CN111443208A - Composition for identifying active tuberculosis and latent tuberculosis - Google Patents
Composition for identifying active tuberculosis and latent tuberculosis Download PDFInfo
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- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
Abstract
The invention relates to the field of molecular biology and immunology, in particular to application of Rv1733c in preparing a kit for identifying active tuberculosis and latent tuberculosis infection, wherein a fluorescence immune spot method is applied to detect specific Th1 cellular immune reaction caused by stimulation of a mycobacterium tuberculosis latent-related antigen Rv1733c and identify the active tuberculosis and the latent tuberculosis infection.
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) seriously threatens human health. Currently about 1/3 people worldwide are latently infected with Mycobacterium tuberculosis (Mtb), of which 5-10% are likely to develop tuberculosis. When the immunity of a latent infected person is reduced, MTB in the body is activated, proliferated and disseminated, and finally active tuberculosis is developed.
Mycobacterium tuberculosis exists mainly in a dormant state during latent infection. Mycobacterium tuberculosis dormant bacteria specifically transcribe and express a group of dormant associated antigens regulated by dormant regulators in order to adapt to host environment and escape from the immune function of the organism. The expression of dormancy associated antigens is of great significance for latent infection and sustained survival of mycobacterium tuberculosis in vivo. The dormancy associated antigen Rv1733c is a protein with high recognition rate of peripheral blood T lymphocytes in Mtb latent infected people, and the protein can effectively stimulate the T lymphocytes to secrete IFN-gamma. Rv1733c is currently used more for the preparation of tuberculosis related vaccines, as described in patent application CN 108239660A; WO2012/210018a 1; WO2006/104389A 1.
At present, in clinical diagnosis and detection, it is difficult to quickly and accurately distinguish Tuberculosis (active Tuberculosis, ATB) from latent Tuberculosis infection (L attention TB infection, L TBI). T-SPOT.TB detection of E L ISPOT technology is widely used for diagnosing Tuberculosis infection clinically, but cannot distinguish and diagnose active Tuberculosis from latent Tuberculosis infection, the main reasons include that 1) antigens applied in T-SPOT.TB detection are only related to virulence of tubercle bacillus, 2) only secretion of a single effector cell factor IFN-gamma is detected, and because of the applied E L ISPOT technology, a chemical color development method is adopted, when multiple factors are detected, the clinical application of the Tuberculosis is influenced because of unidentifiable caused by spot color mixing.
The fluorescence immuno spot assay (FluoroSpot) is currently used to identify active tuberculosis and inactive tuberculosis (L ifan Zhang; Application of IFN-. gamma./I L-2 Fluorospot assay for distinguishing active tuberculosis from non-active tuberculosis: A cohot study; clinical Chimica Acta 499(2019) 64-69), but the stimulus sources used are ESAT-6 antigen and CFP-10 antigen, and the sensitivity and specificity are not ideal.
Disclosure of Invention
In order to improve the sensitivity and specificity of identification of active tuberculosis and latent tuberculosis infection, the invention aims to apply a fluorescence immune spot method (FluoroSpot) to detect specific Th1 cell immune response caused by stimulation of a mycobacterium tuberculosis latent associated antigen Rv1733c and identify ATB and L TBI, experiments show that T cell spot frequency of I L-2 generated by stimulation of a novel latent infection associated antigen Rv1733c has certain value for identifying L TBI and ATB, can assist in identification and improve the identification accuracy.
In particular, the invention relates to a composition comprising Rv1733c antigen, ESAT-6 antigen and CFP-10 antigen; the composition is used for diagnosing tuberculosis; further, it is used for identifying active tuberculosis and latent tuberculosis infection.
Furthermore, the composition can also contain IFN-gamma and I L-2 monoclonal antibodies, and can also contain main reagents used by a fluorescence immune spot method, wherein the reagents can be monoclonal antibodies for resisting IFN-gamma and I L-2, and/or fluorescein-labeled monoclonal antibodies IFN-gamma-FITC and I L-2-biotin, and/or secondary antibodies anti-FITC-490 and SA-550, and the like.
The invention relates to a kit for diagnosing tuberculosis, which contains an Rv1733c antigen, an ESAT-6 antigen and a CFP-10 antigen, wherein the antigens are peptide antigens, and IFN-gamma and an I L-2 monoclonal antibody.
The invention also relates to a kit for identifying active tuberculosis and latent tuberculosis infection, which contains an Rv1733c antigen, an ESAT-6 antigen and a CFP-10 antigen, wherein the antigens are peptide antigens, and IFN-gamma, I L-2 monoclonal antibody and the like.
The invention relates to a kit for diagnosing tuberculosis, which contains Rv1733c antigen and also contains main reagents used by a fluorescence immunospot method, wherein the reagents comprise anti-IFN-gamma and anti-I L-2 monoclonal antibodies, and/or fluorescein-labeled monoclonal antibodies IFN-gamma-FITC and I L-2-biotin, and/or secondary antibodies anti-FITC-490 and SA-550.
The invention relates to a kit for identifying active tuberculosis and latent tuberculosis, which contains Rv1733c antigen and also contains main reagents used by a fluorescence immune spot method, wherein the reagents comprise anti-IFN-gamma and anti-I L-2 monoclonal antibodies, and/or fluorescein-labeled monoclonal antibodies IFN-gamma-FITC and I L-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 further the kit also contains an ESAT-6 antigen and a CFP-10 antigen.
The invention also relates to an application of the Rv1733c in preparing a kit for identifying active tuberculosis and latent tuberculosis infection, and further the kit also contains an ESAT-6 antigen and a CFP-10 antigen.
The invention also relates to the application of the combination of the Rv1733c antigen, the ESAT-6 antigen and the CFP-10 antigen in preparing a kit for identifying active tuberculosis and latent tuberculosis infection.
Further, the Rv1733c antigen is Rv1733c S L P peptide fragment library S L P is synthetic long peptides (synthetic peptides).
Further, each peptide fragment of the Rv1733c S L P peptide fragment library is 28 amino acids long, and the two ends are overlapped by 14 amino acids.
Further, the Rv1733c S L P peptide fragment library is the following peptide fragments or a combination 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 S L P peptide fragment library can also be a combination of any peptide fragments in the Rv1733c S L P peptide fragment library in the prior art or a variant thereof.
Further, the ESAT-6 antigen is an 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: venous blood was collected, peripheral blood mononuclear cells were isolated and cell suspension was prepared using AIM-V medium.
The pre-coating step comprises pre-coating anti-IFN-gamma and anti-I L-2 monoclonal antibodies on the bottom of the reaction plate, using AIM-V cell culture solution as a blank control, and using phytohemagglutinin as a positive control.
The antigen stimulating step comprises: adding ESAT-6 peptide segment library, CFP-10 peptide segment library and Rv1733c peptide segment library; peripheral blood mononuclear cells and anti-CD28 were added.
The incubation detection steps comprise incubation, adding monoclonal antibody IFN-gamma-FITC marked by fluorescein and I L-2-biotin, incubation for 2 hours at room temperature in a dark drying mode, adding secondary antibody anti-FITC-490 and SA-550, incubation for 1 hour at room temperature in a dark drying mode, and counting specific T cells secreting I L-2.
The invention has the following advantages:
firstly, the invention adopts a fluorescence immune spot method, which is to apply an IFN-gamma/I L-2 immune fluorescent spot method to simultaneously detect the secretion of two cytokines of IFN-gamma and I L-2 at the single cell level for joint diagnosis, thereby saving manpower and blood samples to the maximum extent.
Secondly, the invention adopts novel latent infection related antigen, selects representative mycobacterium tuberculosis latent infection antigen Rv1733c, completes synthesis of antigen peptide segment library through cloning expression and purification, and is used as a stimulus for sensitizing T lymphocytes to generate cell factors, and finally, detects the difference of the number of specific T cells of mycobacterium tuberculosis secreting intracellular factors IFN-gamma and I L-2, and proves that the sensitivity and specificity of differential diagnosis of ATB and L I can be improved by jointly applying the T cell spot frequency singly secreting I L-2 after stimulation of the latent infection related antigen Rv1733c on the basis of ESAT-6& CFP-10 Fluorospot.
Drawings
FIG. 1T cell spot frequency of single I L-2 secretion following RV1733S L P antigen stimulation;
FIG. 2T cell spot frequency of single IFN- γ secretion following ESAT-6& CFP-10 antigen stimulation;
FIG. 3 the ratio of IFN- γ monosecretory T cell spots following ESAT-6& CFP-10 antigen stimulation;
FIG. 4T cell spot frequency of single I L-2 secretion following ESAT-6& CFP-10 combined stimulation with Rv1733c S L P antigen
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, 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.
The first embodiment is as follows: sample collection
The screening criteria for pathogen samples are as follows, for patients with active tuberculosis with confirmed diagnosis of the pathogen and asymptomatic latent tuberculosis infections.
1. The inclusion criteria for the active tuberculosis groups were:
1) age 18-75 years;
2) has the symptoms of active tuberculosis such as fever, cough and the like;
3) MTB smear acid fast stain or culture positive, or MTB nucleic acid, or Xpert MTB/RIF detection positive;
4) no antituberculosis treatment was performed.
2. Inclusion criteria for latent tuberculosis infection groups were:
1) age 18-75 years;
2) no fever, cough and other active tuberculosis manifestations;
3) the past has no history of tuberculosis, and the chest film has no old tuberculosis change;
4) positive for T-SPOT.
Exclusion criteria: 1) gestation or lactation; 2) HIV antibody positive.
Example two: latent antigen peptide fragment library synthesis
Referring to the prior art sequence of Rv1733c S L P (Synthetic L ong Peptide) Peptide fragment library (copperoet al; Synthetic L ong Peptide Derived from Mycobacterium Tuberculosis L atencyAntigen Rv1733c Protects against Tuberculosis Tuberculosis; Clin Vaccine Immunol.2015Sep; 22(9):1060-9), the Peptide fragment library of Mycobacterium Tuberculosis hypoxia-associated latency antigen Rv1733c S L P was cloned, expressed and purified, each Peptide fragment 28 amino acids long and 14 amino acids overlapped at both ends, the following Rv1733c S L P 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。
example three: fluorescent immuno spot method (FluoroSpot)
1. Sample collection
The subjects collected 4ml venous blood, anticoagulated with heparin, centrifuged at room temperature for 4 hours by density gradient to obtain Peripheral Blood Mononuclear Cells (PBMC), and treated with AIM-V medium (Gibco)TMAIM V Medium liquid, Invitrogen, USA) was prepared at a concentration of 2.5 × 106PBMCs/ml cell suspension.
2. Pre-coating quilt
A96-well Human IFN-gamma/I L-2 FluoroSpot reaction plate is coated with anti-IFN-gamma and anti-I L-2 monoclonal antibodies at the bottom of the plate, 50ul of AIM-V cell culture solution is added into a single well to serve as a blank control, and Phytohemagglutinin (PHA) with the concentration of 5 mu g/ml is added into a multiple well to serve as a positive control.
3. Antigen stimulation
Adding ESAT-6 peptide segment library, CFP-10 peptide segment library and Rv1733c S L P peptide segment library into each well as stimulating antigen, adding 2.5 × 105Peripheral Blood Mononuclear Cells (PBMC) and anti-CD28 (0.5. mu.g/m L, Stra β berg, Germany).
4. Incubation detection
Placing the reaction plate in a 5% CO2 cell incubator, incubating at 37 ℃ for 16-20h, adding fluorescein-labeled monoclonal antibodies IFN-gamma-FITC and I L-2-biotin (final concentration is 0.5mg/ml), drying in the dark, incubating at room temperature for 2h, adding secondary antibodies anti-FITC-490 and SA-550, incubating at the drying in the dark for 1h, adding a fluorescence enhancer, and counting specific T cells secreting IFN-gamma, I L-2 and IFN-gamma & I L-2 by using a fluorescence iSPOT analyzer.
Example four: statistical analysis
Statistical analysis was performed using SPSS24.0 using a Kolmogorov-Smirnov test whether variables obeyed normal distribution. Normally distributed measurement data are expressed by Mean ± standard deviation (Mean ± SD), and non-normally distributed measurement data are expressed by Median and quartile range (Median, IQR). The data of the counts are expressed as a percentage, 95% confidence interval (%, 95% CI). Comparison of spot-forming cell frequency between groups was performed using two independent sample rank-sum tests.
The spot formation cell frequency of different cytokines secreted after stimulation of Rv1733c S L P peptide library was used to plot a Receiver operating characteristic curve (ROC), compare the areas under the ROC curve (AUROC curve), define the optimal cutoff values for Rv1733c S L P specific FluoroSpot differential diagnosis ATB and L TBI calculate sensitivity, specificity, Positive Predictive Value (PPV), Negative Predictive Value (NPV), positive likelihood ratio (P L R) and negative likelihood ratio (N L R), P value <0.05 was considered statistically significant.
Example five: results of the experiment
By adopting case contrast research design, active tuberculosis patients which are confirmed to be diagnosed by pathogens of Beijing cooperative hospitals and Beijing thoracic hospitals between 2017 and 1-12 months are taken as a case group, and latent tuberculosis infectors at the same period are taken as a contrast group, the secretion of specific T cells IFN-gamma and I L-2 after being stimulated by mycobacterium tuberculosis latent-stage antigen Rv1733c S L P is detected by a fluorescence immune spot method, and the sensitivity, specificity, predicted value and likelihood ratio of the active tuberculosis to the latent tuberculosis infection are evaluated and diagnosed by combining ESAT-6& CFP-10Fluorospot detection.
20 cases of active tuberculosis and 28 cases of latent tuberculosis infection with confirmed pathogen inclusion, a ROC curve is drawn by the single secretion of I L-2T cell spot frequency after the antigen stimulus of mycobacterium tuberculosis latent associated antigen Rv1733c S L P, the AUROC is maximum 0.711(95CI 0.566-0.856), and the diagnostic threshold is 0(SFCs/250,000PBMCs), the sensitivity and specificity of differential diagnosis of ATB and L TBI are respectively 75% (95CI 50.90% to 91.34%) and 60.71% (95CI 40.58% to 78.50%), as shown in figure 1.
ATB and L TBI were differentially diagnosed using the T cell spot frequency of single IFN-. gamma.secretion following ESAT-6& CFP-10 antigen stimulation with sensitivity and specificity of 70% (95% CI 45.72% to 88.11%) and 64.29% (95% CI 44.07% to 81.36%), as shown in FIG. 2.
ATB and L TBI were differentially diagnosed using the ratio of single IFN- γ secreting T cell spots after ESAT-6& CFP-10 antigen stimulation with sensitivity and specificity of 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-10Fluorospot, the T cell spot frequency of singly secreting I L-2 after stimulation by Rv1733c S L P antigen is combined to differentially diagnose ATB and L TBI, and the parallel experiment can improve the sensitivity to 100 percent (95CI83.16 percent to 100.00 percent) and the sequence test can improve the specificity to 92.86 percent (95CI 71.77 percent to 97.73 percent) as shown in figure 4.
TABLE 1
The conclusion is that Rv1733c S L P can be used as a candidate antigen of a T cell-based tuberculosis diagnostic test, is combined with ESAT-6 and CFP-10 antigens to help to identify and diagnose active tuberculosis and latent tuberculosis infection in an auxiliary way, and the sensitivity and specificity of identifying and diagnosing ATB and L TBI can be improved by jointly applying T cell spot frequency of singly secreting I L-2 after being stimulated by latent infection-related antigen Rv1733c S L P on the basis of ESAT-6& CFP-10 FluoroSpot.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (6)
1. A composition comprising Rv1733c antigen, ESAT-6 antigen and CFP-10 antigen.
2. A tuberculosis diagnostic kit is characterized by comprising an Rv1733c antigen, an ESAT-6 antigen and a CFP-10 antigen.
Use of Rv1733c antigen in the preparation of a tuberculosis diagnostic kit.
4. Use according to claim 3, characterized in that the tuberculosis diagnosis is the identification of active tuberculosis and latent tuberculosis infection.
5. The use according to claim 3, wherein the kit further comprises ESAT-6 antigen and CFP-10 antigen.
Use of the combination of Rv1733c antigen, ESAT-6 antigen and CFP-10 antigen in the preparation of a kit for the identification of active tuberculosis and latent tuberculosis infection.
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