A tuberculosis auxiliary diagnostic kit and detection method based on AIE fluorescent microspheres labeled IP10

Abstract

The present application relates to a kind of auxiliary diagnosis kit and detection method of tuberculosis based on AIE fluorescent microsphere labeled IP10, AIE fluorescent immunochromatography kit, containing fluorescent detection test strip and sample diluent, test strip pad is sprayed AIE fluorescent microsphere labeled IP10 antibody, nitrocellulose membrane is equipped with detection line and quality control line.IP10 concentration 300 pg / mL is used as critical value to judge tuberculosis suspected positive and negative;AIE magnetic particle fluorescent immunoassay kit contains IP10 antibody coupled magnetic particle suspension, AIE fluorescent microsphere labeled IP10 antibody working solution, washing liquid, calibrant and quality control product.The advantages are: first, the present application uses aggregation-induced emission (AIE) fluorescent microsphere, which can improve detection accuracy;Second, the present application uses reagent stability, and the storage effective period is more than 12 months at 2-8 ℃, and the anti-aging and freeze-thaw resistance are good;Third, the present application uses serum non-invasive detection, and the operation is simple, short cycle, cost controllable, patient compliance is high, and it has large-scale clinical popularization prospect.

Description

Technical Field

[0001] This invention belongs to the fields of in vitro immunodiagnostic technology, application of nano-fluorescent biomaterials and precise detection of tuberculosis, specifically relating to a tuberculosis auxiliary diagnostic kit and detection method based on AIE fluorescent microspheres labeled with IP10. Background Technology

[0002] Tuberculosis is a major infectious disease that my country focuses on controlling. Early occult infection accounts for a high percentage of cases, and clinical manifestations lack typicality. Existing routine detection techniques have significant limitations. For example, the positive rate of acid-fast sputum staining is only 20%–30%, with extremely low detection sensitivity; tuberculin skin test results are easily affected by BCG vaccination, resulting in a high false-positive rate; the tuberculosis culture cycle is as long as 4–8 weeks, which cannot meet the needs of rapid clinical diagnosis and treatment; the gamma interferon release assay is costly and has a certain proportion of uncertain results, making it difficult to promote its large-scale application in primary care screening (National Center for Respiratory Medicine Medical Quality Control, Chinese Medical Association Tuberculosis Branch, Chinese Anti-Tuberculosis Association Tuberculosis Control Professional Branch, et al. Clinical Practice Guidelines for Early Detection of Pulmonary Tuberculosis in Comprehensive Medical Institutions [J]. Chinese Journal of Anti-Tuberculosis, 2024, 46 (2): 127-140).

[0003] IP10 (CXCL10) is a key cytokine that is highly specifically expressed in the early stage of tuberculosis infection. It can be specifically and highly expressed by mononuclear macrophages and T lymphocytes in the body under the stimulation of tuberculosis antigen. Its expression level is significantly higher than that of traditional interferon indicators. It is currently recognized by the academic community as a non-invasive diagnostic marker for early tuberculosis (Zhang Wei, Li Ming, Zhao Gang, et al. The auxiliary diagnostic significance of serum IP-10 in active pulmonary tuberculosis [J]. Chinese Journal of Tuberculosis and Respiratory Diseases, 2020, 43 (5): 489-493). Existing commercial IP10 detection reagents mostly use traditional fluorescent dyes such as FITC and Rhodamine for labeling, which generally suffer from defects such as fluorescence aggregation quenching, weak resistance to photobleaching, insufficient signal stability, high detection limit, and short reagent shelf life. This leads to insufficient detection sensitivity and poor repeatability, which cannot meet the needs of accurate detection of early tuberculosis infection (Chen Guoxin, Tang Benzhong. Progress in the application of aggregation-induced luminescent microspheres in fluorescence immunochromatography [J]. Analytical Chemistry, 2022, 50 (3): 412-420).

[0004] Aggregation-induced emission (AIE) nanomaterials possess a unique intramolecular motion-restricted emission mechanism: weak fluorescence in the single-molecule state and significantly enhanced fluorescence in the aggregated state. They have advantages such as high quantum yield, strong resistance to photobleaching, low background fluorescence, and good biocompatibility, giving them outstanding technical advantages in the field of immunofluorescence detection (Zhao Xin, Sun Yue, Tang Benzhong. Application of aggregation-induced emission fluorescent probes in biomarker detection [J]. Progress in Biochemistry and Biophysics, 2023, 50(1):78-85). There is currently no integrated tuberculosis detection solution based on AIE fluorescent microsphere labeling of IP10, which simultaneously constructs a rapid screening platform for fluorescence chromatography and a precise quantitative platform for magnetic microparticle fluorescence. This solution cannot simultaneously meet the needs of large-scale primary screening at the grassroots level and the stratified diagnosis and treatment and efficacy monitoring of hospitals. There is a clear technological gap in this field (Wang X, Li Y, Zhang H, et al. Diagnostic performance of IP-10 in childhood tuberculosis:asystematic review and meta-analysis [J]. PLoS One, 2025, 20 (2):e0298145). Summary of the Invention

[0005] Design Objective: To design an AIE fluorescent microsphere-labeled IP10-based auxiliary diagnostic kit for tuberculosis, which is an AIE fluorescent immunochromatographic kit for rapid screening detection. The kit includes a fluorescent test strip and sample diluent. The conjugate pad of the fluorescent test strip is coated with AIE fluorescent microsphere-labeled IP10 antibody. The nitrocellulose membrane of the test strip has a test line and a control line. The test strip uses an IP10 concentration of 300 pg / mL in the sample as a cutoff value to determine whether the tuberculosis is suspected positive or negative. When the IP10 concentration in the sample is greater than or equal to 300 pg / mL, it is considered a suspected positive for tuberculosis; when the IP10 concentration is less than 300 pg / mL, it is considered negative. When the concentration is pg / mL, it is determined to be negative for tuberculosis; or it is an AIE magnetic microparticle fluorescence immunoassay kit for quantitative detection, the AIE magnetic microparticle fluorescence immunoassay kit includes IP10 antibody-conjugated magnetic microparticle suspension, AIE fluorescent microsphere labeled IP10 antibody working solution, washing solution, calibrators and quality control products, the AIE fluorescent microsphere labeled IP10 antibody working solution is prepared from AIE fluorescent microsphere labeled IP10 antibody.

[0006] Preferably, the preparation method of the AIE fluorescent microsphere-labeled IP10 antibody is as follows: Take 1 mL of carboxylated AIE fluorescent microspheres with a mass fraction of 1% and a particle size of 200 nm, and resuspend them in 0.1 mol / L, pH 5.5 MES buffer; add 10 μL each of 10 mg / mL EDC solution and 10 mg / mL NHS solution, and activate for 30 min at room temperature in the dark; then add 200 μg of IP10 monoclonal antibody and couple for 2 h at room temperature in the dark with shaking; add 100 μL of 10% BSA solution and block for 1 h at room temperature; centrifuge at 8000 r / min for 5 min, and wash three times to obtain the AIE fluorescent microsphere-labeled IP10 antibody, resuspend it in antibody preservation solution, and store it at 4°C in the dark for later use.

[0007] Preferably, the concentration of the AIE fluorescent microsphere-labeled IP10 antibody in the working solution is 0.1 mg / mL.

[0008] Preferably, the sample diluent is a 0.01 mol / L PBS buffer containing 0.5% BSA, 0.1% Tween-20, and 0.05% Proclin 300, with a pH of 7.4.

[0009] Preferably, the AIE fluorescent microspheres are carboxylated nanospheres with a particle size range of 100nm-300nm, an excitation wavelength of 365nm, an emission wavelength of 610nm, and a fluorescence quantum yield of not less than 80%. The magnetic microparticles are 3μm superparamagnetic amino magnetic microparticles; the IP10 antibody-coupled magnetic microparticle suspension is prepared by coupling IP10 paired antibody with 3μm amino magnetic microparticles via glutaraldehyde crosslinking, followed by blocking and washing steps.

[0010] Preferably, the AIE magnetic microparticle fluorescence immunoassay kit is used for early screening of pulmonary tuberculosis, identification of latent tuberculosis infection, diagnosis of active pulmonary tuberculosis, and dynamic monitoring of the efficacy of anti-tuberculosis treatment.

[0011] Preferably, the AIE fluorescent microspheres labeled with IP10 antibody are uniformly sprayed onto the conjugate pad at a concentration of 1 μL / cm; the detection line is coated with IP10 paired antibody at a concentration of 1.0 mg / mL; and the control line is coated with goat anti-mouse IgG at a concentration of 0.8 mg / mL.

[0012] A method for auxiliary diagnosis of tuberculosis based on AIE fluorescent microsphere labeled IP10 includes a fully automated fluorescence immunoassay analyzer and quantitative detection using any of the tuberculosis auxiliary diagnostic kits based on AIE fluorescent microsphere labeled IP10 as described in claims 1-5. The detection steps are as follows: Step 1: Add 50 μL of IP10 antibody-conjugated magnetic microparticle suspension, 50 μL of serum sample to be tested, and 50 μL of AIE fluorescently labeled antibody working solution to the reaction tube in sequence, and then incubate with shaking. Step 2: After shaking and incubation, the reaction tube is separated by magnetic field. Then, the supernatant in the reaction tube is discarded, and the tube is washed three times with washing solution. Step 3: Then add 100 μL of buffer solution to the reaction tube and use a fully automated fluorescence immunoassay analyzer to detect the fluorescence intensity under the conditions of 365 nm excitation and 610 nm emission. Step four: The fully automated fluorescence immunoassay analyzer quantitatively calculates the IP10 concentration by fitting a standard curve. The calculated quantitative IP10 concentration can be used in clinical practice as one of the auxiliary diagnostic indicators for determining the grading of tuberculosis infection, as well as as a basis for evaluating the efficacy of tuberculosis treatment.

[0013] Preferably, the oscillation incubation temperature in step one is 37°C and the oscillation incubation time is 25 min; the magnetic field separation time in step two is 3 min.

[0014] A second method for tuberculosis auxiliary diagnostic detection based on AIE fluorescent microsphere labeled IP10 includes a fluorescence immunochromatography system and a rapid screening detection using any one of the tuberculosis auxiliary diagnostic kits based on AIE fluorescent microsphere labeled IP10 as described in claims 1 and 7. The detection steps are as follows: Step 1: Dilute the serum sample to be tested 1:5 with sample diluent to obtain diluted sample solution; Step 2: Add 100 μL of diluted sample solution to the sample pad of the fluorescent test strip and react at room temperature for 15 min. Step 3: Using a fluorescence immunochromatographic analyzer, the ratio of fluorescence signals of the detection line to the control line (T / C ratio) is detected under the parameters of excitation wavelength 365nm and emission wavelength 610nm. Step 4: Use 300 pg / mL as the cutoff value to determine whether the tuberculosis is suspected positive or negative. When the fluorescence immunochromatographic analyzer detects an IP10 concentration in the sample that is greater than or equal to 300 pg / mL, it is determined to be suspected positive for tuberculosis. When the fluorescence immunochromatographic analyzer detects an IP10 concentration in the sample that is less than 300 pg / mL, it is determined to be negative for tuberculosis.

[0015] Compared with the prior art, the present invention has the following advantages: First, both the AIE fluorescence immunochromatographic reagent kit and the AIE magnetic particle fluorescence immunoassay kit constructed in this invention use aggregation-induced emission (AIE) fluorescent microspheres as labeling materials. These materials generate specific fluorescence signals upon excitation by an ultraviolet light source. The detection system constructed in this way replaces the traditional chemiluminescence system, fundamentally solving common industry problems such as aggregation-induced quenching, poor photostability, and strong background interference prevalent in traditional fluorescent dyes. Simultaneously, this system possesses both high fluorescence quantum yield and stable signal output, effectively improving detection accuracy.

[0016] Secondly, both the AIE fluorescence immunochromatographic reagent kit and the AIE magnetic microparticle fluorescence immunoassay kit constructed in this invention have excellent reagent stability. When stored at 2℃ to 8℃, they can be stored for more than 12 months. They also have good anti-aging and freeze-thaw resistance properties, making them suitable for the storage and use conditions of medical institutions at all levels.

[0017] Third, both the AIE fluorescence immunochromatographic reagent kit and the AIE magnetic particle fluorescence immunoassay kit constructed in this invention adopt a non-invasive serum detection mode. This detection method is simple to operate, has a short detection cycle, and is cost-controllable, which can effectively improve patient compliance and has the prospect of wide-ranging clinical application.

[0018] Fourth, the AIE fluorescence immunochromatographic assay kit involved in this invention uses an IP10 concentration of 300 pg / mL in the sample as the cutoff value for determining suspected positive and negative tuberculosis results: when the sample IP10 concentration is ≥300 pg / mL, it is determined to be suspected positive for tuberculosis; when the sample IP10 concentration is <300 pg / mL, it is determined to be negative for tuberculosis. In addition, the detection linear range of this kit is 50 pg / mL-1000 pg / mL, the area under the curve (AUC) is 0.887, and the overall clinical concordance rate can reach 81.7%, which can fully meet the application needs of primary medical institutions to complete large-scale rapid screening within 15 minutes.

[0019] Fifth, the AIE magnetic microparticle fluorescence immunoassay kit involved in this invention has a detection limit as low as 5 pg / mL, and its detection sensitivity is 20 times higher than that of traditional fluorescence reagents. The area under the curve (AUC) of this magnetic microparticle system is 0.912, the diagnostic sensitivity reaches 92.04%, and the specificity is 80.27%. When the AIE magnetic microparticle fluorescence immunoassay kit is used in conjunction with a fully automated fluorescence immunoassay analyzer, the fully automated fluorescence immunoassay analyzer quantifies the IP10 concentration obtained by the AIE magnetic microparticle fluorescence immunoassay kit by fitting a standard curve. This quantitative IP10 concentration can be used as one of the auxiliary diagnostic indicators for tuberculosis infection grading in clinical practice, and can also be used as a basis for efficacy evaluation in the process of tuberculosis treatment. That is, the AIE magnetic microparticle fluorescence immunoassay kit can realize high-precision quantitative detection, infection grading, and dynamic monitoring of efficacy in hospitals, and is fully adapted to the stratified diagnosis and treatment model of tuberculosis.

[0020] Sixth, the AIE magnetic microparticle fluorescence immunoassay kit involved in this invention has an intra-assay coefficient of variation (CV) of no more than 6.50% and an inter-assay coefficient of variation (CV) of no more than 7.80%, indicating that the kit has good repeatability and inter-assay consistency.

[0021] Seventh, the AIE magnetic microparticle fluorescence immunoassay kit involved in this invention exhibits a fluorescence signal attenuation rate of ≤15% after being subjected to accelerated aging treatment at 37°C for 14 days, indicating that the kit possesses good thermal stability and long-term storage potential. Attached Figure Description

[0022] Figure 1 This is a graph showing the detection performance of the AIE magnetic microparticle fluorescent immunoassay kit.

[0023] Figure 2 This is the ROC curve of the AIE magnetic microparticle fluorescence immunoassay kit.

[0024] Figure 3 This is a graph showing the detection performance of the AIE fluorescence immunochromatographic assay kit.

[0025] Figure 4 This is the ROC curve of the AIE fluorescence immunochromatographic assay kit.

[0026] Figure 5 This is a scatter plot of the theoretical concentration versus test results for the linear range of the AIE magnetic microparticle fluorescence immunoassay kit.

[0027] Figure 6 This is a table showing the intra-batch coefficient of variation (CV) of the AIE magnetic microparticle fluorescent immunoassay kit.

[0028] Figure 7 This is a table showing the inter-batch coefficient of variation (CV) of the AIE magnetic microparticle fluorescent immunoassay kit.

[0029] Figure 8This is a graph showing the fluorescence decay of the AIE magnetic microparticle fluorescent immunoassay kit at 37℃ during accelerated aging.

[0030] Figure 9 This is a table showing the linear deviation of the AIE magnetic microparticle fluorescent immunoassay kit. Detailed Implementation

[0031] Example 1: Refer to Appendix Figures 1-9 A tuberculosis auxiliary diagnostic kit based on AIE fluorescent microsphere-labeled IP10 is disclosed. This kit is an AIE fluorescent immunochromatographic assay kit for rapid screening and detection. The kit includes a fluorescent test strip and a sample diluent. The conjugate pad of the fluorescent test strip is coated with AIE fluorescent microsphere-labeled IP10 antibody. The nitrocellulose membrane of the test strip has a test line and a control line. The test strip uses a IP10 concentration of 300 pg / mL in the sample as a cutoff value to determine whether the tuberculosis test is suspected positive or negative. When the IP10 concentration in the sample is greater than or equal to 300 pg / mL, it is considered a suspected positive for tuberculosis; when the IP10 concentration is less than 300 pg / mL, it is considered negative. When the concentration is pg / mL, it is determined to be negative for tuberculosis; or it is an AIE magnetic microparticle fluorescence immunoassay kit for quantitative detection, the AIE magnetic microparticle fluorescence immunoassay kit includes IP10 antibody-conjugated magnetic microparticle suspension, AIE fluorescent microsphere labeled IP10 antibody working solution, washing solution, calibrators and quality control products, the AIE fluorescent microsphere labeled IP10 antibody working solution is prepared from AIE fluorescent microsphere labeled IP10 antibody.

[0032] The preparation method of the AIE fluorescent microsphere-labeled IP10 antibody is as follows: Take 1 mL of carboxylated AIE fluorescent microspheres with a mass fraction of 1% and a particle size of 200 nm, and resuspend them in 0.1 mol / L, pH 5.5 MES buffer; add 10 μL each of 10 mg / mL EDC solution and 10 mg / mL NHS solution, and activate at room temperature in the dark for 30 min; then add 200 μg of IP10 monoclonal antibody and couple at room temperature in the dark with shaking for 2 h; add 100 μL of 10% BSA solution and block at room temperature for 1 h; centrifuge at 8000 r / min for 5 min, and wash three times to obtain the AIE fluorescent microsphere-labeled IP10 antibody, resuspend it in antibody preservation solution, and store it at 4℃ in the dark for later use. The specific information of the IP10 monoclonal antibody used is as follows: the capture antibody was purchased from R&D Company, catalog number MAB2661, and was used for streak coating and magnetic microsphere labeling; the detection antibody was purchased from R&D Company, catalog number AF-266-NA, and was used for fluorescent microsphere labeling.

[0033] The concentration of the AIE fluorescent microsphere-labeled IP10 antibody in the working solution is 0.1 mg / mL.

[0034] The sample diluent was a 0.01 mol / L PBS buffer containing 0.5% BSA, 0.1% Tween-20, and 0.05% Proclin 300, with a pH of 7.4.

[0035] The AIE fluorescent microspheres are carboxylated nanospheres with a particle size range of 100nm-300nm, an excitation wavelength of 365nm, an emission wavelength of 610nm, and a fluorescence quantum yield of not less than 80%. The AIE fluorescent microspheres were purchased from EPRUI Biotechnology Co., Ltd., product code: EPRUI-AIE-RFC-200. The magnetic microparticles are 3μm superparamagnetic amino magnetic microparticles, purchased from EPRUI Biotechnology Co., Ltd., product code: 130300. The IP10 antibody-conjugated magnetic microparticle suspension was prepared by conjugating IP10 paired antibody with 3μm amino magnetic microparticles via glutaraldehyde crosslinking, followed by blocking and washing steps.

[0036] The calibrators refer to six samples of different concentrations used to prepare the standard curve of the kit. The calibrator concentration gradients are 0 pg / mL, 10 pg / mL, 50 pg / mL, 200 pg / mL, 500 pg / mL, and 1000 pg / mL.

[0037] The quality control samples refer to samples used to determine whether reagents and testing instruments are under control. Target concentration parameters for quality control samples (low): 40 pg / mL - 70 pg / mL; Target concentration parameters for quality control samples (medium): 260 pg / mL - 340 pg / mL; Target concentration parameters for quality control samples (high): 550 pg / mL - 650 pg / mL.

[0038] The formula for the detergent is as follows: Purified water: 1000g; Tris(hydroxymethyl)aminomethane (TRIS): 12.15g; Sodium chloride: 8.0 g; Disodium ethylenediaminetetraacetate (EDTA·2Na): 5.0g; Sodium dodecyl sulfate (SDS): 2g; Tween 20: 10g; Proclin 300 preservative: 5g.

[0039] Preparation of AIE Magnetic Particle Fluorescent Immunoassay Kit IP10 paired antibodies were conjugated to 3 μm amino magnetic microparticles using glutaraldehyde crosslinking. After blocking and washing, a working suspension of magnetic microparticles (IP10 antibody-conjugated magnetic microparticle suspension) was prepared. A working solution of 0.1 mg / mL AIE fluorescently labeled antibody was prepared, and BSA and Proclin 300 were added for stabilization and preservation. Washing buffer, gradient calibrators, and quality control samples at high, low, and medium levels were prepared, and then aliquoted and packaged to obtain the AIE magnetic microparticle fluorescent immunoassay kit.

[0040] The AIE magnetic microparticle fluorescent immunoassay kit is used for early screening of pulmonary tuberculosis, identification of latent tuberculosis infection, diagnosis of active pulmonary tuberculosis, and dynamic monitoring of the efficacy of anti-tuberculosis treatment.

[0041] The AIE fluorescent microspheres labeled with IP10 antibody were uniformly sprayed onto the conjugate pad at a concentration of 1 μL / cm; the detection line was coated with IP10 paired antibody at a concentration of 1.0 mg / mL; and the control line was coated with goat anti-mouse IgG at a concentration of 0.8 mg / mL.

[0042] The test strip consists of a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad that are sequentially overlapped, all of which are located on a substrate.

[0043] Preparation of AIE fluorescence immunochromatographic assay kit The glass fiber sample pad and conjugate pad were pretreated; the prepared AIE fluorescently labeled antibody was uniformly sprayed onto the conjugate pad at a concentration of 1 μL / cm and dried at 37°C for 2 hours; 1.0 mg / mL of IP10 paired antibody was coated onto the nitrocellulose membrane as the detection line, and 0.8 mg / mL of goat anti-mouse IgG was coated onto the membrane as the control line, and the membrane was air-dried at room temperature; the sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad were assembled in that order and cut into 4 mm wide test strips; the sample diluent was assembled and packaged to obtain the AIE fluorescent immunochromatographic assay kit.

[0044] During the production and preparation stage of the AIE fluorescence immunochromatographic reagent kit, the preparation of calibrators and quality control samples must be completed simultaneously.

[0045] Calibrators: Used to establish the standard curve for the kit, with a total of 6 concentration gradients set up: 0 pg / mL, 10 pg / mL, 50 pg / mL, 200 pg / mL, 500 pg / mL and 1000 pg / mL.

[0046] Quality control materials: Used to monitor the effectiveness of the testing system and ensure that the testing process is under control. Their target values ​​are set as follows: low-value quality control materials are 40 pg / mL–70 pg / mL, medium-value quality control materials are 260 pg / mL–340 pg / mL, and high-value quality control materials are 550 pg / mL–650 pg / mL.

[0047] If the kit is intended for export, the decision may be made, based on the regulations of the target market and customer needs, to either have the local distributor or end-user prepare the calibrators and quality control products themselves, or to adopt an equivalent internationally accepted quality control solution.

[0048] Example 2: Based on Example 1, a method for auxiliary diagnosis of tuberculosis based on AIE fluorescent microspheres labeled IP10 is provided. The method includes a fully automated fluorescence immunoassay analyzer and uses any one of the tuberculosis auxiliary diagnostic kits based on AIE fluorescent microspheres labeled IP10 as described in claims 1-5 for quantitative detection. The detection steps are as follows: Step 1: Add 50 μL of IP10 antibody-conjugated magnetic microparticle suspension, 50 μL of serum sample to be tested, and 50 μL of AIE fluorescently labeled antibody working solution to the reaction tube in sequence, and then incubate with shaking. Step 2: After shaking and incubation, the reaction tube is separated by magnetic field. Then, the supernatant in the reaction tube is discarded, and the tube is washed three times with washing solution. Step 3: Then add 100 μL of buffer solution to the reaction tube and use a fully automated fluorescence immunoassay analyzer to detect the fluorescence intensity under the conditions of 365 nm excitation and 610 nm emission. Step four: The fully automated fluorescence immunoassay analyzer quantitatively calculates the IP10 concentration by fitting a standard curve. The calculated quantitative IP10 concentration can be used in clinical practice as one of the auxiliary diagnostic indicators for determining the grading of tuberculosis infection, as well as as a basis for evaluating the efficacy of tuberculosis treatment.

[0049] In addition to the results of the AIE magnetic particle fluorescence immunoassay kit, the definitive diagnosis of active tuberculosis still requires a comprehensive assessment combining bacteriological examination, imaging examination, and clinical characteristics; the determination of latent tuberculosis infection requires a comprehensive analysis combining a positive IGRA test, the absence of typical clinical symptoms, and the absence of active lesions on imaging; the definitive diagnosis of non-tuberculous lung lesions requires a comprehensive assessment combining the definitive diagnoses of other lung diseases; and the inclusion of healthy controls requires a comprehensive evaluation considering whether there is a history of tuberculosis exposure. In summary, the results of the AIE magnetic particle fluorescence immunoassay kit can serve as one of the auxiliary diagnostic indicators for the grading of tuberculosis infection in clinical practice.

[0050] During tuberculosis treatment, for confirmed patients, the quantitative detection results of the AIE magnetic microparticle fluorescence immunoassay kit can effectively reflect the current treatment effect and provide an important reference for the formulation of subsequent treatment plans.

[0051] The oscillation incubation temperature in step one is 37℃, and the oscillation incubation time is 25 min; the magnetic field separation time in step two is 3 min.

[0052] Example 3: Based on Example 1, a second method for tuberculosis auxiliary diagnosis based on AIE fluorescent microsphere labeled IP10 is provided, comprising a fluorescence immunochromatography system, using any one of the tuberculosis auxiliary diagnostic kits based on AIE fluorescent microsphere labeled IP10 as described in claims 1 and 7 for rapid screening and detection. The detection steps are as follows: Step 1: Dilute the serum sample to be tested 1:5 with sample diluent to obtain diluted sample solution; Step 2: Add 100 μL of diluted sample solution to the sample pad of the fluorescent test strip and react at room temperature for 15 min. Step 3: Using a fluorescence immunochromatographic analyzer, the ratio of fluorescence signals of the detection line to the control line (T / C ratio) is detected under the parameters of excitation wavelength 365nm and emission wavelength 610nm. Step 4: Use 300 pg / mL as the cutoff value to determine whether the tuberculosis is suspected positive or negative. When the fluorescence immunochromatographic analyzer detects an IP10 concentration in the sample that is greater than or equal to 300 pg / mL, it is determined to be suspected positive for tuberculosis. When the fluorescence immunochromatographic analyzer detects an IP10 concentration in the sample that is less than 300 pg / mL, it is determined to be negative for tuberculosis.

[0053] Example 4: Sample Detection and Performance Verification A total of 260 samples were selected, including 63 patients with clinically confirmed active tuberculosis, 48 ​​patients with latent tuberculosis, 46 patients with non-tuberculous lung disease, and 103 healthy controls.

[0054] Parallel detection was performed using the AIE fluorescence immunochromatographic assay kit and the AIE magnetic particle fluorescence immunochromatographic assay kit, respectively.

[0055] The results show: The AIE fluorescence immunochromatographic assay kit showed a linear detection range of 50 pg / mL to 1000 pg / mL, with an area under the curve (AUC) of 0.887 and an overall clinical concordance rate of 81.7%.

[0056] The AIE magnetic microparticle fluorescence immunoassay kit has a linear detection range of 5 pg / mL to 1000 pg / mL, with a correlation coefficient (R²) of not less than 0.998, an area under the curve (AUC) of 0.912, a sensitivity of 92.04%, and a specificity of 80.27%. The intra-assay coefficient of variation (CV) is no more than 6.50%, and the inter-assay CV is no more than 7.80%. After 14 days of accelerated aging at 37°C, the fluorescence decay rate is no more than 15%. All performance indicators of this kit meet the requirements for clinical diagnosis.

[0057] The linear detection range of the AIE magnetic microparticle fluorescence immunoassay kit is 5 pg / mL to 1000 pg / mL. When the sample detection value exceeds this upper limit of linearity, a gradient dilution should be performed before detection, for example, by diluting the sample with purified water at a ratio of 1:5. The fully automated fluorescence immunoassay analyzer quantifies the IP10 concentration by fitting a standard curve. Finally, the IP10 measurement value output by the instrument is multiplied by the corresponding dilution factor (e.g., ×5) to obtain the actual concentration of IP10 in the sample.

[0058] The kit was used to test samples from each group. The results showed that the IP10 concentration was 1100 pg / mL–4700 pg / mL in the active tuberculosis group, 300 pg / mL–1500 pg / mL in the latent tuberculosis group, 20 pg / mL–100 pg / mL in the non-tuberculous lung disease group, and all were below 90 pg / mL in the healthy control group.

[0059] In summary, the results of the AIE magnetic particle fluorescence immunoassay kit can serve as one of the auxiliary diagnostic indicators for the clinical grading of tuberculosis infection. In practical application, the diagnosis of active tuberculosis still requires a comprehensive assessment combining bacteriological examination, imaging findings, and clinical characteristics; the determination of latent tuberculosis infection requires a comprehensive analysis combining a positive IGRA test, the absence of typical clinical symptoms, and the absence of active lesions on imaging; non-tuberculous lung lesions require a comprehensive assessment combining the definitive diagnoses of other lung diseases; and the inclusion of healthy controls requires a comprehensive evaluation combining the absence of tuberculosis exposure history and negative results for relevant test indicators.

[0060] The AIE magnetic particle fluorescence immunoassay kit demonstrates excellent performance in detection, stability, and clinical discrimination, and can serve as an effective auxiliary diagnostic tool for tuberculosis infection grading, thus possessing significant clinical application value.

[0061] It should be understood that although the above embodiments provide a relatively detailed textual description of the design concept of the present invention, these textual descriptions are merely simple textual descriptions of the design concept of the present invention, and not limitations on the design concept of the present invention. Any combination, addition, or modification that does not exceed the design concept of the present invention falls within the protection scope of the present invention.

Claims

1. A tuberculosis auxiliary diagnostic kit based on AIE fluorescent microspheres labeled with IP10, characterized in that: This is an AIE fluorescence immunochromatographic assay kit for rapid screening detection. The kit includes a fluorescent test strip and sample diluent. The conjugate pad of the test strip is coated with AIE fluorescent microsphere-labeled IP10 antibody. The nitrocellulose membrane of the test strip has a test line and a control line. The test strip uses an IP10 concentration of 300 pg / mL in the sample as a cutoff value to determine whether the tuberculosis test is suspected positive or negative. When the IP10 concentration in the sample is greater than or equal to 300 pg / mL, it is considered a suspected positive for tuberculosis; when the IP10 concentration is less than 300 pg / mL, it is considered a negative for tuberculosis. Alternatively, it is an AIE magnetic microparticle fluorescence immunochromatographic assay kit for quantitative detection. This kit includes an IP10 antibody-conjugated magnetic microparticle suspension, AIE fluorescent microsphere-labeled IP10 antibody working solution, washing solution, calibrators, and quality control materials. The AIE fluorescent microsphere-labeled IP10 antibody working solution is prepared by AIE... It was prepared by labeling IP10 antibody with fluorescent microspheres.

2. The tuberculosis auxiliary diagnostic kit based on AIE fluorescent microspheres labeled IP10 according to claim 1, characterized in that: The preparation method of the AIE fluorescent microsphere labeled IP10 antibody is as follows: 1 mL of carboxylated AIE fluorescent microspheres with a mass fraction of 1% and a particle size of 200 nm were taken and resuspended in 0.1 mol / L, pH 5.5 MES buffer; 10 μL each of 10 mg / mL EDC solution and 10 mg / mL NHS solution were added, and the mixture was activated for 30 min at room temperature in the dark; then 200 μg of IP10 monoclonal antibody was added, and the mixture was coupled for 2 h at room temperature in the dark with shaking. Add 100 μL of 10% BSA solution and block at room temperature for 1 h; centrifuge at 8000 r / min for 5 min and wash three times to obtain AIE fluorescent microsphere labeled IP10 antibody. Resuspend it in antibody preservation solution and store at 4℃ in the dark for later use.

3. The tuberculosis auxiliary diagnostic kit based on AIE fluorescent microspheres labeled IP10 according to claim 1, characterized in that: The concentration of the AIE fluorescent microsphere-labeled IP10 antibody in the working solution is 0.1 mg / mL.

4. The tuberculosis auxiliary diagnostic kit based on AIE fluorescent microspheres labeled IP10 according to claim 1, characterized in that: The sample diluent was a 0.01 mol / L PBS buffer containing 0.5% BSA, 0.1% Tween-20, and 0.05% Proclin 300, with a pH of 7.

4.

5. The tuberculosis auxiliary diagnostic kit based on AIE fluorescent microspheres labeled IP10 according to claim 1, characterized in that: The AIE fluorescent microspheres are carboxylated nanospheres with a particle size range of 100nm-300nm, an excitation wavelength of 365nm, an emission wavelength of 610nm, and a fluorescence quantum yield of not less than 80%. The magnetic microparticles are 3μm superparamagnetic amino magnetic microparticles. The IP10 antibody-coupled magnetic microparticle suspension is prepared by coupling IP10 paired antibody with 3μm amino magnetic microparticles through glutaraldehyde crosslinking, followed by blocking and washing steps.

6. A tuberculosis auxiliary diagnostic kit based on AIE fluorescent microspheres labeled IP10 according to claims 1-5, characterized in that: The AIE magnetic microparticle fluorescent immunoassay kit is used for early screening of pulmonary tuberculosis, identification of latent tuberculosis infection, diagnosis of active pulmonary tuberculosis, and dynamic monitoring of the efficacy of anti-tuberculosis treatment.

7. A tuberculosis auxiliary diagnostic kit based on AIE fluorescent microspheres labeled IP10 according to claim 1, characterized in that: The AIE fluorescent microspheres labeled with IP10 antibody were uniformly sprayed onto the conjugate pad at a concentration of 1 μL / cm; the detection line was coated with IP10 paired antibody at a concentration of 1.0 mg / mL; and the control line was coated with goat anti-mouse IgG at a concentration of 0.8 mg / mL.

8. A method for auxiliary diagnosis and detection of tuberculosis based on AIE fluorescent microspheres labeled with IP10, comprising a fully automated fluorescence immunoassay analyzer, characterized in that: Quantitative detection was performed using any of the tuberculosis auxiliary diagnostic kits based on AIE fluorescent microspheres labeled IP10 as described in claims 1-5, and the detection steps are as follows: Step 1: Add 50 μL of IP10 antibody-conjugated magnetic microparticle suspension, 50 μL of serum sample to be tested, and 50 μL of AIE fluorescently labeled antibody working solution to the reaction tube in sequence, and then incubate with shaking. Step 2: After shaking and incubation, the reaction tube is separated by magnetic field. Then, the supernatant in the reaction tube is discarded, and the tube is washed three times with washing solution. Step 3: Then add 100 μL of buffer solution to the reaction tube and use a fully automated fluorescence immunoassay analyzer to detect the fluorescence intensity under the conditions of 365 nm excitation and 610 nm emission. Step four: The fully automated fluorescence immunoassay analyzer quantitatively calculates the IP10 concentration by fitting a standard curve. The calculated quantitative IP10 concentration can be used in clinical practice as one of the auxiliary diagnostic indicators for determining the grading of tuberculosis infection, as well as as a basis for evaluating the efficacy of tuberculosis treatment.

9. The tuberculosis auxiliary diagnostic detection method based on AIE fluorescent microsphere labeling IP10 according to claim 8, characterized in that: The oscillation incubation temperature in step one is 37℃, and the oscillation incubation time is 25 min; the magnetic field separation time in step two is 3 min.

10. A second method for auxiliary diagnostic detection of tuberculosis based on AIE fluorescent microspheres labeled IP10, comprising a fluorescence immunochromatography instrument, characterized in that: Rapid screening and detection were performed using any of the tuberculosis auxiliary diagnostic kits based on AIE fluorescent microspheres labeled IP10 as described in claims 1 and 7. The detection steps are as follows: Step 1: Dilute the serum sample to be tested 1:5 with sample diluent to obtain diluted sample solution; Step 2: Add 100 μL of diluted sample solution to the sample pad of the fluorescent test strip and react at room temperature for 15 min. Step 3: Using a fluorescence immunochromatographic analyzer, the ratio of fluorescence signals of the detection line to the control line (T / C ratio) is detected under the parameters of excitation wavelength 365nm and emission wavelength 610nm. Step 4: Use 300 pg / mL as the cutoff value to determine whether the tuberculosis is suspected positive or negative. When the fluorescence immunochromatographic analyzer detects an IP10 concentration in the sample that is greater than or equal to 300 pg / mL, it is determined to be suspected positive for tuberculosis. When the fluorescence immunochromatographic analyzer detects an IP10 concentration in the sample that is less than 300 pg / mL, it is determined to be negative for tuberculosis.

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