Kit and method for determining IL-12 based on latex immunoturbidimetry

Abstract

An embodiment of the present invention discloses a kit and method for determining IL-12 based on latex immunoturbidimetry. The present invention uses latex immunoturbidimetry to determine interleukin-12, which is not only simple to operate, convenient and fast to detect, but also has high sensitivity, strong specificity and good repeatability. The present invention is used on an automatic biochemical analyzer, and the detection is carried out automatically, which is more convenient, fast, time-saving, suitable for large-scale detection, and conducive to clinical promotion.

Description

Technical Field

[0001] The embodiments of the present invention relate to the technical field of bioanalysis, and particularly to a kit and method for determining IL-12 based on latex immunoturbidimetry. Background Art

[0002] Interleukin-12, abbreviated as IL-12, is a glycoprotein with a molecular weight of 75KD. It consists of two subunits with molecular weights of 40KD and 35KD respectively, called P40 and P35, which are bound together by two disulfide bonds. IL-12 is mainly produced by activated inflammatory cells (mononuclear macrophages, dendritic cells, and other antigen-presenting cells), and is a small molecule protein with a wide range of biological activities.

[0003] As an important regulatory factor in the process of cellular immunity, IL-12 plays an important role in anti-infection immunity and anti-tumor immunity. IL-12 can induce T cell proliferation, effectively promote the production of TH1, enhance the activity of TH1, and also promote the activities of NK cells and LAK cells. Both can increase the concentrations of TNF, IFN-γ, and IL-2. The cytokines such as TNF, IFN-γ, and IL-12 produced can promote the immune response of cells, and can also assist CTL cells to exert their killing function, thus playing an anti-infection and anti-tumor role. Under normal circumstances, when the body has not experienced viral, bacterial infections, or autoimmune reactions, the antigen-presenting cells of the body are not stimulated, and interleukin 12 is produced less and is in a low level state; when a patient is infected with an inflammation, the immune effector cells are stimulated, and the secretion of IL-12 increases and the concentration will rise. Therefore, IL-12 can be used for the assessment of inflammation, and relevant studies have been conducted on hepatitis, myocarditis, meningitis, asthma, etc. In addition, IL-12 can inhibit angiogenesis and also plays an important role in tumor immunotherapy, and the combined use with tumor surgery has also significantly reduced the recurrence of tumor patients after surgery.

[0004] Currently, the main method for detecting IL-12 is enzyme-linked immunosorbent assay (ELISA). The ELISA method is relatively cumbersome to operate, has a long detection time, and has many interfering factors. However, using latex immunoturbidimetry to determine IL-12 on a biochemical analyzer is simple to operate, convenient and fast to detect, has high sensitivity, strong specificity, and good repeatability. Moreover, this test method can be used on various biochemical analyzers and has strong applicability. Summary of the Invention

[0005] Therefore, the embodiments of the present invention provide a kit and method for determining IL-12 based on latex immunoturbidimetry.

[0006] In order to achieve the above object, the embodiments of the present invention provide the following technical solutions:

[0007] According to the first aspect of the embodiments of the present invention, the present invention provides a method for preparing a latex microsphere solution coated with an IL-12 antibody, comprising the following steps:

[0008] 1) Add latex microspheres to a buffer solution to obtain an activated latex microsphere system;

[0009] 2) Add an IL-12 antibody to the activated latex microsphere system, stir, and then add an EDC·HCl solution and an NHS solution to carry out a coupling reaction to obtain a coupling system;

[0010] 3) Add a blocking solution to the coupling system for blocking, and then carry out centrifugation to obtain a mixed system;

[0011] 4) Add a storage solution to the mixed system and store overnight to obtain the latex microsphere solution coated with the IL-12 antibody;

[0012] Wherein, the buffer solution is composed of a MES buffer solution and a boric acid buffer solution with a volume ratio of 1:3 to 5. The MES buffer solution uses water as a solvent and includes MES·H 2 O 4.5 to 5.0 g / L, NaOH 0.2 to 0.5 g / L, and the pH is 6.0 to 6.5; the boric acid buffer solution uses water as a solvent and includes boric acid 2.5 to 4.5 g / L, sodium tetraborate 0.5 to 1.0 g / L, and the pH is 7.2 to 7.3.

[0013] The present invention discovers that when using EDC as an activator to prepare a conjugate of an IL-12 antibody and latex microspheres by a "one-step method", the latex microspheres aggregate into clusters with low coupling efficiency. Therefore, the inventor uses a "two-step method" for the coupling reaction and studies that the addition method of the buffer solution and materials significantly affects the binding of the IL-12 antibody to the latex microspheres. After the latex microspheres are activated in a specific buffer solution composed of a MES buffer solution and a boric acid buffer solution, adding the IL-12 antibody and then adding EDC and NHS is beneficial to the efficient binding of the IL-12 antibody to the latex microspheres, and the stability of the latex microspheres coated with the IL-12 antibody is greatly improved. Preferably, the volume ratio of the MES buffer solution to the boric acid buffer solution is 1:4. The MES buffer solution uses water as a solvent and includes MES·H 2 O 4.72 g / L, NaOH 0.37 g / L, and the pH is 6.5; the boric acid buffer solution uses water as a solvent and includes boric acid 3.12 g / L, sodium tetraborate 0.88 g / L, and the pH is 7.2.

[0014] As a preferred embodiment of the present invention, the latex microspheres are polystyrene carboxyl particles with a particle size of 220 - 260 nm, preferably 240 nm; the volume ratio of the latex microspheres to the buffer solution is 1:2.5 - 5, preferably 1:4. The present invention further discovers that using the above-mentioned latex microspheres with a larger particle size is more conducive to improving the stability of the latex microspheres coated with IL-12 antibody, thereby improving the detection sensitivity and accuracy of the kit.

[0015] As a preferred embodiment of the present invention, the IL-12 antibody is prepared using an IL-12 mutant as an immunogen, and the IL-12 mutant is obtained by mutating the 66th serine in the amino acid sequence of the IL-12β protein to threonine. The present invention screens for IL-12 antibodies with high titers by mutating and modifying the IL12 antigen. Preferably, the IL-12 antibody is a murine anti-IL-12 monoclonal antibody.

[0016] As a preferred embodiment of the present invention,

[0017] the concentration of the IL-12 antibody is 0.8 - 1.2 mg / ml, preferably 1 mg / ml;

[0018] the final concentration of EDC·HCl is 0.03 - 0.06 g / L, preferably 0.05 g / L;

[0019] the final concentration of NHS is 0.1 - 0.5 g / L, preferably 0.4 g / L;

[0020] The blocking solution uses water as a solvent and includes Tween 20 with a final concentration of 1.5 - 2.5 mL / L and BSA with a final concentration of 0.8 - 1.2 g / L, and the pH is 7.2 - 7.3; preferably, the final concentration of Tween 20 is 2 mL / L, the final concentration of BSA is 1 g / L, and the pH is 7.2;

[0021] The storage solution uses water as a solvent and includes Tween 20 with a final concentration of 1.5 - 2.5 ml / L, BSA with a final concentration of 0.8 - 1.2 g / L, and Proclin300 with a final concentration of 0.3 - 0.6 ml / L, and the pH is 7.2 - 7.3; preferably, the final concentration of Tween 20 is 2 ml / L, the final concentration of BSA is 1 g / L, and the final concentration of Proclin300 is 0.5 ml / L, and the pH is 7.3;

[0022] The volume concentration of the latex microspheres in the latex microsphere solution is 1% - 2%.

[0023] According to the second aspect of the embodiments of the present invention, the present invention provides a latex microsphere solution coated with IL-12 antibody, which is prepared by the method described in any one of the above.

[0024] According to the third aspect of the embodiments of the present invention, the present invention provides a kit for determining IL-12 based on latex immunoturbidimetry, comprising reagent R1 and latex reagent R2.

[0025] The components and contents of the reagent R1 are as follows: buffer 50 - 350 mmol / L; inorganic salt 0.05 - 0.5 mol / L; preservative 0.01 - 5%, and the solvent is pure water.

[0026] The components and contents of the latex reagent R2 are as follows: buffer 50 - 350 mmol / L; stabilizer 0.5 - 5%; surfactant 0.2 - 2%; preservative 0.02 - 5%; latex microsphere solution coated with IL-12 antibody as described in claim 5, 1 - 2%; the balance is pure water.

[0027] As a preferred embodiment of the present invention, the buffer is Tris-HCl buffer; the inorganic salt is sodium chloride; the preservative is Proclin300; the stabilizer is BSA; the surfactant is Tween 20.

[0028] As a preferred embodiment of the present invention, the kit further comprises a calibrator, which contains 50% human serum, 40 mmol / L tris(hydroxymethyl)aminomethane buffer, 5% polyoxyethylene fatty acid ester, 0.15 mol / L sodium chloride, 0.03% Proclin300, and IL-12 with a target concentration range of 1500 - 2500 pg / mL.

[0029] As a preferred embodiment of the present invention, the kit further comprises a quality control product, which contains 50% human serum, 40 mmol / L tris(hydroxymethyl)aminomethane buffer, 5% polyoxyethylene fatty acid ester, 0.15 mol / L sodium chloride, 0.03% Proclin300, and IL-12 with target concentration ranges of 40 - 60 pg / mL and 500 - 1000 pg / mL.

[0030] According to the fourth aspect of the embodiments of the present invention, the present invention provides a method for determining IL-12 using the kit as described above, comprising the following steps:

[0031] Pipette 2 μL of the sample to be tested, add 200 μL of the reagent R1, and incubate at 37°C for 5 min.

[0032] Add 40 μL of the latex reagent R2, incubate at 37°C for 30 s, and read the absorbance A1.

[0033] Continue to incubate at 37°C for 5 min, and read the absorbance A2.

[0034] Calculate ΔA = A2 - A1, and measure the content of IL-12 in the sample to be tested according to ΔA.

[0035] The embodiments of the present invention have the following advantages:

[0036] The present invention uses latex immunoturbidimetry to determine interleukin-12, which is not only simple in operation, convenient and fast in detection, but also high in sensitivity, strong in specificity and good in repeatability.

[0037] The present invention is used on an automatic biochemical analyzer, and the detection is carried out automatically, which is more convenient, fast, time-saving, suitable for large-scale detection, and conducive to popularization in clinical practice. Brief Description of the Drawings

[0038] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only exemplary, and for those of ordinary skill in the art, without creative efforts, other implementation drawings can also be obtained according to the provided drawings.

[0039] Figure 1 It is a preparation method of a latex microsphere solution coated with interleukin-12 antibody provided by an embodiment of the present invention;

[0040] Figure 2 It is a schematic diagram of the fitting curve of a kit provided by an embodiment of the present invention. Detailed Embodiments

[0041] The following specific embodiments illustrate the embodiments of the present invention. Those familiar with this technology can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of them. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0042] Example 1 Preparation of a Latex Microsphere Solution Coated with IL-12 Antibody

[0043] As Figure 1 shown, its preparation method includes the following steps:

[0044] Step 1: Suspend 250 μL of latex microspheres (polystyrene carboxyl particles with a particle size of 240 nm) with 5 ml of reaction buffer (1 ml of the first reaction buffer + 4 mL of the second reaction buffer), and keep the whole reaction system stirred at a constant temperature of 28°C for 90 min to obtain an activated latex microsphere system;

[0045] In this example, the first reaction buffer is selected as MES buffer, including 4.72 g / L of MES·H2 For the first reaction buffer, select a phosphate buffer containing 0.37 g / L of NaOH with a pH value of 6.5; for the second reaction buffer, select a boric acid buffer containing 3.12 g / L of boric acid and 0.88 g / L of sodium tetraborate with a pH value of 7.2.

[0046] Step 2: Add IL-12 antibody to the reaction system of Step 1 with a final concentration of 1 mg / ml, and stir for 90 min under the constant temperature condition of 28°C. Among them, the IL-12 antibody can be a commercially available antibody or obtained through immune screening. The immune screening method is a conventional method in the art. Using IL-12 as a template, amplify and mutate by modifying primers. After detecting successful mutation, transform the cloned strain for plasmid extraction. Subsequently, expression in 293T, animal immunization, and antibody screening are all routine experimental technical operations. Observe various biochemical indexes, and finally select the antibody immunized with the antigen of 66S>T. The IL-12 antibody used in this example is a mouse monoclonal antibody with a high titer screened by ELISA.

[0047] Step 3: Add EDC·HCl solution and NHS solution to the reaction system of Step 2 in sequence. The final concentration of EDC·HCl is 0.05 g / L, and the final concentration of NHS is 0.4 g / L. Stir for 90 min under the constant temperature condition of 28°C. The NHS solution is prepared freshly before use.

[0048] Step 4: Add Tween 20 and BSA to the reaction system of Step 3 for blocking. The final concentration of Tween 20 is 2 mL / L, the final concentration of BSA is 1 g / L, the pH is 7.2. Stir for 90 min at 28°C and then centrifuge for 30 min.

[0049] Step 5: Add a storage solution containing Tween 20, BSA, and Proclin 300 to the reaction system of Step 4. The final concentration of Tween 20 is 2 mL / L, the final concentration of BSA is 1 g / L, and the final concentration of Proclin 300 is 0.5 mL / L, with a pH of 7.3. Stir for 90 min under the constant temperature condition of 28°C.

[0050] Step 6: Store overnight at 37°C to obtain a latex microsphere solution coated with IL-12 antibody, where the volume concentration of the latex microspheres is 1.5%.

[0051] Preparation of the Kit in Example 2

[0052] The main components of the kit are shown in Table 1.

[0053] Table 1 Kit

[0054]

[0055]

[0056] 1) Preparation of R1 and R2

[0057] Prepare Reagent 1 and Reagent 2 according to Table 1.

[0058] 2) Preparation of calibrators

[0059] Prepare a series of calibrators by diluting IL-12 with the calibrator diluent, with concentrations of 0 pg / mL, 125 pg / mL, 250 pg / mL, 500 pg / mL, 1000 pg / mL, and 2000 pg / mL respectively.

[0060] 3) Preparation of quality control samples

[0061] Before use, reconstitute the quality control sample with 1.00 mL of purified water, place it at 10°C to 30°C for 30 minutes. After the lyophilized product is completely dissolved, gently mix it to obtain the quality control sample solution for use.

[0062] 4) Establishment of calibration curve

[0063] React 6 IL-12 calibrators with concentrations ranging from 0 to 2000 pg / mL respectively with the prepared IL-12 latex reagent in the reaction cup, and detect them with a biochemical analyzer, recording the reactivity. The detection wavelength is 570 nm, the transmitted light intensity is detected, and the endpoint method is used for detection.

[0064] Performance detection of the kit in Example 3

[0065] (1) Detection method of the kit

[0066] 1. Reagent preparation

[0067] The liquid R1 and R2 reagents can be used directly after opening the bottle. Before use, reconstitute the calibrators and quality control samples with 1.00 mL of purified water, place them at 10°C to 30°C for 30 minutes. After the lyophilized product is completely dissolved, gently mix them to obtain the original calibrator solution and quality control sample solution for use. Dilute the original calibrator solution with purified water according to the following table (volume ratio) to six concentration gradients, and the dilution method is shown in Table 2:

[0068] Table 2 Dilution ratio of the original calibrator

[0069] Serial number 1 2 3 4 5 6 Purified water: stock solution 1:0 15:1 7:1 3:1 1:1 0:1

[0070] 2. Parameter setting

[0071] Test method: endpoint method Detection temperature: 37 °C Detection wavelength: 570 nm Reaction direction: upward reaction Optical path of cuvette: 1 cm Blank control; deionized water

[0072] 3. Detection steps

[0073]

[0074] 4. Calibration procedure

[0075] Adopt multi-point calibration and correction.

[0076] 5. Procedure

[0077] Before detecting the samples every day, quality control must be carried out to ensure the stability of the test system.

[0078] 6. Calculation

[0079] Sample concentration = (ΔA of sample / ΔA of calibration) × concentration of calibrator

[0080] (2) Detect various indicators of the kit

[0081] I. Verification of linear correlation

[0082] In the range of [10, 2000] pg / mL, dilute the high-value sample close to the upper limit of the linear range with a low-value sample close to the lower limit of the linear range, mix into at least 5 samples with different concentrations, measure each sample 3 times and take the average value, conduct linear regression analysis on the measured values, and calculate that the correlation coefficient of linear regression should be ≥ 0.9900.

[0083] The specific data comparison values are shown in Table 3.

[0084] Table 3 Linear detection results

[0085]

[0086] Figure 2 is the linear regression graph, as Figure 2 shown, the straight line fitting curve is y = 1.0024x + 2.4087, R 2 = 1.0 > 0.9900 and P < 0.05, meeting the requirements.

[0087] II. Verification of precision

[0088] Use the reagent prepared in Example 2 to determine the within-batch precision and between-day precision. Select two levels of quality control sera, low value (50) and high value (800), as evaluation samples. For within-batch precision: continuously repeat the determination 20 times within a short time and under stable conditions, record the determination results each time and calculate the mean (Mean), standard deviation (SD), and coefficient of variation (CV); for between-day precision: measure the evaluation sample once a day, accumulate 20 results, and calculate the mean (Mean), standard deviation (SD), and coefficient of variation (CV). The experimental results are shown in Tables 4-1 and 4-2.

[0089] Table 4-1 Detection results of between-batch repeatability

[0090]

[0091]

[0092] Table 4-2 Results of within-day repeatability test

[0093]

[0094] It can be concluded from Tables 4-1 and 4-2 that the highest within-batch CV of the kit is 1.09%, and the highest between-day CV is 2.04%, meeting the requirement of ≤4%.

[0095] III. Accuracy verification

[0096] Take quality controls at two levels (high and low), perform measurements according to the requirements, measure each level 3 times, and all 3 results should meet the relevant deviation between the measured value and the target value of the quality control product ≤10%. If only 1 or 0 of the 3 results meet the requirements, it indicates that the accuracy of the reagent does not meet the requirements. If all 3 results meet the requirements, the accuracy is verified. If 2 of the 3 results meet the requirements and 1 does not, 20 consecutive measurements should be performed again, and the relative deviation between each measured value and the target value should be calculated. When ≥19 results meet the requirements, the accuracy is also verified. The experimental results are shown in Table 5.

[0097] Table 5 Results of accuracy test

[0098]

[0099] It can be seen from the test results that the highest relative deviation of the low-value quality control is 2.40%, and the highest relative deviation of the high-value quality control is 1.95%, both of which are less than 10%, meeting the requirements.

[0100] IV. Reagent stability after opening the bottle

[0101] Divide two fresh serum specimens (high and low values) without hemolysis, jaundice, and lipemia into 30 portions each, store them frozen at -80°C for later use, take them out of the refrigerator and warm them to room temperature and mix well before testing. Load reagents R1 and R2 into the corresponding reagent compartment of the automatic biochemical analyzer (Toshiba 40), place them with the lid open within the allowable temperature range of the instrument's reagent compartment, do not replace the reagents and calibration after calibration on the same day, measure 1 portion each of the high- and low-value serum specimens after the quality control is qualified every day, repeat 3 times respectively, and calculate the mean value and relative deviation. Use the relative deviation < ±10% as the judgment limit for the stability of the reagent. The experimental results are shown in Table 6.

[0102] Table 6 Results of reagent stability test after opening the kit

[0103]

[0104]

[0105] As can be seen from Table 6, the stability standard of the prepared reagent is far lower than 10%, even less than 2.5%, indicating that the reagent prepared by the present invention has good stability.

[0106] V. Evaluation of Interference Ability

[0107] According to the recommendations of the CLSI EP7-A2 guideline, the anti-interference performance of interleukin-12 was evaluated by adding exogenous interferents. Two fresh serum specimens without hemolysis, jaundice, and lipemia with high and low interleukin-12 concentrations were selected. The interferents (bilirubin, hemoglobin, triglyceride, ascorbic acid, rheumatoid factor, and fat emulsion) and normal saline were mixed with the high-value serum specimen at a ratio of 1:9 to form Specimen A and Specimen B. A and B were mixed at ratios of 3:0, 2:1, 1:2, and 0:3 to form 4 test specimens, and each specimen was measured three times repeatedly. The interference experiment for the low-value serum was performed in the same way. The interference value was obtained by subtracting the mean value of the corresponding group without the interferent from the mean value of each group of each specimen, and the interference rate was calculated. A relative deviation < 10% was used as the judgment limit for the interferent of this detection method. If it was within the range, it was judged as no obvious interference, otherwise it was judged as having obvious interference. The interference rate (%) = interference value / base value × 100%. The experimental results are shown in Tables 7 - 12.

[0108] Table 7 Results of Bilirubin Interference Test

[0109]

[0110] Table 8 Results of Hemoglobin Interference Test

[0111]

[0112] Table 9 Results of Triglyceride Interference Test

[0113]

[0114] Table 10 Results of Ascorbic Acid Interference Test

[0115]

[0116] Table 11 Results of Rheumatoid Factor Interference Test

[0117]

[0118]

[0119] Table 12 Results of Fat Emulsion Interference Test

[0120]

[0121] As can be seen from Table 7-12, when the kit prepared in Example 2 was used to detect samples containing the following concentrations of interferents, it had no effect on the test results: bilirubin ≤ 20 mg / dL, hemoglobin ≤ 3 g / L, triglyceride ≤ 270 mg / dL, ascorbic acid ≤ 20 mg / dL, rheumatoid factor ≤ 500 IU / mL, and fat emulsion ≤ 750 mg / dL.

[0122] In summary, the kit and method for determining interleukin-12 based on latex immunoturbidimetry provided by the present invention can effectively improve the sensitivity and specificity of interleukin-12 detection, have better repeatability, strong anti-interference ability, good reagent stability, realize automatic detection on a biochemical analyzer, are simple to operate, convenient and fast to detect, and are convenient for clinical promotion.

[0123] Although the present invention has been described in detail with general descriptions and specific embodiments above, some modifications or improvements can be made based on the present invention, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of the present invention claimed.

Claims

1. A method for preparing a latex microsphere solution coated with an IL-12 antibody, characterized in that, it comprises the following steps: Step 1: Suspend 250 μL of polystyrene carboxyl particles with a particle size of 240 nm in 1 mL of a first reaction buffer and 4 mL of a second reaction buffer, and stir the entire reaction system at a constant temperature of 28 °C for 90 min to obtain an activated latex microsphere system; The first reaction buffer is selected as MES buffer, including 4.72 g / L of MES·H 2 O, 0.37 g / L of NaOH, with a pH value of 6.5; the second reaction buffer is selected as boric acid buffer, including 3.12 g / L of boric acid, 0.88 g / L of sodium tetraborate, with a pH value of 7.2; Step 2: Add an IL-12 antibody to the reaction system of Step 1 with a final concentration of 1 mg / ml, and stir at a constant temperature of 28 °C for 90 min. The IL-12 antibody is prepared using an IL-12 mutant as an immunogen, and the IL-12 mutant is obtained by mutating serine at position 66 in the amino acid sequence of the IL-12β protein to threonine; Step 3: Add an EDC·HCl solution and an NHS solution to the reaction system of Step 2 successively. The final concentration of EDC·HCl is 0.05 g / L, and the final concentration of NHS is 0.4 g / L, and stir at a constant temperature of 28 °C for 90 min; Step 4: Add Tween 20 and BSA to the reaction system of Step 3 for blocking. The final concentration of Tween 20 is 2 mL / L, the final concentration of BSA is 1 g / L, and the pH is 7.

2. Stir at a constant temperature of 28 °C for 90 min and then centrifuge for 30 min; Step 5: Add a storage solution containing Tween 20, BSA, and Proclin 300 to the reaction system of Step 4. The final concentration of Tween 20 is 2 mL / L, the final concentration of BSA is 1 g / L, and the final concentration of Proclin 300 is 0.5 mL / L, and the pH is 7.

3. Stir at a constant temperature of 28 °C for 90 min; Step 6: Store overnight at 37 °C to obtain a latex microsphere solution coated with an IL-12 antibody, wherein the volume concentration of the latex microspheres is 1.5%.

2. A latex microsphere solution coated with an IL-12 antibody, characterized in that, it is prepared by the method described in claim 1.

3. A kit for determining IL-12 based on latex immunoturbidimetry, characterized in that, it comprises reagent R1 and latex reagent R2, The components and contents of the reagent R1 are: buffer 50 - 350 mmol / L; inorganic salt 0.05 - 0.5 mol / L; preservative 0.01 - 5%, and the solvent is pure water; The components and contents of the latex reagent R2 are: buffer 50 - 350 mmol / L; stabilizer 0.5 - 5%; surfactant 0.2 - 2%; preservative 0.02 - 5%; 1 - 2% of the latex microsphere solution coated with an IL-12 antibody as described in claim 2, and the solvent is pure water.

4. According to the kit described in claim 3, characterized in that, the buffer is Tris-HCl buffer; the inorganic salt is sodium chloride; the preservative is Proclin 300; the stabilizer is BSA; the surfactant is Tween 20.

5. According to the kit described in claim 3, characterized in that, The kit further includes a calibrator, which contains 50% human serum, 40 mmol / L tris(hydroxymethyl)aminomethane buffer, 5% polyoxyethylene fatty acid ester, 0.15 mol / L sodium chloride, 0.03% Proclin 300, and IL-12 with a target concentration range of 1500 - 2500 pg / mL.

6. The kit according to claim 3, wherein, the kit further includes a quality control product, which contains 50% human serum, 40 mmol / L tris(hydroxymethyl)aminomethane buffer, 5% polyoxyethylene fatty acid ester, 0.15 mol / L sodium chloride, 0.03% Proclin 300, and IL-12 with a target concentration range of 40 - 60 pg / mL and 500 - 1000 pg / mL.

7. A method for determining IL-12 using the kit according to claim 3, wherein, it includes the following steps: Aspirate 2 μL of the sample to be tested, add 200 μL of the reagent R1, and incubate at 37°C for 5 min; Add 40 μL of the latex reagent R2, incubate at 37°C for 30 s, and read the absorbance A1; Continue to incubate at 37°C for 5 min, and read the absorbance A2; Calculate ΔA = A2 - A1, and measure the content of IL-12 in the sample to be tested according to ΔA.

Images