Method for amplifying tuberculous hydrothorax mononuclear cells in vitro

Abstract

According to the method for amplifying the tuberculous hydrothorax mononuclear cells in vitro, the number of TB-PFMC cells is successfully increased through a CD3 / CD28 antibody combined IL-2 stimulation method, and the amplified TB-PFMC is suitable for quality control of a TB-IGRA kit. According to the present invention, the TB-PFMC in vitro amplification method is preliminarily established, and the new scheme is provided for the preparation of the TB-IGRA reagent reference material, such that the quality control and the performance evaluation of the TB-IGRA reagent can be effectively performed.

Description

Technical Field

[0001] The invention relates to the field of in vitro diagnostic reagents, and in particular to a method for amplifying tuberculous pleural effusion mononuclear cells in vitro. Background Art

[0002] Tuberculosis is still a global public health problem. TB-IGRA is a method (reagent) for in vitro detection of the specific cellular immune response of Mycobacterium tuberculosis in human body fluid samples, using interferon-gamma (IFN-γ) produced by T cells stimulated by Mycobacterium tuberculosis-specific antigens as the detection target. The detection process is roughly divided into two parts: antigen stimulation and IFN-γ detection. This type of reagent has been widely used in the clinical diagnosis of pulmonary tuberculosis and the screening and monitoring of latent tuberculosis infection, and is one of the key measures to achieve the WHO tuberculosis elimination strategy. With the widespread application of related reagents, clinical medical institutions and drug regulatory agencies at home and abroad have long paid attention to the quality risks of such products, but the lack of reference materials has led to a lack of powerful tools for the quality control and performance evaluation of the reagents.

[0003] Quality control samples in in vitro diagnostic reagents refer to substances, materials or articles that are intended by their manufacturers to verify the performance characteristics of in vitro diagnostic reagents. The basic characteristics of quality control samples should meet the following requirements: first, the matrix should be as consistent as possible with the specimens to be tested in actual clinical tests to avoid the possible existence of "matrix effects"; second, the concentration of the analyte contained in it should be as close as possible to the cut-off value of the test or the clinical decisive level; third, it should have good stability and repeatability. At present, freshly collected whole blood from tuberculosis patients is often used as a reference material for product development, quality control and performance evaluation of TB-IGRA kits. However, the source of freshly collected whole blood with appropriate reactivity as a reference material is limited and it is difficult to use it for large-scale preparation of reference materials, and it is also difficult to store it by freezing or freeze-drying like serum and plasma. Due to the problems of unstable source (donor flow), difficulty in long-term storage, and limited single collection volume of fresh whole blood samples, it is difficult to use the same sample for a large number of repeated tests under different conditions, thus limiting the stable and comprehensive quality control of this type of in vitro diagnostic reagents and the unified evaluation of the performance of different in vitro diagnostic reagents.

[0004] Based on the above background, the present invention aims to provide a method for amplifying tuberculous pleural effusion mononuclear cells in vitro, thereby solving the problem of insufficient source of reference material raw material samples and difficulty in repeatability, and providing a basis for the development of reference materials for TB-IGRA quality control and performance evaluation. Summary of the invention

[0005] In order to make up for the deficiencies of the prior art, the object of the present invention is to provide a method for amplifying tuberculous pleural effusion mononuclear cells in vitro, and a method and application thereof for preparing quality control samples for in vitro diagnostic reagents.

[0006] In order to achieve the above object, the present invention adopts the following technical solution: The first aspect of the present invention provides a method for preparing a reference substance for an in vitro diagnostic reagent for tuberculosis, the method comprising: in vitro amplifying mononuclear cells in tuberculous pleural effusion, and replacing the culture medium with a non-T cell amplification culture medium on the fourth day of culture.

[0007] Furthermore, the non-T cell expansion medium is an RPMI-1640 medium containing a mixture of 10% fetal bovine serum and 1% penicillin-streptomycin (100×).

[0008] In the present invention, reference material refers to a stable substance used to check the performance of analytical instruments, methods or reagents. It is usually not specially set or adjusted and optimized for a specific test kit or instrument, and can provide a type of quality control material for performance monitoring of test kits and detection systems. The composition of the reference material should be similar or the same as the matrix of the patient sample tested. The ideal matrix is ​​human serum, urine, or cerebrospinal fluid. The reference material should be uniform and stable, and if conditions permit, it can be stored for one year. The variability between bottles should be less than the variability of the analytical system. The reference material can be a commercial reference material or a reference material that can be made by the laboratory. It can be a liquid or freeze-dried material containing one or more components of known concentration or known amount.

[0009] In the present invention, pleural effusion is a common clinical symptom characterized by pathological fluid accumulation in the pleural cavity, and can be transudative pleural effusion or exudative pleural effusion.

[0010] Furthermore, the tuberculosis includes diseases caused by infection with Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium voles or Mycobacterium tuberculosis complex.

[0011] Furthermore, the tuberculosis includes respiratory system tuberculosis, urinary system tuberculosis, digestive system tuberculosis, reproductive system tuberculosis, and bone and joint tuberculosis.

[0012] Further, the respiratory system tuberculosis is usually pulmonary tuberculosis, which may be primary pulmonary tuberculosis; hematogenously disseminated pulmonary tuberculosis; secondary pulmonary tuberculosis; tracheal and bronchial tuberculosis; tuberculous pleurisy. The urinary system tuberculosis may be renal tuberculosis; ureteral tuberculosis; bladder tuberculosis; urethral tuberculosis; urinary system tuberculosis secondary to pulmonary tuberculosis. The digestive system tuberculosis may be ulcerative intestinal tuberculosis; hyperplastic intestinal tuberculosis; extraintestinal tuberculosis; tuberculous peritonitis; digestive system tuberculosis secondary to pulmonary tuberculosis. The reproductive system tuberculosis may be fallopian tube tuberculosis; endometrial tuberculosis; cervical tuberculosis; ovarian tuberculosis; tuberculous endosalpingitis; reproductive system tuberculosis secondary to pulmonary tuberculosis. The bone and joint tuberculosis may be spinal tuberculosis; hip joint tuberculosis; knee joint tuberculosis; bone and joint tuberculosis secondary to pulmonary tuberculosis.

[0013] Furthermore, the tuberculosis in vitro diagnostic reagent is a reagent for in vitro diagnosis through an IGRA test.

[0014] The IGRA test, also known as TB-IGRA, is a method for in vitro detection of the specific cellular immune response of Mycobacterium tuberculosis in human body fluid samples, using interferon-gamma (IFN-γ) produced by T cells stimulated by Mycobacterium tuberculosis specific antigens as the detection target. It is an in vitro immunological method recommended by the World Health Organization for the diagnosis of Mycobacterium tuberculosis infection, and is also one of the means for auxiliary diagnosis of active tuberculosis in the diagnostic criteria for pulmonary tuberculosis in my country.

[0015] Furthermore, the tuberculosis in vitro diagnostic reagent includes reagents used for in vitro diagnosis via ELISPOT and / or ELISA.

[0016] Furthermore, the tuberculosis in vitro diagnostic reagent includes one or more of T-SPOT.TB and QuantiFERON.TB.

[0017] The second aspect of the present invention provides a method for in vitro expansion of mononuclear cells of tuberculous pleural effusion, the steps of the method comprising: treating tuberculous pleural effusion with lymphocyte separation fluid, isolating mononuclear cells, resuspending with T cell expansion culture medium, and subsequently culturing in a culture dish coated with CD3 monoclonal antibody.

[0018] Furthermore, the treatment with lymphocyte separation fluid refers to slowly adding tuberculous pleural effusion to the upper layer of lymphocyte separation fluid, the volume ratio of tuberculous pleural effusion and lymphocyte separation fluid is 4:1, collecting the buffy coat layer after centrifugation to obtain mononuclear cells.

[0019] Furthermore, the centrifugation condition is 800 g, 30 min.

[0020] Furthermore, the mononuclear cells can be directly cultured after separation, or can be revived after being frozen for subsequent culture.

[0021] Furthermore, the freezing step includes: diluting with freezing solution, adjusting cell concentration, and programmed cooling.

[0022] Furthermore, the components of the freezing solution are fetal bovine serum and DMSO.

[0023] Furthermore, the content of fetal bovine serum and DMSO in the components of the freezing solution is 4:1.

[0024] Furthermore, the cell concentration is adjusted to 1×10 7 / mL.

[0025] Furthermore, the T cell expansion medium includes a basal medium and additives.

[0026] Furthermore, the basic culture medium is selected from at least one of RPMI-1640 culture medium, AIM-V culture medium, X-VIVO culture medium, T-VIVO culture medium, and DMEM / F12 culture medium.

[0027] Furthermore, the basic culture medium is RPMI-1640 culture medium.

[0028] Furthermore, the additives include one or more of serum, antibiotics, growth factors, functional antibodies, inorganic salts, reducing agents, insulin, and amino acids.

[0029] Furthermore, the additives include serum, antibiotics, growth factors, and functional antibodies.

[0030] In the present invention, additives refer to supplements, nutrients and other enrichment preparations designed to promote the cultivation of various microorganisms. Serum is one of the key additives in the culture medium, usually animal serum, such as human serum, bovine serum. In some embodiments, serum can be human serum, fetal bovine serum, newborn calf serum or calf serum.

[0031] Growth factors are cell signaling proteins that play an important role in cell proliferation, differentiation, and survival. Growth factors required in cell culture can be provided in the form of exogenous added serum or purified recombinant proteins. High-quality growth factors can maintain the health of cells and provide stable and reproducible conditions for in vitro studies of mammalian cells. In some embodiments, the growth factor can be epidermal growth factor, fibroblast growth factor, brain-derived neurotrophic factor, vascular endothelial growth factor, granulocyte macrophage colony stimulating factor, interleukin.

[0032] Functional antibodies refer to antibodies that show activator or antagonist activity. In some embodiments, the functional antibodies may be CD3, CD28, and in a specific embodiment of the present invention, the functional antibodies are anti-human T cell CD3 (OKT3) and CD28 (15E8) mouse monoclonal antibodies.

[0033] Furthermore, the serum is fetal bovine serum.

[0034] Furthermore, the antibiotic is a penicillin-streptomycin mixture.

[0035] Furthermore, the growth factor is IL-2.

[0036] Furthermore, the functional antibodies are CD3 monoclonal antibodies and CD28 monoclonal antibodies.

[0037] The third aspect of the present invention provides in vitro expanded mononuclear cells obtained by culture using the method described in the second aspect of the present invention.

[0038] The fourth aspect of the present invention provides a reference substance for an in vitro diagnostic reagent for tuberculosis, wherein the reference substance is prepared by the method described in the first aspect of the present invention, or the reference substance comprises the mononuclear cells described in the third aspect of the present invention.

[0039] The fifth aspect of the present invention provides the use of the mononuclear cell described in the third aspect of the present invention or the reference substance described in the fourth aspect of the present invention, wherein the use includes any one of the following: 1) Application in the preparation of quality control samples for in vitro diagnostic reagents for tuberculosis.

[0040] 2) Application in the preparation of in vitro diagnostic products for tuberculosis.

[0041] Furthermore, the tuberculosis in vitro diagnostic product may be a test kit, a diagnostic instrument, or a microfluidic chip.

[0042] Advantages and beneficial effects of the present invention: The present invention provides a method for amplifying tuberculous pleural effusion mononuclear cells in vitro, and the amplified TB-PFMC can be used as a reference material raw material for TB-IGRA reagents, thereby solving the problems of insufficient source of original reference material raw material samples, difficulty in long-term storage and limited single collection volume, and providing a basis for the development of quality control samples for TB-IGRA quality control and performance evaluation. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is a cell morphology image of TB-PFMC expanded in vitro observed under a microscope (magnification 40×).

[0044] Figure 2 This is the spot result of ELISPOT reagent for in vitro expansion of TB-PFMC. DETAILED DESCRIPTION

[0045] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvement by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0046] Example 1 In vitro expansion of tuberculous pleural effusion mononuclear cells 1. Materials and equipment 1.1 Materials: Tuberculous pleural effusion (PEF) was obtained from a tuberculosis patient by thoracentesis at Guangzhou Chest Hospital. The patient was confirmed to have tuberculous pleurisy by etiology, and the clinical pleural effusion TB-IGRA result was positive. Ethical and informed consent were completed in accordance with regulations. RPMI-16440 culture medium, fetal bovine serum (FBS) (ThermoFisher Scientific, USA); Penicillin-Streptomycin Liquid (100×), P / S (Beijing Solebao Technology Co., Ltd.); recombinant human interleukin 2 (IL-2), anti-human T cell CD3 (OKT3) and CD28 (15E8) mouse monoclonal antibodies (Aibixin (Shanghai) Biotechnology Co., Ltd.); lymphocyte separation fluid (Shenzhen Dakoway Biotechnology Co., Ltd.); AO / PI cell staining fluid (Shanghai Ruiyu Biotechnology Co., Ltd.).

[0047] 1.2 Instruments and equipment: Countstar Mira FL cell counter (Shanghai Ruiyu Biotechnology Co., Ltd.); DMIL LED inverted microscope (Leica, Germany); Echo Revovle upright and inverted integrated microscope (Echo, USA).

[0048] 2. Methods 2.1 TB-PFMC separation and cryopreservation: Slowly add pleural effusion to the upper layer of lymphocyte separation solution (the volume ratio of pleural effusion to lymphocyte separation solution is 4:1) and centrifuge (800 g, 30 min). Discard the upper layer of pleural effusion, collect the mononuclear cells in the middle buffy coat layer, dilute with RPMI-1640 medium, centrifuge (300 g, 10 min) and remove the supernatant. Dilute TB-PFMC with cryopreservation solution (80% FBS + 20% DMSO) to adjust the cell concentration to 1×10 7 / mL was dispensed into cryopreservation tubes at 0.5 mL per tube and stored in a liquid nitrogen tank using programmed cooling.

[0049] 2.2 TB in vitro expansion: On the day of culture, anti-human T cell CD3 mouse monoclonal antibody was first coated into a 6-well plate at a concentration of 1 μg / mL. Then, TB-PFMC cells were resuscitated in a 37°C water bath, washed with RPMI-1640 medium, centrifuged at 300g for 10 min, and the supernatant was removed. The cells were resuspended in T cell expansion medium containing 10% FBS, 1% P / S, 1 μg / mL anti-human T cell CD28 mouse monoclonal antibody, and 10 ng / mL IL-2, and the culture medium was diluted to 1×10 6 The cells were cultured continuously for 3 days at a cell density of 100 μg / mL in a 6-well plate coated with the antibody. On the 4th day of culture, the cells were cultured in RPMI-1640 medium containing 10% FBS and 1% P / S. The cell morphology was observed under an optical microscope on the day of culture (day 0), day 2, day 4, and day 8. The cells were dispersed by pipetting and counted using AO / PI cell staining solution and a cell counter.

[0050] 3. Results 3.1 In vitro expansion of TB-PFMC: Microscopic observation results ( Figure 1 ) It can be seen that on the 0th day of culture, TB-PFMCs were scattered individually in the culture plate. The cells were round, translucent and shiny, and in good condition. On the second day, a small number of cells aggregated into clusters, indicating proliferation, but some cells shrank, indicating cell death, and the cell viability was 68.47%. On the fourth day, the cell clusters began to increase in size, the cells began to proliferate rapidly, and some cells died at the same time. The cell viability increased, and the number of live cells increased from 1.00×10 6 / mL increased to 1.58×10 6 / mL. After culturing for 8 days, the number of viable TB-PFMC cells increased to 1.88×10 6 / mL, the expansion multiple reached 177.36%, and the cell viability also increased to 79.88%. It can be seen that TB-PFMC successfully achieved in vitro expansion (Table 1).

[0051] Table 1. Number of TB-PFMC cells and expansion times in vitro

[0052] Example 2 Preparation of quality control reference material for TB-IGRA reagent 1. Materials and equipment 1.1 Materials: TB-PFMC cells amplified in Example 1; AIM-V serum-free culture medium (ThermoFisher Scientific, USA); T-SPOT.TB reagent (Oxford Immunotec, UK).

[0053] 1.2 Instruments: ImmunoSpot S6 Entry M2 enzyme-linked immunosorbent assay analyzer (CTL, USA).

[0054] 2. Methods 2.1 ELISPOT detection: On the day of culture (day 0), day 4 and day 8, take some TB-PFMC cells amplified in Example 1, adjust the cell concentration with AIM-V serum-free medium, and use T-SPOT.TB reagent to perform ELISPOT detection with 100,000 cells (cells / well) per test well. For the specific experimental operation and result interpretation criteria of ELISPOT, please refer to the T-SPOT.TB reagent manual. The main steps are as follows: 50 μL of AIM-V serum-free medium (blank control, N well), Mycobacterium tuberculosis specific stimulation antigens (ESAT-6 and CFP 10 antigens, A well and B well) and phytohemagglutinin PHA (positive control, P well) are added to the 96-well microwell culture plate coated with IFN-γ capture antibody, and 100 μL of the above cell suspension is added to each well, and then the microwell culture plate is placed in a 5% CO2 incubator for 18 h to 20 h. After the culture is completed, washing, enzyme-labeled secondary antibody incubation, and alkaline phosphatase substrate color development are performed in sequence. The microwell culture plate corresponding to each test was photographed and the spots were counted using an ELISA spot analyzer.

[0055] 3. Results 3.1 Tuberculosis-specific cellular immune response of TB-PFMC after in vitro expansion: ELISPOT results showed ( Figure 2 ), on the 0th day of culture, TB-PFMC had not yet undergone obvious activation, and the number of spots in the N, A, B and P wells were 0, 102, 245 and 435, respectively, which were judged as "positive" according to the instructions of the T-SPOT.TB kit. On the 4th day of amplification, TB-PFMC cells were in a highly activated state, and a large amount of IFN-γ was released non-specifically, and the number of spots in the N well increased to 202. Due to the excessive generation of spots in the N wells, the ELISPOT experiment of TB-PFMC will produce invalid results. If there are more than 10 spots, the result will be judged as "uncertain" according to the instructions of the kit. On the 4th day of culture, the culture medium of TB-PFMC was replaced with a complete culture medium without amplification reagent. On the 8th day of culture, no large number of nonspecific spots appeared in the N wells, indicating that the T cells stopped non-specifically releasing a large amount of IFN-γ at this time, and the judgment result became a normal "positive" ( Figure 2). Comparing the tuberculosis-specific cellular immune response before and after in vitro expansion of TB-PFMC, 102 ESAT-6-specific spots and 245 CFP 10-specific spots were detected in 100,000 TB-PFMC cells on day 0 of culture, and 119 and 123 ESAT-6 and CFP 10-specific spots were still detected on day 8 of culture (Table 2). It is worth noting that, combined with the amplification multiple (Table 1), the number of ESAT-6-specific T cells in TB-PFMC increased by about 1 times after amplification culture, while the number of CFP 10-specific T cells did not change significantly. In addition, the number of spots generated in the positive quality control wells of TB-PFMC after culture on day 8 was significantly more than that before expansion, indicating that the cells maintained strong activity at this time.

[0056] Table 2. ELISPOT spot numbers and interpretation results of TB-PFMCs at 0, 4, and 8 days of in vitro expansion

[0057] CD3 / CD28 antibody-activated immune cells are often used as a model for T cell activation, in which the release of cytokines such as IFN-γ is an important feature of T cell activation. However, the large amount of nonspecific IFN-γ released by amplified TB-PFMC will lead to abnormal interpretation results of commercial kits. In order to avoid invalid results of ELISPOT test caused by nonspecific release of IFN-γ by T cells, TB-PFMC was terminated on the 4th day of culture and entered the "resting" period. After the 4th day of "resting", almost no nonspecific release of IFN-γ was observed in TB-PFMC, while the specific release of IFN-γ by T cells sensitized by tuberculosis antigens ESAT-6 and CFP10 antigens was still clearly observed. At this time, TB-PFMC can be correctly interpreted by ELISPOT reagents and has been verified to have good applicability. Therefore, unlike tumor adoptive T cell immunotherapy, which requires continuous stimulation and amplification of T cells, T cell amplification conditions and culture time need to be appropriately adjusted when preparing reference materials for TB-IGRA reagent quality control.

[0058] ESAT-6 and CFP 10 are two antigens expressed by the gene difference region 1 of Mycobacterium tuberculosis. They are not expressed in Mycobacterium bovis and other non-tuberculous mycobacteria used in BCG. Therefore, most manufacturers of TB-IGRA reagents use these two tuberculosis-specific antigens as stimulating antigens. In the present invention, ESAT-6 and CFP 10 specific T cells in the initial TB-PFMC are amplified and show high reactivity through in vitro amplification by CD3 / CD28 antibody combined with IL-2 strategy. This not only better meets the needs of preparing a large number of reference materials, but can also be used for quality control of a single tuberculosis-specific antigen test well of the reagent.

[0059] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A method for preparing a reference material for an in vitro diagnostic reagent for tuberculosis, characterized in that: The method comprises: amplifying mononuclear cells in tuberculous pleural effusion in vitro, and replacing the culture medium with a non-T cell amplification medium on the fourth day of culture; Preferably, the non-T cell expansion medium is an RPMI-1640 medium containing a mixture of 10% fetal bovine serum and 1% penicillin-streptomycin (100×).

2. The method according to claim 1, characterized in that: The tuberculosis includes diseases caused by infection with Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium microti or Mycobacterium tuberculosis complex; Preferably, the tuberculosis includes respiratory system tuberculosis, urinary system tuberculosis, digestive system tuberculosis, reproductive system tuberculosis, and bone and joint tuberculosis; Preferably, the tuberculosis in vitro diagnostic reagent is a reagent for in vitro diagnosis by IGRA test; Preferably, the tuberculosis in vitro diagnostic reagent includes reagents used for in vitro diagnosis by ELISPOT and / or ELISA; Preferably, the tuberculosis in vitro diagnostic reagents include one or more of T-SPOT.TB and QuantiFERON.TB.

3. A method for in vitro expansion of mononuclear cells of tuberculous pleural effusion, characterized in that: The method comprises the following steps: treating tuberculous pleural effusion with lymphocyte separation fluid, separating mononuclear cells, resuspending the cells with T cell expansion culture medium, and subsequently culturing the cells in a culture dish coated with CD3 monoclonal antibody.

4. The method according to claim 3, characterized in that: The treatment with lymphocyte separation fluid refers to slowly adding tuberculous pleural effusion to the upper layer of lymphocyte separation fluid, the volume ratio of tuberculous pleural effusion to lymphocyte separation fluid is 4:1, collecting the buffy coat layer after centrifugation, and obtaining mononuclear cells; Preferably, the centrifugation condition is 800 g, 30 min.

5. The method according to claim 4, characterized in that The mononuclear cells can be directly cultured after separation, or revived after freezing for subsequent culture; Preferably, the freezing step includes: diluting with freezing solution, adjusting cell concentration, and programmed cooling; Preferably, the components of the freezing solution are fetal bovine serum and DMSO; Preferably, the content of fetal bovine serum and DMSO in the components of the freezing solution is 4:1; Preferably, the cell concentration is adjusted to 1×10 7 / mL.

6. The method according to claim 3, characterized in that The T cell expansion medium includes a basal medium and additives; Preferably, the basal culture medium is selected from at least one of RPMI-1640 culture medium, AIM-V culture medium, X-VIVO culture medium, T-VIVO culture medium, and DMEM / F12 culture medium; Preferably, the basal culture medium is RPMI-1640 culture medium; Preferably, the additives include one or more of serum, antibiotics, growth factors, functional antibodies, inorganic salts, reducing agents, insulin, and amino acids; Preferably, the additives include serum, antibiotics, growth factors, and functional antibodies; Preferably, the serum is fetal bovine serum; Preferably, the antibiotic is a mixture of penicillin and streptomycin; Preferably, the growth factor is IL-2; Preferably, the functional antibody is CD3 monoclonal antibody or CD28 monoclonal antibody.

7. In vitro expanded mononuclear cells obtained by culturing using the method according to any one of claims 3 to 6.

8. A reference material for an in vitro diagnostic reagent for tuberculosis, characterized in that: The reference substance is prepared by the method of claim 1 or 2, or the reference substance comprises the mononuclear cells of claim 7.

9. Use of the mononuclear cell according to claim 7 or the reference substance according to claim 8, characterized in that: The application includes any of the following: 1) Application in the preparation of quality control samples for in vitro diagnostic reagents for tuberculosis; 2) Application in the preparation of in vitro diagnostic products for tuberculosis.

10. The use according to claim 9, characterized in that: The tuberculosis in vitro diagnostic product can be a test kit, a diagnostic instrument, or a microfluidic chip.

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