Kit for detecting interleukin 23 as well as use method and application of kit

Abstract

The invention provides a kit for detecting interleukin 23 as well as a use method and application of the kit, and relates to the field of in-vitro detection. The kit for detecting the interleukin 23, provided by the invention, comprises a pre-excitation liquid and an excitation liquid, the pre-excitation liquid is prepared from 0.03 mol / L to 0.15 mol / L of hydrogen peroxide and 0.01 mol / L to 0.05 mol / L of benzoic acid The excitation liquid comprises 0.5 to 1.5 mol / L of sodium hydroxide and 3 to 8 g / L of hexadecyl trimethyl ammonium bromide; the kit can be used for measuring interleukin 23 in serum, and is high in accuracy, high in signal-to-noise ratio and strong in stability.

Description

Technical Field

[0001] The present invention belongs to the field of in vitro detection, relates to interleukin 23, and particularly relates to a kit for detecting interleukin 23 and a use method and application thereof. Background Art

[0002] Interleukins (ILs) are a class of cytokines secreted by immune cells (such as T cells and macrophages) that are primarily involved in immune regulation, inflammatory responses, and hematopoiesis. Currently, at least 29 interleukin members have been discovered, including IL-1, IL-2, IL-6, IL-10, and IL-23. Among them, interleukin 23 (IL-23) is a member of the IL-12 cytokine family. It is composed of the p19 subunit and the 40kD subunit of IL-12 and is primarily secreted by activated dendritic cells, macrophages, and monocytes. Its structural characteristics give IL-23 a unique role in immune regulation, particularly in the differentiation and inflammatory response of Th17 cells. As a key proinflammatory cytokine, IL-23 is involved in the development of multiple chronic inflammatory diseases. Studies have found that IL-23 levels are significantly increased in biopsies from patients with Crohn's disease, ulcerative colitis, and psoriasis. IL-23 shows potential in disease treatment, but the concentration level of IL-23 in serum is increasingly affected by the disease, and a rapid detection method is needed. Domestic interleukin-23 detection technology is mainly ELISA, and there is still a lack of an effective detection kit.

[0003] Chemiluminescence is a luminescent phenomenon that first occurred in living organisms. In 1888, Wiedemann of Germany first described the basic principle of chemiluminescence, namely that chemiluminescence is the result of a chemical reaction. Chemiluminescence can be divided into two main categories, gas-phase and liquid-phase, depending on the state of the reaction medium, with liquid-phase chemiluminescence being the most widely used. Common luminescent reagents include luminol, acridinium esters, 1,2-dioxetanes, and peroxyoxalates. Acridiniums are chemiluminescent reagents with very high quantum yields. Their molecular structure consists of at least two components: a luminescent group and a leaving group. The most widely used are acridinium esters and acridinium sulfonamides. The general reaction mechanism involves the addition of hydrogen peroxide to the 9-carbon atom of acridinium under alkaline conditions. The resulting peroxide anion then nucleophilically attacks the carbonyl carbon. The leaving group then leaves and forms an unstable four-membered ring intermediate. The ring opens to form an excited acridone, which releases photons as it returns to the ground state. As a technology for quantitative detection of trace substances, chemiluminescence immunoassays have become advanced and mature, playing a crucial role in human disease screening and health monitoring. Based on the different luminescence systems, chemiluminescence can be divided into direct chemiluminescence immunoassays, luminescent oxygen channel immunoassays, enzymatic chemiluminescence immunoassays, and electrochemiluminescence immunoassays. Magnetic particle chemiluminescence technology has gradually become a leading technology in the field of clinical testing in China.

[0004] Chemiluminescence is used in serum or plasma sample testing. To enhance the chemiluminescence intensity of acridinium esters or acridinium sulfonamides and stabilize H₂O₂, nitric acid, hydrochloric acid, sulfuric acid, and other substances are typically added to the pre-stimulation solution. Nitric acid, hydrochloric acid, and sulfuric acid are all added as concentrated acids during use, posing a safety hazard. High acid concentrations place high demands on testing equipment and can easily corrode it over extended periods. Therefore, there is a need for a new interleukin-23 detection kit with an stimulating solution system that improves detection efficiency and reduces damage to testing equipment.

[0005] Prior art CN107817354A discloses a chemiluminescent detection kit for interleukin-6 and its preparation method. The kit includes: a sample diluent, magnetic microparticles coated with an interleukin-6 monoclonal antibody, an interleukin-6 monoclonal antibody labeled with an acridinium ester, a series of interleukin-6 standard solutions, chemiluminescent excitation solution A, chemiluminescent excitation solution B, and a cleaning solution. Prior art CN115586331A also discloses adding an acidic excitation solution to liberate the acridinium ester and adding an alkaline excitation solution to induce photon emission. Both of these techniques use nitric acid in the excitation solution, which is highly corrosive to the instrument and poses a safety hazard to the operator. Summary of the Invention

[0006] The present invention addresses the problems of the prior art and provides a kit for detecting interleukin-23. The kit comprises magnetic beads, an interleukin-23 antibody, an acridinium ester, a magnetic bead storage solution, an acridinium ester storage solution, a diluent, a pre-stimulation solution, and a stimulation solution. The pre-stimulation solution comprises 0.03-0.15 mol / L hydrogen peroxide and 0.01-0.05 mol / L benzoic acid. The stimulation solution comprises 0.5-1.5 mol / L sodium hydroxide and 3-8 g / L hexadecyltrimethylammonium bromide. The kit can be used to measure interleukin-23 in serum with high accuracy, a high signal-to-noise ratio, and strong stability.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] On the one hand, the present invention provides a kit for detecting interleukin-23, the kit comprising a pre-excitation solution and an excitation solution; the pre-excitation solution comprises 0.03-0.15 mol / L hydrogen peroxide and 0.01-0.05 mol / L benzoic acid; the excitation solution comprises 0.5-1.5 mol / L sodium hydroxide and 3-8 g / L hexadecyltrimethylammonium bromide.

[0009] Preferably, the pre-stimulation solution comprises 0.05-0.10 mol / L hydrogen peroxide and 0.01-0.05 mol / L benzoic acid; and the stimulation solution comprises 0.8-1.2 mol / L sodium hydroxide and 3-8 g / L hexadecyltrimethylammonium bromide.

[0010] Preferably, the pre-stimulation solution comprises 0.08 mol / L hydrogen peroxide and 0.01-0.05 mol / L benzoic acid; and the stimulation solution comprises 1.0 mol / L sodium hydroxide and 3-8 g / L hexadecyltrimethylammonium bromide.

[0011] Preferably, the kit further comprises magnetic beads, interleukin-23 antibody, acridinium ester, magnetic bead preservation solution and acridinium ester preservation solution.

[0012] Preferably, the magnetic bead storage solution comprises 70%-80% phosphate buffer, 5%-15% calf serum, 5%-15% glycerol, 0.05%-0.15% Proclin 300 and 0.05%-0.15% Tween 20 by volume.

[0013] Specifically, the magnetic bead storage solution contains 79.8% phosphate buffer, 10% calf serum, 10% glycerol, 0.1% Proclin 300 and 0.1% Tween 20 by volume.

[0014] Preferably, the acridinium ester preservation solution comprises 60%-80% phosphate buffer, 20%-40% glycerol and 0.05%-0.15% Proclin 300 by volume, and BSA is added to make the mass fraction in the solution 0.5%-1.5%.

[0015] Specifically, the acridinium ester preservation solution contains 69.9% by volume of phosphate buffer, 30% by volume of glycerol, and 0.1% by volume of Proclin 300, and BSA is added to make the mass fraction of BSA in the solution 1.0%.

[0016] Preferably, the kit further comprises a diluent.

[0017] Preferably, the diluent is a phosphate buffer solution supplemented with 0.5%-1.5% BSA, 0.05%-0.15% TritonX-100 and 0.1%-0.5% Proclin300 in mass fractions.

[0018] Specifically, the diluent is a phosphate buffer solution containing 1% BSA, 0.1% Triton X-100 and 0.3% Proclin 300 in mass fraction.

[0019] Preferably, the concentration of the phosphate buffer is 0.005-0.1M.

[0020] Specifically, the concentration of the phosphate buffer is 0.01M.

[0021] Preferably, the pH of the phosphate buffer is 7.0-8.0.

[0022] Specifically, the pH of the phosphate buffer is 7.4.

[0023] In another aspect, the present invention provides a use of the above-mentioned kit in in vitro sample detection, and the application is a non-disease diagnosis or treatment application.

[0024] Preferably, the in vitro sample comprises a body fluid sample.

[0025] Preferably, the body fluid sample includes serum or plasma.

[0026] In another aspect, the present invention provides a method for using the above kit.

[0027] Preferably, the method of use comprises preparing and detecting using the above-mentioned magnetic beads, interleukin-23 antibody, acridinium ester, magnetic bead storage solution, acridinium ester storage solution, pre-excitation solution or excitation solution.

[0028] Preferably, the method of use comprises the following steps:

[0029] Antibody-coated magnetic beads, acridinium ester-labeled antibodies, and detection of interleukin-23 content in samples.

[0030] Preferably, the antibody-coated magnetic beads include the following steps:

[0031] (1) Add magnetic beads to a centrifuge tube, remove the supernatant after magnetic separation; add magnetic bead cleaning solution to the centrifuge tube, mix well, remove the supernatant after magnetic separation, and wash;

[0032] (2) Prepare MES (2-morpholinoethanesulfonic acid) buffer, EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) solution, and NHS (N-hydroxysuccinimide) solution. Add EDC solution and NHS solution to a centrifuge tube and mix well for activation. After activation, wash with MES buffer and remove the supernatant.

[0033] (3) Add interleukin-23 antibody to the centrifuge tube, couple, remove the supernatant after magnetic separation, add magnetic bead blocking solution for blocking, wash after blocking, and remove the supernatant;

[0034] (4) Add magnetic bead preservation solution to wash, remove the supernatant, and then resuspend with magnetic bead preservation solution to obtain magnetic bead coating working solution.

[0035] Preferably, the acridinium ester-labeled antibody comprises the following steps:

[0036] (1) Take interleukin-23 antibody, add acridinium ester at a molar ratio of antibody to acridinium ester of 1:5-1:15, and the final concentration of the interleukin-23 antibody is 1-3 mg / mL, and react at 25°C for 2-3 hours;

[0037] (2) blocking with lysine at a molar ratio of 1:120-1:160 for acridinium ester:lysine and reacting for 15-30 min;

[0038] (3) After blocking, use a 40-60 kD dialysis bag to perform buffer exchange, dialyzing 3-5 times, each dialysis for 2-3 hours; after dialysis, add glycerol to a final antibody concentration of 0.3-0.8 mg / mL and set aside;

[0039] (4) When using, add acridinium ester preservation solution to make the final concentration of the antibody 6-10 μg / mL to obtain acridinium ester labeled antibody working solution.

[0040] Preferably, the detection of the interleukin-23 content in the sample comprises the following steps:

[0041] (1) Take the sample, add the magnetic bead coating working solution, mix well and incubate at 37°C for 3-10 minutes, then perform magnetic separation, wash, and remove the supernatant to obtain the magnetic bead-antigen complex;

[0042] (2) Adding acridine lipid-labeled antibody working solution to the reaction cup containing the magnetic bead-antigen complex, mixing and incubating at 37°C for 3-10 minutes, then magnetically separating, washing, and removing the supernatant to obtain the magnetic bead-antigen-detection antibody complex;

[0043] (3) Adding the pre-excitation solution and the excitation solution to the reaction cup containing the magnetic bead-antigen-detection antibody complex, mixing and measuring the maximum luminescence intensity;

[0044] (4) The luminescence intensity of the standard sample is fitted into a standard curve, and the interleukin-23 content in the sample to be tested is calculated using the standard curve.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] 1. The present invention provides a kit for detecting interleukin-23, comprising magnetic beads, an interleukin-23 antibody, an acridinium ester, a magnetic bead storage solution, an acridinium ester storage solution, a diluent, a pre-stimulation solution, and a stimulation solution. This kit can be used to measure interleukin-23 in serum with high accuracy and a high signal-to-noise ratio.

[0047] 2. Benzoic acid in the pre-excitation solution of the present invention is a weak acid, which replaces conventional strong acids such as nitric acid. It has low corrosiveness to the detection instrument pipeline and is highly safe for operators, while ensuring long-term stability. DETAILED DESCRIPTION

[0048] Unless otherwise specified, the raw materials used in the present invention are all common commercially available products, and their sources are not specifically limited.

[0049] Example 1: Preparation and application of the kit

[0050] (1) Preparation of basic solution

[0051] 1.1. Preparation of magnetic bead storage solution: 79.8% by volume of 0.01 M phosphate buffer, 10% calf serum, 10% glycerol, 0.1% Proclin 300, and 0.1% Tween 20;

[0052] 1.2. Preparation of acridinium ester preservation solution: 69.9% by volume of 0.01 M phosphate buffer, 30% by volume of glycerol, and 0.1% by volume of Proclin 300, followed by 1% by volume of BSA.

[0053] 1.3. Preparation of pre-stimulation solution: Add 0.08 mol / L hydrogen peroxide and 0.02 mol / L benzoic acid to purified water and mix well.

[0054] 1.4. Preparation of the excitation solution: Add 1.0 mol / L sodium hydroxide and 4.5 g / L hexadecyltrimethylammonium bromide to purified water and mix well.

[0055] 1.5. Preparation of magnetic bead washing solution: Prepare 0.2 M MES buffer and add 0.02% (v / v) Proclin 300.

[0056] 1.6. Preparation of magnetic bead blocking solution: 99.4% by volume of 0.01 M phosphate buffer and 0.6% by volume of Tween 20, followed by 0.5% by volume of BSA.

[0057] In a specific embodiment, the phosphate buffer refers to PBS buffer.

[0058] (2) Preparation of magnetic bead-coated antibodies

[0059] 2.1. Add 200 μL of magnetic beads (EM1-100 / 40 (high carboxyl) magnetic microspheres, Cat. No. 23710087) to a 2 mL centrifuge tube, perform magnetic separation for 3 min, and then remove the supernatant.

[0060] 2.2. Add 400 μL of magnetic bead washing solution to the centrifuge tube, shake to mix, remove the supernatant by magnetic separation, and wash twice.

[0061] 2.3. Weigh a certain amount of EDC and add a certain volume of 0.02M MES buffer to prepare a 50mg / ml EDC solution; weigh a certain amount of NHS and add a certain volume of 0.02M MES buffer to prepare a 50mg / ml NHS solution.

[0062] 2.4. Add 100 μL of EDC solution and 100 μL of NHS solution to the centrifuge tube and shake to mix.

[0063] 2.5. Place the two centrifuge tubes on a mixer and activate for 30 minutes. Adjust the speed appropriately so that the liquid flows steadily when inverted.

[0064] 2.6. After activation, wash twice with 2 volumes of 0.02 M MES buffer and discard the supernatant.

[0065] 2.7. Add 120 μg of interleukin-23 antibody (purchased from abcam, cat. no. ab314486) to a centrifuge tube, dilute to 300 μL with 0.01 M phosphate buffer, and couple for 3 h.

[0066] 2.8. Wash twice with 600 μL of magnetic bead blocking buffer and discard the supernatant.

[0067] 2.9. Add 600 μL of magnetic bead blocking solution to the centrifuge tube, shake and mix for 30 minutes, and remove the supernatant.

[0068] 2.10. Wash twice with 600 μl of magnetic bead storage solution, remove the supernatant, and then transfer to 30 mL of magnetic bead storage solution to obtain magnetic bead coating working solution.

[0069] (3) Preparation of acridinium ester-labeled antibodies

[0070] 3.1. Dissolve 200 μg of interleukin-23 antibody (purchased from abcam, cat. no. ab190356) in 0.01 M phosphate buffer and add 2 mg of acridinium ester (NSP-SA-NHS, with a molar ratio of antibody to acridinium ester of 1:10) to a final concentration of 2 mg / mL.

[0071] 3.2. Incubate on a constant temperature shaker at 25°C for 3 h.

[0072] 3.3. Add lysine (acridinium ester: lysine = 1:140, molar concentration ratio) for blocking and react for 20 minutes.

[0073] 3.4. After the reaction, a 50KD dialysis bag was used for buffer exchange. The dialysis buffer was 0.01M phosphate buffer. The dialysis was performed 4 times, each time for 3 hours.

[0074] 3.5. After dialysis is completed, add glycerol to a final antibody concentration of 0.5 mg / mL and set aside.

[0075] 3.6. Dilute the antibody to a final concentration of 8 μg / mL using acridinium ester storage solution to obtain acridinium ester-labeled antibody working solution.

[0076] (4) Detection and calculation of interleukin-23 content in the sample to be tested

[0077] The detection was performed using a fully automatic chemiluminescence analyzer. The specific operation was as follows:

[0078] 4.1. Take 5 μL of the sample to be tested, add 50 μL of the magnetic bead coating working solution, mix well, and incubate at 37°C for 5 minutes. Then, perform magnetic separation, wash to remove unbound substances, and remove the supernatant to obtain the magnetic bead-antigen complex;

[0079] 4.2. Add 50 μL of acridine-labeled antibody solution to the reaction cup containing the magnetic bead-antigen complex, mix well, and incubate at 37°C for 5 minutes. Then, perform magnetic separation, wash to remove unbound substances, and remove the supernatant to obtain the magnetic bead-antigen-detection antibody complex;

[0080] 4.3. Add 100 μL of pre-stimulation solution and 100 μL of stimulation solution to the reaction cup containing the magnetic bead-antigen-detection antibody complex, mix thoroughly, and measure the maximum luminescence intensity;

[0081] 4.4. Fit the luminescence intensity of the standard sample to a standard curve, and calculate the interleukin-23 content in the sample to be tested using the standard curve.

[0082] Example 2: Preparation and application of the kit

[0083] The method of Example 1 was followed, except that the concentration of benzoic acid in the pre-stimulation solution was 0.01 mol / L, and the concentration of cetyltrimethylammonium bromide in the stimulation solution was 8 g / L.

[0084] Example 3: Preparation and application of the kit

[0085] The method of Example 1 was followed, except that the concentration of benzoic acid in the pre-stimulation solution was 0.05 mol / L, and the concentration of cetyltrimethylammonium bromide in the stimulation solution was 3 g / L.

[0086] Comparative Example 1: Preparation and Application of Kit

[0087] The method of Example 1 was followed, except that benzoic acid was not added to the pre-excitation solution.

[0088] Comparative Example 2: Preparation and Application of Kit

[0089] The method of Example 1 was followed, except that hexadecyltrimethylammonium bromide was not added to the exciting solution.

[0090] Comparative Example 3: Preparation and Application of Kit

[0091] The method of Example 1 was followed, except that benzoic acid was not added to the pre-excitation solution, and cetyltrimethylammonium bromide was not added to the excitation solution.

[0092] Comparative Example 4: Preparation and Application of Kit

[0093] The method of Example 1 was followed, except that the concentration of benzoic acid in the pre-stimulation solution was 0.07 mol / L.

[0094] Comparative Example 5: Preparation and Application of Kit

[0095] The method of Example 1 was followed, except that the concentration of cetyltrimethylammonium bromide in the stimulating solution was 10 g / L.

[0096] Comparative Example 6: Preparation and Application of Kit

[0097] The method of Example 1 was followed, except that the benzoic acid in the pre-excitation solution was replaced with an equal molar amount of nitric acid.

[0098] Comparative Example 7: Preparation and Application of Kit

[0099] The method of Example 1 was followed, except that the hexadecyltrimethylammonium bromide in the exciting solution was replaced with an equimolar amount of hexadecyltrimethylammonium chloride.

[0100] Effect Example 1: Verification of the test kit's detection efficiency

[0101] In this effect example, interleukin 23 antigen (purchased from Beijing Biolabs, product number JN0403) was used as a standard, PBS buffer containing 10% calf serum was used as a matrix liquid, and the interleukin 23 antigen was diluted in a certain proportion to obtain a series of concentrations of quality control products; the kits prepared in the examples and comparative examples were tested for the following items.

[0102] SPSS 21.0 software was used for data statistics and analysis. The t test was used for statistical analysis, and P < 0.05 indicated a significant difference.

[0103] (1) Signal-to-noise ratio

[0104] Detection sample: dilute acridinium ester (NSP-SA-NHS) to 1.5×10 -7 mol / L, referred to as the acridinium ester sample); the diluent was used as a blank test, referred to as the blank sample. The diluent was prepared by adding 1% BSA, 0.1% Triton X-100, and 0.3% Proclin 300 to 0.01 M phosphate buffer at pH 7.4.

[0105] Determination method: 50 μL test sample + 100 μL pre-excitation solution + 100 μL excitation solution, react for 2 min, and test the luminescence value (RLU) in a fully automatic chemiluminescence immunoassay. In this experiment, the test sample includes an acridinium ester sample (S) and a blank sample (N). Each parallel determination is performed 3 times to obtain the luminescence value (S) of the acridinium ester sample and the luminescence value (N) of the blank sample. The ratio (S / N) of the two is the signal-to-noise ratio, and the performance of the signal-to-noise ratio is better. The results are shown in Table 1. The luminescence value data in Table 1 are the average values after 3 parallel determinations. It can be seen that the combined use of benzoic acid and CTAB of the present invention (Examples 1-3) has a synergistic effect compared to the use of benzoic acid (Comparative Example 1) and CTAB (Comparative Example 2) alone, and the signal-to-noise ratio is significantly improved.

[0106] Table 1 Comparison of signal-to-noise ratios of different kits

[0107]

[0108]

[0109] Note: The same letters in the same column of data indicate no significant difference between the groups (P>0.05), and different letters indicate significant difference between the groups (P<0.05).

[0110] (2) Stability of the excitation liquid system

[0111] The excitation liquid system consists of pre-excitation liquid and excitation liquid.

[0112] Detection sample: dilute acridinium ester (NSP-SA-NHS) to 1.5×10 -7 mol / L, referred to as acridinium ester sample); the diluent was used as the blank test, referred to as the blank sample.

[0113] Open the bottle: Open the caps of the pre-stimulation solution and stimulation solution bottles and store them in a cool, dark place at room temperature.

[0114] Determination method: 50 μL test sample + 100 μL pre-excitation liquid + 100 μL excitation liquid, react for 2 minutes, test the luminescence value (RLU) in the fully automatic chemiluminescence immunoassay, the acridinium ester sample (S) and the blank sample (N) are measured in parallel 3 times, and the luminescence value (S) of the acridinium ester sample and the luminescence value (N) of the blank sample are measured. The ratio (S / N) of the two is the signal-to-noise ratio. The excitation liquid system with different opening times is tested, and the statistical results are shown in Table 2. The data in Table 2 are the average values after 3 parallel measurements. It can be seen that the pre-excitation liquid and the excitation liquid (Examples 1-3) provided by the present invention can maintain good stability when opened at room temperature for 60 days.

[0115] Table 2 Test opening stability of the excitation liquid system

[0116]

[0117]

[0118] (3) Minimum detection limit

[0119] The IL-23 quality control product at 0.50 pg / mL was measured and repeated three times. The results and analysis are shown in Table 3. It can be seen that Examples 1-3 of the present invention have a lower detection limit of no more than 1.50 pg / mL.

[0120] Table 3 Comparison of the minimum detection limits of different kits

[0121]

[0122] (4) Accuracy

[0123] The accuracy of the high and low concentration quality control products (500 pg / mL and 20 pg / mL) was tested and repeated three times. The results and analysis are shown in Table 4. It can be seen that the kit provided by the present invention (Examples 1-3) has high accuracy and the relative deviation is significantly reduced compared with other kits.

[0124] Table 4 Comparison of the accuracy of different kits

[0125]

[0126]

[0127] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A kit for detecting interleukin-23, characterized in that: The kit comprises a pre-stimulation solution and a stimulation solution; the pre-stimulation solution comprises 0.03-0.15 mol / L hydrogen peroxide and 0.01-0.05 mol / L benzoic acid; and the stimulation solution comprises 0.5-1.5 mol / L sodium hydroxide and 3-8 g / L hexadecyltrimethylammonium bromide.

2. The kit according to claim 1, wherein The pre-stimulation solution contains 0.05-0.10 mol / L hydrogen peroxide and 0.01-0.05 mol / L benzoic acid; the stimulation solution contains 0.8-1.2 mol / L sodium hydroxide and 3-8 g / L hexadecyltrimethylammonium bromide.

3. The kit according to claim 2, wherein The pre-stimulation solution contains 0.08 mol / L hydrogen peroxide and 0.01-0.05 mol / L benzoic acid; the stimulation solution contains 1.0 mol / L sodium hydroxide and 3-8 g / L hexadecyltrimethylammonium bromide.

4. The kit according to claim 1, wherein The kit further comprises magnetic beads, interleukin-23 antibody, acridinium ester, magnetic bead preservation solution and acridinium ester preservation solution.

5. The kit according to claim 4, characterized in that The magnetic bead storage solution contains 70%-80% phosphate buffer, 5%-15% calf serum, 5%-15% glycerol, 0.05%-0.15% Proclin 300 and 0.05%-0.15% Tween 20 according to volume fraction.

6. The kit according to claim 5, characterized in that The magnetic bead storage solution contains 79.8% phosphate buffer, 10% calf serum, 10% glycerol, 0.1% Proclin 300 and 0.1% Tween 20 according to volume fraction.

7. The kit according to claim 4, characterized in that The acridinium ester preservation solution contains 60%-80% phosphate buffer, 20%-40% glycerol and 0.05%-0.15% Proclin 300 in volume percentage, and then 0.5%-1.5% BSA is added.

8. The kit according to claim 7, characterized in that The acridinium ester preservation solution contains 69.9% by volume of phosphate buffer, 30% by volume of glycerol, and 0.1% by volume of Proclin 300, and then 1.0% by volume of BSA is added.

9. The kit according to any one of claims 5 to 8, characterized in that The concentration of the phosphate buffer is 0.005-0.1M.

10. The kit according to claim 9, characterized in that The concentration of the phosphate buffer is 0.01M.

11. Use of the kit according to any one of claims 1 to 10 in in vitro sample detection, characterized in that: The application is non-disease diagnosis or treatment application.

12. The use according to claim 11, characterized in that The in vitro sample includes a body fluid sample.

13. The use according to claim 12, characterized in that The body fluid sample includes serum or plasma.

14. The method for using the kit according to claim 1, characterized in that: The method of use comprises the steps of preparing and detecting the magnetic beads, interleukin-23 antibody, acridinium ester, magnetic bead preservation solution, acridinium ester preservation solution, the pre-excitation solution and the excitation solution.

15. The method of use according to claim 14, characterized in that: The following steps are involved: Antibody-coated magnetic beads, acridinium ester-labeled antibodies, and detection of interleukin-23 content in samples.

16. The method of use according to claim 15, characterized in that: The antibody-coated magnetic beads comprise the following steps: S1. Add magnetic beads to a centrifuge tube, remove the supernatant after magnetic separation; add magnetic bead cleaning solution to the centrifuge tube, mix well, remove the supernatant after magnetic separation, and wash; S2. Prepare MES buffer, EDC solution, and NHS solution. Add EDC solution and NHS solution to a centrifuge tube and mix well for activation. After activation, wash with MES buffer and remove the supernatant. S3, add interleukin-23 antibody to the centrifuge tube, couple, remove supernatant after magnetic separation, add magnetic bead blocking solution for blocking, wash after blocking, and remove supernatant; S4. Add magnetic bead preservation solution to wash, remove the supernatant, and then resuspend with magnetic bead preservation solution to obtain magnetic bead coating working solution.

17. The method of use according to claim 15, characterized in that: The acridinium ester-labeled antibody comprises the following steps: a. Take interleukin-23 antibody, add acridinium ester at a molar ratio of antibody to acridinium ester of 1:5-1:15, and the final concentration of the interleukin-23 antibody is 1-3 mg / mL. React at 25°C for 2-3 hours; b. Add lysine at a molar ratio of 1:120-1:160 of acridinium ester to lysine for blocking and react for 15-30 minutes; c. After blocking, use a 40-60KD dialysis bag to perform buffer exchange, dialyzing 3-5 times, each dialysis for 2-3 hours; after dialysis, add glycerol to a final antibody concentration of 0.3-0.8mg / mL; d. When using, add acridinium ester preservation solution to make the final antibody concentration 6-10 μg / mL to obtain acridinium ester-labeled antibody working solution.

18. The method of use according to claim 15, characterized in that: The detection of interleukin-23 content in the sample comprises the following steps: (1) Take the sample, add the magnetic bead coating working solution, mix well and incubate at 37°C for 3-10 minutes, then perform magnetic separation, wash, and remove the supernatant to obtain the magnetic bead-antigen complex; (2) Adding acridine lipid-labeled antibody working solution to the reaction cup containing the magnetic bead-antigen complex, mixing and incubating at 37°C for 3-10 minutes, then magnetically separating, washing, and removing the supernatant to obtain the magnetic bead-antigen-detection antibody complex; (3) Adding the pre-excitation solution and the excitation solution to the reaction cup containing the magnetic bead-antigen-detection antibody complex, mixing and measuring the maximum luminescence intensity; (4) The luminescence intensity of the standard sample is fitted into a standard curve, and the interleukin-23 content in the sample to be tested is calculated using the standard curve.

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