WO2019109321A1 - Detection system for memory immunity cells, and applications thereof - Google Patents

Abstract

The present invention provides a detection system for memory immunity cells, and applications thereof. The quantity of samples in the detection system ranges from 0.3 ml to 0.8 ml.

Description

Detection system of memory immune cells and application thereof Technical field

The invention belongs to the field of medical detection applications, and relates to a detection system of memory immune cells and an application thereof.

Background technique

Cytokine refers to a small class of polypeptide glycoproteins that are closely related to the body's inflammation and immune response. When the body is infected with a disease, a series of related cells participate in the immune response process by coordinating the body. Taking tuberculosis immunity as an example, Mycobacterium tuberculosis induces type IV hypersensitivity through T lymphocyte mediation. The body plays a role in T cell immunity, and multiple cytokines participate in the immune response, mainly by CD4+ T cells. Helper type 1 cells (Th1) and CD8+ T cells, T helper type 2 cells (Th2), are mediated. Related cytokines include interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-10 (IL-10), interferon-γ (IFN-γ), and the like.

It has been reported that by detecting the degree of response of memory T cells to specific antigen stimulation, it is usually expressed by the secretion of various cytokines, which can reveal the state of the body's infectious diseases. This is a reliable detection method, such as γ-interferon release assay. (IGRAs). IGRAs are techniques for detecting the ability of T cells to produce and release gamma-interferon after stimulation with tuberculosis-specific antigens in vitro, thereby determining cell-mediated immune responses. IGRAs stimulates the whole blood or peripheral blood mononuclear cells of the subject with two kinds of antigens specific to E. coli, ESAT-6 and CFP-10, to produce a large amount of γ-interferon by T lymphocytes, and then use enzyme-linked immunosorbent assay or Enzyme-linked immunospot assay for detecting gamma-interferon concentration or counting methods for secreting γ-interferon cells.

The internationally widely used IGRAs commercial kits are the following: QuantiFERON-TB Gold In-Tube (QFT-GIT) manufactured by Cellistis, Australia, QFT, QFT-Gold from Qiagen, Germany; T-produced by Oxford Immunotec, UK SPOT.TB, TB-IGRA of China Wantai.

QuantiFERON-TB Gold has been approved by the US FDA as an in vitro diagnostic for the detection of M. tuberculosis infection. It uses a mixture of analog ESAT-6, CFP-10 and TB7.7 (p4) proteins to stimulate heparinized cells in whole blood, and IFN-interferon is detected by ELISA to identify in vitro responses to these peptide antigens. The response is associated with M. tuberculosis infection.

QFT is the first IGRAs product approved for FDA listing and is a preferred alternative to TST in many national guidelines and recommendations. QFT is a highly specific, controlled hematology test that can be used to aid in the diagnosis of TB infections and provides results to show individuals' T cell responses that are highly specific for M. tuberculosis antigens. QFT uses an enzyme-linked immunosorbent assay (ELISA) to detect gamma-interferon responses in whole blood samples after incubation with TB-specific antigens.

Both QFT and QuantiFERON-TB Gold and TB-IGRA use 0.8-1.2 mL of blood for stimulation experiments. QFT and QuantiFERON-TB Gold are directly collected into the stimulation tube for subsequent testing. TB-IGRA is a stimulation experiment using precise blood separation of 1 mL of blood. T-Spot TB was performed after isolation of PBMC cells from 8 mL of adult blood, requiring 250,000 PBMC cells per well.

The method of diagnosing disease states by cytokine changes relies on the detection of high sensitivity of the target cytokine. Taking the commercial kit of IGRAs as an example, T-SPOT.TB and QFT-GIT detect changes in the secretion of IFN-γ or the number of IFN-γ secreted by memory T cells stimulated by tuberculosis antigen, thereby judging whether the body is Infected with M. tuberculosis. According to reports, the sensitivity of T-SPOT.TB in the diagnosis of tuberculosis is 90%, but T-SPOT.TB needs to separate human peripheral blood mononuclear cells (PBMC), which is cumbersome. The QFT-GIT method is relatively easy to operate, but the sensitivity to assist in the diagnosis of tuberculosis is only 70%. At present, there are many reports aimed at improving the overall sensitivity of QFT-GIT through the adjustment of experimental methods, such as increasing the incubation temperature to 39 ° C, incubating venous blood, etc., but the T cells will undergo non-specific activation at high temperatures, increasing the detection. False positive rate.

CN 102680684 A discloses the technical field of detection of Mycobacterium tuberculosis, in particular to a method for detecting whole blood T cells specific to tuberculosis antigen, which incubates a tuberculosis specific epitope peptide, an MHC donor and a peripheral whole blood of a subject, directly Signals for detection of complexes formed by MHC donor-Mycobacterium tuberculosis epitope-specific T cells. CN 101446585A discloses an agent and method for detecting Mycobacterium tuberculosis infection in vitro, comprising the M233 polypeptide represented by SEQ ID NO. 1; by contacting with T cells of Mycobacterium tuberculosis host by using M233 polypeptide or the like, detecting The cytokine released by the T cell determines whether the T cell recognizes the M233 polypeptide analog. The detection sample of the above detection method has a volume of 1-5 mL, and the detection sensitivity thereof needs to be further improved.

Therefore, it is of great significance to develop a detection method capable of further improving the sensitivity of detecting memory immune cells.

Summary of the invention

In view of the prior art, the present patent provides a detection system for memory immune cells and an application thereof, which can improve the sensitivity of detecting cytokines secreted by memory T cells after being stimulated again, and improve the detection of cytokines secreted by memory T cells. The basic auxiliary diagnostic kit has overall sensitivity and reduces the possibility of missed detection.

In a first aspect, an object of the present invention is to provide a detection system for memory immune cells, wherein the sample amount in the detection system is 0.3-0.8 mL.

In the present invention, the inventors have found that by stimulating the incubation time of the antigen, the incubation temperature, and the amount of blood to be incubated, it is found that controlling the amount of blood to be incubated can help the memory of the immune cells to be stimulated again, and the amount of secretion is significantly increased, and the amount of secretion is increased. The experimental verification found that when the sample size is 0.3-0.8 mL, the sensitivity of detecting secreted substances can be significantly improved under the condition that the final concentration of the stimulating antigen is consistent.

The sample amount may be, for example, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, or 0.8 mL, preferably 0.6 mL.

In the present invention, the inventors have further found that when the sample volume is 0.6 mL, the secretion amount of the secretory substance of the memory immune cells is the highest, and the detection sensitivity is the highest.

According to the present invention, the target of the detection system is a substance secreted by the cells after stimulation, and the substance secreted by the cells after stimulation is any one of cytokines, chemokines or proteins or a combination of at least two.

According to the invention, the cytokine is a cytokine which is stimulated and secreted by immune cells and/or non-immune cells, preferably a cytokine secreted by immune cells.

In the present invention, the immune cells may be selected from, but not limited to, T cells, B cells, NK cells or macrophages, and the immune cells pass through specific cell stimulators, and after stimulation, secrete corresponding cytokines, thereby detecting whole blood. The cell secretions in the test are achieved.

According to the invention, the detection system further comprises a stimulating antigen and/or a positive control antigen.

According to the invention, the stimulating antigen is a specific antigen that stimulates memory T cells.

According to the present invention, the specific antigen for stimulating memory T cells is any one or a combination of at least two of a natural protein, a polypeptide, a nucleic acid, a polysaccharide, a small molecule compound, a virus-specific antigen or a bacterial-specific antigen, A bacterial specific antigen is preferred, and a Mycobacterium tuberculosis specific antigen is further preferred.

The natural protein, polypeptide, nucleic acid, polysaccharide, small molecule compound may be an allergen capable of stimulating a cellular response.

The virus-specific antigen or the bacteria-specific antigen is feasible as long as it can stimulate the antigen of the memory T cell, including the virus or bacterial surface, internal antigenic protein, nuclear protein, virus or bacterial secreted protein, and is not special here. The virus-specific antigen may be, for example, a CMV virus, and the bacterial-specific antigen may be, for example, a Mycobacterium tuberculosis-specific antigen.

According to the invention, the positive control antigen is a non-specific stimulator that stimulates immune cells.

According to the present invention, the non-specific stimulating agent for stimulating immune cells is selected from, but not limited to, concanavalin A (Concanavalin A, ConA), Phytohemagglutinin (PHA), human luciferin (PWM), peanut agglutinin (PNA), soybean agglutinin (SBA), staphylococcal A protein (SAC), Any one or a combination of at least two of lipopolysaccharide (LPS) or tumor stimulating agent (PMA) is preferably phytohemagglutinin.

According to the present invention, the cytokine is cytokine which is stimulated and secreted by immune cells or non-immune cells, and can be selected by a person skilled in the art according to experimental needs. The cytokines in the present invention are IL-1, IL- 2. IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-17, IFN-α, IFN-β, IFN-γ, TNF-β, SCF, G-CSF, M-CSF, GM-CSF, neuroleukocytidine, IgA, IgG, perforin, granulin, IP10 or Any one or a combination of at least two of TGF-β is preferably any one of IL-2, IL-6, IFN-γ, IP10 or TGF-β or a combination of at least two, and further preferably IFN - γ.

In the present invention, most of the cytokines are glycoproteins having a mass of less than 25 kDa. The present application has verified that the control sample volume is 0.3-0.8 mL by verifying three cytokines such as IL-2, IL-6 and IFN-γ. It does improve the detection sensitivity. The detection of other cytokines is also carried out using the corresponding kit. However, the detection system of the present application can be used for the detection of most cytokines, and the volume of the test sample is controlled to be 0.3-0.8 mL. The sensitivity of the assay is further increased, and the volume of a particular sample can be fine-tuned by each cytokine by those skilled in the art.

According to the invention, the test sample is heparin anticoagulated blood.

According to the present invention, the detection system further comprises detecting a stimulating antigen of the sample, the stimulating antigen being added to the sample to stimulate secretion of the cytokine for detection.

According to the present invention, the detection system further comprises a conventional reagent for detecting a sample, and the conventional reagent refers to a conventional detection reagent used for detecting a cytokine. For example, the detection of the cytokine IFN-γ can be detected by using an IFN-γ detection kit. For example, IL-2 can be detected by IL-2 test kit. The kit is a commercially available kit.

In a second aspect, the present invention provides a detection method using the detection system according to the first aspect, comprising the steps of:

(1) collecting blood samples into heparin anticoagulation blood collection tubes, mixing, and dispensing into 0.3-0.8 mL;

(2) adding a corresponding stimulating antigen to the sample of step (1), culturing;

(3) Detecting the content of the cytokine in the supernatant.

According to the invention, the culture temperature is 30-40 ° C, for example 30 ° C, 31 ° C, 32 ° C, 33 ° C, 34 ° C, 35 ° C, 36 ° C, 37 ° C, 38 ° C, 39 ° C or 40 ° C It is preferably 35-38 ° C, and more preferably 37 ° C.

According to the invention, the culture time is 10-35 h, for example, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h, 25 h, 26 h. 27h, 28h, 29h, 30h, 31h, 32h, 33h, 34h or 35h, preferably 18-24h.

In a third aspect, the invention provides the detection system of the first aspect for detecting the function of a memory immune cell.

In a fourth aspect, the invention provides the use of a detection system according to the first aspect in the manufacture of a kit for disease detection and/or diagnosis.

In the present invention, the disease is a disease such as tuberculosis, and any disease that can be detected by detecting a secretory substance of a memory immune cell, particularly a cytokine, is feasible, and is not particularly limited herein.

Compared with the prior art, the present invention has the following beneficial effects:

(1) The detection system provided by the present invention can significantly improve the sensitivity of detecting cytokines when the final concentration of the stimulating antigen is uniform, but when the sample is reduced to 0.3-0.8 mL, compared with the conventional 1-5 mL sample. Volume, the secretion of cytokines is significantly increased, and the detection sensitivity is more than doubled, which significantly improves the sensitivity of detection and reduces the possibility of missed detection;

(2) The detection system provided by the invention is simple, rapid and efficient;

(3) The detection system provided by the invention has a wide application range and has great market value.

DRAWINGS

1 is a view showing the detection result of detecting INF-γ by the detection system of the present invention, wherein LD represents a Mycobacterium tuberculosis-specific stimulating antigen, and TB represents a patient number;

2 is a view showing the detection result of detecting INF-γ by the detection system of the present invention, wherein P represents phytoagglutinin and TB represents a patient number;

3 is a graph showing the effect of the detection system of the present invention on blood sample detection in healthy humans, wherein LD represents Mycobacterium tuberculosis-specific stimulating antigen, P represents phytohemagglutinin, N represents a blank control, and WT represents a control tuberculosis-specific stimulating antigen;

4 is a graph showing the effect of oxygen content on the detection system of the present invention, wherein LD represents a Mycobacterium tuberculosis-specific stimulating antigen, and WT represents a control tuberculosis-specific stimulating antigen;

Figure 5 is a graph showing the results of detecting the IL-2 sensitivity of a patient sample by the detection system of the present invention, wherein LD represents a Mycobacterium tuberculosis-specific stimulating antigen;

6 is a diagram showing the results of detecting the IL-2 sensitivity of a healthy human sample by the detection system of the present invention, wherein LD represents a Mycobacterium tuberculosis-specific stimulating antigen;

Figure 7 is a graph showing the results of detecting the IL-6 sensitivity of a patient sample by the detection system of the present invention, wherein LD represents a Mycobacterium tuberculosis-specific stimulating antigen;

Fig. 8 is a graph showing the results of detecting the IL-6 sensitivity of a healthy human sample by the detection system of the present invention, wherein LD represents a specific stimulation resistance of Mycobacterium tuberculosis.

Detailed ways

In order to further illustrate the technical means and effects of the present invention, the technical solutions of the present invention will be further described below in conjunction with the drawings and embodiments of the present invention, but the present invention is not limited to the scope of the embodiments. Inside.

The specific techniques or conditions are not indicated in the examples, according to the techniques or conditions described in the literature in the art, or in accordance with the product specifications. The reagents or instruments used are not specified by the manufacturer, and are conventional products that are commercially available through formal channels.

material:

IFN-γ detection kit (ELISA): Guangzhou Reid Biotechnology Co., Ltd.;

IGRA kit (ELISA): Wantai Biopharmaceutical Co., Ltd.;

IL-2 Test Kit (ELISA): Guangzhou Reid Biotechnology Co., Ltd.;

IL-6 Test Kit (ELISA): Guangzhou Reid Biotechnology Co., Ltd.;

raw material:

Specific Mycobacterium tuberculosis stimulating antigen (LD, Guangzhou Reid Biotechnology Co., Ltd.):

Positive stimulator phytohemagglutinin (P, Sigma's PHA):

Specific Mycobacterium tuberculosis stimulating antigen (WT, Wantai Biopharmaceutical Co., Ltd.):

Pre-preparation: Recruit 18-45 year old patient volunteers and healthy volunteers to collect blood samples.

Example 1: Effect of different volume detection samples on the detection of IFN-γ

The blood samples of the collected patients will be subjected to the following experimental steps within 4 hours:

(1) Mix the fresh human venous blood upside down 5 times, add 0.3, 0.6, 1.0 mL blood samples to 3 positive tubes (P), and add 6 0.3, 0.4, 0.5, 0.6 to each of the 6 detection tubes (T). , 0.8, 1.0mL blood sample, 6 negative tubes (N) were added 0.3, 0.4, 0.5, 0.6, 0.8, 1.0mL blood samples respectively, different volumes of blood were controlled to add stimulation antigen (LD), the end of positive stimuli (P) Consistent concentration;

(2) The step (1) mixture is placed in a 37 ° C incubator, cultured for 20 ± 2h;

(3) Aspirate the plasma, place it in a 1.5 mL EP tube, and use the γ-interferon ELISA kit. IFN-γ detection was performed.

The results of the six test tubes (T) are shown in Figure 1. The results of the three positive tubes (P) are shown in Figure 2. As can be seen from Figure 1, there is no non-specific stimulation in the negative control tubes in the range of 0.3-1 mL. As the volume of the blood sample increases, the detection sensitivity first increases and then decreases. As can be seen from Figure 2, the three volumes of 0.3mL, 0.6mL and 1.0mL are further compared. The sensitivity of the 0.6mL volume sample is the highest. It can be seen that in the range of 0.3mL-0.6mL, the smaller the stimulated blood volume, the stimulating antigen and The stronger the stimuli stimulated by memory T cells, the stronger the IFN-γ secretion.

Example 2: Effect of 0.6 mL and 1.0 mL Volume Detection Samples on Detection of IFN-γ

The blood samples of 32 patients (TB8-TB39) collected will be subjected to the following experimental steps within 4 hours:

(1) Mix the fresh human venous blood upside down 5 times, mix and add 0.6, 1.0mL blood sample to 2 test tubes (T), and control the addition of stimulating antigen (LD) to make the final concentration consistent;

(2) The step (1) mixture is placed in a 37 ° C incubator, cultured for 20 ± 2h;

(3) The plasma was aspirated and placed in a 1.5 mL EP tube, and IFN-γ was detected using a γ-interferon ELISA kit.

The test results are shown in Table 1.

Table 1

It can be seen from Table 1 that the same stimulating antigen concentration, compared with the 1.0 mL group, significantly increased the response level of antigen-stimulated T cells, and the content of cytokine IFN-γ was significantly increased in 0.6 mL group. Above the 1.0 mL group, the secretion amount of 0.6 mL group/secretion amount 1.0 mL group ≥ 2 percentage reached 81.25%.

Comparative Example 1 Detection of IFN-γ in healthy volunteers

The collected blood samples of healthy people will be subjected to the following experimental steps within 4 hours:

(1) Mix the fresh human venous blood upside down 5 times, mix and add 1.0 blood sample to the negative tube (N), positive tube (P), and 2 test tubes (T) respectively to add 0.6mL, 1.0mL blood sample. The antigen (WT) was detected by the IGRA kit as a control group;

(2) The step (1) mixture is placed in a 37 ° C incubator, cultured for 20 ± 2h;

(3) The plasma was aspirated and placed in a 1.5 mL EP tube, and IFN-γ was detected using a γ-interferon ELISA kit.

The results of the test are shown in Fig. 3. It can be seen that the amount of IFN-γ secreted by the 0.6 mL blood sample group in healthy people is not significantly different from that of the negative control group and the WT control group, indicating that the method has no specific stimulation on healthy human blood samples.

Comparative Example 2 Effect of oxygen content on the detection of IFN-γ

The collected blood samples of healthy people will be subjected to the following experimental steps within 4 hours:

(1) Mix the fresh human venous blood upside down 5 times, mix and add 1.0mL blood sample to the negative tube (N), positive tube (P), control tube (WT), and 2 test tubes (T) respectively. mL, 1.0mL Blood sample, tighten the lid of the incubation tube, and use the IGRA kit to detect the antigen (WT) as the control group;

(2) According to the step (1), lead another set of experiments, and loosen the incubation tube cover to keep the oxygen content sufficient;

(3) The two sets of experiments were placed in a 37 ° C incubator and cultured for 20 ± 2 h;

(4) The plasma was aspirated and placed in a 1.5 mL EP tube, and IFN-γ was detected using a gamma interferon-ELISA test kit.

The test results are shown in Figure 4. It can be seen that the 0.6mL blood volume increases the stimulation level of the stimulating antigen regardless of the oxygen content of the incubation tube.

Example 3 Effect of different volume detection samples on IL-2 detection

The collected blood samples of the patient and healthy person will be subjected to the following experimental steps within 4 hours:

(1) Mix the fresh human venous blood upside down 5 times, and then mix and add 0.6, 1.0mL patients, blood samples from healthy people to 2 test tubes (T), control the addition of stimulating antigen (LD), so that the final concentration is consistent. ;

(2) The step (1) mixture is placed in a 37 ° C incubator, cultured for 20 ± 2h;

(3) Aspirate the plasma, place it in a 1.5 mL EP tube, and use the IL-2 ELISA test kit for IL-2 detection.

The results of the test are shown in Fig. 5 - Fig. 6. It can be seen that under the condition that the concentration of the stimulating antigen is uniform, the 0.6 mL group is compared with the 1.0 mL group to increase the degree of response of the stimulating antigen to stimulate the memory T cells. In the patient sample, the 0.6 mL group is secreted. The content of cytokine IL-2 was significantly higher than that of the 1.0 mL group. In healthy patient samples, IL-2 was detected as 0 pg/mL.

Example 4 Effect of different volume test samples on IL-6 detection

The collected blood samples of the patient and healthy person will be subjected to the following experimental steps within 4 hours:

(1) Mix the fresh human venous blood upside down 5 times, and then mix and add 0.6, 1.0mL patients, blood samples from healthy people to 2 test tubes (T), control the addition of stimulating antigen (LD), so that the final concentration is consistent. ;

(2) The step (1) mixture is placed in a 37 ° C incubator, cultured for 20 ± 2h;

(3) Aspirate the plasma, place it in a 1.5 mL EP tube, and use the IL-6 ELISA test kit for IL-6 detection;

The results of the test are shown in Fig. 7 to Fig. 8. It can be seen that under the condition that the concentration of the stimulating antigen is uniform, the 0.6mL group is compared with the 1.0mL group to increase the degree of stimulation of the antigen-stimulated memory T cells. In the patient sample, the 0.6mL group is secreted. The content of cytokine IL-6 was significantly higher than that of the 1.0 mL group. In the healthy patient samples, the secretion of cytokine IL-6 in the 0.6 mL group was also significantly higher than that in the 1.0 mL group.

In summary, the detection system provided by the present invention can significantly improve the sensitivity of detecting cytokines when the final concentration of the stimulating antigen is uniform, but when the sample is reduced to 0.3-0.8 mL, compared with the conventional 1-5 mL. The sample volume, the secretion of cytokines is significantly increased, and the detection sensitivity is more than doubled, which significantly improves the sensitivity of detection and reduces the possibility of missed detection; the detection system of the invention has a wide application range and has great market value.

The Applicant declares that the present invention is described by the above-described embodiments, but the present invention is not limited to the above detailed methods, that is, it does not mean that the present invention must be implemented by the above detailed methods. It should be apparent to those skilled in the art that any modifications of the present invention, equivalent substitution of the various materials of the products of the present invention, addition of auxiliary components, selection of specific means, and the like, are all within the scope of the present invention.

Claims (13)

A detection system for memory immune cells, characterized in that the sample amount in the detection system is 0.3-0.8 mL. The detection system according to claim 1, wherein the sample amount in said detection system is 0.6 mL. The detection system according to claim 1 or 2, wherein the target of the detection system is a substance secreted by the cells after being stimulated; Preferably, the substance secreted by the cell after stimulation is any one of a cytokine, a chemokine, an antibody or a protein or a combination of at least two; Preferably, the cytokine is a cytokine stimulated and secreted by immune cells and/or non-immune cells, preferably a cytokine secreted by immune cells. The detection system according to any one of claims 1 to 3, wherein the detection system further comprises a stimulating antigen and/or a positive control antigen; Preferably, the stimulating antigen is a specific antigen that stimulates memory T cells; Preferably, the specific antigen for stimulating memory T cells is any one of natural proteins, polypeptides, nucleic acids, polysaccharides, small molecule compounds, virus-specific antigens or bacterial-specific antigens, or a combination of at least two, preferably Further, a bacterial specific antigen is more preferably a Mycobacterium tuberculosis specific antigen. The detection system according to claim 4, wherein said positive control antigen is a non-specific stimulating agent that stimulates immune cells; Preferably, the non-specific stimulating agent for stimulating immune cells is concanavalin A, phytohemagglutinin, human porphyrin, peanut agglutinin, soybean lectin, staphylococcal A protein, lipopolysaccharide or tumor Any one or a combination of at least two of the stimulating agents is preferably phytohemagglutinin. The detection system according to any one of claims 1 to 5, wherein the cytokines are IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL. -7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-17, IFN-α, IFN-β, IFN-γ, TNF-β, SCF, G-CSF, M-CSF, GM-CSF, neuroleukocytidine, IgA, IgG, Any one or a combination of at least two of perforin, granullin, IP10 or TGF-β, preferably any one or at least two of IL-2, IL-6, IFN-γ, IP10 or TGF-β The combination of the species is further preferably IFN-γ. The detection system according to any one of claims 1 to 6, wherein the sample is heparin anticoagulated blood. The detection system according to any one of claims 1 to 7, wherein the detection system further comprises detecting a stimulating antigen of the sample; Preferably, the detection system further comprises a conventional reagent for detecting a sample. A detection method using the detection system according to any one of claims 1 to 7, characterized in that it comprises the following steps: (1) Collect samples and pack them into 0.3-0.8 mL; (2) adding a corresponding stimulating antigen to the sample of step (1), culturing; (3) Detecting the content of the cytokine in the supernatant. The detecting method according to claim 9, wherein the culture temperature is 30 to 40 ° C, preferably 35 to 38 ° C, and more preferably 37 ° C. The detection method according to claim 9, wherein the culture is carried out for a period of 10 to 35 h, preferably 18 to 24 h. The detection system according to any one of claims 1 to 7 for detecting the function of a memory immune cell. Use of a detection system according to any of claims 1-7 in the preparation of a kit for disease detection and/or diagnosis.

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