Interleukin 33 detection kit, preparation method and application

Abstract

The invention provides an interleukin 33 detection kit as well as a preparation method and application thereof. The interleukin 33 detection kit comprises a first antibody coupled to a magnetic fluorescent light-emitting microsphere, a second antibody labeled with biotin and fluorescein-labeled streptavidin, the first antibody strain and the second antibody strain are monoclonal antibodies for resisting interleukin 33, and interleukin 33 specific binding sites recognized by the first antibody strain and the second antibody strain are different. According to the kit disclosed by the invention, the interleukin 33 antigen is specifically combined with the magnetic fluorescent luminous microsphere formed by coupling the interleukin 33 capture antibody, then the magnetic fluorescent luminous microsphere is specifically combined with the biotin-labeled interleukin 33 detection antibody, the conjugate is combined with fluorescein-labeled streptavidin, fluorescence is emitted, and the content of the interleukin 33 in a sample to be detected is determined. The method is high in sensitivity, good in repeatability, short in reaction time, simple to operate and high in anti-interference performance.

Description

Technical Field

[0001] The invention belongs to a medical detection method and relates to an interleukin-33 microsphere flow immunofluorescence luminescence detection kit, a preparation method and an application. Background Art

[0002] Interleukin 33 (IL-33), also known as IL-1F11, is produced by Th2 cells, mast cells, and innate lymphocytes. It primarily participates in Th2 cell-mediated immune responses and regulates mast cell function. It also acts as an intracellular nuclear factor, regulating gene transcription. IL-33 is a proinflammatory cytokine with multiple effects, playing a crucial role in regulating innate immune responses, particularly mast cell function. As a proinflammatory cytokine, it can cause tissue immunopathological damage. IL-33 can induce and regulate various inflammatory responses and may play a role in chronic inflammatory and autoimmune diseases. Therefore, the combined detection of IL-33 with other immunological markers has important diagnostic value in vascular, allergic, autoimmune, and inflammatory diseases.

[0003] IL-33 can be detected using immunoassays. Common immunoassay techniques include enzyme immunoassay, fluorescence chromatography immunoassay, immunoturbidimetric assay, colloidal gold immunoassay, and luminescence immunoassay. The concentration of IL-33 in healthy human blood is reported to be less than 100 pg / mL. Therefore, common enzyme immunoassays, fluorescence chromatography immunoassays, immunoturbidimetric assays, and colloidal gold immunoassays lack the required sensitivity for IL-33 detection. While luminescence immunoassays have sufficient sensitivity, they cannot perform multi-item combined detection, limiting their application prospects.

[0004] Flow cytometry is a luminescence detection method that has developed rapidly in recent years. It has high sensitivity and high precision, can simultaneously realize the joint detection of multiple indicators, and has the advantages of high throughput and rapid detection. This technology is based on the basic principles of immunological analysis and adopts a detection mode similar to the direct sandwich method to identify and quantify the substance to be tested. By using fluorescent microspheres as carriers of immune reactions, the level of target protein in biological samples can be effectively measured by combining immunoassay with flow cytometry. Its working principle is as follows: the capture antibody coupled to the magnetic fluorescent microspheres forms a "sandwich" complex with the biotin-labeled detection antibody and the antigen to be tested in the sample, and then reacts with fluorescein-labeled streptavidin. The intensity of the fluorescent signal is detected by flow cytometry, thereby accurately determining the concentration of the antibody to be tested in the sample, and the detection sensitivity can reach the pg / mL level. Summary of the Invention

[0005] The purpose of the present invention is to provide a detection kit for interleukin-33, which has better sensitivity and is capable of conducting joint detection of multiple items.

[0006] Another object of the present invention is to provide a method for preparing an interleukin-33 detection kit.

[0007] In addition, the present invention also provides the application of the above kit.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] In one technical solution, the present invention provides an interleukin-33 detection kit, which includes a first antibody coupled to a magnetic fluorescent microsphere, a second antibody labeled with biotin, and fluorescein-labeled streptavidin, wherein the first antibody and the second antibody are monoclonal antibodies against interleukin-33, and the interleukin-33 specific binding sites recognized by the first antibody and the second antibody are different.

[0010] In a specific embodiment provided by the present invention, the magnetic fluorescent microspheres are selected from a complex of nano-sized Fe2O3 and / or Fe3O4 magnetic particles and polymer materials, and the average particle size of the magnetic fluorescent microspheres is 2-8 μm.

[0011] In a specific embodiment provided by the present invention, multiple active groups can be added to the surface of the magnetic fluorescent microspheres. In an exemplary embodiment, the active groups can be selected from -CHO and / or -COOH.

[0012] In a specific embodiment provided by the present invention, the fluorescein includes one or more of fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), and Cy5; optionally, the fluorescein is phycoerythrin, and the fluorescein-labeled streptavidin is phycoerythrin-labeled streptavidin (SA-PE).

[0013] In a specific embodiment provided by the present invention, the ratio of the magnetic fluorescent microspheres to the first antibody is 1×10 6 The ratio of the number of biotin molecules to the number of second antibody molecules is 50-200:1.

[0014] In a specific embodiment provided by the present invention, the kit includes a diluent of fluorescein-labeled streptavidin; optionally, the diluent contains phosphate.

[0015] In a specific embodiment provided by the present invention, the diluent further comprises a preservative commonly used in the art. In an optional embodiment, the preservative is Proclin 300; preferably, the content of Proclin 300 in the diluent is 0.1%.

[0016] In a specific embodiment provided by the present invention, the kit further comprises a coupling diluent and a labeling diluent.

[0017] In a specific embodiment provided by the present invention, the coupling diluent is Tris buffer. Preferably, the coupling diluent includes a preservative.

[0018] In a specific embodiment provided by the present invention, the labeling diluent is a PBS buffer solution. Preferably, the coupling diluent includes a preservative.

[0019] In another technical solution, the present invention also provides a method for preparing the interleukin-33 detection kit, wherein the method comprises the steps of coupling the first antibody to magnetic fluorescent microspheres and labeling the second antibody with biotin.

[0020] In a specific embodiment provided by the present invention, 2-(N-morpholino)ethanesulfonic acid (ie, MES) buffer is used in the step of coupling the first antibody to magnetic fluorescent microspheres.

[0021] In a specific embodiment provided by the present invention, a blocking solution is further used in the step of coating the magnetic fluorescent microspheres with the first antibody. The blocking solution is a borate mixture of BSA and ethanolamine, the blocking solution includes 0.1% Proclin 300 preservative, and is ultrafiltered using a 0.45 μm membrane.

[0022] In a specific embodiment provided by the present invention, PBS buffer is used in the step of labeling the second antibody with biotin.

[0023] In a specific embodiment provided by the present invention, the second antibody is labeled with biotin for 1-2 hours; optionally, the second antibody is labeled with biotin for 2 hours.

[0024] In another technical solution, the present invention also provides a first antibody coupled to magnetic fluorescent microspheres, a second antibody labeled with biotin, and fluorescein-labeled streptavidin, for use in preparing a reagent or kit for detecting the presence of interleukin-33 in a test sample. In an optional embodiment, the first and second antibodies are monoclonal antibodies against interleukin-33, and the first and second antibodies recognize different interleukin-33-specific binding sites.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The interleukin-33 microsphere flow immunofluorescence luminescence detection kit provided by the present invention utilizes the specific binding of magnetic fluorescent luminescent microspheres formed by coupling interleukin-33 antigen with interleukin-33 capture antibody, which is then specifically bound to an interleukin-33 detection antibody labeled with biotin. The above-mentioned conjugate binds to fluorescein-labeled streptavidin and emits fluorescence to determine the interleukin-33 content in the sample to be tested. The kit has high sensitivity, good repeatability, short reaction time, simple operation, and high anti-interference ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a reaction principle diagram of the detection method of the present invention for detecting interleukin-33 in blood using microsphere flow immunofluorescence luminescence.

[0028] Figure 2 It is a calibration curve diagram of the detection kit involved in the present invention.

[0029] Figure 3 It is a linear correlation diagram of the detection kit involved in the present invention. DETAILED DESCRIPTION

[0030] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below with reference to the accompanying drawings. Numerous specific details are provided in the following detailed description to better illustrate the present invention. Those skilled in the art will appreciate that the present invention may be practiced without certain specific details. In other instances, methods, means, equipment, and steps well known to those skilled in the art are not described in detail in order to highlight the main points of the present invention.

[0031] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0032] Myoglobin detection kit:

[0033] See also Figure 1 The present invention provides a detection kit for interleukin-33, which comprises a first antibody coupled to a magnetic fluorescent microsphere, a second antibody labeled with biotin, and fluorescein-labeled streptavidin, wherein the first antibody and the second antibody are monoclonal antibodies against interleukin-33, and the interleukin-33 specific binding sites recognized by the first antibody and the second antibody are different.

[0034] The present invention's method for detecting interleukin-33 uses a double-antibody sandwich method, primarily utilizing two different specific monoclonal antibodies. The biotin-labeled antibody has a binding site distinct from that of the antibody coupled to the magnetic fluorescent microspheres. These binding sites not only facilitate biotin labeling or coupling to the magnetic fluorescent microspheres, but also prevent the antibody from binding to the antigen to form a sandwich complex, thereby enhancing the specificity and sensitivity of the reaction.

[0035] In one embodiment, in order to improve the signal and sample consistency of the detection sample, preferably, the first antibody of the present invention is antibody 3: IL33-CAP2 (Abcam, Cat: ab244668), and the second antibody is antibody 4: IL33-DET2 (Abcam, Cat: ab244933).

[0036] There are various fluoresceins suitable for use in the present invention, such as fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), Cy5, and the like.

[0037] Among them, preferably, the fluorescent pigment is phycoerythrin, which has the following characteristics: phycoerythrin has excellent light absorption properties and high quantum yield, strong and stable fluorescence, and high sensitivity; it has a wide absorption spectrum in a wide pH range, and it is relatively easy to select a suitable excitation wavelength to obtain efficient fluorescence emission; the fluorescence background is small, not easy to quench, and the fluorescence shelf life is long; it has excellent water solubility, is easy to cross-link with other molecules, and has little non-specific adsorption.

[0038] In an exemplary embodiment, the fluorescein is selected from phycoerythrin-labeled streptavidin (SA-PE).

[0039] In one embodiment, the magnetic fluorescent microspheres suitable for use in the present invention, referred to as magnetic beads or magnetic particles, can be any commonly used magnetic beads in the art. Exemplarily, the magnetic beads used in the present invention are micron-sized solid-phase microspheres with paramagnetic properties and a high protein adsorption capacity, formed by combining nanoscale Fe2O3 and / or Fe3O4 magnetic particles with a polymer material. These magnetic microspheres can be rapidly magnetized under an external magnetic field and have zero remanence after the magnetic field disappears. There is no limitation on the type of polymer material used for the composite.

[0040] In one embodiment, the magnetic fluorescent microspheres used in the present invention have an average particle size ranging from 2 to 8 μm. Furthermore, the magnetic microspheres can be surface-modified to incorporate multiple reactive groups, including but not limited to -CHO and -COOH. Furthermore, given that incomplete solid phase sedimentation before use can affect accuracy, magnetic beads with good dispersibility and slow sedimentation rates should be selected.

[0041] In one embodiment, the core of the magnetic beads is selected as Fe3O4. The magnetic particles can be directly coupled to the interleukin-33 capture antibody, or the magnetic particles can be coupled to streptavidin and the interleukin-33 capture antibody can be labeled with biotin.

[0042] In one embodiment, the present invention provides a microsphere flow immunofluorescence luminescence detection kit for interleukin-33, comprising an interleukin-33 capture antibody coupled to micromagnetic particles, a biotin-labeled interleukin-33 detection antibody, and phycoerythrin-labeled streptavidin (SA-PE).

[0043] In an exemplary embodiment, in the process of preparing the micromagnetic particle-coupled capture antibody of the present invention, the buffer is MES buffer. Optionally, the buffer has a pH of 5.0 and a concentration of 0.05 mol / L.

[0044] In an exemplary embodiment, in the process of preparing the biotin-labeled detection antibody of the present invention, the buffer is 1×PBS buffer. Optionally, the buffer is at pH 7.4.

[0045] In another exemplary embodiment, the calibrator of the present invention further contains a preservative. In an optional embodiment, the preservative is Proclin 300. In an optional embodiment, the preservative contains 0.1% Proclin 300.

[0046] Preparation method of interleukin-33 detection kit:

[0047] In another embodiment, the present invention provides a method for preparing an interleukin-33 detection kit.

[0048] In one embodiment, the method provided by the present invention comprises the steps of coupling the first antibody to magnetic fluorescent microspheres and labeling the second antibody with biotin.

[0049] In another embodiment, the method provided by the present invention comprises the following steps: preparing a suspension of magnetic fluorescent microspheres coupled to interleukin-33 monoclonal antibody; preparing a biotin-labeled interleukin-33 monoclonal antibody solution; and preparing phycoerythrin-labeled streptavidin.

[0050] Application of Interleukin-33 Detection Kit:

[0051] In another embodiment, the present invention provides a use of an interleukin-33 detection kit.

[0052] In one embodiment, the present invention provides a use of a first antibody coupled to magnetic fluorescent microspheres and a second antibody labeled with biotin in preparing a kit for detecting the content of interleukin-33 in a sample to be tested.

[0053] In another embodiment, the present invention provides a detection kit for detecting interleukin-33, which can be used to detect whether a subject has the risk of suffering from vascular diseases, allergic diseases, autoimmune diseases and inflammatory diseases.

[0054] Example

[0055] The technical solutions of the present invention will be further described below in conjunction with specific examples. It should be understood that the following examples are only used to explain and illustrate the present invention, and are not intended to limit the scope of protection of the present invention.

[0056] Unless otherwise stated, all reagents and equipment mentioned in the examples can be purchased from commercial sources.

[0057] Example 1

[0058] This example provides a method for preparing an interleukin-33 detection kit.

[0059] Preparation 1: Magnetic fluorescent microsphere suspension coupled with interleukin-33 monoclonal antibody:

[0060] (1) Take 100 μL (concentration is 1×10 7 To the solution of magnetic fluorescent microspheres (particles / mL), add 1 mL of 0.01 M MES buffer, vortex to mix, and adsorb on a magnet for 5-10 min until the magnetic beads are completely adsorbed to one side of the tube wall. Discard the supernatant, repeat the above washing steps 3-5 times, add 1 mL of 0.01 M MES, and vortex to mix.

[0061] (2) Add 20 μg of the first monoclonal antibody against interleukin-33, vortex and mix thoroughly, and incubate at 37°C for 30 min.

[0062] (3) Add 10 μL of 10 mg / mL 1-(3-dimethylaminopropyl)-3-ethyldiimide hydrochloride (EDC), vortex to mix, place in a 37°C incubator, and incubate for 2 h.

[0063] (4) Add 200 μL of borate solution containing 1% BSA and 0.25% ethanolamine and block for 2 h.

[0064] (5) Add 1 mL of coupling diluent (0.5 g of bovine serum albumin + 6.06 g of Tris + 5‰ of Tween-20 + 1000 mL of purified water) to the blocked magnetic bead suspension, adsorb with a magnet, remove the supernatant, and repeat the above washing steps 3 to 5 times.

[0065] (6) Add 1 mL of coupling diluent to the prepared magnetic beads and store at 2-8°C for a long time.

[0066] Preparation 2: Preparation of biotin-labeled interleukin-33 monoclonal antibody solution:

[0067] (1) Place 24 μg of the second monoclonal antibody against interleukin-33 in a dialysis bag and dialyze for 24 h using no less than 2 L of 1× PBS (pH = 7.4), changing the solution four times during the process.

[0068] (2) The dialyzed IL-33 monoclonal antibody was placed in a 0.5 mL centrifuge tube, and 1.6 μL of a 10 mM biotin-NHS solution dissolved in DMF was added to make the ratio of biotin-NHS to IL-33 monoclonal antibody about 100:1. After mixing, the mixture was reacted at room temperature (protected from light) for 2 h.

[0069] (3) Wash the ultrafiltration tube twice by centrifugation (4000 g, 10 min), and filter to remove unreacted biotin.

[0070] (4) Dilute to 0.5 mL with labeled diluent and store at 2-8°C for a long time.

[0071] Preparation 3: Phycoerythrin-labeled streptavidin: Dilute SA-PE to 2000 ng / mL with SA-PE diluent (1X PBS, pH 7.2) and set aside.

[0072] Example 2

[0073] This example provides a method for using the interleukin-33 detection kit.

[0074] The kit prepared in Example 1 was used to detect the concentration of interleukin-33 in the sample by flow cytometry. Specifically, the flow cytometer was used according to the following steps:

[0075] (1) The magnetic beads-IL-33 antibody prepared in Preparation 1 of Example 1 was diluted with coupling diluent at 1:25 and 1:50 for use.

[0076] The biotin-labeled antibody prepared in Preparation 2 of Example 1 was diluted with labeling diluent at 1:100 and 1:200 for use.

[0077] (2) Add 50 μL of sample and 10 μL of Reagent 1 (diluted Preparation 1 product) to the reaction cup and react for 30 minutes.

[0078] (3) Add 50 μl of Reagent II (diluted Preparation 2 product) to the reaction cup and react for 30 minutes.

[0079] (4) Add 50 μl of reagent III (to prepare product 3) to the reaction cup and react for 15 minutes.

[0080] (5) Detecting the photon value. Based on the light intensity detected in the reaction cup, the instrument automatically calculates the concentration of interleukin-33 in the sample to be tested.

[0081] Example 3

[0082] This example provides a panel screening of antibodies used in the interleukin-33 detection kit.

[0083] (1) Antibody pairing screening

[0084] There are four interleukin-33 antibodies used in the present invention: Antibody 1: IL33-CAP1 (Ibotek, Cat: A22621), Antibody 2: IL33-DET1 (Ibotek, Cat: A8096); Antibody 3: IL33-CAP2 (Abcam, Cat: ab244668), Antibody 4: IL33-DET2 (Abcam, Cat: ab244933).

[0085] Antibody 1 and antibody 3 were coated with magnetic microspheres respectively to form antibody 1-magnetic microspheres and antibody 3-magnetic microspheres. Meanwhile, the other two antibodies were labeled with biotin to form antibody 2-biotin and antibody 4-biotin.

[0086] The above antibodies were combined and paired, serum samples were tested, and the optimal combination was screened, see Table 1.

[0087] Table 1. Antibody pair combination screening results

[0088]

[0089] As shown in Table 1, the overall signal values ​​of myoglobin detected by the pairings in combination 1, combination 2, and combination 3 were relatively low; the overall signal value of the pairing in combination 4 was relatively good.

[0090] Example 4

[0091] This example provides a technical effect test of the interleukin detection kit

[0092] (1) Interleukin-33 antigen was used to prepare the standard product, and the test was performed twice at the indicated concentration. The average value was taken to prepare the calibration curve. The results are shown in the table. Figure 2With Table 2.

[0093] Table 2. Results of the initial calibration curve

[0094] Labeled concentration Signal value 1 Signal value 2 Signal value mean 0 1531 1653 1592 50 54513 53548 54031 100 184029 174604 179317 200 317073 301676 309375 400 529652 510109 519881 800 643665 623716 633691

[0095] (2) Blank limit test: The test kit is tested using a blank (zero-value enterprise calibrator) as a sample. The test is repeated 20 times. The mean X1 of the blank response and the standard deviation (SD) of the blank response are calculated. X1 (mean of the blank response) + 2SD (standard deviation of the blank response) is the blank limit.

[0096] Use the company's calibrator to dilute 5 low-value samples with a concentration close to the detection limit (10pg / mL) for testing. Each sample is tested 5 times. The test results are sorted by size. The results should meet the following requirements: the number of test results less than the blank limit should be less than or equal to three.

[0097] The results are shown in Tables 3 and 4.

[0098] Table 3. Detection blank limits

[0099] 1971 2547 1734 2070 2191 1883 2068 1875 1965 1883 1604 2426 2131 1802 1740 2390 1984 2160 1860 1848 AVE 2006.60 SD 245.28 AVE+2SD 2497.16

[0100] Table 4. Detection blank limits

[0101]

[0102] The number of test results less than the blank limit is 0, and there is a significant difference between the blank limit and the detection limit test results, indicating that the sensitivity of the present invention is good.

[0103] (3) Precision test: Use the same batch of kit to test two samples with concentrations in the high value quality control range (400-600) and the low value quality control range (10-30). Repeat the test 10 times for each sample and calculate the CV value. The result should be ≤10%. See Table 5 for the results.

[0104] Calculation formula:

[0105] Description: Standard deviation is the average value of the test data.

[0106] Table 5. Precision

[0107]

[0108] The CVs of the high and low quality control measurement results were both less than 10%, indicating that the present invention has good precision.

[0109] (4) Accuracy Test: A known high-level analyte A was added to a low-concentration serum B at a volume ratio of 1:9. The test was repeated three times, and the average value was calculated. The recovery rate was calculated according to the formula and should be between 85% and 115%. The results are shown in Table 6.

[0110] Calculation formula:

[0111] Where:

[0112] R——recovery rate;

[0113] C is the average value of the concentration detected after adding solution A to solution B;

[0114] V0——volume of liquid B;

[0115] Vs——volume of liquid A;

[0116] C0——average value of concentration of liquid B;

[0117] Cs——the average value of concentration of liquid A.

[0118] Table 6. Accuracy

[0119] A concentration B concentration Mixed concentration Recovery rate 243.20 5.10 29.50 102％

[0120] The recovery rate is within 85-115%, indicating that the accuracy of the present invention is good.

[0121] (5) Linear range test: Dilute a high-concentration sample close to the upper limit of the linear range (800 pg / mL) by 2 times, then 3 times, and then 5 times the gradient dilution to 5 concentrations. The low-concentration sample should be close to the lower limit of the linear range. Repeat the measurement twice for each concentration of the sample, calculate the average value, and use the least squares method to perform a straight line fit between the average value of the measured concentration and the theoretical concentration or dilution ratio to obtain a linear regression equation and calculate the linear correlation coefficient r, which should be no less than 0.975. The results are shown in Figure 3 With Table 7.

[0122] Correlation coefficient calculation formula:

[0123] Note: In the formula, -1≤γ≤1,

[0124] Table 7. Linear range

[0125]

[0126]

[0127] As shown in Table 7, the linear correlation coefficient is greater than 0.975, indicating that the linear range of the present invention is good.

[0128] The present invention relates to the field of immunodiagnosis. Aiming at the problem that interleukin-33 requires high sensitivity and requires joint detection of multiple items, a microsphere flow immunofluorescence luminescence detection technology for joint diagnosis of multiple antigens is provided, and a related kit is designed.

[0129] Clinical application and evaluation of interleukin-33 microsphere flow cytometry fluorescence luminescence detection method: Collect qualified clinical specimens, compare the established new method with the existing gold standard method, statistically analyze the results, and combine clinical indications, imaging, etc. to demonstrate that this method can sensitively detect antigens.

[0130] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form or substance. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention. Any equivalent changes, modifications and evolutions made by technicians familiar with this profession without departing from the spirit and scope of the present invention by using the technical content disclosed above are all equivalent embodiments of the present invention; at the same time, any equivalent changes, modifications and evolutions made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A microsphere flow immunofluorescence luminescence detection kit for interleukin-33, comprising a first antibody coupled to magnetic fluorescent microspheres, a second antibody labeled with biotin, and fluorescein-labeled streptavidin, wherein the first and second antibodies are monoclonal antibodies against interleukin-33 and the first and second antibodies recognize different interleukin-33 specific binding sites; characterized in that: The first antibody and the second antibody are IL33-CAP2 and IL33-DET2, respectively; the magnetic fluorescent microspheres are selected from a complex of nano-sized Fe2O3 and / or Fe3O4 magnetic particles and polymer materials; the ratio of the magnetic fluorescent microspheres to the first antibody is 1×106:10-50 μg, and the ratio of the number of biotin molecules to the second antibody is 50-200:

1.

2. The interleukin-33 microsphere flow immunofluorescence luminescence detection kit according to claim 1, characterized in that: The average particle size of the magnetic fluorescent microspheres is 2-8 μm, and a plurality of active groups can be added to the surface of the magnetic fluorescent microspheres.

3. The interleukin-33 microsphere flow immunofluorescence luminescence detection kit according to claim 1, characterized in that: The kit includes a diluent of fluorescein-labeled streptavidin and / or a coupling diluent and / or a labeling diluent.

4. The interleukin-33 microsphere flow immunofluorescence luminescence detection kit according to any one of claims 1 to 3, characterized in that: The fluorescein includes one or more of fluorescein isothiocyanate, phycoerythrin, allophycocyanin, and Cy5.

5. The method for preparing the detection kit according to any one of claims 1 to 3, wherein: The method comprises: 1) The first antibody is coated with magnetic fluorescent microspheres, and the second antibody is labeled with biotin; 2) The fluorescein-labeled streptavidin is phycoerythrin-labeled streptavidin.

6. The method for preparing the detection kit according to claim 5, characterized in that: In step 1), the buffer used in biotin labeling of the second antibody is PBS buffer; and the buffer used in magnetic fluorescent microsphere coating of the first antibody is 2-(N-morpholino)ethanesulfonic acid.

7. The method for preparing the detection kit according to claim 5, characterized in that: Step 1) The first antibody is coated on magnetic fluorescent microspheres, and a blocking solution is used. The blocking solution is a borate mixture of BSA and ethanolamine, containing 0.1% Proclin 300 preservative, and is ultrafiltered using a 0.45 μm membrane.

8. The method for preparing the detection kit according to claim 5, wherein: The labeling time of the second antibody-labeled tracer marker is 1-2 hours.

9. The method for preparing the detection kit according to claim 5, wherein: The labeling time of the second antibody-labeled tracer marker is 2 hours.

10. Use of the detection kit according to any one of claims 1 to 4 or the detection kit prepared by the method according to any one of claims 6 to 9, characterized in that: The detection kit is used in the detection of interleukin-33, wherein the first antibody and the second antibody are monoclonal antibodies against interleukin-33, and the interleukin-33 specific binding sites recognized by the first antibody and the second antibody are different.

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