dormant bacteria specifically transcribe and express a group of dormancy related antigens regulated by dormancy modulators in order to adapt to host environment and evade organism immunity. Expression of dormancy-associated antigens is important for latent infection and persistent survival of Mycobacterium tuberculosis in vivo. Wherein the dormancy related antigen Rv1733c is a protein with higher peripheral blood T lymphocyte recognition rate in people with Mtb latent infection, and the protein can effectively stimulate T lymphocytes to secrete IFN-gamma. Rv1733c is currently used more for the preparation of tuberculosis-related vaccines, as in patent application CN108239660a; WO2012/210018A1; WO2006/104389A1.
At present, it is difficult to rapidly and accurately distinguish tuberculosis (active Tuberculosis, ATB) from latent tuberculosis infection (Latent TB infection, LTBI) in clinical diagnosis and detection. T-SPOT.TB tests such as ELISPOT technology have been widely used clinically to diagnose tuberculosis infection, but they fail to differentially diagnose active tuberculosis and latent tuberculosis infection. The main reasons include 1) that the antigen used for T-SPOT.TB test is only associated with tuberculous virulence; 2) Only secretion of the single effector cytokine IFN- γ was detected. Because of the ELISPOT technology, the detection of multiple factors by adopting a chemical color development method can not be recognized due to spot color mixing, and the clinical application of the ELISPOT technology is affected.
Fluorescent immunospot (FluoSpot) is currently used to identify active and inactive tuberculosis (Lifan Zhang; application of IFN-gamma/IL-2 FluoroSpot assay for distinguishing active tuberculosis from non-active tuberculosis: A method student; clinica Chimica Acta 499 (2019) 64-69), but uses stimulation sources that are ESAT-6 antigen and CFP-10 antigen, and the sensitivity and specificity are not ideal. Therefore, there is a need for a kit and method that can accurately and sensitively identify active tuberculosis and latent tuberculosis infection.
Disclosure of Invention
In order to improve the sensitivity and specificity of identification of active tuberculosis and latent tuberculosis infection, the invention aims at detecting specific Th1 cell immune response caused by stimulation of mycobacterium tuberculosis latent related antigen Rv1733c by using a fluorescence immune spot method (FluoSpot) to identify ATB and LTBI. Experiments show that the spot frequency of the T cells which generate IL-2 after being stimulated by the novel latent infection related antigen Rv1733c has a certain value for identifying LTBI and ATB, can assist in identification and improves the identification accuracy.
In particular, the present invention relates to a composition comprising an Rv1733c antigen, an ESAT-6 antigen and a CFP-10 antigen; the composition is useful for diagnosing tuberculosis; further, it is used to identify active tuberculosis and latent tuberculosis infection.
Furthermore, the composition can also contain IFN-gamma and IL-2 monoclonal antibodies; may also contain the main reagents used in the fluorescent immunoblots; the reagent can be anti-IFN-gamma and anti-IL-2 monoclonal antibodies, and/or fluorescein labeled monoclonal antibodies IFN-gamma-FITC and IL-2-biotin, and/or secondary antibodies anti-FITC-490 and SA-550, etc.
The invention relates to a kit for diagnosing tuberculosis, which contains an Rv1733c antigen; further, the kit can also contain ESAT-6 antigen and CFP-10 antigen; the antigen is a peptide fragment antigen; furthermore, the kit also contains IFN-gamma and IL-2 monoclonal antibodies.
The invention also relates to a kit for identifying active tuberculosis and latent tuberculosis infection, wherein the kit contains an Rv1733c antigen; further, the kit can also contain ESAT-6 antigen and CFP-10 antigen; the antigen is a peptide fragment antigen; furthermore, the kit can also contain IFN-gamma, IL-2 monoclonal antibodies and the like.
The invention relates to a kit for diagnosing tuberculosis, which contains an Rv1733c antigen and a main reagent used by a fluorescence immune spot method; the reagent comprises anti-IFN-gamma and anti-IL-2 monoclonal antibodies, and/or fluorescein labeled monoclonal antibodies IFN-gamma-FITC and IL-2-biotin, and/or secondary antibodies anti-FITC-490 and SA-550.
The invention relates to a kit for distinguishing active tuberculosis from latent tuberculosis, which contains an Rv1733c antigen and a main reagent used by a fluorescence immune spot method; the reagent comprises anti-IFN-gamma and anti-IL-2 monoclonal antibodies, and/or fluorescein labeled monoclonal antibodies IFN-gamma-FITC and IL-2-biotin, and/or secondary antibodies anti-FITC-490 and SA-550.
The invention also relates to application of the Rv1733c in preparing a kit for diagnosing tuberculosis, and the kit further comprises ESAT-6 antigen and CFP-10 antigen.
The invention also relates to application of the Rv1733c in preparing a kit for identifying active tuberculosis and latent tuberculosis infection, and the kit further comprises ESAT-6 antigen and CFP-10 antigen.
The invention also relates to the use of the combination of the Rv1733c antigen, ESAT-6 antigen and CFP-10 antigen for the preparation of a kit for the identification of active tuberculosis and latent tuberculosis infection.
Further, the Rv1733c antigen is an Rv1733c SLP peptide fragment library. SLP is synthetic long peptide (Synthetic long peptides).
Further, each peptide fragment of the Rv1733c SLP peptide fragment library is 28 amino acids long, and 14 amino acids are overlapped at both ends.
Further, the Rv1733c SLP peptide fragment library is the following peptide fragments or combinations thereof:
Rv1733c p1-28 MIATTRDREGATMITFRLRLPCRTILRV；
Rv1733c p16-43 FRLRLPCRTILRVFSRNPLVRGTDRLEA；
Rv1733c p29-56 FSRNPLVRGTDRLEAVVMLLAVTVSLLT；
Rv1733c p43-70 AVVMLLAVTVSLLTIPFAAAAGTAVQDS；
Rv1733c p57-84 IPFAAAAGTAVQDSRSHVYAHQAQTRHP；
Rv1733c p71-98 RSHVYAHQAQTRHPATATVIDHEGVIDS；
Rv1733c p85-112 ATATVIDHEGVIDSNTTATSAPPRTK IT；
Rv1733c p99-126 NTTATSAPPRTKITVPARWVVNGIERSG；
Rv1733c p113-140 VPARWVVNGIERSGEVNAKPGTKSGDRV；
Rv1733c p125-152 SGEVNAKPGTKSGDRVGIWVDSAGQLVD；
Rv1733c p141-168 GIWVDSAGQLVDEPAPPARAIADAALAA；
Rv1733c p169-196 LGLWLSVAAVAGALLALTRAILIRVRNA。
further, the Rv1733c SLP peptide fragment library may also be a combination of any peptide fragment in the Rv1733c SLP peptide fragment library in the prior art or a variant thereof.
Further, the ESAT-6 antigen is ESAT-6 peptide fragment library.
Further, the CFP-10 antigen is a CFP-10 peptide fragment library.
The invention also relates to a method for identifying active tuberculosis and latent tuberculosis infection, which comprises the steps of sample collection, pre-coating, antigen stimulation and incubation detection.
The sample collection step comprises the following steps: venous blood is collected, peripheral blood mononuclear cells are obtained through separation, and AIM-V culture medium is used for preparing cell suspension.
The pre-coating step comprises the following steps: the bottom of the reaction plate is pre-coated with anti-IFN-gamma and anti-IL-2 monoclonal antibodies; AIM-V cell culture broth was used as a blank; phytohemagglutinin was used as a positive control.
The antigen stimulation step comprises: adding ESAT-6 peptide fragment library, CFP-10 peptide fragment library and Rv1733c peptide fragment library; peripheral blood mononuclear cells and anti-CD28 were added.
The incubation detection step comprises: incubating; adding fluorescein labeled monoclonal antibody IFN-gamma-FITC and IL-2-biotin; incubating for 2 hours at the room temperature after light-shielding drying; adding secondary antibodies anti-FITC-490 and SA-550; incubating for 1h at the room temperature after light-shielding drying; specific T cells secreting IL-2 were counted.
The invention has the following advantages:
firstly, the invention adopts a fluorescence immune spot method: IFN-gamma/IL-2 immunofluorescence spot method is used to detect secretion of two cytokines of IFN-gamma and IL-2 at single cell level, and joint diagnosis is performed, so that manpower and blood sample are saved to the greatest extent.
Secondly, the invention adopts novel latent infection related antigen, selects a representative latent tuberculosis infection related antigen Rv1733c, completes the synthesis of an antigen peptide fragment library through clone expression and purification, is used as a stimulus antigen sensitized T lymphocyte to generate cytokines, and finally proves that the sensitivity and specificity of differential diagnosis ATB and LTBI can be improved by detecting the difference of specific T cell numbers of the mycobacterium tuberculosis secreting intracellular factors IFN-gamma and IL-2 on the basis of ESAT-6& CFP-10fluoroSpot and jointly using the T cell spot frequency of single secretion IL-2 after the stimulus of the latent tuberculosis related antigen Rv1733 c.
Drawings
FIG. 1T cell spot frequency of monosecreteing IL-2 following RV1733SLP antigen stimulation;
FIG. 2T cell spot frequency of single secretion IFN-gamma following ESAT-6& CFP-10 antigen stimulation;
FIG. 3T cell spot ratio of single secretion IFN-. Gamma.following ESAT-6& CFP-10 antigen stimulation;
FIG. 4T cell spot frequency of single secreted IL-2 after stimulation with Rv1733c SLP antigen in combination with ESAT-6& CFP-10
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art, and the reagents used are commercially available.
Embodiment one: sample collection
Active tuberculosis patients with confirmed pathogen diagnosis and asymptomatic latent tuberculosis patients are included, and the following are screening criteria for pathogen samples.
1. The inclusion criteria for active tuberculosis groups were:
1) Age 18-75 years old;
2) Active tuberculosis manifestations such as fever, cough and the like;
3) Acid fast staining or culture positive of MTB smear, or MTB nucleic acid, or Xpert MTB/RIF detection positive;
4) Is not treated by anti-tuberculosis treatment.
2. The inclusion criteria for the group of latent tuberculosis infection were:
1) Age 18-75 years old;
2) No active tuberculosis such as fever, cough and the like;
3) No history of tuberculosis and no change of old tuberculosis in chest film;
4) T-SPOT. TB positive.
Exclusion criteria: 1) Gestation or lactation; 2) HIV antibody positive.
Embodiment two: latent antigen peptide fragment library synthesis
Referring to the sequence of Rv1733c SLP (Synthetic Long Peptide) peptide fragment library in the prior art (copola et al; synthetic Long Peptide Derived from Mycobacterium tuberculosis Latency Antigen Rv1733c Protects against Tuberculosis; clin Vaccine immunol.20150ep; 22 (9): 1060-9), clone expression and purification are completed to obtain the Mycobacterium tuberculosis hypoxia-related latency antigen Rv1733c SLP peptide fragment library, each peptide fragment is 28 amino acids long and 14 amino acids are overlapped at both ends. The following Rv1733c SLP peptide fragment library was synthesized:
Rv1733c p1-28 MIATTRDREGATMITFRLRLPCRTILRV；
Rv1733c p16-43 FRLRLPCRTILRVFSRNPLVRGTDRLEA；
Rv1733c p29-56 FSRNPLVRGTDRLEAVVMLLAVTVSLLT；
Rv1733c p43-70 AVVMLLAVTVSLLTIPFAAAAGTAVQDS；
Rv1733c p57-84 IPFAAAAGTAVQDSRSHVYAHQAQTRHP；
Rv1733c p71-98 RSHVYAHQAQTRHPATATVIDHEGVIDS；
Rv1733c p85-112 ATATVIDHEGVIDSNTTATSAPPRTK IT；
Rv1733c p99-126 NTTATSAPPRTKITVPARWVVNGIERSG；
Rv1733c p113-140 VPARWVVNGIERSGEVNAKPGTKSGDRV；
Rv1733c p125-152 SGEVNAKPGTKSGDRVGIWVDSAGQLVD；
Rv1733c p141-168 GIWVDSAGQLVDEPAPPARAIADAALAA；
Rv1733c p169-196 LGLWLSVAAVAGALLALTRAILIRVRNA。
embodiment III: fluorescent immunospot method (FluoSpot)
1. Sample collection
The subjects were harvested for 4ml venous blood, heparin anticoagulated, and Peripheral Blood Mononuclear Cells (PBMC) were obtained by density gradient centrifugation at room temperature for 4 hours, using AIM-V medium (Gibco TM AIM V Medium liquid, invitrogen, USA) was configured at a concentration of 2.5X10 6 Cell suspensions of PBMCs/ml.
2. Pre-coating
96-well Human IFN-gamma/IL-2 FluoSpot reaction plates, the bottoms of which are pre-coated with anti-IFN-gamma and anti-IL-2 monoclonal antibodies. 50ul of AIM-V cell culture medium was added to the single well as a blank; phytohemagglutinin (PHA) was added to the wells at a concentration of 5. Mu.g/ml as a positive control.
3. Antigen stimulation
The ESAT-6 peptide fragment library, the CFP-10 peptide fragment library and the Rv1733c SLP peptide Duan Ku are respectively added into the compound wells to serve as stimulating antigens. 2.5X10 of each well 5 Peripheral Blood Mononuclear Cells (PBMC) and anti-CD28 (0.5. Mu.g/mL, AID, stra. Beta. Berg, germany).
4. Incubation detection
The reaction plate was placed in a 5% CO2 cell incubator and incubated at 37℃for 16-20h. Fluorescein labeled monoclonal antibody IFN-gamma-FITC and IL-2-biotin (final concentration 0.5 mg/ml) were added. Incubate 2h at room temperature with drying in the dark. Secondary antibodies anti-FITC-490 and SA-550 were added. Incubate for 1h at room temperature with drying in the dark. Adding a fluorescence enhancer. Specific T cells secreting IFN-gamma, IL-2, IFN-gamma & IL-2 were counted separately using a fluorescence iSPOT analyzer.
Embodiment four: statistical analysis
Statistical analysis was performed using SPSS24.0 and Kolmogorov-Smirnov test variables were subject to normal distribution. The normal distribution of the measurement data is represented by mean±standard deviation (mean±sd), and the abnormal distribution of the measurement data is represented by Median, and quartile range (Median, IQR). The count data is expressed in percent, 95% confidence interval (%, 95% ci). The cell frequency comparisons between the two groups were performed using two independent sample rank sums.
The frequency of spot formation cell counts secreting different cytokines after stimulation by Rv1733c SLP peptide pool was plotted on the subject operating characteristics (Receiver operating characteristic, ROC) and the area under the ROC curve was compared (area under the ROC curve, AUROC). The optimal cutoff values for the differential diagnosis of ATB and LTBI for FluoSpot specific to Rv1733c SLP are defined. Sensitivity, specificity, positive Predictive Value (PPV), negative Predictive Value (NPV), positive Likelihood Ratio (PLR) and Negative Likelihood Ratio (NLR) were calculated. p-values <0.05 were considered statistically significant.
Fifth embodiment: experimental results
The case control study design is adopted, and pathogenically-diagnosed active tuberculosis patients admitted to Beijing synergetic hospitals and Beijing pectoral hospitals in 2017 and 1 to 12 months are taken as case groups, and contemporaneous latent tuberculosis infected persons are taken as control groups. The secretion of specific T cells IFN-gamma and IL-2 after stimulation by the mycobacterium tuberculosis latency antigen Rv1733c SLP is detected by using a fluorescence immune spot method. And (3) evaluating sensitivity, specificity, predictive value and likelihood ratio of differential diagnosis of active tuberculosis and latent tuberculosis infection by combining ESAT-6& CFP-10fluoroSpot detection.
The study included 20 cases of active tuberculosis with confirmed diagnosis of the pathogen and 28 cases of latent tuberculosis infection. ROC curves were plotted with T cell spot frequency of single secreted IL-2 after stimulation with Mycobacterium tuberculosis latency associated antigen Rv1733c SLP antigen, with AUROC of 0.711 (95 CI 0.566-0.856) at maximum and diagnostic threshold of 0 (SFCs/250,000 PBMCs) with sensitivity and specificity of 75% (95CI 50.90%to 91.34%) and 60.71% (95CI 40.58%to 78.50%), respectively, for differential diagnosis of ATB and LTBI, as shown in FIG. 1.
The T cell spot frequency of single secretion IFN-gamma following ESAT-6& CFP-10 antigen stimulation was used to differentially diagnose ATB and LTBI with sensitivities and specificities of 70% (95%CI 45.72%to 88.11%) and 64.29% (95%CI 44.07%to 81.36%), as shown in FIG. 2.
The differential diagnosis of ATB and LTBI using the proportional number of T cell spots that monosecrete IFN-gamma following stimulation with ESAT-6& CFP-10 antigen was 85% (95%CI 62.11%to 96.79%) and 71.43% (95%CI 51.33%to 86.78%), as shown in FIG. 3.
Based on the early ESAT-6& CFP-10fluoro spot, the T cell spot frequency of single secretion IL-2 after the stimulation of Rv1733c SLP antigen is used for differential diagnosis of ATB and LTBI, the sensitivity can be improved to 100% (95CI 83.16%to 100.00%) by parallel experiments, and the specificity can be improved to 92.86% (95CI 71.77%to 97.73%) by serial experiments, as shown in FIG. 4.
TABLE 1
Conclusion: rv1733c SLP can be used as an alternative antigen of a tuberculosis diagnostic test based on T cells, and is combined with ESAT-6 and CFP-10 antigens to help to differential diagnosis of active tuberculosis and latent tuberculosis infection. On the basis of ESAT-6& CFP-10fluoroSpot, the sensitivity and specificity of differential diagnosis of ATB and LTBI can be improved by jointly applying T cell spot frequency of single secretion IL-2 after the stimulation of latent infection related antigen Rv1733c SLP.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (2)
1. An application of a composition in preparing a reagent for identifying latent tuberculosis infection and active tuberculosis, which is characterized in that the composition is an Rv1733c SLP antigen, an ESAT-6 antigen and a CFP-10 antigen, the composition realizes the application by a fluorescence immunospot method of T cell spot frequency of single secretion IL-2 after stimulation, the combined application of the T cell spot frequency of single secretion IL-2 after stimulation of the Rv1733c SLP can improve sensitivity and specificity of identifying and diagnosing the active tuberculosis infection and the latent tuberculosis infection on the basis of the frequency and proportion of the single secretion IFN-gamma spot after stimulation of the ESAT-6 antigen and the CFP-10 antigen, the Rv1733c SLP is a latent tuberculosis infection related antigen, and the Rv1733c SLP peptide segment library is as follows:
Rv1733c p1-28 MIATTRDREGATMITFRLRLPCRTILRV；
Rv1733c p16-43 FRLRLPCRTILRVFSRNPLVRGTDRLEA；
Rv1733c p29-56 FSRNPLVRGTDRLEAVVMLLAVTVSLLT；
Rv1733c p43-70 AVVMLLAVTVSLLTIPFAAAAGTAVQDS；
Rv1733c p57-84 IPFAAAAGTAVQDSRSHVYAHQAQTRHP；
Rv1733c p71-98 RSHVYAHQAQTRHPATATVIDHEGVIDS；
Rv1733c p85-112 ATATVIDHEGVIDSNTTATSAPPRTKIT；
Rv1733c p99-126 NTTATSAPPRTKITVPARWVVNGIERSG；
Rv1733c p113-140 VPARWVVNGIERSGEVNAKPGTKSGDRV；
Rv1733c p125-152 SGEVNAKPGTKSGDRVGIWVDSAGQLVD；
Rv1733c p141-168 GIWVDSAGQLVDEPAPPARAIADAALAA；
Rv1733c p169-196 LGLWLSVAAVAGALLALTRAILIRVRNA。
2. the use according to claim 1, wherein the fluorescent immunoblotch method comprises the steps of:
(1) Sample collection
Collecting 4ml venous blood from a subject, anticoagulating heparin, centrifuging at room temperature for 4 hours by using a density gradient to obtain peripheral blood mononuclear cell PBMC, and preparing the peripheral blood mononuclear cell PBMC with AIM-V culture medium with the concentration of 2.5X10 6 Cell suspensions of PBMCs/ml;
(2) Pre-coating
The 96-well Human IFN-gamma/IL-2 fluoroSpot reaction plate, the plate bottom is pre-coated with anti-IFN-gamma and anti-IL-2 monoclonal antibodies, and 50ul AIM-V cell culture solution is added into a single well as a blank control; adding phytohemagglutinin PHA with the concentration of 5 mug/ml into the compound holes to serve as a positive control;
(3) Antigen stimulation
The ESAT-6 peptide fragment library, the CFP-10 peptide fragment library and the Rv1733c SLP peptide Duan Ku are respectively added into the compound wells to serve as stimulating antigens, and 2.5 multiplied by 10 is added into each well 5 Peripheral blood mononuclear cells PBMC and 0.5 μg/mL anti-CD28;
(4) Incubation detection
The reaction plate was placed on 5% CO 2 Incubating for 16-20h at 37 ℃, adding fluorescein labeled monoclonal antibody IFN-gamma-FITC and IL-2-biotin, incubating for 2h at room temperature in a dark place, adding secondary antibody anti-FITC-490 and SA-550, incubating for 1h at room temperature in a dark place, adding a fluorescence enhancer, and counting specific T cells secreting IFN-gamma, IL-2, IFN-gamma and IL-2 by using a fluorescence iSPOT analyzer.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101203239A (en) * | 2005-03-31 | 2008-06-18 | 莱顿大学医药中心 | Methods and means for diagnostics, prevention and treatment of mycobacterium infections and tuberculosis disease |
| CN104640564A (en) * | 2012-07-10 | 2015-05-20 | 特兰斯吉恩股份有限公司 | Mycobacterial antigen vaccine |
| CN104990905A (en) * | 2015-06-30 | 2015-10-21 | 曲凯 | Kit for diagnosis of hepatocellular carcinoma metastasis based on solid-phase enzyme immunoassay fluorescence spots |
| CN107976543A (en) * | 2017-11-23 | 2018-05-01 | 苏州因湃生物科技有限公司 | A kind of diagnosis kit and detection method |
| CN109991417A (en) * | 2019-04-16 | 2019-07-09 | 上海市肺科医院 | A kind of immunological marker object lungy and application |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013175459A2 (en) * | 2012-05-25 | 2013-11-28 | Stellenbosch University | Method for diagnosing tuberculosis disease |
-
2020
- 2020-03-23 CN CN202010206015.9A patent/CN111443208B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101203239A (en) * | 2005-03-31 | 2008-06-18 | 莱顿大学医药中心 | Methods and means for diagnostics, prevention and treatment of mycobacterium infections and tuberculosis disease |
| CN104640564A (en) * | 2012-07-10 | 2015-05-20 | 特兰斯吉恩股份有限公司 | Mycobacterial antigen vaccine |
| CN104990905A (en) * | 2015-06-30 | 2015-10-21 | 曲凯 | Kit for diagnosis of hepatocellular carcinoma metastasis based on solid-phase enzyme immunoassay fluorescence spots |
| CN107976543A (en) * | 2017-11-23 | 2018-05-01 | 苏州因湃生物科技有限公司 | A kind of diagnosis kit and detection method |
| CN109991417A (en) * | 2019-04-16 | 2019-07-09 | 上海市肺科医院 | A kind of immunological marker object lungy and application |
Non-Patent Citations (3)
| Title |
|---|
| Application of IFN-γ/IL-2 FluoroSpot assay for distinguishing active tuberculosis from non-active tuberculosis: A cohort study;Lifan Zhang et.al;Clinica Chimica Acta;第499卷;第1-4节 * |
| Humoral Responses to Rv1733c, Rv0081, Rv1735c, and Rv1737c DosR Regulon-Encoded Proteins of Mycobacterium tuberculosis in Individuals with Latent Tuberculosis Infection;Simon G.Kimuda et.al.;《Journal of Immunology Research》;20170201;全文 * |
| Synthetic Long Peptide Derived from Mycobacterium tuberculosis Latency Antigen Rv1733c Protects against Tuberculosis;Mariateresa Coppola et. al.;Clin Vaccine Immunol;第22卷(第9期);INTRODUCTION、MATERIALS and METHODS节 * |
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