Tuberculosis infection T cell immune evaluation system and evaluation method

Abstract

The tuberculosis infection T cell immune evaluation system comprises a microfluidic immune reactor, a pipettor, a detection antibody, a detection tracer SA-PolyHRP, a luminol chemiluminescence substrate, a chemiluminescence detection unit and a computer, the microfluidic immune reactor is coated with an interferon capture antibody, and the microfluidic immune reactor is coated with a chemiluminescence detection unit. The pipettor is used for sucking a blood sample, a detection antibody, a detection tracer SA-PolyHRP and a luminol chemiluminescent substrate into the microfluidic immune reactor; the chemiluminescence detection unit collects a chemiluminescence image signal and converts the chemiluminescence image signal into a quantitative value signal, and the quantitative value signal is converted into an equivalent standard interferon concentration through a standard curve so as to evaluate the T cell immune protection capability of the sample; according to the tuberculosis infection T cell immune evaluation system and evaluation method provided by the invention, through a high-sensitivity detection platform, the stimulation incubation in a trace blood sample can be realized, the incubation time can be shortened, and the clinical demand on the blood volume of a patient can be reduced, so that some special crowds can also perform IGRA detection, and the system and the method are rapid, flexible, stable and reliable.

Description

Technical Field

[0001] The present application relates to the field of biological detection technology, and in particular to a tuberculosis-infected T cell immunity assessment system and assessment method. Background Art

[0002] Tuberculosis (TB) is an airborne infectious disease caused by Mycobacterium tuberculosis (Mtb) that not only causes localized pneumonia but also affects multiple organs. As highlighted in the Global Tuberculosis Report 2023, TB causes an estimated 1.3 million deaths worldwide and poses a persistent threat to individual health and healthcare systems worldwide. The key to fighting TB is accurate and timely diagnosis, which is essential for disease treatment and transmission control.

[0003] Among the commonly used T cell immunoassays for tuberculosis infection, the interferon-γ release assay (IGRA) can highly specifically and quantitatively detect the activation response of T cells to tuberculosis pathogen-specific peptides, detect the potential immune response of individuals to tuberculosis-related pathogens, and predict the protective immunity of individuals. IGRA measures the absolute amount of interferon-γ (IFNγ) released after stimulation with antigens or specific peptides or the number of cells that secrete IFNγ after stimulation through an enzyme-linked immunosorbent assay (ELISA), but its detection process is long and requires a lot of manpower, material resources, professional technology and equipment. The second method uses the enzyme-linked immunosorbent spot (ELISPOT) assay to detect the release and binding of IFNγ by activated T cells fixed on the detection membrane. Its operation process is complicated, the stimulation incubation process requires incubation for 3 days, and the observation results after incubation need to be interpreted by microscope, which is very cumbersome. IGRA has certain limitations, especially the large sample volume required (3-5mL whole blood) and the need for venous blood collection by professionals. Due to the large blood collection volume requirement, this method is difficult to apply in people with difficulty in blood collection, such as infants and the elderly. Summary of the invention

[0004] In view of this, it is necessary to provide a tuberculosis infection T cell immunity assessment system and method that can quickly, flexibly, stably and reliably detect tuberculosis infection, in order to address the defects of the existing technology of tuberculosis infection T cell immunity detection, such as long cycle and low feasibility.

[0005] To solve the above problems, this application adopts the following technical solutions:

[0006] One of the purposes of the present application is to provide a tuberculosis infection T cell immune assessment system, including a microfluidic immunoreactor, a pipette, a detection antibody, a detection tracer SA-PolyHRP, a luminol chemiluminescent substrate and a chemiluminescent detection unit, wherein:

[0007] The microfluidic immunoreactor is coated with interferon capture antibodies, and the microfluidic immunoreactor is tightly fitted with the pipette;

[0008] The detection antibody is coupled with biotin and can specifically detect interferon, and the pipette is used to absorb the blood sample, the detection antibody, the detection tracer SA-PolyHRP and the luminol chemiluminescent substrate into the microfluidic immunoreactor;

[0009] The chemiluminescence detection unit can fix the pipette that is tightly attached to the microfluidic immunoreactor, collect chemiluminescence image signals and convert the chemiluminescence image signals into quantitative numerical signals. The quantitative numerical signals are converted into equivalent standard interferon concentrations through a standard curve to evaluate the T cell immune protection ability of the sample.

[0010] In some of the embodiments, there are multiple microfluidic immunoreactors, and any one of the microfluidic immunoreactors is tightly fitted with any one of the pipettes or its adapter.

[0011] In some embodiments, the pipette is multi-channel or single-channel, and the pipette includes but is not limited to a multi-channel pipette gun or a liquid pump.

[0012] In some embodiments, the interferon capture antibody is diluted in a phosphate buffer solution to a target concentration of 5 to 15 μg / mL of the interferon capture antibody.

[0013] In some embodiments, the detection antibody is diluted to 800-1000 ng / mL using 1% casein buffer.

[0014] In some embodiments, the detection tracer SA-PolyHRP is diluted about 3000 to 6000 times using 1% casein buffer.

[0015] In some embodiments, the chemiluminescence detection unit includes a light-shielding box, a CMOS camera arranged at the bottom of the light-shielding box, a limiting device arranged opposite to the CMOS camera, and the computer connected to the CMOS camera signal, the limiting device fixes the pipette tightly fitted with the microfluidic immunoreactor, the CMOS camera collects the chemiluminescent image signal, the computer converts the chemiluminescent image signal into a quantitative numerical signal, and the quantitative numerical signal is converted into an equivalent standard interferon concentration through a standard curve to evaluate the T cell immune protection ability of the sample.

[0016] In some embodiments, the blood sample includes venous blood or fingertip blood.

[0017] The second object of the present application is to provide a method for evaluating the tuberculosis infection T cell immune evaluation system, comprising the following steps:

[0018] Using a pipette, aspirate the supernatant of the blood sample into a microfluidic immunoreactor, and incubate for 20 to 30 minutes, wherein the microfluidic immunoreactor is coated with an interferon capture antibody;

[0019] Using the pipette to aspirate the washing solution and discharge the washing solution to remove unstable non-specific binding;

[0020] Using the pipette to suck the detection antibody into the microfluidic immunoreactor and incubate for 10 to 20 minutes, the detection antibody is coupled with biotin and can specifically detect interferon;

[0021] Using the pipette to aspirate the washing solution and discharge the washing solution to remove unstable non-specific binding;

[0022] Using the pipette, the detection tracer SA-PolyHRP is sucked into the microfluidic immunoreactor and incubated for 3 to 5 minutes;

[0023] Using the pipette to aspirate the washing solution and discharge the washing solution to remove unstable non-specific binding;

[0024] Using the pipette to suck the luminol chemiluminescent substrate into the microfluidic immunoreactor;

[0025] The chemiluminescence detection unit collects chemiluminescence image signals in the microfluidic immunoreactor;

[0026] The computer converts the chemiluminescent image signal into a quantitative numerical signal, and the quantitative numerical signal is converted into an equivalent standard interferon concentration through a standard curve to evaluate the T cell immune protection ability of the sample.

[0027] In some embodiments, the blood sample includes venous blood or fingertip blood;

[0028] When the blood sample is venous blood, the blood sample further includes the following treatment before being sucked into the microfluidic immunoreactor: adding 10% v / v stimulant to the blood sample, and placing it in a 37° C. cell incubator for 10 to 20 hours, and the volume of each venous blood incubation is 50 to 1000 μL;

[0029] When the blood sample is fingertip blood, solid lithium heparin particles are dissolved in physiological saline and the target concentration of the lithium heparin solution is 30 IU / mL. The fingertip blood and the lithium heparin solution are mixed at a volume ratio of 1:1, and then 10% v / v stimulant is added. The mixture is placed in a 37°C cell incubator for incubation for 10 to 20 hours. The volume of each fingertip blood incubation is 10 to 50 μL.

[0030] In some embodiments, the interferon capture antibody is diluted in a phosphate buffer solution to a target concentration of 5 to 15 μg / mL of the interferon capture antibody.

[0031] In some embodiments, the detection antibody is diluted to 800-1000 ng / mL using 1% casein buffer.

[0032] In some embodiments, the detection tracer SA-PolyHRP is diluted 3000-6000 times using 1% casein buffer.

[0033] This application adopts the above technical solution, and its beneficial effects are as follows:

[0034] The tuberculosis infection T cell immunity evaluation system and evaluation method provided by the present application include a microfluidic immunoreactor, a pipette, a detection antibody, a detection tracer SA-PolyHRP, a luminol chemiluminescent substrate, a chemiluminescent detection unit and a computer. The supernatant of the blood sample is sucked into the microfluidic immunoreactor with a pipette and incubated for 30 minutes. The microfluidic immunoreactor is coated with an interferon capture antibody; the cleaning solution is sucked in and discharged by the pipette to remove unstable non-specific binding; the detection antibody is sucked into the microfluidic immunoreactor with a pipette and incubated for 15 minutes. The detection antibody is coupled with biotin and can specifically detect interferon; the cleaning solution is sucked in and discharged by the pipette to remove unstable non-specific binding; the detection tracer is sucked in and discharged by the pipette to remove unstable non-specific binding; the detection tracer is sucked in and discharged by the pipette to remove unstable non-specific binding. SA-PolyHRP is sucked into the microfluidic immunoreactor and incubated for 3 minutes; the cleaning solution is sucked and discharged by the pipette to remove unstable non-specific binding; the luminol chemiluminescent substrate is sucked into the microfluidic immunoreactor by the pipette; the chemiluminescent detection unit collects the chemiluminescent image signal in the microfluidic immunoreactor; the computer converts the chemiluminescent image signal into a quantitative numerical signal, and the quantitative numerical signal is converted into an equivalent standard interferon concentration through a standard curve to evaluate the T cell immune protection ability of the sample. The present application provides a T cell immune evaluation system and an evaluation method for evaluating tuberculosis infection. The system completes the T cell immune evaluation of tuberculosis infection through two indicators of T and N, excluding immunodeficient patients such as AIDS. The system relies on a high-sensitivity detection platform, can stimulate and incubate in trace blood samples, and significantly shorten the incubation time, thereby reducing the demand for the amount of patient blood samples. This enables special populations such as the elderly and children to also perform IGRA testing. The evaluation system not only has the advantages of fast detection speed and high flexibility, but also performs well in stability and reliability. In addition, the tuberculosis infection T cell immunity assessment system and method provided in the present application can not only evaluate the T cell immune response of tuberculosis, but also increase the test group indicators to evaluate the T cell immune protection ability of other pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments of the present application or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0036] Figure 1 This is a schematic diagram of the structure of the tuberculosis infection T cell immune assessment system provided in Example 1 of the present application.

[0037] Figure 2 This is a schematic diagram of the structure of the chemiluminescence detection unit provided in Example 1 of the present application.

[0038] Figure 3 Schematic diagram of the principle of the method for assessing T cell immunity of tuberculosis infection provided in Example 2 of the present application.

[0039] Figure 4 This is a flowchart of the steps of the method for evaluating T cell immunity of tuberculosis infection provided in Example 2 of the present application.

[0040] Figure 5 The standard curve of the standard interferon protein solution in plasma was constructed using 2-fold diluted negative plasma provided in Example 2.

[0041] Figure 6 This is a schematic diagram of the experimental process for reducing the incubation volume of venous blood and the relationship between IFN-γ concentration and incubation volume provided in Example 2 of the present application.

[0042] Figure 7 This is a schematic diagram of the experimental process for shortening the venous blood incubation time and the relationship between IFN-γ concentration and time analysis provided in Example 2 of the present application.

[0043] Figure 8 A schematic diagram comparing 10 μL of fingertip blood and 15 μL of fingertip blood anticoagulant provided in Example 2 of the present application. DETAILED DESCRIPTION

[0044] Embodiments of the present application are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present application, and should not be construed as limiting the present application.

[0045] In the description of the present application, it should be understood that the terms "upper", "lower", "horizontal", "inside", "outside", etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present application and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present application.

[0046] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of this application, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0047] In order to make the objectives, technical solutions and advantages of the present application more clearly understood, the present application is further described in detail below in conjunction with the accompanying drawings and embodiments.

[0048] Example 1

[0049] See also Figure 1 to Figure 2 , is a schematic diagram of the structure of the tuberculosis infection T cell immune assessment system provided in Example 1 of the present application, including: a microfluidic immunoreactor 100, a pipette 200, a detection antibody, a detection tracer SA-PolyHRP, a luminol chemiluminescent substrate, a chemiluminescent detection unit 300 and a computer. The specific structure of each component and its connection relationship are described in detail below.

[0050] The microfluidic immunoreactor 100 is coated with interferon capture antibodies. The microfluidic immunoreactor 100 is tightly fitted with the pipette 200 , and the pipette 200 can control the liquid entering and exiting the microfluidic immunoreactor 100 .

[0051] In this embodiment, there are multiple microfluidic immunoreactors 100, and any one of the microfluidic immunoreactors 100 is closely attached to any one of the pipettes 200 or its adapter. Since the microfluidic immunoreactor 100 can be closely attached to the pipette 200, and no auxiliary materials such as glue are needed after attachment, airtightness can be naturally achieved, which is conducive to batch processing and application.

[0052] In this embodiment, any of the pipettes 200 is a multi-channel or single-channel pipette. The pipette 200 includes but is not limited to a multi-channel pipette gun or a liquid pump.

[0053] Furthermore, the interferon capture antibody needs to be diluted in a phosphate buffer solution before use to a target concentration of 5 to 15 μg / mL of the interferon capture antibody, and incubated for 30 to 60 minutes.

[0054] The detection antibody is coupled with biotin and can specifically detect interferon.

[0055] Furthermore, the detection antibody is diluted to 800-1000 ng / mL using 1% casein buffer.

[0056] The pipette is used to suck the blood sample, the detection antibody, the detection tracer SA-PolyHRP and the luminol chemiluminescent substrate into the microfluidic immunoreactor 100 .

[0057] Furthermore, the detection tracer SA-PolyHRP is diluted 3000 to 6000 times using 1% casein buffer.

[0058] The chemiluminescence detection unit 300 can fix the pipette 200 that is tightly attached to the microfluidic immunoreactor 100 and collect chemiluminescence image signals, and at the same time convert the chemiluminescence image signals into quantitative numerical signals. The quantitative numerical signals are converted into equivalent standard interferon concentrations through a standard curve to evaluate the T cell immune protection ability of the sample.

[0059] Please refer to 2. The chemiluminescence detection unit 300 includes a light-shielding box 310, a CMOS camera 320 arranged at the bottom of the light-shielding box 310, a limiting device 330 arranged opposite to the CMOS camera 320, and a computer (not shown) connected to the signal of the CMOS camera 320. The limiting device 330 fixes the pipette 200 that is tightly fitted with the microfluidic immunoreactor 100. The CMOS camera 320 collects the chemiluminescence image signal, and the computer converts the chemiluminescence image signal into a quantitative numerical signal. The quantitative numerical signal is converted into an equivalent standard interferon concentration through a standard curve to evaluate the T cell immune protection ability of the sample.

[0060] Furthermore, the chemiluminescence detection unit 300 further includes a slidable protective cover 340 , and the protective cover 340 can seal the pipette 200 tightly attached to the microfluidic immunoreactor 100 in the light shielding box 310 .

[0061] It should be noted that, in this embodiment, the blood sample includes venous blood or fingertip blood.

[0062] Specifically, when the blood sample is venous blood, it is necessary to add 10% v / v stimulant to the venous blood and then place it in a 37° C. cell incubator for incubation for 10 to 20 hours.

[0063] Specifically, when the blood sample is fingertip blood, solid lithium heparin particles are dissolved using physiological saline and the target concentration of the lithium heparin solution is 30 IU / mL, the fingertip blood and the lithium heparin solution are mixed in a volume ratio of one to one, and then 10% v / v stimulant is added to the mixed solution and placed in a 37°C cell incubator for incubation for 10 to 20 hours.

[0064] The tuberculosis infection T cell immune assessment system provided in the above-mentioned Example 1 of the present application has a simple structure. Through a highly sensitive detection platform, it can achieve stimulation and incubation in a tiny whole blood sample and shorten the incubation time, which can reduce the clinical demand for the amount of patient blood, so that special populations such as the elderly and children can also undergo IGRA testing. The assessment system is not only fast and flexible, but also stable and reliable.

[0065] Example 2

[0066] See also Figure 3 A schematic diagram of the principle of a method for evaluating the immunity of tuberculosis infected T cells is provided for this embodiment 2. The implementation method thereof is described in detail below.

[0067] The blood samples used in this embodiment include venous blood or fingertip blood. Since the cell components and contents of fingertip blood and venous blood are basically the same, this embodiment first uses venous blood to conduct a large number of preliminary optimization tests, and finally performs verification on fingertip blood.

[0068] In this embodiment, when venous blood samples are used, the sample stimulation incubation experimental process is: collect 30μL, 40μL, and 50μL of venous blood; add 10% v / v stimulant (N, T, P) to the collected venous blood; place in a 37°C cell incubator for 10 hours, and the volume of each venous blood incubation is 50 to 1000μL.

[0069] In this embodiment, when the blood sample is fingertip blood, solid lithium heparin particles are dissolved with physiological saline and the target concentration of the lithium heparin solution is 30 IU / mL. The fingertip blood and the lithium heparin solution are mixed at a volume ratio of 1:1, and then 10% v / v stimulant (N, T, P) is added, and the mixture is placed in a 37°C cell incubator for 10 hours. The volume of each fingertip blood incubation is 10 to 50 μL.

[0070] In this embodiment, before using the tuberculosis-infected T cell immune assessment, the following operations are required: 1. Dilute the interferon capture antibody in phosphate buffered solution (PBS) to a target concentration of 10 μg / mL, and allow it to stay in the reactor for incubation for 30-60 minutes. 2. Use a multi-channel pipette to aspirate and discharge the cleaning solution. The cleaning solution uses 0.05% Tween20 dissolved in PBS to remove unstable non-specific binding. 3. Use a multi-channel pipette to aspirate the blocking liquid system for about 20 minutes of blocking. This blocking process can be repeated once to further improve the signal-to-noise ratio. The blocking liquid system contains 3% bovine serum albumin and 1% casein. 4. Use a multi-channel pipette to aspirate and discharge the cleaning solution. The cleaning solution uses 0.05% Tween20 dissolved in PBS.

[0071] See also Figure 4 The steps of using the tuberculosis infection T cell immunity assessment method are as follows:

[0072] Step S110: using a pipette to suck the supernatant of the blood sample into a microfluidic immunoreactor, and incubate for 20 to 30 minutes, wherein the microfluidic immunoreactor is coated with an interferon capture antibody, so that the interferon in the sample binds to the capture antibody on the surface of the immunoreactor;

[0073] Step S120: using the pipette to aspirate the cleaning solution and discharge the cleaning solution to remove unstable non-specific binding;

[0074] Step S130: using the pipette to absorb the detection antibody into the microfluidic immunoreactor and incubate for 10 to 20 minutes, wherein the detection antibody is coupled with biotin and can specifically detect interferon;

[0075] Furthermore, the interferon capture antibody is diluted in a phosphate buffer solution to a target concentration of 5 to 15 μg / mL. The detection antibody is diluted to 800-1000 ng / mL using a 1% casein buffer.

[0076] Step S140: Use the pipette to suck in the washing solution and discharge the washing solution to remove unstable non-specific binding. The washing process is repeated three times to achieve a better signal-to-noise ratio.

[0077] Step S150: using the pipette to aspirate the detection tracer SA-PolyHRP into the microfluidic immunoreactor and incubate for 3 to 5 minutes.

[0078] Furthermore, the detection tracer SA-PolyHRP is diluted 3000 to 6000 times using 1% casein buffer.

[0079] Step S160: using the pipette to aspirate the cleaning solution and discharge the cleaning solution to remove unstable non-specific binding.

[0080] Step S170: using the pipette to suck the luminol chemiluminescent substrate into the microfluidic immunoreactor.

[0081] Step S180: the chemiluminescence detection unit collects chemiluminescence image signals in the microfluidic immunoreactor.

[0082] Step S190: The computer converts the chemiluminescent image signal into a quantitative numerical signal, and the quantitative numerical signal is converted into an equivalent standard interferon concentration through a standard curve to evaluate the T cell immune protection ability of the sample.

[0083] See also Figure 5 , the standard curve of the standard interferon protein solution in plasma was constructed using 2-fold diluted negative plasma provided in this example.

[0084] This example adopts the above-mentioned tuberculosis infection T cell immunity assessment method, and the concentrations of the selected standard interferon proteins are 9000, 3000, 1000, 333.3, 111.1, 37.0, 12.3, 4.1 and 1.37 pg / mL. The above interferon concentrations cover the interferon concentration range of normal people and tuberculosis patients, and 5 repeated experiments are performed. The chemiluminescent image signal is converted into a quantitative numerical signal by a computer, and the quantitative numerical signal is converted into an equivalent standard interferon concentration through a standard curve.

[0085] Specifically, x is the interferon protein concentration value of the standard, y is the quantitative numerical signal, and x and y are taken as 10 logarithms to fit the standard curve log(y)=0.9054log(x)+0.5904. When the real sample is subsequently detected, the signal value of the real sample is taken as 10 logarithms, substituted into the above curve equation to calculate x, and then x is converted back to a constant by 10. At this time, x is the absolute concentration value of the patient's interferon, R 2 Can reach 0.998, R 2 A high value indicates that there is a strong linear relationship between the concentration of the standard and its detection signal, which helps to more accurately quantify the target substance in the unknown sample. This shows that its detection performance is very good and can achieve absolute quantification of interferon concentration in patient samples.

[0086] See also Figure 6 , which is a schematic diagram of the experimental process for reducing the venous blood incubation volume and the relationship between IFN-γ concentration and incubation volume provided in this embodiment.

[0087] in, Figure 6 (b) is represented as the "test group (T)", whose T cells in the blood sample secrete IFN-γ (γ interferon), and the T tube contains specific Mycobacterium tuberculosis antigens (such as ESAT-6 and CFP-10) to stimulate the immune cells of the subjects; the "positive control group (P)" is stimulated with mitogens such as phytohemagglutinin (PHA) to verify the function of T cells; the "negative control group (N)" is used as a baseline calibration to measure the basal level of interferon-γ (IFN-γ) release of the subjects in the absence of specific stimulation, and also as a quality control to ensure the stability and reliability of experimental operations and reagents. By comparing the interferon-γ (IFN-γ) levels in the N tube, T tube, and P tube, it can be determined whether there is tuberculosis infection.

[0088] Figure 6(c) shows a schematic diagram of data analysis of T / N and T / P of different volumes. It can be seen from the figure that the reduction of the culture volume from the standard 1mL to 50μL did not affect the positive and negative discrimination of the diagnostic results. From the distribution of positive and negative points in the figure, it can be seen that there is an obvious distribution threshold for positive and negative, and several points have false negatives, which may be caused by problems with sample collection or interference caused by other medical histories of patients.

[0089] See also Figure 7 , which is a schematic diagram of the experimental process for shortening the venous blood incubation time and the relationship between IFN-γ concentration and time analysis provided in this embodiment.

[0090] in, Figure 7 (b) is represented as the "test group (T)", whose T cells in the blood sample secrete IFN-γ (γ interferon), and the T tube contains specific Mycobacterium tuberculosis antigens (such as ESAT-6 and CFP-10) to stimulate the immune cells of the subjects; the "positive control group (P)" is stimulated with mitogens such as phytohemagglutinin (PHA) to verify the function of T cells; the "negative control group (N)" is used as a baseline calibration to measure the basal level of interferon-γ (IFN-γ) release of the subjects in the absence of specific stimulation, and also as a quality control to ensure the stability and reliability of experimental operations and reagents. By comparing the interferon-γ (IFN-γ) levels in the N tube, T tube, and P tube, it can be determined whether the patient has tuberculosis infection.

[0091] Figure 7 Part (c) shows the heat map of T / N and T / P data at different incubation times. Through the heat map analysis, it can be seen how the interferon release of different patients changes over time. It is observed that at 10 hours of incubation, the negative and positive status of patients can be clearly distinguished, instead of the 20 hours required by the traditional method. Please refer to Figure 8 , which is a comparative schematic diagram of 10 μL fingertip blood and 15 μL fingertip blood anticoagulant provided in this embodiment.

[0092] from Figure 8 As can be seen in Figure a, this embodiment explores two aspects of the anticoagulant, one is the concentration of the anticoagulant, and the other is the volume of the anticoagulant, to explore the interference degree of volume and concentration on the anticoagulation of fingertip blood or the degree of anticoagulation. Figure 7 b is a graph showing the results of fingertip blood incubation provided in this embodiment. Figure 7 As can be seen in b, the anticoagulant concentration of 15 IU / mL has the best stability. The volume of the anticoagulant was selected to take into account the difficulty of extracting the supernatant at the end, so an anticoagulant volume of 15 μL was selected.

[0093] It should be noted that the volume here is the volume without adding fingertip blood, 10 / 15μL of anticoagulant alone, and the concentration below is the final concentration after the anticoagulant is added to the fingertip blood. (The previous application mentioned an anticoagulant concentration of 30IU / mL, because fingertip blood will be added 1:1, which is equivalent to a 2-fold dilution of the anticoagulant).

[0094] The present application provides a T cell immunity method for evaluating tuberculosis infection, which completes the T cell immunity evaluation of tuberculosis infection through two indicators, T and N, while excluding immunodeficient patients such as AIDS. This method uses a high-sensitivity detection platform, which can not only stimulate and incubate in trace whole blood samples, but also shorten the incubation time. This reduces the clinical demand for the amount of blood of patients, so that special populations such as the elderly and children can also receive IGRA testing. The method is fast, flexible, stable and reliable. In addition, the tuberculosis infection T cell immunity evaluation system and method provided in this application can not only evaluate the T cell immune response of tuberculosis, but also increase the test group indicators to evaluate the T cell immune protection ability of other pathogens.

[0095] It can be understood that the technical features of the above-described embodiments can be arbitrarily combined. In order to make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0096] The above are only preferred embodiments of the present application, and only specifically describe the technical principles of the present application. These descriptions are only for explaining the principles of the present application and cannot be interpreted as limiting the scope of protection of the present application in any way. Based on the explanation here, any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application, and other specific implementation methods of the present application that can be associated with the technicians in this field without creative work, should be included in the scope of protection of the present application.

Claims

1. A tuberculosis infection T cell immune assessment system, characterized in that: It includes a microfluidic immunoreactor, a pipette, a detection antibody, a detection tracer SA-PolyHRP, a luminol chemiluminescent substrate, and a chemiluminescent detection unit, wherein: The microfluidic immunoreactor is coated with interferon capture antibodies, and the microfluidic immunoreactor is tightly fitted with the pipette; The detection antibody is coupled with biotin and can specifically detect interferon, and the pipette is used to absorb the blood sample, the detection antibody, the detection tracer SA-PolyHRP and the luminol chemiluminescent substrate into the microfluidic immunoreactor; The chemiluminescence detection unit can fix the pipette that is tightly attached to the microfluidic immunoreactor, collect chemiluminescence image signals and convert the chemiluminescence image signals into quantitative numerical signals. The quantitative numerical signals are converted into equivalent standard interferon concentrations through a standard curve to evaluate the T cell immune protection ability of the sample.

2. The tuberculosis infection T cell immune assessment system according to claim 1, characterized in that: There are multiple microfluidic immunoreactors, and any one of the microfluidic immunoreactors is tightly fitted with any one of the pipettes or its adapter.

3. The tuberculosis infection T cell immune assessment system according to claim 1, characterized in that: The pipette is multi-channel or single-channel, and includes but is not limited to a multi-channel pipette gun or a liquid pump.

4. The tuberculosis infection T cell immune assessment system according to claim 1, characterized in that: The interferon capture antibody is diluted in a phosphate buffer solution to a target concentration of 5 to 15 μg / mL.

5. The tuberculosis infection T cell immune assessment system according to claim 1, characterized in that: The detection antibody was diluted to 800-1000 ng / mL using 1% casein buffer.

6. The tuberculosis infection T cell immune assessment system according to claim 1, characterized in that: The detection tracer SA-PolyHRP was diluted 3000-6000 times using 1% casein buffer.

7. The tuberculosis infection T cell immune assessment system according to claim 1, characterized in that: The chemiluminescence detection unit includes a light-shielding box, a CMOS camera arranged at the bottom of the light-shielding box, a limiting device arranged opposite to the CMOS camera, and the computer connected to the CMOS camera signal, wherein the limiting device fixes the pipette tightly fitted with the microfluidic immunoreactor, the CMOS camera collects the chemiluminescent image signal, the computer converts the chemiluminescent image signal into a quantitative numerical signal, and the quantitative numerical signal is converted into an equivalent standard interferon concentration through a standard curve to evaluate the T cell immune protection ability of the sample.

8. The tuberculosis infection T cell immune assessment system according to claim 1, characterized in that: The blood sample includes venous blood or fingertip blood.

9. An evaluation method using the tuberculosis infection T cell immunity evaluation system according to any one of claims 1 to 8, characterized in that: The steps include: Using a pipette, aspirate the supernatant of the blood sample into a microfluidic immunoreactor, and incubate for 20 to 30 minutes, wherein the microfluidic immunoreactor is coated with an interferon capture antibody; Using the pipette to aspirate the washing solution and discharge the washing solution to remove unstable non-specific binding; Using the pipette to suck the detection antibody into the microfluidic immunoreactor and incubate for 10 to 20 minutes, the detection antibody is coupled with biotin and can specifically detect interferon; Using the pipette to aspirate the washing solution and discharge the washing solution to remove unstable non-specific binding; Using the pipette, the detection tracer SA-PolyHRP is sucked into the microfluidic immunoreactor and incubated for 3 to 5 minutes; Using the pipette to suck in the cleaning solution and discharge the cleaning solution to remove unstable non-specific binding; using the pipette to suck the luminol chemiluminescent substrate into the microfluidic immunoreactor; The chemiluminescence detection unit collects chemiluminescence image signals in the microfluidic immunoreactor; The computer converts the chemiluminescent image signal into a quantitative numerical signal, and the quantitative numerical signal is converted into an equivalent standard interferon concentration through a standard curve to evaluate the T cell immune protection ability of the sample.

10. The evaluation method of the tuberculosis infection T cell immune evaluation system according to claim 9, characterized in that: The blood sample includes venous blood or fingertip blood; When the blood sample is venous blood, the blood sample further includes the following treatment before being sucked into the microfluidic immunoreactor: adding 10% v / v stimulant to the blood sample, and placing it in a 37° C. cell incubator for 10 to 20 hours, and the volume of each venous blood incubation is 50 to 1000 μL; When the blood sample is fingertip blood, solid lithium heparin particles are dissolved in physiological saline and the target concentration of the lithium heparin solution is 30 IU / mL. The fingertip blood and the lithium heparin solution are mixed at a volume ratio of 1:1, and then 10% v / v stimulant is added. The mixture is placed in a 37°C cell incubator for incubation for 10 to 20 hours. The volume of each fingertip blood incubation is 10 to 50 μL.

11. The method for evaluating the tuberculosis infection T cell immunity evaluation system according to claim 9, characterized in that: The interferon capture antibody is diluted in a phosphate buffer solution to a target concentration of 5 to 15 μg / mL.

12. The evaluation method of the tuberculosis infection T cell immunity evaluation system according to claim 9, characterized in that: The detection antibody was diluted to 800-1000 ng / mL using 1% casein buffer.

13. The evaluation method of the tuberculosis infection T cell immunity evaluation system according to claim 9, characterized in that: The detection tracer SA-PolyHRP was diluted 3000-6000 times using 1% casein buffer.

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