Triple detection method for cell factors IL-1beta, IL-6 and IL-8 and application of triple detection method

Abstract

The invention provides a triple detection method for cell factors IL-1beta, IL-6 and IL-8 and application of the triple detection method, and belongs to the technical field of biological detection. According to the present invention, the digital fluorescence immunoassay technology is adopted, the antibodies are immobilized on the detection plate, and after the antigen in the sample is combined with the immobilized antibodies, the fluorescein-labeled antibody is combined with the other antigen determinant on the antigen so as to detect the antigen in the sample; the fluorescein labeled antibody and the solid-phase antibody are combined, so that a sandwich type compound structure is constructed between the solid-phase antibody and the fluorescein labeled antibody, the higher the antigen concentration is, the more the fluorescent particles are, and the content of the antigen is quantified according to the number of the fluorescent particles, so that high-sensitivity and high-precision detection of the antigen can be realized; moreover, the content of the antigen can be accurately quantified by accurately counting the number of the fluorescent particles, triple detection of cell factors IL-1beta, IL-6 and IL-8 is realized, the detection range is wide, the sensitivity is high, the accuracy is high, the detection is automatic, and convenience and rapidness are realized.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a triple detection method for cytokines IL-1β, IL-6, and IL-8 and applications thereof. Background Art

[0002] Interleukins (ILs) are common cytokines that play an important role in regulating and modulating human immunity, inflammation, and hematopoiesis. The IL family comprises 38 members, including IL-1, IL-2, IL-6, IL-10, IL-12, and IL-17, based on their structure and function. Each family member possesses unique biological activities and mechanisms of action. Among these, the inflammatory factors IL-1β, IL-6, and IL-8 play key regulatory roles in pathological processes such as infection, autoimmune diseases, and cancer. Their accurate detection is crucial for disease diagnosis and efficacy evaluation.

[0003] IL-1 is a common inflammatory cytokine in the IL family. Its biological activity manifests in two forms: interleukin-1α and interleukin-1β. Systemic inflammatory diseases characterized by fever, anemia, and elevated acute phase protein levels are associated with excessive production and biological activity of IL-1β. Under normal physiological conditions, IL-1β is essentially not expressed. However, under pathological conditions (such as rheumatoid arthritis, neuropathic pain, inflammatory bowel disease, osteoarthritis, vascular disease, multiple sclerosis, and Alzheimer's disease), IL-1β expression is increased.

[0004] IL-6 is a multifunctional cytokine with a wide range of functions. IL-6 is rapidly produced during acute inflammatory responses to internal and external trauma, surgery, stress, infection, brain death, tumor formation, and other conditions. After an inflammatory response occurs, IL-6 serum concentrations increase earlier than other biomarkers. IL-6 concentrations in surgical patients can predict whether surgical complications will occur. Continuous monitoring of IL-6 levels in the serum or plasma of intensive care unit (ICU) patients can effectively assess the severity of systemic inflammatory response syndrome (SIRS), the prognosis of sepsis and septic shock, and can also serve as an early warning indicator of sepsis. IL6 also plays an important role in chronic inflammatory responses (such as rheumatoid arthritis).

[0005] IL-8 is a potent neutrophil chemotactic factor. Macrophages, epithelial cells, and other related cells secrete small amounts of IL-8, making it virtually undetectable in healthy tissues. However, its secretion can increase 10- to 100-fold in response to proinflammatory cytokines, bacterial or viral products, and cellular stress. Studies have shown that IL-8 has a higher diagnostic sensitivity and negative predictive value than PCT for the early prediction of sepsis. IL-8 can also predict the potential for multi-organ dysfunction in patients with sepsis and serve as a risk marker for mortality. IL-8 overexpression is also closely associated with malignant biological behaviors such as tumor cell proliferation, invasion, and metastasis.

[0006] Clinically, cytokine testing can provide information for early diagnosis of the disease. Significant increases in IL-1β, IL-6, and IL-8 may indicate inflammation or bacterial infection. Therefore, testing multiple cytokines allows for timely monitoring of dynamic changes in a patient's cytokine levels and quantitative analysis. This is crucial for assessing disease severity, treatment efficacy, and prognosis. This allows doctors to obtain richer information and more accurately assess the course of the disease.

[0007] At present, single-index detection technologies such as enzyme-linked immunosorbent assay (ELISA) are still the mainstream methods with high sensitivity and specificity, but each indicator needs to be tested independently, resulting in large sample consumption, long time and high cost, and is greatly affected by human operation and prone to errors; although immunofluorescence technology (such as flow cytometry) can improve throughput, it has strict requirements on sample processing and is easily interfered by fluorescence crosstalk, with low sensitivity, affecting accuracy and large deviations in detection results from different manufacturers; although electrochemiluminescence method (ECLIA) has a wide linear range and low detection limit, it relies on expensive equipment and is difficult to achieve multiplexing, which limits its clinical use and promotion in hospitals; although mass spectrometry technology can perform high-throughput analysis, its pre-treatment is complex, the equipment cost is high, and its clinical application is limited; the detection results of the colloidal gold method can only be used for preliminary qualitative analysis and cannot be used for quantitative analysis; and although the emerging biosensor technology can detect quickly, its stability and repeatability still need to be optimized, and it has not yet been maturely promoted.

[0008] In response to the demand for simultaneous detection of multiple indicators, microsphere-based multiple immunoassay technology (such as Luminex) has achieved some breakthroughs and can detect multiple factors at the same time. However, problems such as antibody cross-reactivity, signal interference and insufficient standardization still restrict its detection accuracy, and high costs and complex data analysis limit its popularization. Therefore, the development of efficient, accurate and economical simultaneous detection methods has become an urgent need. It is necessary to overcome the sensitivity, specificity and standardization bottlenecks of existing technologies while increasing throughput to meet the precise needs of dynamic monitoring of inflammatory factors in clinical and scientific research. Summary of the Invention

[0009] The purpose of the present invention is to provide a triple detection method for cytokines IL-1β, IL-6, and IL-8 and its application, which can realize the triple detection of cytokines IL-1β, IL-6, and IL-8, with a wide detection range, high sensitivity, high accuracy, automated detection, and convenience and speed.

[0010] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0011] The present invention provides a method for triple detection of cytokines IL-1β, IL-6, and IL-8, comprising the following steps:

[0012] (1) Antibody-coated test plate

[0013] Cytokine IL-1β, IL-6, and IL-8 antibodies and coating solution were added to the wells of the test plate for coating, and then blocking solution was added to block the wells.

[0014] (2) Fluorescent microspheres coupled to antibodies

[0015] Activating the fluorescent microspheres and coupling them with antibodies, followed by blocking to obtain antibody-coupled fluorescent microspheres;

[0016] (3) adding the sample to be tested to the detection well sealed in step (1) and incubating, and adding the antibody-coupled fluorescent microspheres in step (2) and incubating, and then detecting.

[0017] Preferably, in step (1), the concentrations of the cytokine IL-1β, IL-6, and IL-8 antibodies are 0.4-0.6 μg / T, respectively, the coating temperature is 5-9° C., and the coating time is 13-15 h.

[0018] Preferably, in step (1), the detection plate comprises a polystyrene ELISA plate, a carboxyl plate or a PMMA plate.

[0019] Preferably, the detection plate includes a calibration area, a quality control area, and a sample detection area; the sample detection area includes detection areas for cytokines IL-1β, IL-6, and IL-8.

[0020] Preferably, the conditions for activating the fluorescent microspheres in step (2) are: avoiding light, 36-38° C., and time for 25-35 min.

[0021] Preferably, the coupling conditions in step (2) are: protected from light, 36-38° C., and time is 2-3 h.

[0022] Preferably, in step (3), the sample to be tested is added to the detection well sealed in step (1) and incubated for 40-60 minutes; and the antibody-coupled fluorescent microspheres in step (2) are added and incubated for 40-60 minutes.

[0023] The present invention also provides an application of a cytokine IL-1β, IL-6, IL-8 triple detection method in preparing a cytokine IL-1β, IL-6, IL-8 triple detection kit.

[0024] The beneficial effects of the present invention compared with the prior art are:

[0025] (1) The present invention uses digital fluorescent immunoassay technology to immobilize antibodies on a test plate. When the antigen in the sample binds to these immobilized antibodies, the fluorescein-labeled antibody binds to another antigenic determinant on the antigen, thereby constructing a "sandwich" complex structure between the immobilized antibody and the fluorescein-labeled antibody. The higher the antigen concentration, the more fluorescent particles there are, and the antigen content is quantified by the number of fluorescent particles. This unique design enables the present invention to achieve high-sensitivity and high-precision detection of antigens, and can also accurately quantify the antigen content by precisely counting the number of fluorescent particles.

[0026] (2) The present invention determines the antigen concentration by the number of fluorescent particles, which can avoid the influence of lipemia / hemolysis on the test results. The prepared solid-phase detection plate is coated with three-item antibodies, different item calibration areas, quality control areas, and sample detection areas, which can realize the triple detection of cytokines IL-1β, IL-6, and IL-8, with a wide detection range, high sensitivity, high accuracy, automated detection, and convenient and fast detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0028] Figure 1 This is the 96-well assay plate used in Example 1 of the present invention, wherein S0-S5 are calibration areas; Q1-Q2 are quality control areas; P1-P16 are sample areas;

[0029] Figure 2 The clinical correlation between the IL-1β detection results and R in Example 1 of the present invention, wherein A is the IL-1β calibrator and the concentration of fluorescent particles; B is the clinical correlation between the IL-1β physics institute and XMZ;

[0030] Figure 3 The clinical correlation between the IL-6 test results and R in Example 1 of the present invention, where A is the IL-6 calibrator and the fluorescent particle concentration; B is the clinical correlation between the IL-6 physics institute and XMZ;

[0031] Figure 4 The clinical correlation between the IL-8 test results and R in Example 1 of the present invention, where A is the IL-8 calibrator and the fluorescent particle concentration; B is the clinical correlation between the IL-8 physics institute and XMZ;

[0032] Figure 5 This is the linear range of IL-8 detection in Test Example 1 of the present invention. DETAILED DESCRIPTION

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0035] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0036] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0037] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0038] Example 1

[0039] Example 1 of the present invention provides a method for triple detection of cytokines IL-1β, IL-6, and IL-8, and the specific steps are as follows:

[0040] (1) Solution preparation:

[0041] Coupling buffer: 10 mM MES (pH 6.2 ± 0.05), containing 0.05% ProClin 300;

[0042] EDC solution: 10 mg / mL, prepared with coupling buffer, ready for use;

[0043] NHS solution: 10 mg / mL, prepared with coupling buffer, ready for use;

[0044] Fluorescent microsphere blocking solution: borate buffer (5 mM boric acid, 11.2 mM sodium tetraborate decahydrate, 0.05% Tween-20, pH 9.0 ± 0.05), 1% BSA, 0.24% ethanolamine;

[0045] Fluorescent microsphere washing solution: 50 mM Tris (pH 8.0 ± 0.05), 0.5% BSA, 0.05% Tween-20, 0.03% ProClin 300;

[0046] Fluorescent microsphere storage solution: 25 mM Tris (PH 7.2±0.05), 150 mM NaCI, 0.05% Tween-20, 1% BSA, 5% trehalose, 0.1% ProClin 300.

[0047] (2) Activation of fluorescent microspheres

[0048] A. Add 0.05 mL of fluorescent microsphere suspension (1% solid content) to a 2 mL centrifuge tube containing 1 mL of coupling buffer, mix thoroughly by ultrasonication, centrifuge at 15°C, 20,000 g for 10 min, and remove the supernatant. Add another 1 mL of coupling buffer, mix thoroughly by ultrasonication, centrifuge at 15°C, 20,000 g for 10 min, and remove the supernatant.

[0049] B. Take the precipitate from A and add 1 mL of coupling buffer. After ultrasonic mixing, add 3.5 μL of EDC solution and vortex mix. Then add 33 μL of NHS solution and ultrasonic mix. Place the centrifuge tube on a turntable and incubate at 38°C, 40 rpm, in the dark for 30 min. After activation, centrifuge at 15°C, 20,000 g for 10 min and remove the supernatant.

[0050] C. Take the precipitate from B and add 1.5 mL of coupling buffer, mix by ultrasonication, centrifuge at 15°C and 20,000 g for 10 min, remove the supernatant, add 1.5 mL of coupling buffer, and repeat the washing step once.

[0051] (3) Coupling of fluorescent microspheres and antibodies

[0052] Add 0.75 mL of coupling buffer and mix thoroughly by ultrasonication. Add 50 μg of the cytokine IL-1β, IL-6, and IL-8 labeled antibodies to 0.25 mL of coupling buffer to prepare the coupling solution. Add the antibody-containing coupling solution to the mixed fluorescent microspheres and vortex to mix thoroughly. Place the centrifuge tube on a turntable and incubate at 38°C, 40 rpm, in the dark for 2.5 h.

[0053] (4) Sealing and preservation of fluorescent microspheres

[0054] Add 0.5 mL of fluorescent microsphere blocking solution to the centrifuge tube and vortex mix; place the centrifuge tube on a turntable, block at 37°C, 40 r / min, and protect from light for 1 hour; centrifuge at 15°C, 20,000 g for 10 minutes, remove the supernatant; add 1.5 mL of fluorescent microsphere washing solution, ultrasonically mix, centrifuge at 15°C, 20,000 for 10 minutes, remove the supernatant; add 1.5 mL of fluorescent microsphere washing solution, repeat the washing steps once; finally, add 0.5 mL of fluorescent microsphere preservation solution (final concentration of fluorescent microspheres 1 mg / mL) to the centrifuge tube, ultrasonically mix, and store at 2-8°C in the dark for later use.

[0055] (5) Antibody-coated solid-phase detection plate

[0056] A. Solution preparation

[0057] Coating solution: sodium carbonate (Na2CO3) 0.1M, sodium bicarbonate (NaHCO3) 0.1M, pH 9.6.

[0058] Blocking solution: 0.05-0.1M PBS (pH 7.4) + 1-2% BSA + 0.1% ProClin 300 + 0.1% protein protectant.

[0059] Calibrator diluent: 0.1 M PBS buffer, 1% protein (bovine serum albumin), 0.1% protein protectant and 0.2% ProClin 300.

[0060] Preparation of quality control / calibrators: Use the above calibrator diluents to prepare cytokine IL-1β, IL-6, and IL-8 calibrators according to the table below.

[0061]

[0062] B. Antibody coating of the test plate

[0063] According to the antibody concentration of 0.5 μg / T, cytokine IL-1β, IL-6, and IL-8 antibodies were taken respectively. Figure 1 The polystyrene ELISA plate was coated with 100 μL / T coating solution at 9° C. for 15 h, washed five times with PBS+TWEEN (0.05% wt), and patted dry.

[0064] Add 150 μL / well blocking solution, block at 37° C. for 2 h, wash 5 times with PBS+TWEEN (0.05% wt), pat dry, put into desiccant, seal in tin foil bag and set aside for use.

[0065] (6) Add the quality control products, calibrators and body fluid samples containing cytokines IL-1β, IL-6 and IL-8 into the coated corresponding ELISA plate wells, place on a shaker, react at room temperature for 1 hour, wash five times with PBS + TWEEN (0.05% wt) and pat dry.

[0066] (7) After diluting the corresponding blocked fluorescent microspheres 25 times with microsphere storage solution, 50 μL was added to the wells of the ELISA plate containing the quality control products, calibrators and body fluid samples of cytokines IL-1β, IL-6, and IL-8, and placed on a shaker. After reacting at room temperature for 1 hour, the microspheres were washed 5 times with PBS + TWEEN (0.05% wt) and patted dry.

[0067] (8) Place the above enzyme-labeled plate on the self-developed instrument of the Institute of Physics of Henan Academy of Sciences, the digital fluorescence immunoassay analyzer: FX100. The instrument is calibrated according to the information on the reagent calibration card. After successful calibration, the quality control test is within the range before subsequent clinical sample testing can be carried out. The results are as follows Figures 2-4 shown.

[0068] Test results: Correlation R between the test results of cytokines IL-1β, IL-6, and IL-8 and the test results of different well-known listed companies 2 All of them are greater than 0.95, indicating accurate results and high reliability.

[0069] Example 2

[0070] Example 2 of the present invention uses the reagents of Example 1 to provide a triple detection method for cytokines IL-1β, IL-6, and IL-8, and the specific steps are as follows:

[0071] (1) Activation of fluorescent microspheres

[0072] A. Add 0.05 mL of fluorescent microsphere suspension (1% solid content) to a 2 mL centrifuge tube containing 1 mL of coupling buffer, mix thoroughly by ultrasonication, centrifuge at 15°C, 20,000 g for 10 min, and remove the supernatant. Add another 1 mL of coupling buffer, mix thoroughly by ultrasonication, centrifuge at 15°C, 20,000 g for 10 min, and remove the supernatant.

[0073] B. Add 1 mL of coupling buffer to the precipitate from A, mix thoroughly by ultrasonication, then add 3.5 μL of EDC solution, vortex mix thoroughly, and then add 33 μL of NHS solution, and mix thoroughly by ultrasonication. Place the centrifuge tube on a turntable and incubate at 36°C, 40 rpm, in the dark for 35 min. After activation, centrifuge at 15°C, 20,000 g for 10 min, and remove the supernatant.

[0074] C. Take the precipitate from B and add 1.5 mL of coupling buffer, mix by ultrasonication, centrifuge at 15°C and 20,000 g for 10 min, remove the supernatant, add 1.5 mL of coupling buffer, and repeat the washing step once.

[0075] (2) Coupling of fluorescent microspheres and antibodies

[0076] Add 0.75 mL of coupling buffer and mix thoroughly by ultrasonication. Add 50 μg of the cytokine IL-1β, IL-6, and IL-8 labeled antibodies to 0.25 mL of coupling buffer to prepare the coupling solution. Add the antibody-containing coupling solution to the mixed fluorescent microspheres and vortex to mix thoroughly. Place the centrifuge tube on a turntable and incubate at 36°C, 40 rpm, and protect from light for 3 h.

[0077] (3) Sealing and preservation of fluorescent microspheres

[0078] Add 0.5 mL of fluorescent microsphere blocking solution to the centrifuge tube and vortex mix; place the centrifuge tube on a turntable, block at 37°C, 40 r / min, and protect from light for 1 hour; centrifuge at 15°C, 20,000 g for 10 minutes, remove the supernatant; add 1.5 mL of fluorescent microsphere washing solution, ultrasonically mix, centrifuge at 15°C, 20,000 for 10 minutes, remove the supernatant; add 1.5 mL of fluorescent microsphere washing solution, repeat the washing steps once; finally, add 0.5 mL of fluorescent microsphere preservation solution (final concentration of fluorescent microspheres 1 mg / mL) to the centrifuge tube, ultrasonically mix, and store at 2-8°C in the dark for later use.

[0079] (4) Antibody-coated solid-phase detection plate

[0080] A. Activation of the test plate

[0081] Use carboxylated plates, add 50 μL of 20 mg / ml EDC and NHS solution, activate in a 37°C incubator for 1 h, wash with PBS and pat dry;

[0082] B. Antibody coating of the test plate

[0083] According to the antibody concentration of 0.5 μg / T, cytokine IL-1β, IL-6, and IL-8 antibodies were taken respectively. Figure 1The carboxylation plates were coated with 100 μL / T coating solution at 7° C. for 15 h, washed five times with PBS+TWEEN (0.05% wt), and patted dry.

[0084] Add 150 μL / well blocking solution, block at 37° C. for 2 h, wash 5 times with PBS+TWEEN (0.05% wt), pat dry, put into desiccant, seal in tin foil bag and set aside for use.

[0085] (5) Add the quality control products, calibrators and body fluid samples containing cytokines IL-1β, IL-6 and IL-8 into the coated corresponding ELISA plate wells, place on a shaker, react at room temperature for 1 hour, wash with PBS + TWEEN (0.05% wt) 5 times, and pat dry.

[0086] (6) After diluting the corresponding blocked fluorescent microspheres 25 times with microsphere storage solution, 50 μL was added to the wells of the ELISA plate containing the quality control products, calibrators and body fluid samples of cytokines IL-1β, IL-6, and IL-8, and placed on a shaker. After reacting at room temperature for 1 hour, the microspheres were washed 5 times with PBS + TWEEN (0.05% wt) and patted dry.

[0087] Test Example 1

[0088] Test Example 1 of the present invention tested the effect of the detection method of Example 1, and the specific steps are as follows:

[0089] (1) High sensitivity:

[0090] Assessment method:

[0091] Limit of Blank (LOB): Take 5 samples with a value of 0 (samples without the substance to be tested) and test according to the steps in Example 1. Repeat the test 3 times and evaluate for 4 days;

[0092] Limit of Detection (LOD): Take 5 samples with 1 to 4 times the LoB and test according to the steps in Example 1. Repeat the test 3 times and evaluate for 4 days.

[0093] Taking the IL-8 project as an example for verification, the test data are shown in Table 2 below.

[0094] Table 2 IL-8 Item Sensitivity

[0095]

[0096] (2) Wide linear range:

[0097] The linear range is selected from samples close to 100% to 130% of the upper limit of the calibrator, and one sample close to 0 value, mixed in different proportions, with at least 4 to 6 gradients (the number of points can also be appropriately increased according to the width of the linear range), and the correlation between the measured value and the theoretical value is required to be r>0.99. Taking the IL-8 project as an example for verification, the test data are shown in Table 3 below. The upper limit of linearity can be achieved at 8397.31pg / mL, and the upper limit of linearity of the commercial product is 7500pg / mL. The explicit results are as follows Figure 5 shown.

[0098] Table 3 IL-8 project linear range verification data

[0099]

[0100] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A triple detection method for cytokines IL-1β, IL-6, and IL-8, characterized in that: The steps include: (1) Antibody-coated test plate Cytokine IL-1β, IL-6, and IL-8 antibodies and coating solution were added to the wells of the test plate for coating, and then blocking solution was added to block the wells. (2) Fluorescent microspheres coupled to antibodies Activating the fluorescent microspheres and coupling them with antibodies, followed by blocking to obtain antibody-coupled fluorescent microspheres; (3) adding the sample to be tested to the detection well sealed in step (1) and incubating the well, and then adding the antibody-coupled fluorescent microspheres in step (2) for detection after incubation.

2. The triple detection method of cytokines IL-1β, IL-6, and IL-8 according to claim 1, characterized in that: In step (1), the concentrations of the cytokine IL-1β, IL-6, and IL-8 antibodies are 0.4-0.6 μg / T, respectively, the coating temperature is 5-9° C., and the coating time is 13-15 h.

3. The triple detection method of cytokines IL-1β, IL-6, and IL-8 according to claim 1, characterized in that: In step (1), the detection plate includes a polystyrene ELISA plate, a carboxyl plate or a PMMA plate.

4. The triple detection method of cytokines IL-1β, IL-6, and IL-8 according to claim 3, characterized in that: The detection plate includes a calibration area, a quality control area, and a sample detection area; the sample detection area includes detection areas for cytokines IL-1β, IL-6, and IL-8.

5. The triple detection method of cytokines IL-1β, IL-6, and IL-8 according to claim 1, characterized in that: The conditions for activating the fluorescent microspheres in step (2) are: avoiding light, 36-38° C., and time for 25-35 min.

6. The triple detection method of cytokines IL-1β, IL-6, and IL-8 according to claim 1, characterized in that: The coupling conditions in step (2) are: protected from light, 36-38° C., and time is 2-3 h.

7. The triple detection method of cytokines IL-1β, IL-6, and IL-8 according to claim 1, characterized in that: In step (3), the sample to be tested is added to the detection well sealed in step (1) and incubated for 40-60 minutes; the antibody-coupled fluorescent microspheres in step (2) are added and incubated for 40-60 minutes.

8. Use of the cytokine IL-1β, IL-6, IL-8 triple detection method according to any one of claims 1 to 7 in the preparation of a cytokine IL-1β, IL-6, IL-8 triple detection kit.

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