Kits for Interleukin-23 Detection, Usage Methods, and Applications

Abstract

This invention provides a kit for the detection of interleukin-23, its method of use, and its application, relating to the field of in vitro detection. The kit for interleukin-23 detection provided by this invention comprises a pre-activation solution and an activation solution; the pre-activation solution comprises 0.03-0.15 mol / L hydrogen peroxide and 0.01-0.05 mol / L benzoic acid; the activation solution comprises 0.5-1.5 mol / L sodium hydroxide and 3-8 g / L hexadecyltrimethylammonium bromide; this kit can be used to determine interleukin-23 in serum, exhibiting high accuracy, high signal-to-noise ratio, and strong stability.

Description

Technical Field

[0001] This invention belongs to the field of in vitro detection, and relates to interleukin-23, specifically to a kit for the detection of interleukin-23, its usage method, and its application. Background Technology

[0002] Interleukins (ILs) are a class of cytokines secreted by immune cells (such as T cells and macrophages), primarily involved in immune regulation, inflammatory responses, and hematopoiesis. At least 29 interleukin members have been identified, including IL-1, IL-2, IL-6, IL-10, and IL-23. Interleukin-23 (IL-23) is a member of the IL-12 cytokine family, composed of the p19 subunit and the 40kD subunit of IL-12. It is mainly secreted by activated dendritic cells, macrophages, and monocytes. Its structural characteristics give IL-23 a unique role in immune regulation, particularly in the differentiation of Th17 cells and inflammatory responses. As a key pro-inflammatory cytokine, IL-23 participates in the development of several chronic inflammatory diseases. Studies have found significantly increased IL-23 levels in biopsies of patients with Crohn's disease, ulcerative colitis, and psoriasis. IL-23 has shown potential in disease treatment, but the concentration of IL-23 in serum is greatly affected by the disease, requiring a rapid detection method. In China, the main detection technology for interleukin-23 is the ELISA method, and there is still a lack of an effective detection kit.

[0003] Chemiluminescence was first observed in living organisms. In 1888, Wiedemann of Germany first described the basic principle of chemiluminescence, stating that it is the result of a chemical reaction. Chemiluminescence can be broadly classified into gas-phase and liquid-phase based on the state of the reaction medium, with liquid-phase chemiluminescence being the most widely used. Common luminescent reagents include luminol derivatives, acridine esters, 1,2-dioxane derivatives, and peroxate esters. Acridine derivatives are chemiluminescent reagents with very high quantum yields; their molecular structure consists of at least two parts: a luminescent group and a leaving group. Acridine esters and acridine sulfonamides are the most widely used. The conventional reaction mechanism involves the addition of hydrogen peroxide to the 9-carbon atom of acridine under alkaline conditions. The addition product, a peroxide anion formed under alkaline conditions, then nucleophilically attacks the carbonyl carbon, causing the leaving group to leave and further forming an unstable four-membered ring intermediate. After ring opening, an excited-state acridine ketone is formed, which releases photons during its return to the ground state. Chemiluminescence immunoassay, as a quantitative detection technique for trace substances, has become advanced and mature, playing a vital role in human disease screening and health monitoring. Based on different luminescence systems, chemiluminescence can be categorized into: direct chemiluminescence immunoassay, luminescent oxygen channel immunoassay, enzyme-catalyzed chemiluminescence immunoassay, and electrochemiluminescence immunoassay. Among these, magnetic microparticle chemiluminescence technology is gradually becoming the dominant technique in domestic clinical testing.

[0004] Chemiluminescence is used in the detection of serum or plasma samples. To enhance the chemiluminescence intensity of acridine esters or acridine sulfonamides and stabilize H₂O₂, nitric acid, hydrochloric acid, or sulfuric acid are typically added to the pre-excitation solution. However, nitric acid, hydrochloric acid, and sulfuric acid are added as concentrated acids, posing safety hazards. High-concentration acids place high demands on detection equipment, and prolonged use can corrode the equipment. Therefore, a new kit for interleukin-23 detection is needed, in which the excitation solution system can improve detection efficiency and reduce damage to the detection equipment.

[0005] Existing technology CN107817354A discloses a chemiluminescence detection kit for interleukin-6 and its preparation method. This kit includes: sample diluent, magnetic microparticles coated with interleukin-6 monoclonal antibody, interleukin-6 monoclonal antibody labeled with acridine ester, interleukin-6 series standard solutions, chemiluminescence excitation solution A, chemiluminescence excitation solution B, and washing solution. Existing technology CN115586331A also discloses adding an acidic excitation solution to release acridine ester; and adding an alkaline excitation solution to make acridine ester emit photons. Both of these technologies use nitric acid in the excitation solution, which is highly corrosive to the instrument and poses a safety hazard to operators. Summary of the Invention

[0006] This invention addresses the problems existing in the prior art by providing a kit for the detection of interleukin-23. The kit comprises magnetic beads, interleukin-23 antibody, acridine ester, magnetic bead preservation solution, acridine ester preservation solution, diluent, pre-activation solution, and activation solution. The pre-activation solution contains 0.03-0.15 mol / L hydrogen peroxide and 0.01-0.05 mol / L benzoic acid. The activation solution contains 0.5-1.5 mol / L sodium hydroxide and 3-8 g / L hexadecyltrimethylammonium bromide. This kit can be used to determine interleukin-23 in serum with high accuracy, high signal-to-noise ratio, and strong stability.

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

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

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

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

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

[0012] Preferably, the magnetic bead preservation solution contains, by volume fraction, 70%-80% phosphate buffer, 5%-15% fetal bovine serum, 5%-15% glycerol, 0.05%-0.15% Proclin 300 and 0.05%-0.15% Tween 20.

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

[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 then BSA is added to make the mass fraction of the solution 0.5%-1.5%.

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

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

[0017] Preferably, the diluent is a phosphate buffer solution with a mass fraction of 0.5%-1.5% BSA, 0.05%-0.15% Triton X-100 and 0.1%-0.5% Proclin 300.

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

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

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

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

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

[0023] On the other hand, the present invention provides the application of the above-mentioned kit in in vitro sample detection, and the application is not a disease diagnosis or treatment application.

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

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

[0026] On the other hand, the present invention provides a method for using the above-mentioned reagent kit.

[0027] Preferably, the method of use includes preparation and detection using the above-mentioned magnetic beads, interleukin-23 antibody, acridine ester, magnetic bead preservation solution, acridine ester preservation solution, pre-activation solution, or activation solution.

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

[0029] Detection of antibody-coated magnetic beads, acridine ester-labeled antibodies, and 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, separate magnetically and remove the supernatant; add magnetic bead cleaning solution to the centrifuge tube, mix well, separate magnetically and remove the supernatant, 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 centrifuge tubes, mix well and activate. After activation, wash with MES buffer and remove supernatant.

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

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

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

[0036] (1) Take interleukin-23 antibody and add acridine ester at a molar ratio of antibody to acridine ester of 1:5-1:15. The final concentration of the interleukin-23 antibody is 1-3 mg / mL. React at 25°C for 2-3 h.

[0037] (2) Add lysine at a molar ratio of acridine ester to lysine of 1:120-1:160 for blocking, and react for 15-30 min;

[0038] (3) After the blocking is completed, use a 40-60KD dialysis bag to replace the buffer solution. Dialyze 3-5 times in total, each time for 2-3 hours. After dialysis, add glycerol to the final antibody concentration of 0.3-0.8mg / mL and set aside.

[0039] (4) 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.

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

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

[0042] (2) Add acridine ester labeled antibody working solution to the reaction cup containing magnetic bead-antigen complex, mix well and incubate at 37°C for 3-10 min, then perform magnetic separation, wash, remove supernatant to obtain magnetic bead-antigen-detection antibody complex;

[0043] (3) Add the pre-excitation solution and the excitation solution to the reaction cup containing the magnetic bead-antigen-detection antibody complex, mix well and then measure the maximum luminescence intensity;

[0044] (4) A standard curve is fitted based on the luminescence intensity detected by the standard sample, and the interleukin-23 content in the sample to be tested is calculated through the standard curve.

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

[0046] 1. This invention provides a kit for the detection of interleukin-23, comprising magnetic beads, interleukin-23 antibody, acridine ester, magnetic bead preservation solution, acridine ester preservation solution, diluent, pre-activation solution, and activation solution. This kit can be used to determine interleukin-23 in serum with high accuracy and high signal-to-noise ratio.

[0047] 2. The benzoic acid in the pre-activation solution of this invention is a weak acid, which replaces conventional strong acids such as nitric acid. It has low corrosiveness to the pipeline of the detection instrument, high safety for operators, and ensures long-term stability. Detailed Implementation

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

[0049] Example 1: Preparation and Application of the Reagent Kit

[0050] (1) Preparation of basic solution

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

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

[0053] 1.3 Preparation of pre-activation 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 activation 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 cleaning solution: Prepare 0.2M MES buffer and add 0.02% (v / v) Proclin300.

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

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

[0058] (2) Preparation of antibodies coated with magnetic beads

[0059] 2.1 Take 200 μL of magnetic beads (EM1-100 / 40 (high carboxyl) magnetic microspheres, catalog number 23710087) and add them to a 2 mL centrifuge tube. Perform magnetic separation for 3 min and then discard the supernatant.

[0060] 2.2 Add 400 μL of magnetic bead cleaning solution to the centrifuge tube, shake to mix, magnetically separate to remove the supernatant, 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 to ensure the liquid flows steadily downwards when inverted.

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

[0065] 2.7 Add 120 μg of interleukin-23 antibody (purchased from ABCAM, catalog number AB314486) to a centrifuge tube, and bring the volume to 300 μL with 0.01 M phosphate buffer. Couple for 3 h.

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

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

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

[0069] (3) Preparation of acridinium ester labeled antibody

[0070] 3.1 Take 200 μg of interleukin-23 antibody (purchased from abcam, catalog number ab190356) and dissolve it in 0.01M phosphate buffer. Add 2 mg of acridine ester (NSP-SA-NHS, with a molar ratio of antibody to acridine ester of 1:10) to achieve a final antibody concentration of 2 mg / mL.

[0071] 3.2. React at 25°C for 3 hours on a constant temperature shaker;

[0072] 3.3 Add lysine (acridinium ester: lysine = 1:140, molar concentration ratio), block the reaction, and react for 20 min.

[0073] 3.4 After the reaction was completed, the buffer solution was replaced using a 50KD dialysis bag. The dialysis buffer was 0.01M phosphate buffer. Dialysis was performed 4 times in total, with each dialysis lasting 3 hours.

[0074] 3.5 After dialysis, add glycerol until the final antibody concentration is 0.5 mg / mL, and set aside for use.

[0075] 3.6 Dilute with acridine ester preservation solution to a final antibody concentration of 8 μg / mL to obtain acridine 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 automated chemiluminescence analyzer, and the specific operation is 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, incubate at 37℃ for 5 min, 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 ester labeled antibody working solution to the reaction vessel containing the magnetic bead-antigen complex, mix well, incubate at 37°C for 5 min, 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-excitation solution and 100 μL of excitation solution to the reaction vessel containing the magnetic bead-antigen-detection antibody complex, mix thoroughly, and then measure the maximum luminescence intensity.

[0081] 4.4. Based on the luminescence intensity obtained from the standard sample, a standard curve is fitted, and the interleukin-23 content in the sample to be tested is calculated through the standard curve.

[0082] Example 2: Preparation and Application of the Reagent Kit

[0083] The procedure was carried out according to Example 1, except that the concentration of benzoic acid in the pre-activation solution was 0.01 mol / L and the concentration of hexadecyltrimethylammonium bromide in the activation solution was 8 g / L.

[0084] Example 3: Preparation and Application of the Reagent Kit

[0085] The procedure was carried out according to Example 1, except that the concentration of benzoic acid in the pre-activation solution was 0.05 mol / L and the concentration of hexadecyltrimethylammonium bromide in the activation solution was 3 g / L.

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

[0087] The procedure was carried out according to Example 1, except that benzoic acid was not added to the pre-activation solution.

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

[0089] The procedure was carried out according to Example 1, except that cetyltrimethylammonium bromide was not added to the activating solution.

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

[0091] The procedure was carried out according to Example 1, except that benzoic acid was not added to the pre-activation solution and hexadecyltrimethylammonium bromide was not added to the activation solution.

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

[0093] The procedure was carried out according to Example 1, except that the concentration of benzoic acid in the pre-activation solution was 0.07 mol / L.

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

[0095] The procedure was carried out according to Example 1, except that the concentration of hexadecyltrimethylammonium bromide in the activating solution was 10 g / L.

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

[0097] The procedure was carried out according to Example 1, except that benzoic acid in the pre-activation solution was replaced with an equimolar amount of nitric acid.

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

[0099] The procedure was carried out according to Example 1, except that the hexadecyltrimethylammonium bromide in the activating solution was replaced with an equimolar amount of hexadecyltrimethylammonium chloride.

[0100] Example 1: Validation of the reagent kit's detection efficacy

[0101] In this example, interleukin-23 antigen (purchased from Beijing Bio-Rad Laboratories, catalog number JN0403) was used as the standard, and PBS buffer containing 10% fetal bovine serum was used as the matrix solution. Interleukin-23 antigen was diluted at a certain ratio to obtain a series of quality control samples of different concentrations. 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 was considered to be statistically significant.

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

[0104] Test sample: Acridinium ester (NSP-SA-NHS) was diluted to 1.5 × 10⁻⁶ with diluent. -7 The sample concentration was mol / L (abbreviated as acridinium ester sample); the diluent was used as a blank sample. The diluent was prepared by adding 1% BSA, 0.1% Triton X-100 and 0.3% Proclin 300 to a 0.01M phosphate buffer at pH 7.4.

[0105] Measurement method: 50 μL of test sample + 100 μL of pre-activation solution + 100 μL of activation solution, react for 2 min, and test the luminescence value (RLU) in a fully automated chemiluminescence immunoassay analyzer. In this experiment, the test samples included acridinium ester sample (S) and blank sample (N), each measured in triplicate. The luminescence value (S) of the acridinium ester sample and the luminescence value (N) of the blank sample were measured, and the ratio of the two (S / N) is the signal-to-noise ratio. The higher the S / N value, the better the performance. The results are shown in Table 1, where the luminescence value data are the average values ​​after three parallel measurements. It can be seen that the combined use of benzoic acid and CTAB in this invention (Examples 1-3) has a synergistic effect and significantly improves the signal-to-noise ratio compared with the use of benzoic acid (Comparative Example 1) and CTAB (Comparative Example 2) alone.

[0106] Table 1 compares the signal-to-noise ratios of different reagent kits.

[0107]

[0108]

[0109] Note: The same letter in the same column indicates no significant difference between groups (P>0.05), while different letters indicate significant differences between groups (P<0.05).

[0110] (2) Stability of the activating fluid system

[0111] The activation liquid system consists of a pre-activation liquid and an activation liquid.

[0112] Test sample: Acridinium ester (NSP-SA-NHS) was diluted to 1.5 × 10⁻⁶ with diluent. -7 The concentration of acridine ester (mol / L) is used as a blank sample; the diluted solution is used as a blank sample.

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

[0114] Measurement method: 50 μL of test sample + 100 μL of pre-activation solution + 100 μL of activation solution, react for 2 min, and test the luminescence value (RLU) in a fully automated chemiluminescence immunoassay analyzer. Acridinium ester sample (S) and blank sample (N) were measured in parallel three times. The ratio of the luminescence value (S) of the acridinium ester sample and the luminescence value (N) of the blank sample (S / N) is the signal-to-noise ratio. Activation solution systems with different opening times were tested, and the statistical results are shown in Table 2. The data in Table 2 are the average values ​​after three parallel measurements. It can be seen that the pre-activation solution and activation solution (Examples 1-3) provided by this invention maintain good stability after 60 days of opening at room temperature.

[0115] Table 2. Opening stability of the test activation fluid system

[0116]

[0117]

[0118] (3) Limit of detection

[0119] The IL-23 quality control sample with a concentration of 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, which is no greater than 1.50 pg / mL.

[0120] Table 3 compares the limits of detection for different kits.

[0121]

[0122] (4) Accuracy

[0123] Accuracy tests were performed on high and low concentration quality control samples (500 pg / mL and 20 pg / mL), and the tests were repeated three times consecutively. 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 compares the accuracy of different reagent 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, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart 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 contains a pre-activation solution and an activation solution; the pre-activation solution contains 0.03-0.15 mol / L hydrogen peroxide and 0.01-0.05 mol / L benzoic acid; the activation solution contains 0.5-1.5 mol / L sodium hydroxide and 3-8 g / L cetyltrimethylammonium bromide; the kit also contains magnetic beads, interleukin-23 antibody, acridine ester, magnetic bead preservation solution, and acridine ester preservation solution.

2. The reagent kit according to claim 1, characterized in that, The pre-activation solution contains 0.05-0.10 mol / L hydrogen peroxide and 0.01-0.05 mol / L benzoic acid; the activation solution contains 0.8-1.2 mol / L sodium hydroxide and 3-8 g / L cetyltrimethylammonium bromide.

3. The reagent kit according to claim 2, characterized in that, The pre-activation solution contains 0.08 mol / L hydrogen peroxide and 0.01-0.05 mol / L benzoic acid; the activation solution contains 1.0 mol / L sodium hydroxide and 3-8 g / L cetyltrimethylammonium bromide.

4. The reagent kit according to claim 1, characterized in that, The magnetic bead preservation solution contains, by volume fraction, 70%-80% phosphate buffer, 5%-15% fetal bovine serum, 5%-15% glycerol, 0.05%-0.15% Proclin 300 and 0.05%-0.15% Tween 20.

5. The reagent kit according to claim 4, characterized in that, The magnetic bead preservation solution contains, by volume fraction, 79.8% phosphate buffer, 10% fetal bovine serum, 10% glycerol, 0.1% Proclin 300 and 0.1% Tween 20.

6. The reagent kit according to claim 1, characterized in that, The acridinium ester preservation solution contains 69.9% phosphate buffer, 30% glycerol and 0.1% Proclin 300 by volume, plus 1.0% BSA by mass.

7. The reagent kit according to any one of claims 4-6, characterized in that, The concentration of the phosphate buffer solution is 0.005-0.1M.

8. The reagent kit according to claim 7, characterized in that, The concentration of the phosphate buffer solution is 0.01M.

9. The application of the kit according to any one of claims 1-8 in in vitro sample detection, characterized in that, The application in question is not for disease diagnosis or treatment.

10. The application according to claim 9, characterized in that, The in vitro samples include body fluid samples.

11. The application according to claim 10, characterized in that, The body fluid sample includes serum or plasma.

12. The method of using the reagent kit according to claim 1, characterized in that, The method of use includes preparation and detection using magnetic beads, interleukin-23 antibody, acridine ester, magnetic bead preservation solution, acridine ester preservation solution, the pre-activation solution, and the activation solution; and the method of use is applied in non-disease diagnosis or treatment fields.

13. The method of use according to claim 12, characterized in that, Includes the following steps: Detection of antibody-coated magnetic beads, acridine ester-labeled antibodies, and interleukin-23 content in samples.

14. The method of use according to claim 13, characterized in that, The antibody-coated magnetic beads include the following steps: S1. Add magnetic beads to a centrifuge tube, perform magnetic separation, and discard the supernatant; add magnetic bead cleaning solution to the centrifuge tube, mix well, perform magnetic separation, discard the supernatant, and wash. S2. Prepare MES buffer, EDC solution and NHS solution. Add EDC solution and NHS solution to centrifuge tube, mix well and activate. After activation, wash with MES buffer and remove supernatant. S3. Add interleukin-23 antibody to centrifuge tube, couple, magnetically separate and remove supernatant, add magnetic bead blocking solution for blocking, wash after blocking and remove supernatant. S4. Add magnetic bead preservation solution to wash, remove supernatant, and then resuspend in magnetic bead preservation solution to obtain magnetic bead coated working solution.

15. The method of use according to claim 13, characterized in that, The acridine ester-labeled antibody includes the following steps: a. Take interleukin-23 antibody and add acridine ester at a molar ratio of antibody to acridine ester of 1:5-1:

15. 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 acridine ester to lysine of 1:120-1:160 for blocking, and react for 15-30 min; c. After the blocking is completed, use a 40-60KD dialysis bag to replace the buffer solution, and dialyze 3-5 times in total, each time for 2-3 hours; after dialysis, add glycerol until the final antibody concentration is 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 the acridinium ester labeled antibody working solution.

16. The method of use according to claim 13, characterized in that, The detection of interleukin-23 content in the sample includes the following steps: (1) Take a sample, add magnetic bead coating working solution, mix well and incubate at 37°C for 3-10 min, then separate by magnetic separation, wash, remove supernatant to obtain magnetic bead-antigen complex; (2) Add acrid ester labeled antibody working solution to the reaction cup containing magnetic bead-antigen complex, mix well and incubate at 37°C for 3-10 min, then separate magnetically, wash, remove supernatant to obtain magnetic bead-antigen-detection antibody complex; (3) Add the pre-excitation solution and the excitation solution to the reaction cup containing the magnetic bead-antigen-detection antibody complex, mix well and then measure the maximum luminescence intensity; (4) A standard curve is fitted based on the luminescence intensity detected by the standard sample, and the content of interleukin-23 in the sample to be tested is calculated through the standard curve.

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