Kit for chemiluminiscence detection of interleukin 13 and application of kit

Abstract

The invention discloses a kit for chemiluminiscence detection of interleukin 13 and application of the kit. The kit comprises a reagent R1, a reagent R2 and a reagent R3, the reagent R1 comprises a magnetic bead, and the surface of the magnetic bead is coated with streptavidin; the reagent R2 and the reagent R3 respectively comprise an interleukin 13 monoclonal antibody 1 and an interleukin 13 monoclonal antibody 2, and the interleukin 13 monoclonal antibody 1 and the interleukin 13 monoclonal antibody 2 are respectively coupled with biotin and alkaline phosphatase. The kit has the advantages of being high in sensitivity, wide in linear range, simple in sample treatment and high in automation degree when being applied to detection of the interleukin 13, and has the application prospect of being applied to clinical determination of the concentration of the interleukin 13 in a serum or plasma sample.

Description

Technical Field

[0001] This invention belongs to the field of detection technology, specifically relating to a kit for the chemiluminescence detection of interleukin-13 and its application. Background Technology

[0002] Interleukin-13 (IL-13) is an important member of the interleukin family. It is a protein encoded by the IL-13 gene, with a molecular weight of approximately 10 kDa. IL-13 can be secreted by various cells, such as CD4+ T cells, CD8+ T cells, mast cells, basophils, eosinophils, and natural killer cells, especially type 2 helper T cells (Th2). IL-13 has various biological functions on monocytes / macrophages, B lymphocytes, NK cells, and vascular endothelial cells, and is a mediator of the body's immune response.

[0003] IL-13 can inhibit and regulate the activity of immune cells, playing a particularly important role in allergic reactions and autoimmune diseases. Excessive IL-13 production may lead to the development of allergic diseases such as asthma, rhinitis, and dermatitis. In asthma, IL-13 can stimulate airway epithelial cells to produce various inflammatory mediators, promoting the accumulation of inflammatory cells and leading to airway hyperresponsiveness and allergic symptoms. IL-13 participates in regulating autoimmune responses; excessive IL-13 production may lead to the development of autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus.

[0004] Currently, clinical detection of interleukin-13 can be achieved through enzyme-linked immunosorbent assay (ELISA) and multifactor microsphere detection technology. ELISA involves binding the analyte to an antibody on a solid-phase carrier, adding an enzyme-labeled antibody, and then detecting the antibody concentration by substrate-catalyzed enzyme activity. Multifactor microsphere detection involves covalently coupling the analyte to an antibody on the surface of fluorescently encoded microspheres, adding a fluorescent probe to label the antibody, and then detecting the labeled antibody by excitation light to determine the antibody concentration.

[0005] The disadvantages of enzyme-linked immunosorbent assay (ELISA) are that it is cumbersome to operate, time-consuming, and dependent on manual operation. It also has low sensitivity, narrow linear range, and poor accuracy. Multifactor microsphere detection technology relies on expensive equipment, has complex sample pretreatment, and there are large differences in reagent kits from different manufacturers, lacking a unified technical standard. Summary of the Invention

[0006] Because existing technologies lack chemiluminescence detection kits for interleukin-13 that are highly sensitive, have a wide linear range, and are easy to use, this invention provides a chemiluminescence detection kit that has the advantages of high sensitivity, a wide linear range, simple sample processing, and a high degree of automation. It is valuable for clinical application in determining the concentration of interleukin-13 (IL-13) in serum or plasma samples.

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0008] The first aspect of the present invention provides a kit for the chemiluminescence detection of interleukin-13, the kit comprising reagent R1, reagent R2 and reagent R3;

[0009] Reagent R1 contains magnetic beads coated with streptavidin.

[0010] Reagents R2 and R3 contain interleukin-13 monoclonal antibodies 1 and 2, respectively, which are conjugated with biotin and alkaline phosphatase, respectively.

[0011] In some embodiments of the present invention, the interleukin-13 monoclonal antibody 1 is Abcam's Anti-IL-13 antibody [EPR20465-131] (catalog number ab285053); the interleukin-13 monoclonal antibody 2 is Abcam's Anti-IL-13 antibody [EPR20465-55] (catalog number ab285159).

[0012] In some embodiments of the present invention, reagent R1, reagent R2 and / or reagent R3 further comprise:

[0013] (1) Stabilizer; and / or, (2) Buffer; and / or, (3) Surfactant; and / or, (4) Protectant; In some optional embodiments of the present invention, reagent R1, reagent R2 and / or reagent R3 further include: (5) Preservative.

[0014] The percentages mentioned above are mass-volume percentages in the reagent, in g / mL.

[0015] In some preferred embodiments of the present invention, reagent R1, reagent R2, and / or reagent R3 further include:

[0016] (1) A stabilizer, wherein the concentration of the stabilizer is 1-10%, and the stabilizer is a chloride and / or a disaccharide; and / or,

[0017] (2) A buffer having a concentration of 0.01 M-1 M, wherein the buffer is one or more selected from PBS, triethanolamine hydrochloride, MES, Tris, MOPS, and HEPES; and / or,

[0018] (3) A surfactant, wherein the concentration of the surfactant is 0.01%-1%, and the surfactant is a nonionic surfactant; and / or,

[0019] (4) a protective agent, wherein the protective agent is BSA or ethanolamine; and / or, (5) a preservative, wherein the concentration of the preservative is 0.01%-0.5%, and the preservative is one or more of ProClin300, ProClin950, gentamicin sulfate, sodium azide, 2-hydroxypyridine-N-oxide, chloroacetamide, imidazolidinyl urea, thimerosal, and 2-methyl-3(2H)-isothiazolidinone 5-bromo-5-nitro-1,3-dioxane;

[0020] The percentages mentioned above are mass-volume percentages in the reagent, in g / mL.

[0021] In some preferred embodiments of the present invention, the stabilizer is one or more selected from sodium chloride, potassium chloride and magnesium chloride; the disaccharide is one or more selected from sucrose, maltose and trehalose; and / or, the buffer is one or more selected from PBS, triethanolamine hydrochloride, MES, Tris, MOPS and HEPES; and / or, the surfactant is Triton X-100; and / or, the protectant is BSA.

[0022] In some further preferred embodiments of the present invention, the stabilizer is sodium chloride; the disaccharide is trehalose; and / or the buffer is PBS or triethanolamine hydrochloride; and / or the preservative is ProClin 300 or ProClin 950.

[0023] In some embodiments of the present invention, the concentration of the magnetic beads is 0.25-1 mg / mL; and / or the concentration of interleukin-13 monoclonal antibody 1 is 0.1-1 μg / mL; and / or the concentration of interleukin-13 monoclonal antibody 2 is 0.1-1 μg / mL.

[0024] In some specific embodiments of the present invention, the sodium chloride is 0.5-5%, the trehalose is 0.5-5%, the PBS is 0.01 M-0.1 M, the triethanolamine hydrochloride is 0.05 M-0.2 M, the Triton X-100 is 0.2%-0.1%, the BSA is 0.2%-0.1%, the ProClin300 is 0.05-0.2%, and / or the ProClin950 is 0.05-0.2%.

[0025] In some preferred embodiments of the present invention, the concentration of the magnetic beads is 0.25-0.8 mg / mL; and / or, the particle size of the magnetic beads is 3 μm; and / or, the concentration of interleukin-13 monoclonal antibody 1 is 0.1-0.6 μg / mL; and / or, the concentration of interleukin-13 monoclonal antibody 2 is 0.1-0.6 μg / mL.

[0026] In some embodiments of the present invention, reagent R1 comprises: 0.25-0.8 mg / mL magnetic beads coated with streptavidin, 0.5-2% sodium chloride, 0.01 M-0.5 M PBS, and 0.2%-0.08% Triton X; and / or,

[0027] The reagent R2 comprises: 0.1-0.6 μg / mL interleukin-13 antibody 1, 0.5-2% sodium chloride, 0.01 M-0.5 M PBS, and 0.2%-0.08% Triton X; and / or,

[0028] The reagent R3 comprises: 0.1-0.6 μg / mL interleukin-13 antibody 2, 0.5-2% sodium chloride, 0.5-2% trehalose, 0.01 M-0.5 M PBS, and 0.2%-0.08% Triton X;

[0029] In some preferred embodiments of the present invention, reagent R1, reagent R2 and / or reagent R3 respectively further include 0.2%-0.5% BSA and / or 0.5-2% trehalose.

[0030] In some optional embodiments of the present invention, reagent R1 and reagent R2 further comprise 0.1-0.2 μg / mL ProClin300; and / or, reagent R3 further comprises 0.1-0.2 μg / mL ProClin950.

[0031] In some embodiments of the present invention, the kit further includes reagent R4, which contains a chemiluminescent substrate.

[0032] In some preferred embodiments of the present invention, the chemiluminescent substrate is AMPPD, CDP-Star, or APS-5.

[0033] A second aspect of the present invention provides a method for preparing a reagent kit as described in the first aspect, the method comprising the steps of preparing the reagent R1, the reagent R3 and the reagent R3.

[0034] A third aspect of the present invention provides a method for detecting interleukin-13, the method comprising the step of detecting a sample using a kit as described in the first aspect.

[0035] In some embodiments of the present invention, the test sample is a serum sample or a plasma sample.

[0036] In some embodiments of the present invention, the method includes:

[0037] S1: Add reagent R2 and reagent R3 to the sample to be tested and incubate;

[0038] S2: Add reagent R1 and incubate;

[0039] S3: Add reagent R4 to develop color;

[0040] In some preferred embodiments of the present invention, the volume ratio of the sample to be tested, the reagent R2 and the reagent R3 is (2-5):(8-12):(8-12).

[0041] In some preferred embodiments of the present invention, the volume ratio of the test sample, the reagent R2 and the reagent R3 is (3-5):(10-12):(10-12).

[0042] In some preferred embodiments of the present invention, the volume ratio of the test sample to the reagent R1 is (2-5):(8-12).

[0043] In some preferred embodiments of the present invention, the volume ratio of the test sample to the reagent R1 is (3-5):(10-12).

[0044] In some preferred embodiments of the present invention, the incubation time in S2 and S3 is 5-30 min.

[0045] In some preferred embodiments of the present invention, the incubation time is 5-15 min.

[0046] In some preferred embodiments of the present invention, the incubation temperature is 30-37°C.

[0047] In some preferred embodiments of the present invention, the incubation temperature is 35-37°C.

[0048] In some preferred embodiments of the present invention, the incubation step S2 further includes a washing step.

[0049] In some preferred embodiments of the present invention, the number of washing cycles is 3-5.

[0050] A fourth aspect of the present invention provides the application of the kit as described in the first aspect in the detection of interleukin-13.

[0051] The technical solution of this invention:

[0052] A chemiluminescent detection kit for interleukin-13 and its application, the kit comprising the following components:

[0053] Magnetic bead reagent: Contains magnetic beads, the surface of which is coated with streptavidin;

[0054] Biotin-antibody solution: Contains an antibody that specifically binds to interleukin-13, said antibody being biotin-labeled;

[0055] Alkaline phosphatase-labeled solution: containing an antibody that specifically binds to interleukin-13, said antibody being labeled with alkaline phosphatase;

[0056] Calibration solution: a solution containing interleukin-13.

[0057] The preparation method of the above-mentioned chemiluminescent detection kit for interleukin-13 includes the following steps:

[0058] 1) Preparation of magnetic bead reagent: After magnetic separation of the magnetic bead solution coated with streptavidin, remove the supernatant, wash 3 times with PBS buffer containing 0.5% BSA, remove the supernatant, add PBS buffer containing 0.5% BSA and 0.1% Proclin300 to adjust the volume, and finally store the magnetic bead reagent at 2-8℃.

[0059] 2) Preparation of Biotin-Antibody Solution: Take 50 μg of interleukin-13 monoclonal antibody and add 2 μL of 1 mg / mL biotin aqueous solution. Mix and react at room temperature in the dark for 30 min. Place the reaction mixture in a dialysis bag containing 20 mM PBS buffer (pH 7.4) and dialyze at 2-8℃ in the dark. Change the PBS buffer every 2 hours, for at least 5 times. Finally, dialyze overnight at 2-8℃ in the dark to remove unreacted biotin. After removing the biotin-antibody solution, add 10% BSA to a final concentration of 0.5% according to the solution volume. Add an equal volume of glycerol and mix well. Dilute the biotin-antibody solution with PBS buffer containing 0.5% BSA and 0.1% Proclin 300.

[0060] 3) Preparation of alkaline phosphatase-labeled solution: Take 50 μg of interleukin-13 monoclonal antibody and add 1 μL of 1 mg / mL SMCC (PEG)8 aqueous solution, and let stand at room temperature in the dark for 30 min; simultaneously, take 50 μg of alkaline phosphatase and add 1 μL of 1 mg / mL 2-IT aqueous solution, and let stand at room temperature in the dark for 30 min; add 1 μL of 1 mol / L glycine aqueous solution to the antibody and alkaline phosphatase solutions respectively, and let stand at room temperature in the dark for 10 min; mix the above solutions and let stand at room temperature in the dark for 60 min; add 1 μL of 1 mg / mL NEM DMF solution to the mixed solution and let stand at room temperature in the dark for 10 min; add 1 μL of 1 mol / L ethanolamine aqueous solution to the above solution and let stand at room temperature in the dark for 10 min; place the mixed solution in a desalting column and add 20 mM PBS buffer (pH 7.4) for desalting and purification. After removing the alkaline phosphatase-antibody solution, add 10% BSA to a final concentration of 0.5% according to the solution volume, then add an equal volume of glycerol and mix well. Dilute the biotin-antibody solution with PBS buffer containing 0.5% BSA and 0.05% Proclin 950.

[0061] 4) Preparation of interleukin-13 antigen calibrator solutions: Dilute interleukin-13 antigen to concentrations of 0 pg / mL, 5 pg / mL, 10 pg / mL, 50 pg / mL, 250 pg / mL, 1000 pg / mL, and 2500 pg / mL with triethanolamine hydrochloride buffer containing 0.5% BSA, 0.1% Proclin 300, and pH 7.4.

[0062] The inventive point of this invention is:

[0063] A novel enzymatic chemiluminescence assay kit for detecting interleukin-13 was developed, employing magnetic microparticle chemiluminescence technology as the detection method, along with alkaline phosphatase labeling and a streptavidin-biotin system. Alkaline phosphatase offers advantages such as stable luminescence, low background interference, high sensitivity, and a wide linear range; the streptavidin-biotin system can enrich and amplify the signal, further enhancing the detection sensitivity and linear range.

[0064] This invention has low requirements for sample pretreatment. Serum / plasma samples only need to be centrifuged after acquisition before testing, which facilitates high-throughput and fully automated detection.

[0065] In summary, it features high sensitivity, good specificity, wide linear range, simple sample processing, high degree of automation, and convenient detection, and can be applied to clinical determination of interleukin-13 (IL-13) concentration in serum / plasma samples.

[0066] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0067] The reagents and raw materials used in this invention are all commercially available.

[0068] The significant advantages of this invention are as follows: The kit provided utilizes a chemiluminescent immunoassay platform to detect interleukin-13, offering advantages such as high sensitivity, good specificity, wide linear range, simple sample processing, high automation, and convenient detection. It avoids the cumbersome operation and long reaction time of enzyme-linked immunosorbent assay (ELISA) used in clinical practice, and also avoids the disadvantages of multifactor microsphere detection technology, such as complex sample processing, expensive equipment, and the requirement for multiple indicator simultaneous detection. It can be used to assist in the early diagnosis, efficacy evaluation, and prognostic monitoring of various inflammatory diseases, including allergic reactions and autoimmune diseases, and is suitable for widespread application. Attached Figure Description

[0069] Figure 1 The correlation results are for the interleukin-13 detection kit.

[0070] Figure 2 The results are for the linearity validation of the interleukin-13 detection kit. Detailed Implementation

[0071] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0072] Example 1 Preparation of buffer solution

[0073] For the screening and optimization of buffer 1 and buffer 2, please refer to Example 10. The determined buffer formulations are shown below:

[0074] Buffer 1: Prepared from 0.02 M PBS, 0.9% sodium chloride, 0.5% BSA, 1% trehalose, 0.5% Triton X-100, and 0.1% ProClin300. Used for further preparation of magnetic bead reagents and biotin-antibody solutions.

[0075] Buffer 2: Prepared from 0.02 M PBS, 0.9% sodium chloride, 0.5% BSA, 1% trehalose, 0.5% Triton X-100, and 0.1% ProClin950. Used for further preparation of alkaline phosphatase-labeled solutions.

[0076] Buffer 3: Prepared from 0.1 M triethanolamine hydrochloride, 0.9% sodium chloride, 0.5% BSA, 1% trehalose, 0.5% Triton X-100, and 0.1% ProClin300. Used for further preparation of interleukin-13 antigen calibrator dilution.

[0077] Example 2: Preparation of a chemiluminescent detection kit for interleukin-13

[0078] The interleukin-13 chemiluminescence detection kit provided in this embodiment includes magnetic bead reagent, biotin-antibody solution, alkaline phosphatase-labeled solution, and interleukin-13 antigen calibrator solution. The biotin-antibody solution and alkaline phosphatase-labeled solution contain antibodies for detection; the screening of antibody pairs is described in Example 9.

[0079] The interleukin-13 chemiluminescence detection kit provided in this embodiment can be prepared through the following steps:

[0080] 1) Preparation of magnetic bead reagent: After magnetic separation of the magnetic bead solution (particle size 3 μm, purchased from Beijing Bomei Biotechnology Co., Ltd., catalog number MS300 / Streptavidin) coated with streptavidin, remove the supernatant, wash 3 times with PBS buffer containing 0.5% BSA, remove the supernatant, add buffer 1 prepared in Example 1 to make up the volume, and finally store the magnetic bead reagent at 2-8℃.

[0081] 2) Preparation of Biotin-Antibody Solution: Take 50 μg of interleukin-13 monoclonal antibody 1 (Anti-IL-13 antibody [EPR20465-131] - BSA and Azide free (Capture), purchased from Abcam, catalog number TM285-D), add 2 μL of 1 mg / mL biotin (purchased from Thermo Fisher, catalog number 20217) aqueous solution, mix and react at room temperature in the dark for 30 min. Place the reaction mixture in a dialysis bag containing 20 mM PBS buffer at pH 7.4, and dialyze at 2-8°C in the dark. Change the PBS buffer every 2 hours, for at least 5 times. For the last dialyze, dialyze overnight at 2-8°C in the dark to remove unreacted biotin. After removing the biotin-antibody solution, add 10% BSA to a final concentration of 0.5% according to the solution volume, add an equal volume of glycerol and mix well. Dilute the biotin-antibody solution using buffer 1 prepared in Example 1.

[0082] 3) Preparation of alkaline phosphatase-labeled solution: Take 50 μg of interleukin-13 monoclonal antibody 2 (Anti-IL-13 antibody [EPR20465-55] - BSA and Azide free (Detector), purchased from Abcam, catalog number ab285159), add 1 μL of 1 mg / mL SMCC (PEG) 8 aqueous solution, and let stand at room temperature in the dark for 30 min; simultaneously, take 50 μg of alkaline phosphatase (purchased from Thermo Fisher, catalog number 31392), add 1 μL of 1 mg / mL 2-IT (purchased from Thermo Fisher, catalog number 26101) aqueous solution, and let stand at room temperature in the dark for 30 min; add 1 μL of 1 mol / L glycine aqueous solution to the antibody and alkaline phosphatase solutions respectively, and let stand at room temperature in the dark for 10 min; mix the above solutions well, and let stand at room temperature in the dark for 60 min; add 1 μL of 1 mol / L glycine aqueous solution to the mixed solution after the reaction. A 1 mg / mL solution of NEM in DMF was added and allowed to stand at room temperature in the dark for 10 min. 1 μL of a 1 mol / L aqueous ethanolamine solution was added and allowed to stand at room temperature in the dark for 10 min. The resulting mixture was then placed in a desalting column and desalted using 20 mM PBS buffer (pH 7.4). 10% BSA was added to the desalted and purified solution to a final concentration of 0.5%, followed by the addition of an equal volume of glycerol and mixing. The biotin-antibody solution was diluted using buffer 2 prepared in Example 1.

[0083] 4) Preparation of interleukin-13 antigen calibrator dilution solution: Dilute interleukin-13 antigen with buffer 3 prepared in Example 1 to concentrations of 0 pg / mL, 5 pg / mL, 10 pg / mL, 50 pg / mL, 250 pg / mL, 1000 pg / mL, and 2500 pg / mL interleukin-13 antigen calibrator dilution solution.

[0084] Example 3: Detection steps of interleukin-13 in actual samples

[0085] The screening of detection methods and steps is described in Example 11. The final detection method determined is the one-step delayed method, and the specific steps are as follows:

[0086] 1) Add calibrator (for calibration) and sample to the reaction tube in sequence, with a sample volume of 30 μL / tube for each.

[0087] 2) Add 100 μL of biotin-antibody solution and 100 μL of alkaline phosphatase-labeled solution to each tube; wherein the antibody concentration in the biotin-antibody solution is 0.6 μg / mL and the antibody concentration in the alkaline phosphatase-labeled solution is 0.6 μg / mL.

[0088] 3) After mixing, incubate at 37°C for 15 minutes.

[0089] 4) Add 100 μL of magnetic bead reagent to each tube, wherein the concentration of magnetic microparticles in the magnetic bead reagent is 0.8 mg / mL.

[0090] 5) After mixing, incubate at 37°C for 15 minutes.

[0091] 6) Wash 5 times with cleaning solution (0.1M Tris, 0.9% NaCl, 0.5% Tween and 0.5% Proclin 300).

[0092] 7) Add 200 μL of luminescent substrate solution (AMPPD; purchased from Guangdong Feipeng Biotechnology Co., Ltd.) to each well.

[0093] 8) Detect the luminescence value 1-5 minutes after mixing.

[0094] 9) Calculation of results.

[0095] Choose an appropriate curve fitting method for result calculation. This kit recommends using a four-parameter fitting method, establishing a calibration curve with the calibrator concentration value on the x-axis and the log value of the calibrator luminescence value on the y-axis, and calculating the corresponding concentration value based on the luminescence value of the sample to be tested. The fully automated TK2000 chemiluminescence analyzer's automatic operating system can automatically calculate the sample test results based on the stored calibration curve and the luminescence values ​​obtained from sample testing.

[0096] Example 4: Clinical performance evaluation of the interleukin-13 detection kit

[0097] Two hundred clinical samples, including 100 negative samples, were simultaneously tested using the kit of this invention and the Human IL-13 Quantikine ELISA Kit (enzyme-linked immunosorbent assay kit) manufactured by R&D Systems. All samples were obtained from Tongji Hospital, Shanghai, and the concentration range was 3 pg / mL to 2000 pg / mL. Concentrations exceeding the reference range (5 pg / mL) were considered positive. Clinical correlation analysis and concordance rate analysis were performed on the test results. The concordance rate results are shown in Table 1, and the correlation results are shown in... Figure 1 As shown.

[0098] Table 1. Results of Compliance Rate Analysis

[0099]

[0100] Example 5: Sensitivity evaluation of the kit of the present invention

[0101] Interleukin-13 antigen calibrator dilution buffer (interleukin-13 antigen concentration of 0 pg / mL, i.e., the zero-concentration calibrator described below) was used as the sample for testing. The assay was repeated 20 times, and the RLU values ​​of the 20 assays were obtained. The mean M and standard deviation SD of these 20 measurements were calculated, and the RLU value corresponding to (M+2SD) was obtained. Three measurements were taken with adjacent concentration calibrators (interleukin-13 antigen concentration of 5.37 pg / mL), and their RLU values ​​were recorded. The average value was calculated. A two-point regression was performed based on the concentration-RLU values ​​between the zero-concentration calibrator and the adjacent concentration calibrators to obtain a linear equation. The RLU value corresponding to (M+2SD) was substituted into the above equation to calculate the corresponding concentration, which is the blank limit. The experimental results show that the blank limit of this kit is 0.09 pg / mL, as shown in Table 2.

[0102] Table 2 RLU values ​​for sensitivity testing

[0103]

[0104] Example 6: Evaluation of the linear range of the kit of the present invention

[0105] One high-value sample H (>2500 pg / mL) and one low-value sample L (<2.5 pg / mL) were taken and mixed to form six concentration samples (5H, 4H+1L, 3H+2L, 2H+3L, 1H+5L, and 5L) according to the following proportions. Each concentration sample was tested three times, and the mean was calculated. The test values ​​are shown in Table 3. The theoretical values ​​and the measured mean were used to perform a linear regression analysis of the equation y = kx + b using EXCEL software. The slope k and correlation R of the factor equation were statistically analyzed. 2 Given that k = 1 ± 0.1, R 2 >0.99. The result is as follows: Figure 2 As shown, the slope k = 1.0098, and the correlation coefficient R 2= The value of 0.9995 indicates that the linear range of the kit of the present invention is 2.5-2500 pg / mL, and the linearity is good within the linear range.

[0106] Table 3 Test Values

[0107]

[0108] Note: The units for the test values ​​in Table 3 are all pg / mL.

[0109] Example 7 Precision evaluation of the kit of the present invention

[0110] This kit was used to measure samples at two concentrations (20 pg / mL and 1000 pg / mL) 10 times each. The mean (M) and standard deviation (SD) of the 10 measurements were calculated, and the coefficient of variation (CV) was calculated using the formula. The experimental results are shown in Table 4. The coefficients of variation were all less than 3%, indicating that this kit has good precision.

[0111] Table 4 Precision Test

[0112]

[0113] Example 8: Accuracy evaluation of the reagent kit of the present invention

[0114] This kit was used to test two concentrations (international standards prepared at 20 pg / mL and 1000 pg / mL). Each sample was tested three times, and the relative deviation was calculated. The experimental results are shown in Table 5 below. The deviations were all less than 5%, which shows that the kit has good accuracy.

[0115] Table 5 Accuracy Test

[0116]

[0117] Example 9 Screening of antibody pairs in the kit

[0118] Different detection antibody pairs were used to detect gradient standards S1-S6, and the detection results are shown in Table 6.

[0119] The experimental group using antibody pair 1 is the interleukin-13 chemiluminescence detection kit provided in Example 2 of this invention. In the experimental groups using antibody pairs 2 and 3, except for the antibody pairs which are different from those in Example 2, the other components and preparation methods are the same as those in Example 1. The steps for detecting interleukin-13 are as described in Example 3.

[0120] Table 6. Detection results (RLU values) for different antibody pairs

[0121]

[0122] As shown in Table 6, when antibody pair 1 was tested with gradient samples, the background (i.e., 0 concentration calibrator S0) was low and the signal values ​​of the gradient calibrators (i.e., S1-S6) were high, indicating that antibody pair 1 had high overall reactivity and low background, and good signal-to-noise ratio. Therefore, antibody pair 1 was selected for the detection of interleukin-13 in this invention.

[0123] Example 10 Screening of reagent kit components

[0124] 1. Buffer 1

[0125] 1.1 Screening of different buffer solutions

[0126] PBS, MES, and HEPES buffers were selected for reactivity testing, and the results are shown in Table 7.

[0127] The experimental group using PBS buffer is the interleukin-13 chemiluminescence detection kit provided in Example 2 of this invention. The experimental group using MES buffer and HEPES buffer is the same as that in Example 2 except for the antibody pair. The steps for detecting interleukin-13 are as described in Example 3.

[0128] Table 7. Detection results (RLU values) for different buffer solutions

[0129]

[0130] As shown in Table 7, when the experimental group using PBS buffer tested gradient samples, the background (i.e., 0 concentration calibrator S0) was low, and the signal values ​​of the gradient calibrators (i.e., S1-S6) were high, indicating that the experimental group containing PBS buffer had high overall reactivity and low background, and good signal-to-noise ratio.

[0131] 1.2 Formulation Screening

[0132] The stability of the reagent was tested using the following formulation, and the RLU values ​​were recorded.

[0133] Formula 1: 0.02 M PBS + 0.9% sodium chloride + 0.5% Triton X-100 + 0.1% ProClin 300;

[0134] Formula 2: 0.02 M PBS + 0.9% sodium chloride + 0.5% Triton X-100 + 0.1% ProClin300 + 0.5% BSA;

[0135] Formula 3: 0.02 M PBS + 0.9% sodium chloride + 0.5% Triton X-100 + 0.1% ProClin300 + 1% trehalose;

[0136] Formula 4: 0.02 M PBS + 0.9% sodium chloride + 0.5% Triton X-100 + 0.1% ProClin300 + 0.5% BSA + 1% trehalose.

[0137] The stability assessment procedure is as follows: After storing the prepared reagents at 2-8℃ and 37℃ for 7 days respectively, use the reagents under the corresponding storage conditions to test the signal values ​​of the gradient calibrators (i.e., S1-S6), and calculate the signal value decrease as follows:

[0138] Table 8. Stability of buffer solutions with different formulations

[0139]

[0140] The stability test results of different buffer formulations (Table 8) show that the addition of BSA and trehalose can improve reagent stability. The combination of the two can synergistically improve reagent stability (compare formulation 4 and formulation 1). Therefore, formulation 4 is selected.

[0141] 2. Buffer 2

[0142] 2.1 Screening with different buffer solutions

[0143] PBS, Tris, and MOPS buffers were selected for reactivity testing, and the results are shown in Table 9.

[0144] Table 9. Detection results (RLU values) for different buffer solutions

[0145]

[0146] As shown in Table 9, when the experimental group using PBS buffer tested gradient samples, the background (i.e., 0 concentration calibrator S0) was low, and the signal values ​​of the gradient calibrators (i.e., S1-S6) were high, indicating that the overall reactivity was high and the background was low, and the signal-to-noise ratio was good.

[0147] 2.2 Formulation Screening

[0148] The reagent stability was tested using the following formulation, and the RLU values ​​were recorded.

[0149] Formula 1: 0.02 M PBS + 0.9% sodium chloride + 0.5% Triton X-100 + 0.1% ProClin950;

[0150] Formula 2: 0.02 M PBS + 0.9% sodium chloride + 0.5% Triton X-100 + 0.1% ProClin950 + 0.5% BSA;

[0151] Formula 3: 0.02 M PBS + 0.9% sodium chloride + 0.5% Triton X-100 + 0.1% ProClin950 + 1% trehalose;

[0152] Formula 4: 0.02 M PBS + 0.9% sodium chloride + 0.5% Riton X-100 + 0.1% ProClin950 + 0.5% BSA + 1% trehalose.

[0153] Table 10 Stability of buffer solutions with different formulations

[0154]

[0155] The stability test results of different buffer formulations (Table 10) show that the addition of BSA and trehalose can improve reagent stability, and the combination of the two can synergistically improve reagent stability (compare formulation 4 and formulation 1).

[0156] Example 11 Comparison of Interleukin-13 Detection Methods

[0157] 1.1 Reaction Mode Screening

[0158] Mode 1: One-step method (sample + biotin-antibody solution + alkaline phosphatase-antibody solution + magnetic bead reagent → incubation → washing → + substrate → detection)

[0159] The specific steps are as follows:

[0160] 1) Add calibrator (for calibration) and sample to the reaction tube in sequence, with a sample volume of 30 μL / tube for each.

[0161] 2) Add 100 μL of biotin-antibody solution, 100 μL of alkaline phosphatase-labeled solution and 100 μL of magnetic bead reagent to each tube respectively; wherein, the antibody concentration in the biotin-antibody solution is 0.6 μg / mL and the antibody concentration in the alkaline phosphatase-labeled solution is 0.6 μg / mL.

[0162] 3) After mixing, incubate at 37°C for 30 minutes.

[0163] 4) Wash 5 times with cleaning solution (0.1M Tris, 0.9% NaCl, 0.5% Tween, 0.5% Proclin 300).

[0164] 5) Add 200 μL of luminescent substrate solution (AMPPD; purchased from Guangdong Feipeng Biotechnology Co., Ltd.) to each well.

[0165] 6) Detect the luminescence value 1-5 minutes after mixing.

[0166] 7) Calculation of results.

[0167] Mode 2: Delayed one-step method (sample + biotin-antibody solution + alkaline phosphatase-antibody solution → incubation → + magnetic bead reagent → incubation → washing → + substrate → detection)

[0168] For the specific steps of Mode 2, please refer to Example 3.

[0169] Mode 3: Two-step method (sample + magnetic bead reagent + biotin-antibody solution → incubation → washing → + alkaline phosphatase-antibody solution → incubation → washing → + substrate → detection)

[0170] 1) Add calibrator (for calibration) and sample to the reaction tube in sequence, with a sample volume of 30 μL / tube for each.

[0171] 2) Add 100 μL of biotin-antibody solution and 100 μL of magnetic bead reagent to each tube; wherein the concentration of antibody in biotin-antibody solution is 0.6 μg / mL and the concentration of magnetic microparticles in magnetic bead reagent is 0.8 mg / mL.

[0172] 3) After mixing, incubate at 37°C for 15 minutes.

[0173] 4) Wash 5 times with cleaning solution.

[0174] 5) Add 100 μL of alkaline phosphatase-labeled solution to each tube, wherein the antibody concentration in the alkaline phosphatase-labeled solution is 0.6 μg / mL.

[0175] 6) After mixing, incubate at 37°C for 15 minutes.

[0176] 7) Wash 5 times with cleaning solution (0.1M Tris, 0.9% NaCl, 0.5% Tween, 0.5% Proclin 300).

[0177] 8) Add 200 μL of luminescent substrate solution (AMPPD; purchased from Guangdong Feipeng Biotechnology Co., Ltd.) to each well.

[0178] 9) Detect the luminescence value 1-5 minutes after mixing.

[0179] 10) Calculation of results.

[0180] The test results are shown in Table 11.

[0181] Table 11 Detection results (RLU values) of different detection methods

[0182]

[0183] As shown in Table 11, when gradient samples were tested under reaction mode 2, the background (i.e., 0 concentration calibrator S0) was low, and the signal values ​​of gradient calibrators (i.e., S1-S6) were high, indicating that reaction mode 2 has high overall reactivity and low background.

[0184] 1.2 Reaction Time Screening

[0185] Under the extended one-step method, the combination of incubation reaction times after adding reagents in steps 3) and 5) was screened as follows.

[0186] Combination 1: 10 min + 10 min;

[0187] Combination 2: 15 min + 15 min;

[0188] Combination 3: 20 min + 20 min.

[0189] The test results are shown in Table 12.

[0190] Table 12 Detection results (RLU values) for different combinations of incubation times

[0191]

[0192] As shown in Table 12, combination 1 has low reactivity (i.e., high and low signal values ​​of S1-S6), while combination 2 has low background (i.e., 0 concentration calibrator S0) and high signal values ​​of gradient calibrators (i.e., S1-S6). Combination 3 does not significantly improve reactivity and gradient. Therefore, considering both optimizing reactivity and saving reaction time, combination 2 with incubation time is selected.

Claims

1. A kit for the chemiluminescence detection of interleukin-13, characterized in that, The kit contains reagent R1, reagent R2, and reagent R3; Reagent R1 contains magnetic beads coated with streptavidin. Reagents R2 and R3 contain interleukin-13 monoclonal antibodies 1 and 2, respectively, which are conjugated with biotin and alkaline phosphatase, respectively.

2. The kit according to claim 1, characterized in that, The interleukin-13 monoclonal antibody 1 is Abcam's EPR20465-131; the interleukin-13 monoclonal antibody 2 is Abcam's EPR20465-55.

3. The kit according to claim 1 or 2, characterized in that, The reagent R1, the reagent R2 and / or the reagent R3 further include: (1) A stabilizer, preferably a chloride and / or a disaccharide, wherein the chloride is selected, for example, from one or more of sodium chloride, potassium chloride, and magnesium chloride, more preferably sodium chloride; the disaccharide is selected, for example, from one or more of sucrose, maltose, and trehalose, preferably trehalose; and / or, (2) A buffer, preferably one or more selected from PBS, triethanolamine hydrochloride, MES, Tris, MOPS and HEPES, preferably PBS or triethanolamine hydrochloride; and / or, (3) A surfactant, preferably a nonionic surfactant, such as Triton X-100; and / or, (4) Protective agent, wherein the protective agent is preferably BSA or ethanolamine, and more preferably BSA; Preferably, reagent R1, reagent R2 and / or reagent R3 further comprise: (5) Preservative, wherein the preservative is preferably one or more of ProClin 300, ProClin 950, gentamicin sulfate, sodium azide, 2-hydroxypyridine-N-oxide, chloroacetamide, imidazolidinyl urea, thimerosal and 2-methyl-3(2H)-isothiazolidinone 5-bromo-5-nitro-1,3-dioxane; The percentages mentioned above are mass-volume percentages in the reagent, in g / mL.

4. The kit according to any one of claims 1-3, characterized in that, The concentration of the magnetic beads is 0.25-1 mg / mL; and / or the concentration of interleukin-13 monoclonal antibody 1 is 0.1-1 μg / mL; and / or the concentration of interleukin-13 monoclonal antibody 2 is 0.1-1 μg / mL.

5. The kit according to claim 4, characterized in that, The reagent R1 comprises: 0.25-0.8 mg / mL magnetic beads coated with streptavidin, 0.5-2% sodium chloride, 0.01 M-0.5 M PBS, and 0.2%-0.08% Triton X; and / or, The reagent R2 comprises: 0.1-0.6 μg / mL interleukin-13 antibody 1, 0.5-2% sodium chloride, 0.01 M-0.5 M PBS, and 0.2%-0.08% Triton X; and / or, The reagent R3 comprises: 0.1-0.6 μg / mL interleukin-13 antibody 2, 0.5-2% sodium chloride, 0.5-2% trehalose, 0.01M-0.5M PBS, and 0.2%-0.08% Triton X; More preferably, reagent R1, reagent R2 and / or reagent R3 further include 0.2%-0.5% BSA and / or 0.5-2% trehalose; Optionally, reagent R1 and reagent R2 further include 0.1-0.2 μg / mL ProClin300; and / or, reagent R3 further includes 0.1-0.2 μg / mL ProClin950.

6. A reagent kit according to any one of claims 1-5, characterized in that, The kit also includes reagent R4, which contains a chemiluminescent substrate; Preferably, the chemiluminescent substrate is AMPPD, CDP-Star, or APS-5.

7. A method for preparing the kit according to any one of claims 1-6, characterized in that, The method includes the steps of preparing reagent R1, reagent R3 and reagent R3.

8. A method for detecting interleukin-13, characterized in that, The method includes the step of testing the sample using the kit as described in any one of claims 1-6; Preferably, the test sample is a serum sample or a plasma sample.

9. The method as described in claim 8, characterized in that, The method includes: S1: Add reagent R2 and reagent R3 to the sample to be tested and incubate; S2: Add reagent R1 and incubate; S3: Add reagent R4 to develop color; Preferably, the volume ratio of the test sample, reagent R2, and reagent R3 is (2-5):(8-12):(8-12); and / or, the volume ratio of the test sample and reagent R1 is (2-5):(8-12); and / or, the incubation time in S2 and S3 is 5-30 min, and / or the incubation temperature is 30-37°C; and / or, the incubation in S2 further includes a washing step, wherein the washing is preferably performed 3-5 times. More preferably, the volume ratio of the test sample, reagent R2 and reagent R3 is (3-5):(10-12):(10-12); and / or, the volume ratio of the test sample and reagent R1 is (3-5):(10-12); and / or, the incubation time is 5-15 min, and / or the incubation temperature is 35-37℃.

10. The use of the kit according to any one of claims 1-6 in the detection of interleukin-13.

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