CN110702918A - Kit for rapidly detecting active tuberculosis - Google Patents
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Abstract
The invention belongs to the field of detection, and particularly relates to a kit for rapidly detecting active tuberculosis, which comprises a stimulus protein and a stimulant, wherein a mixed stimulant consisting of the stimulus protein and the stimulant is used as a specific stimulus antigen to stimulate peripheral blood lymphocytes to generate cell factors IFN-gamma and IL-2, and the secretion concentration of the IFN-gamma and the IL-2 is detected to achieve the purpose of judging whether the active tuberculosis exists. The inventive stimulating protein comprises CFP-10, ESAT-6 and Rv1985c, and the stimulating agent comprises phorbol ester polyclonal stimulating agent and ionomycin. Wherein the stimulatory protein stimulates the secretion of cytokines by the tuberculosis-specific effector T-cells in the lymphocytes, and the stimulatory agent stimulates the activated lymphocytes to enhance the ability to synthesize and secrete cytokines. The invention can greatly improve the specificity of the detection result and more accurately judge whether the tuberculosis is active tuberculosis.
Description
Technical Field
The invention belongs to the field of detection, and particularly relates to a kit for rapidly detecting active tuberculosis.
Background
Tuberculosis is a common chronic infectious disease caused by mycobacterium tuberculosis, can invade many organs, and is the most common pulmonary tuberculosis infection. At present, people near 1/3 worldwide infect tubercle bacillus, and statistical data show that 150 million people die from tuberculosis in 2013, 900 million new cases, and tuberculosis becomes one of the main diseases of adults dying due to infectious diseases all over the world. China is one of 22 tuberculosis high-load countries in the world, and the number of active tuberculosis is the second place in the world. Tuberculosis infection occurs mainly before patients are not found and treated, and up to 10-15 people can be infected by close contact in 1 tuberculosis patient in one year. Tuberculosis is transmitted through respiratory tract, a large number of tubercle bacilli exist in the lungs of tuberculosis patients, and if the tuberculosis patients cough or sneeze to healthy people, the tuberculosis is likely to transmit germs to the healthy people. Before the patient is found, the patient does not take any preventive measures, and the contact person is easily infected by tubercle bacillus in the process of being closely contacted with family members, colleagues, classmates and the like. At present, the main treatment method of tuberculosis is mainly drug therapy, but the treatment cannot completely prevent the spread of tuberculosis. The BCG vaccination is the most effective immunization method for preventing tuberculosis internationally recognized at present, and the planned immunization program in China stipulates that newborn babies need to be vaccinated with BCG vaccines, but the protection efficiency does not reach 100 percent, so the primary measure for preventing tuberculosis is to discover new patients hidden in the crowd as soon as possible.
Active tuberculosis refers to tuberculosis, which proves that mycobacterium tuberculosis is discharged, the focus belongs to the active stage, and the chest film is often provided with speckled shadow or tuberculosis cavity or spreads the focus, which indicates that the mycobacterium tuberculosis is actively bred and has strong toxicity. Active tuberculosis patients are the main infectious sources of tuberculosis, if diagnosis and treatment cannot be carried out in time, large-scale spread of the tuberculosis can be caused, and the rapid and accurate detection of the active tuberculosis patients is the key for reducing the morbidity and the fatality rate of the tuberculosis. The existing clinical method commonly used for active tuberculosis diagnosis has poor specificity, the diagnosis process is closely related to the experience of doctors, and the diagnosis result has strong subjectivity; the sputum culture method is the standard of tuberculosis diagnosis gold, but has low sensitivity and long time consumption (2-8 weeks), and is not beneficial to early diagnosis of tuberculosis; the sensitivity of the acid-fast staining method is only 20-30%; the molecular diagnosis method (such as PCR) has poor repeatability and complex operation, and has certain requirements on the laboratory environment; the tuberculin test (TST) is susceptible to vaccination with bacillus calmette-guerin (BCG), resulting in false positives of the test results. In recent years, a T cell-based gamma interferon release test has been developed, but this method can only determine whether a patient is infected with Mycobacterium tuberculosis (too) and cannot determine whether the patient is active tuberculosis.
Mycobacterium tuberculosis is a typical intracellular infectious bacterium, and the immune mechanism of a human body is mainly cell immunity taking T cells as main factors. After the mycobacterium tuberculosis invades a human body, when the immunity of the human body is low, macrophages can not prevent the phagocytosed mycobacterium tuberculosis from growing, but can bring the phagocytosed mycobacterium tuberculosis to other places to present antigens, at the moment, the mycobacterium tuberculosis proliferates in a large quantity to sensitize peripheral T lymphocytes, the sensitized T lymphocytes can generate various cytokines (such as INF-gamma, IL-2, TNF-alpha and the like), and the cytokines act together to kill the mycobacterium tuberculosis in focuses. The gamma interferon release assay is based on the use of this property of using antigens expressed by Mycobacterium tuberculosis to stimulate peripheral blood mononuclear cells to produce gamma interferon. The stimulation antigens used by the existing gamma-interferon release test detection kit are CFP-10 and ESAT-6, and the CFP-10 and ESAT-6 are proteins expressed by a mycobacterium tuberculosis RD region, so that the kit has tuberculosis specificity. However, the existing gamma-interferon release test kit has low specificity which is only about 70% -85%, and further promotion possibility and demand still exist.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a kit for rapidly detecting active tuberculosis, and the kit can greatly improve the specificity of a detection result and more accurately judge whether the active tuberculosis exists.
In order to achieve the purpose, the invention adopts the technical scheme that:
the invention provides a kit for rapidly detecting active tuberculosis, which comprises:
a) stimulating protein: CFP-10, ESAT-6 and Rv1985c, CFP-10, ESAT-6 and Rv1985c are specific surface antigens of non-pathogenic Mycobacterium tuberculosis;
b) a stimulant; phorbol ester polyclonal stimulator and ionomycin, and phorbol ester polyclonal stimulator (PMA) and ionomycin (ionomycin) have the ability to stimulate activated lymphocytes to enhance the synthesis and secretion of cytokines.
Preferably, the CFP-10, ESAT-6 and Rv1985c are linked in any order by a linking polypeptide to form a recombinant fusion protein.
More preferably, the linker polypeptide has the amino acid sequence shown in SEQ ID NO. 1.
Preferably, the detection method of the kit comprises an enzyme-linked immunosorbent assay, an enzyme-linked immunospot assay, an intracellular cytokine staining assay, a Luminex assay, a PCR/RT-PCR assay, an immunofluorescence assay, an immunochromatography assay, an immunohybridization assay, an immunoblotting assay and a cell proliferation assay.
Preferably, the detection sample of the kit is peripheral blood or lymphocytes separated from peripheral blood.
Preferably, the detection index of the kit is the secretion amount of the cytokines IFN-gamma and IL-2.
The criteria for determining whether active tuberculosis is present are: based on the IFN-gamma secretion of more than 20pg/mL and the IL-2 secretion of more than 15pg/mL, when the detection results of the IFN-gamma and the IL-2 are positive, the active tuberculosis can be judged. Compared with a kit for a gamma-interferon release test, the kit for the gamma-interferon release test has the advantages that the detection of interleukin 2 is added, so that the specificity of a detection result is greatly improved, and whether the tuberculosis is active or not can be more accurately judged.
One of the using methods of the kit is as follows:
s1, separating lymphocytes from peripheral blood samples, and diluting to the concentration of 2.5X 106Cell solution per mL;
lymphocytes in the peripheral blood sample were separated using a commercially available lymphocyte separation medium, and 4mL of physiological salt was collectedMixing water with 4mL peripheral blood, slowly adding into lymphocyte separation solution, centrifuging at 1800r/min for 20min at room temperature, sucking cloud layer cells, supplementing to 12mL with 1640 culture medium (RPMI-1640 culture medium), centrifuging at 600r/min at room temperature for 10min, discarding supernatant, supplementing precipitate to 5mL with 1640 culture medium, mixing, centrifuging at 350r/min for 10min at room temperature, resuspending cells with 1640 culture medium or PBS phosphate buffer solution, separating and purifying to obtain lymphocytes, counting the purified viable lymphocytes, and diluting with AIM culture medium (AIM V culture medium of Gibco) to concentration of 2.5 × 106Cell solution per mL.
S2, adding the kit as a specific stimulating antigen into the cell solution, mixing uniformly, and placing in 5% CO2Culturing in incubator at 37 deg.C for 16h, and collecting culture;
mixing 50 μ L of mixed stimulant solution (mixed stimulant solution prepared from stimulant protein and stimulant in kit with physiological saline) and 100 μ L of the above cell solution, adding into the same cell culture well, setting 10 multiple wells, and placing in 5% CO2Culturing in incubator at 37 deg.C for 16h, and collecting culture.
S3, centrifuging the culture, collecting supernatant, detecting the concentration of IFN-gamma and IL-2 in the supernatant, and judging whether the tuberculosis is active according to the concentration of the IFN-gamma and the IL-2.
The other using method of the kit comprises the following steps:
s1, adding the kit as specific stimulating antigen into peripheral blood, mixing, and placing in 5% CO2Culturing in incubator at 37 deg.C for 16h, and collecting plasma;
mixing 50 μ L of mixed stimulant solution (mixed stimulant solution prepared from stimulant protein and stimulant in kit with physiological saline) and 1mL of peripheral blood, and adding 5% CO2Culturing in incubator at 37 deg.C for 16h, and collecting plasma.
S2, detecting the concentration of IFN-gamma and IL-2 in the blood plasma, and judging whether the tuberculosis is active or not according to the concentration of the IFN-gamma and the IL-2.
Preferably, in the above two methods of use, the final concentration of the stimulatory protein is 1.5-2.5. mu.g/mL, the final concentration of the phorbol ester polyclonal stimulatory agent is 250-350pg/mL, and the final concentration of the ionomycin is 0.5-1.5. mu.g/mL.
More preferably, in both of the above methods of use, the final concentration of the stimulatory protein is 2. mu.g/mL, the final concentration of the phorbol ester polyclonal stimulatory agent is 300pg/mL, and the final concentration of ionomycin is 1. mu.g/mL.
Compared with the two methods, the whole blood sample has the advantages of being more convenient and quicker compared with the separation of the lymphocyte sample, and has the defect that misdiagnosis is more likely to occur to patients with abnormal immune systems.
The kit can be applied to the preparation of a kit for assisting in the diagnosis of active tuberculosis or latent tuberculosis infection.
The kit can also be applied to the preparation of the kit for evaluating the clinical anti-tuberculosis treatment effect.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a kit for rapidly detecting active tuberculosis, which comprises a stimulating protein and a stimulating agent, wherein a mixed stimulant consisting of the stimulating protein and the stimulating agent is taken as a specific stimulating antigen to stimulate peripheral blood lymphocytes to generate cell factors IFN-gamma and IL-2, and the goal of judging whether the active tuberculosis exists is achieved by detecting the secretion concentration of the IFN-gamma and the IL-2.
The kit is a more efficient specific stimulant for the mycobacterium tuberculosis, and is a mixture of specific stimulant protein for the mycobacterium tuberculosis and a stimulant, wherein the stimulant protein is nonpathogenic specific surface antigens CFP-10, ESAT-6 and Rv1985c of the mycobacterium tuberculosis, and can stimulate the tubercle specific effector T cells in lymphocytes to secrete cytokines, and compared with the antigen of a gamma-interferon release test kit, the newly added Rv1985c protein can improve the specificity of a detection system; the stimulator is phorbol ester polyclonal stimulator (PMA) and ionomycin (ionomycin), and can stimulate activated lymphocyte to enhance the ability of synthesizing and secreting cytokine, thereby improving the ability of stimulator. Phorbol ester polyclonal stimulant (PMA) and ionomycin (ionomycin) are generally used for detecting Th1/Th2 cells by flow cytometry, the using concentration of the phorbol ester polyclonal stimulant is generally 0.5-150ng/mL, the using concentration of the ionomycin is generally 1-2 mug/mL, and the invention finds that when the stimulator is used as an antigen in combination with the stimulator protein disclosed by the invention, lymphocytes activated by the antigen can be stimulated to strengthen the secretion of relevant cytokines without influencing the secretion of other lymphocytes. The concentration of the phorbol ester polyclonal stimulant used in the invention is 250-300pg/ml, and the ionomycin is 0.5-1.5 mu g/ml, so that the ability of activated lymphocytes to secrete cytokines under the stimulation is enhanced, and the specificity of the detection system is further improved.
Meanwhile, according to a large number of screening experiments, IFN-gamma and IL-2 in the cytokines secreted by the peripheral blood mononuclear cells induced by the specific stimulating antigen (the kit of the invention) are closely related to the disease state of the patient, so that whether the patient is the active tuberculosis can be accurately judged by detecting the secretion of the two factors, and the method is more beneficial for a clinician to timely diagnose the disease state of the patient with the tuberculosis. The detection range of the kit provided by the invention comprises all active tuberculosis including pulmonary tuberculosis and extrapulmonary tuberculosis (such as lymphoid tuberculosis, intestinal tuberculosis, bone tuberculosis, renal tuberculosis and the like), and the coverage and the practicability are wider.
Drawings
FIG. 1 shows the results of detecting the concentrations of IFN-. gamma.and IL-2 in the supernatants of patients with active tuberculosis under the stimulation of two different stimulators (a is the secretion value of IFN-. gamma.; b is the secretion value of IL-2);
FIG. 2 shows the results of measuring the concentrations of IFN-. gamma.and IL-2 in the supernatants of healthy persons under the stimulation of two different stimuli (a is the secretion value of IFN-. gamma.; b is the secretion value of IL-2);
FIG. 3 shows the results of detecting the concentrations of IFN-gamma and IL-2 in the supernatants of patients with active tuberculosis, tuberculosis-infected patients and healthy persons under the stimulation of two different stimulators by enzyme-linked immunosorbent assay (a is IFN-gamma secretion value; b is IL-2 secretion value);
FIG. 4 shows the results of detecting the concentrations of IFN-gamma and IL-2 in the supernatants of patients with active tuberculosis, tuberculosis-infected patients and healthy persons under the stimulation of two different stimulators by ELISA (a is IFN-gamma secretion value; b is IL-2 secretion value);
FIG. 5 shows the results of detecting the concentrations of IFN-. gamma.and IL-2 in the plasma (peripheral blood) of patients with active tuberculosis, tuberculosis-infected patients and healthy persons with two different stimulators by enzyme-linked immunosorbent assay (a is the IFN-. gamma.secretion value; b is the IL-2 secretion value).
Detailed Description
The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
Example 1
(1) Collecting samples:
in the embodiment, 1 active tuberculosis patient is taken as an experimental subject, peripheral blood of not less than 15mL is collected by using a heparin sodium blood collection tube, and the peripheral blood is reversed and mixed uniformly for standby;
(2) lymphocyte separation: separating lymphocytes in peripheral blood sample with commercially available lymphocyte separation solution, mixing 4mL physiological saline with 4mL peripheral blood, slowly adding into lymphocyte separation solution, centrifuging at 1800r/min for 20min at room temperature, sucking cloud layer cells, supplementing to 12mL 1640 medium (RPMI-1640 medium), centrifuging at 600r/min for 10min at room temperature, discarding supernatant, supplementing to 5mL sediment with 1640 medium, gently mixing, centrifuging at 350r/min for 10min at room temperature, resuspending cells with 1640 medium or PBS phosphate buffer solution, separating and purifying to obtain lymphocytes, counting the purified viable lymphocytes, and diluting with AIM medium (AIM V medium of Gibco) to 2.5 × 106Cell solution per mL;
(3) collecting 50 μ L of mixed stimulator solution (stimulator protein concentration of 6 μ g/mL, phorbol esters)The concentration of the polyclonal stimulant is 900pg/mL, the final concentration of the ionomycin is 3 mug/mL) and 100 muL of the cell solution are evenly mixed, added into the same cell culture hole, provided with 10 multiple holes and then placed in 5 percent CO2Culturing in incubator at 37 deg.C for 16h, and collecting culture;
using stimulus only added with stimulating protein as control, namely preparing stimulating protein solution (the concentration of stimulating protein is 6 μ g/mL) from stimulating protein with physiological saline, mixing 50 μ L of stimulating protein solution and 100 μ L of the above cell solution, adding into the same cell culture well, setting 10 multiple wells, and placing in 5% CO2Culturing in incubator at 37 deg.C for 16h, and collecting culture;
(4) and (3) centrifuging the culture, collecting supernatant, detecting the concentrations of IFN-gamma and IL-2 in the supernatant by using an enzyme-linked immunosorbent assay, and researching the statistical difference of the concentrations of the IFN-gamma and the IL-2 in the supernatant under the stimulation of two different stimulators by using statistical analysis software. The data show (Table 1) that the concentrations of IFN-. gamma.and IL-2 in the supernatants with the mixed stimuli were significantly higher than those in the supernatants with the stimulator, and that the samples secreted IFN-. gamma.at an average concentration of 137.9. + -. 20.6pg/mL (95% confidence interval, the same applies below) and IL-2 at 100.7. + -. 15.7pg/mL under the mixed stimuli; the average IFN-gamma concentration secreted by the stimulation protein is 108.5 +/-15.1 pg/mL, and the IL-2 concentration is 81.5 +/-11.0 pg/mL. Indicating that the mixed stimulant can effectively improve the secretion amount of the relevant cytokines of the activated lymphocytes.
In this example, mixed stimuli and single stimulatory proteins were evaluated for stimulating equal amounts of lymphocytes from multiple samples from the same tuberculosis patient for 16 h. The used stimulating proteins are the same, and the final concentration is 2 mug/mL; the final concentration of phorbol ester polyclonal stimulant (PMA) in the mixed stimulant is 300pg/mL, and the final concentration of ionomycin (ionomycin) is 1 mug/mL. The average IFN-gamma concentration stimulated secretion by the mixed stimulators is 137.9 +/-20.6 pg/mL, while the concentration under stimulation of the stimulating protein is only 108.5 +/-15.1 pg/mL; the mean concentration of IL-2 under mixed stimulus was 100.7 + -15.7 pg/mL, while the concentration under stimulus of the stimulator protein was only 81.5 + -11.0 pg/mL, indicating that there was a significant difference between IFN-. gamma.and IL-2 produced by stimulation of activated lymphocyte secretion with different stimuli. This protocol demonstrates that the use of the kit of the invention (i.e., the mixed stimulus) is effective in increasing the secretion of the relevant cytokine from activated lymphocytes.
TABLE 1 statistics of IFN-. gamma.and IL-2 concentrations in supernatants under stimulation with two different stimuli
Example 2
(1) Collecting samples:
in the embodiment, 1 example of healthy human experimental subjects with a negative PPD detection result is collected by a heparin sodium blood collection tube, and the peripheral blood is reversed and mixed uniformly for later use;
(2) lymphocyte separation: separating lymphocytes in peripheral blood sample with commercially available lymphocyte separation solution, mixing 4mL physiological saline with 4mL peripheral blood, slowly adding into lymphocyte separation solution, centrifuging at 1800r/min for 20min at room temperature, sucking cloud layer cells, supplementing to 12mL 1640 medium (RPMI-1640 medium), centrifuging at 600r/min for 10min at room temperature, discarding supernatant, supplementing to 5mL sediment with 1640 medium, gently mixing, centrifuging at 350r/min for 10min at room temperature, resuspending cells with 1640 medium or PBS phosphate buffer solution, separating and purifying to obtain lymphocytes, counting the purified viable lymphocytes, and diluting with AIM medium (AIM V medium of Gibco) to 2.5 × 106Cell solution per mL;
(3) mixing 50 μ L of mixed stimulant solution (with stimulating protein concentration of 6 μ g/mL, phorbol ester polyclonal stimulant concentration of 900pg/mL, and ionomycin final concentration of 3 μ g/mL) and 100 μ L of the above cell solution, adding into the same cell culture well, setting 10 multiple wells, and placing in 5% CO2Culturing in incubator at 37 deg.C for 16h, and collecting culture;
using stimulus with only stimulating protein as control, i.e. preparing stimulating protein into stimulating protein solution with physiological salineMixing 50 μ L of stimulating protein solution and 100 μ L of the above cell solution, adding into the same cell culture well, setting 10 multiple wells, and placing in 5% CO2Culturing in incubator at 37 deg.C for 16h, and collecting culture;
taking cell culture medium without stimulus as blank control, adding 50 μ L AIM culture medium and 100 μ L above cell solution into the same cell culture well, setting 10 multiple wells, and placing in 5% CO2Culturing in incubator at 37 deg.C for 16h, and collecting culture;
(4) and (3) centrifuging the culture, collecting supernatant, detecting the concentrations of IFN-gamma and IL-2 in the supernatant by using an enzyme-linked immunosorbent assay, and researching the statistical difference of the concentrations of the IFN-gamma and the IL-2 in the supernatant under the stimulation of two different stimulators by using statistical analysis software. The data show (Table 2) that there is no significant difference between the concentration of IFN-gamma and IL-2 in the supernatant using the stimulator protein and the supernatant using the mixed stimulator, and the average concentration of IFN-gamma secreted by the sample under the stimulation of the mixed stimulator is 18.2 +/-3.2 pg/mL, and IL-2 is 8.0 +/-2.2 pg/mL; the average IFN-gamma concentration secreted by the stimulation protein is 17.2 +/-2.5 pg/mL, and the IL-2 concentration is 7.5 +/-3.0 pg/mL. The added stimulant in the mixed stimulant can not stimulate the unactivated lymphocyte to secrete the relevant cytokine.
In this example, equal numbers of lymphocytes from the same healthy human specimen (negative for PPD test) were evaluated in cell culture media, mixed stimulators and single stimulators for 16 h. The used stimulating proteins are the same, and the final concentration is 2 mug/mL; the final concentration of phorbol ester polyclonal stimulant (PMA) in the mixed stimulant is 300pg/mL, and the final concentration of ionomycin (ionomycin) is 1 mug/mL. The average concentration of IFN-gamma stimulated secretion by the mixed stimulators is 18.2 +/-3.2 pg/mL, and the concentration of IFN-gamma under stimulation of the stimulating protein is 17.2 +/-2.5 pg/mL; the mean concentration of IL-2 under mixed stimulus was 8.0. + -. 2.2pg/mL and only 7.5. + -. 3.0pg/mL for the stimulated protein, indicating that there was no significant difference between IFN-. gamma.and IL-2 produced by stimulation of unactivated lymphocyte secretion with two different stimuli. This protocol demonstrates that the use of the kit of the invention (i.e., mixed stimuli) does not stimulate unactivated lymphocytes to secrete the relevant cytokines.
TABLE 2 statistics of IFN-. gamma.and IL-2 concentrations in supernatants under stimulation with two different stimuli
Example 3
(1) Collecting samples:
in the embodiment, 30 samples in total of 10 active tuberculosis patients, 10 tuberculosis infected persons (positive PPD detection result) and 10 healthy persons (negative PPD detection result) are taken as experimental objects, and at least 12mL of peripheral blood is collected by using a heparin sodium blood collection tube, and is reversely mixed for standby;
(2) lymphocyte separation: separating lymphocytes in peripheral blood sample with commercially available lymphocyte separation solution, mixing 4mL physiological saline with 4mL peripheral blood, slowly adding into lymphocyte separation solution, centrifuging at 1800r/min for 20min at room temperature, sucking cloud layer cells, supplementing to 12mL 1640 medium (RPMI-1640 medium), centrifuging at 600r/min for 10min at room temperature, discarding supernatant, supplementing to 5mL sediment with 1640 medium, gently mixing, centrifuging at 350r/min for 10min at room temperature, resuspending cells with 1640 medium or PBS phosphate buffer solution, separating and purifying to obtain lymphocytes, counting the purified viable lymphocytes, and diluting with AIM medium (AIM V medium of Gibco) to 2.5 × 106Cell solution per mL;
(3) mixing 50 μ L of mixed stimulant solution (with stimulator protein concentration of 6 μ g/mL, phorbol ester polyclonal stimulant concentration of 900pg/mL, and ionomycin final concentration of 3 μ g/mL) and 100 μ L of the above cell solution, adding into cell culture hole and ELISPOT pre-coated plate hole, setting 2 multiple holes, and placing in 5% CO2Culturing in incubator at 37 deg.C for 16h, and collecting culture;
using stimulus with stimulating protein as control, i.e. adding stimulating protein with physiological salinePreparing stimulating protein solution (the concentration of stimulating protein is 6 μ g/mL), mixing 50 μ L of stimulating protein solution and 100 μ L of the above cell solution, adding into cell culture hole and ELISPOT pre-coated detection plate hole, setting 2 multiple holes, and placing in 5% CO2Culturing in incubator at 37 deg.C for 16h, and collecting culture;
taking cell culture medium without adding stimulator as blank control, namely taking 50 μ L AIM culture medium and 100 μ L above cell solution, mixing well, adding into cell culture well and ELISPOT pre-coated detection plate well, setting 2 multiple wells, and placing in 5% CO2Culturing in incubator at 37 deg.C for 16h, and collecting culture;
(4) centrifuging the culture, collecting supernatant, and detecting IFN-gamma and IL-2 concentration in the culture supernatant by enzyme-linked immunosorbent assay (ELISA), the results are shown in Table 3; meanwhile, an ELISPOT pre-coating detection plate is detected by using an enzyme-linked immunosorbent assay, and the concentration of IFN-gamma and IL-2 in culture supernatant is detected, and the result is shown in a table 3; ROC analysis was performed on each test result and the area under the curve was calculated, and the area under each set of curves is shown in table 4.
As shown in tables 3 and 4, the results of the tests using both of the test methods showed that the test results using the mixed stimuli were superior to the test results using the single stimulatory proteins.
In this example, the mixed stimulators and the stimulator protein were evaluated for stimulating equal amounts of lymphocytes for multiple tuberculosis patients, tuberculosis infected patients (positive PPD test result), and healthy human samples (negative PPD test result) for 16 h. The used stimulator protein concentration is 2 μ g/mL, the phorbol ester polyclonal stimulator (PMA) concentration is 300pg/mL, and the ionomycin (ionomycin) concentration is 1 μ g/mL. The IFN-gamma and the IL-2 generated by secretion are detected by using an enzyme-linked immunosorbent assay and an enzyme-linked immunosorbent assay, ROC curve analysis is used for finding that the IFN-gamma and the IL-2 generated by two stimulators and stimulated by the two stimulators are related to whether a sample has tuberculosis, and the areas under ROC curves of two cytokines generated by stimulation of mixed stimulators are higher than those of the stimulated protein, so that the kit (namely the mixed stimulators) has higher relevance to whether the sample has the tuberculosis and has higher diagnostic value.
TABLE 3 results of IFN-. gamma.and IL-2 concentration detection in culture supernatants by enzyme-linked immunosorbent assay and ELISA Spot assay
TABLE 4 area table under each test result curve of PBMC (human peripheral blood mononuclear cell) cell culture method
Note: higher lower area tables indicate more valuable diagnostic reagents.
Example 4
(1) Collecting samples:
in the embodiment, 30 samples in total of 10 active tuberculosis patients, 10 tuberculosis infected persons (positive PPD detection result) and 10 healthy persons (negative PPD detection result) are taken as experimental objects, and at least 5mL of peripheral blood is collected by using a heparin sodium blood collection tube, and is reversely mixed for standby;
(2) mixing 50 μ L of mixed stimulator solution (stimulator protein concentration of 40 μ g/mL, phorbol ester polyclonal stimulator concentration of 6ng/mL, and ionomycin final concentration of 20 μ g/mL) and 1mL of peripheral blood, adding into cell culture well, setting 2 multiple wells, and placing in 5% CO2Culturing in incubator at 37 deg.C for 16h, and collecting plasma;
taking stimulus only added with stimulating protein as control, namely preparing stimulating protein solution (the concentration of stimulating protein is 40 μ g/mL) from stimulating protein with physiological saline, mixing 50 μ L of stimulating protein solution and 1mL of peripheral blood, adding into cell culture well, setting 2 multiple wells, and placing in 5% CO2Culturing in incubator at 37 deg.C for 16h, and collecting plasma;
taking cell culture medium without addition of stimulator as blank control, namely taking 50 μ L AIM culture medium and 1mL peripheral blood, mixing, adding into cell culture well, setting 2 multiple wells, and placing in 5% CO2Culturing in incubator at 37 deg.C for 16h, and collecting plasma;
(3) the concentrations of IFN-gamma and IL-2 in the blood plasma were measured by enzyme-linked immunosorbent assay, and the results are shown in Table 5; ROC analysis was performed on each test result and the area under the curve was calculated, and the area under each set of curves is shown in table 6.
The results of the tests shown in tables 5 and 6 show that the test results using the mixed stimulators are superior to the test results using the single stimulators.
In this example, the amount of peripheral blood stimulated by the mixed stimulators and the stimulator protein was evaluated for 16h in a plurality of tuberculosis patients, tuberculosis infected patients (positive PPD test result), and healthy human specimens (negative PPD test result). The used stimulator protein concentration is 2 μ g/mL, the phorbol ester polyclonal stimulator (PMA) concentration is 300pg/mL, and the ionomycin (ionomycin) concentration is 1 μ g/mL. The IFN-gamma and the IL-2 generated by secretion are detected by using an enzyme linked immunosorbent assay, and ROC curve analysis is used for finding that the IFN-gamma and the IL-2 generated by two stimulators and stimulated by the two stimulators are related to whether a sample has tuberculosis, and the areas under ROC curves of two cytokines generated by stimulation of a mixed stimulator are higher than those of the stimulator, so that the kit (namely the mixed stimulator) is higher in correlation with whether the sample has the tuberculosis, and has higher diagnostic value.
TABLE 5 results of IFN-. gamma.and IL-2 concentration assay in plasma by ELISA
TABLE 6 area table under curve of peripheral blood cytokine detection results
Note: higher area under the curve indicates more valuable diagnostic reagents.
Example 5
In order to verify the detection performance of the kit, the stimulus protein is used as a control, the detection performance of the mixed stimulus (comprising the stimulus protein, the phorbol ester polyclonal stimulus and the ionomycin) is detected, 200 samples in total of 100 active tuberculosis patients, 50 tuberculosis infected persons (the PPD detection result is positive) and 50 healthy persons (the PPD detection result is negative) are selected as experimental objects, wherein 100 cases of active tuberculosis are used as a clinical positive group, 50 cases of tuberculosis infected persons and 50 cases of healthy persons are used as a clinical negative group, the detection method in the embodiment 3 is adopted for detection, the ROC curve is used for analyzing the optimal cut-off value of two cytokines, and (3) counting the sensitivity and specificity of diagnosis by taking the optimal cut-off value as a threshold value and positive gamma-interferon and/or interleukin-2 as a positive value, wherein the detection results are shown in tables 7-9.
The results shown in Table 9 show that the specificity of the mixed stimulus detection is 92% higher than that of the conventional stimulus protein, which indicates that the kit of the invention has good specificity and high detection precision.
TABLE 7 four-grid Table of mixed stimuli and clinical diagnostic results
| Clinical positive | Clinical negativity | Total up to | |
| Mixed thornLaser positive | 82 | 8 | 90 |
| Mixed stimulus negativity | 18 | 92 | 110 |
| Total up to | 100 | 100 | 200 |
TABLE 8 four-panel table of stimulatory proteins and clinical diagnostic results
| Clinical positive | Clinical negativity | Total up to | |
| Stimulation protein positivity | 83 | 19 | 102 |
| Stimulation protein negativity | 17 | 81 | 98 |
| Total up to | 100 | 100 | 200 |
TABLE 9 comparison of the Performance of the kit of the present invention and that of the common stimulator protein kit
| The kit of the invention | Stimulator protein kit | |
| Sensitivity of the probe | 82％ | 83％ |
| Degree of specificity | 92％ | 81％ |
The embodiments of the present invention have been described in detail, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.
Claims (10)
1. A kit for rapidly detecting active tuberculosis is characterized in that: the kit comprises:
a) stimulating protein: CFP-10, ESAT-6 and Rv1985 c;
b) a stimulant; phorbol ester polyclonal stimulator and ionomycin.
2. The kit of claim 1, wherein: the CFP-10, ESAT-6 and Rv1985c are connected into a recombinant fusion protein through connecting polypeptides in any order.
3. The kit of claim 2, wherein: the connecting polypeptide has an amino acid sequence shown in SEQ ID NO. 1.
4. The kit of claim 1, wherein: the detection method comprises an enzyme-linked immunosorbent assay, an enzyme-linked immunospot assay, an intracellular cytokine staining method, a Luminex assay, a PCR/RT-PCR assay, an immunofluorescence assay, an immunochromatography assay, an immunohybridization assay, an immunoblotting assay and a cell proliferation assay.
5. The kit of claim 1, wherein: the detection sample is peripheral blood or lymphocyte separated from peripheral blood.
6. The kit of claim 1, wherein: the detection indexes are the secretion amounts of the cytokines IFN-gamma and IL-2.
7. The kit of claim 1, wherein: when the kit is used, the final concentration of the stimulating protein is 1.5-2.5 mug/mL, the final concentration of the phorbol ester polyclonal stimulating agent is 250-350pg/mL, and the final concentration of the ionomycin is 0.5-1.5 mug/mL.
8. The kit of claim 1, wherein: the final concentration of the stimulating protein is 2 mug/mL, the final concentration of the phorbol ester polyclonal stimulating agent is 300pg/mL, and the final concentration of the ionomycin is 1 mug/mL.
9. Use of a kit according to any one of claims 1 to 8 for the manufacture of a kit for the auxiliary diagnosis of active tuberculosis or latent tuberculosis infection.
10. Use of a kit according to any one of claims 1 to 8 in the manufacture of a kit for the evaluation of the efficacy of a clinical anti-tuberculosis treatment.
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Inventor after: Hu Pengnan Inventor after: Ge Taihong Inventor after: He Xin Inventor after: Xiao Yanwen Inventor before: Hu Pengnan Inventor before: Ge Taihong Inventor before: He Xin Inventor before: Xiao Yanwen |
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