Application of buffer solution in preparation of kit for detecting cytokines based on flow cytometry

Abstract

The invention discloses application of a buffer solution in preparation of a kit for detecting cytokines based on a flow cytometer, and belongs to the technical field of flow cytometry analysis. The buffer solution is a Tris buffer solution, the Tris buffer solution further contains 0.1%-0.3% v / v of a preservative, 0.05%-0.15% m / v of inorganic salt, 0.1%-0.5% m / v of a first surfactant, 0.3%-1% v / v of a second surfactant and 0.05%-0.5% m / v of BSA (Bovine Serum Albumin), and the first surfactant is domiphen. When the buffer solution is used as a microsphere diluent for detecting cytokines based on a flow cytometer, the buffer solution still has better linearity and high sensitivity for a low-concentration sample, so that the low-concentration sample can be detected, the final detection accuracy is high, random sample abnormity can be eliminated, and the buffer solution has very important clinical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of flow cytometric analysis, and in particular relates to an application of a buffer in preparing a kit for detecting cytokines based on a flow cytometer. Background Art

[0002] Cytokines are a class of small molecule proteins with extensive biological activity that are synthesized and secreted by immune cells and certain non-immune cells after stimulation. Cytokines can be divided into interleukins, interferons, tumor necrosis factor superfamily, colony stimulating factors, chemokines, growth factors, etc. The secretion of cytokines will change under pathological conditions, such as various infectious diseases. The detection of cytokines provides a scientific auxiliary basis for the prevention, diagnosis and treatment of diseases in clinical practice.

[0003] Helper T cells (Th) are one of the subpopulations of T cells, usually referring to a type of T cell subpopulation that has the ability to assist humoral immunity and cellular immune responses. According to the different cytokines secreted, Th cells can be divided into Th1, Th2 and other cell subpopulations. At present, the cells that secrete cytokines that are studied more are Th1 and Th2 cells. Th1 cells mainly secrete proinflammatory cytokines IFN-γ, IL-2 and TNF-α, which are beneficial for B cells to produce opsonizing antibodies (IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, IgA) and complement-binding antibodies (IgM, IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ), which can stimulate macrophages, NK cells, and CD8+ cytotoxic T cells to fight intracellular pathogens, causing T lymphocyte-mediated cytotoxicity and cellular immunity. However, for patients with autoimmune diseases and underlying diseases, autoimmune antibodies in the human body (such as rheumatoid factor, anti-mitochondrial antibodies, HAMA, etc.) often interfere with the test results and produce non-specific immune reactions, that is, false positives, which may cause the test results to be different from the actual situation, thereby affecting the clinical judgment of the disease.

[0004] However, the existing kits for detecting cytokines such as IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-17A, IFN-γ, TNF-α, IFN-α, etc. have low accuracy and low sensitivity, and many positive cases cannot be detected, which is not conducive to the detection of rare samples. Summary of the invention

[0005] In order to solve at least one of the above technical problems, the inventors conducted a series of explorations on the detection reagents and detection conditions, and unexpectedly found that when domiphene is added to the microsphere diluent, the method can solve the microsphere aggregation, improve the sensitivity and accuracy of simultaneous detection of 12 cytokines, and improve the stability of the reagents, thereby completing the present invention. On this basis, the technical solution adopted by the present invention is as follows: A first aspect of the present invention provides an application of a buffer in the preparation of a kit for detecting 12 cytokines based on flow cytometry, wherein the buffer is a Tris buffer, and the Tris buffer also contains 1-3‰ v / v preservative, 5-15‰ m / v inorganic salt, 1-5‰ m / v first surfactant, 0.3-1‰ v / v second surfactant and 20-50‰ m / v BSA, wherein the first surfactant is domiphene, and the cytokines include IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, IL-17A, IL-1β, IL-5, IL-12p70, IFN-α and IL-8.

[0006] Domiphene is a quaternary ammonium cationic surfactant, which is commonly used as a chemical preservative or antibacterial agent. In the present invention, when domiphene is added to the buffer solution, it can prevent the microspheres from aggregating when used as a microsphere diluent.

[0007] In some embodiments of the present invention, the concentration of the Tris buffer is 80-150 mmol / L.

[0008] In some embodiments of the present invention, the inorganic salt is NaCl, KCl, MgCl2 and / or ZnCl2.

[0009] In some embodiments of the present invention, the preservative is at least one of the KroVin series of preservatives.

[0010] In some specific embodiments of the present invention, the KroVin series preservatives include KroVin 100, KroVin 400, KroVin 500 and KroVin 750.

[0011] In some embodiments of the present invention, the preservative is at least one of the ProClin series of preservatives.

[0012] In some specific embodiments of the present invention, the ProClin series preservatives include ProClin 50, ProClin 150, ProClin 200, ProClin 300, ProClin 950 and ProClin 5000.

[0013] In the present invention, there is no special requirement for the second surfactant. In some embodiments of the present invention, the second surfactant is selected from at least one of SDS, Triton X-100, Tween 80 and Tween 20. In some preferred embodiments of the present invention, the second surfactant is Tween 20.

[0014] In some embodiments of the present invention, the kit includes an antibody solution coupled to fluorescent microspheres, a fluorescently labeled antibody solution, and a microsphere buffer, wherein: The antibody solution coupled with fluorescent microspheres is prepared by adding IL-2 first antibody, IL-4 first antibody, IL-6 first antibody, IL-10 first antibody, TNF-α first antibody, IFN-γ first antibody, IL-17A first antibody, IL-1β first antibody, IL-5 first antibody, IL-8 first antibody, IL-12P70 first antibody and IFN-a first antibody coupled with fluorescent microspheres to the Tris buffer, and the fluorescent microspheres coupled with each first antibody have different fluorescence intensities; The fluorescently labeled antibody solution includes fluorescently labeled IL-2 second antibody, IL-4 second antibody, IL-6 second antibody, IL-10 second antibody, TNF-α second antibody, IFN-γ second antibody, IL-17A second antibody, IL-1β second antibody, IL-5 second antibody, IL-8 second antibody, IL-12P70 second antibody and IFN-a second antibody; The microsphere buffer includes 0.2% to 0.3% m / v KH2PO4, 3% to 4% m / v Na2HPO4·12H2O, 0.5% to 1.0% m / v NaCl, 0.1% to 0.5% m / v KCl, 2% to 4% m / v BSA, 0.1% to 0.2% m / v ProClin300, 0.05% to 0.1% v / v Tween-20 and 0.1% to 0.3% m / v PVP-k30.

[0015] In the present invention, the microsphere buffer is used to replace the Tris buffer of the antibody solution coupled with fluorescent microspheres when the kit is used to detect 12 cytokines in a liquid biological sample. The specific steps are: centrifuging the antibody solution coupled with fluorescent microspheres to remove the supernatant, adding the microsphere buffer, and then adding the liquid biological sample and the fluorescently labeled antibody solution simultaneously or sequentially, incubating at room temperature in the dark, and obtaining a solution to be detected.

[0016] The second aspect of the present invention provides a kit for detecting 12 cytokines based on flow cytometry, comprising an antibody solution coupled to fluorescent microspheres, a fluorescently labeled antibody solution and a microsphere buffer, wherein: The antibody solution coupled with fluorescent microspheres is prepared by adding IL-2 first antibody, IL-4 first antibody, IL-6 first antibody, IL-10 first antibody, TNF-α first antibody, IFN-γ first antibody, IL-17A first antibody, IL-1β first antibody, IL-5 first antibody, IL-8 first antibody, IL-12P70 first antibody and IFN-a first antibody coupled with fluorescent microspheres to the Tris buffer described in the first aspect of the present invention, and the fluorescent microspheres coupled with each first antibody have different fluorescence intensities; The fluorescently labeled antibody solution includes fluorescently labeled IL-2 second antibody, IL-4 second antibody, IL-6 second antibody, IL-10 second antibody, TNF-α second antibody, IFN-γ second antibody, IL-17A second antibody, IL-1β second antibody, IL-5 second antibody, IL-8 second antibody, IL-12P70 second antibody and IFN-a second antibody; The microsphere buffer includes 0.2% to 0.3% m / v KH2PO4, 3% to 4% m / v Na2HPO4·12H2O, 0.5% to 1.0% m / v NaCl, 0.1% to 0.5% m / v KCl, 2% to 4% m / v BSA, 0.1% to 0.2% m / v ProClin300, 0.05% to 0.1% v / v Tween-20 and 0.1% to 0.3% m / v PVP-k30.

[0017] In the present invention, the buffer in the fluorescent microsphere-coupled antibody solution is replaced with the microsphere buffer before detection. Specifically, the fluorescent microsphere-coupled antibody solution is centrifuged to remove the supernatant, and then an equal volume of the microsphere buffer is added.

[0018] The third aspect of the present invention provides a method for detecting 12 cytokines in a liquid biological sample using the kit described in the second aspect of the present invention, comprising the following steps: S1, centrifuging the antibody solution coupled to fluorescent microspheres to remove the supernatant, adding the microsphere buffer, the liquid biological sample and the fluorescently labeled antibody solution, incubating at room temperature in the dark, to obtain a solution to be detected; S2, detecting the fluorescence intensity of the fluorescent marker in the solution to be tested to calculate the content of different cytokines in the sample to be tested.

[0019] Compared with the prior art, the present invention has the following beneficial effects: When the buffer of the present invention is used to detect cytokines based on a flow cytometer, it still has good linearity for low-concentration samples, high sensitivity, and can detect low-concentration samples, and the final detection accuracy is high, and the random sample abnormality can be eliminated, which has very important clinical application value. It can solve the aggregation of microspheres, thereby improving the accuracy and sensitivity of the reagent. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The linear relationship between the MFI values ​​and the concentrations of the cytokine TNF-α and IFN-γ standard products detected in the microsphere diluent of formula #1 in Example 1 of the present invention is shown.

[0021] Figure 2 The distribution diagram of microspheres when the microsphere dilution solution of formula #1 is used for detection in Example 9 of the present invention is shown, A: P1=47.51% represents the proportion of normal microspheres, and P2=48.46% represents the proportion of aggregated microspheres; B: P3, P4, P5, P6, P7, P8, and P9 are the distribution diagrams of P1 in A, respectively, and the percentages are between 1.52% and 51.38%; C: P10, P11, P12, P13, P14, P15, and P16 are the distribution diagrams of P2 in A, respectively, and the percentages are between 0.19% and 23.76%.

[0022] Figure 3 The distribution diagram of microspheres when the microsphere dilution solution of formula #9 is used for detection in Example 9 of the present invention is shown, A: P1=95.93% represents the proportion of normal microspheres, and P2=3.33% represents the proportion of aggregated microspheres; B: P3, P4, P5, P6, P7, P8, and P9 are the distribution diagrams of P1 in A, respectively, and the percentages are between 10.86% and 17.24%; C: P10, P11, P12, P13, P14, P15, and P16 are the distribution diagrams of P2 in A, respectively, and the percentages are between 0.68% and 4.11%.

[0023] Figure 4 The deviation of the MFI values ​​of the microsphere diluent of formula #9 in Example 12 of the present invention detected at different concentrations of various cytokine standards after being stored at two storage temperatures is shown.

[0024] Figure 5 The detection concentration deviation of 15 clinical samples obtained after the microsphere diluent of formula #9 in Example 12 of the present invention was stored at two storage temperatures is shown. DETAILED DESCRIPTION

[0025] Unless otherwise indicated, implied from the context, or customary in the prior art, all parts and percentages in this application are based on weight, and the tests and characterization methods used are all synchronized with the filing date of this application. Where applicable, the contents of any patent, patent application or publication involved in this application are fully incorporated herein by reference, and their equivalent patent families are also introduced as references, especially the definitions of relevant terms in the art disclosed in these documents. If the definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition of the term provided in this application shall prevail.

[0026] Numerical ranges in this application are approximate values, so unless otherwise specified, they may include values ​​outside the range. Numerical ranges include all values ​​from the lower limit to the upper limit in increments of 1 unit, provided that there is an interval of at least 2 units between any lower value and any higher value. For a range containing a value less than 1 or containing a fraction greater than 1 (e.g., 1.1, 1.5, etc.), 1 unit is appropriately considered to be 0.0001, 0.001, 0.01 or 0.1. For a range containing a single digit less than 10 (e.g., 1 to 5), 1 unit is generally considered to be 0.1. These are only specific examples of what is intended to be expressed, and all possible combinations of the values ​​between the lowest and highest values ​​listed are considered to be clearly recorded in this application.

[0027] The terms "comprising", "including", "having" and their derivatives do not exclude the presence of any other components, steps or processes, and are irrelevant to whether these other components, steps or processes are disclosed in this application. To eliminate any doubt, unless explicitly stated, all compositions using the terms "comprising", "including", or "having" in this application may include any additional additives, adjuvants or compounds. On the contrary, except for those necessary for operating performance, the term "essentially consisting of..." excludes any other components, steps or processes from the scope of any description of the term below. The term "consisting of..." does not include any components, steps or processes that are not specifically described or listed. Unless explicitly stated, the term "or" refers to the listed individual members or any combination thereof.

[0028] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments.

[0029] The following examples are used to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present invention and therefore can be considered as preferred embodiments of the present invention. However, it will be appreciated by those skilled in the art based on this specification that many modifications may be made to the specific embodiments disclosed herein and still achieve the same or similar results without departing from the spirit or scope of the present invention.

[0030] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the disclosure and materials cited therein are hereby incorporated by reference.

[0031] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many technical equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0032] The experimental methods in the following examples are conventional methods unless otherwise specified. The instruments and equipment used in the following examples are conventional laboratory instruments and equipment unless otherwise specified; the experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0033] Example 1 Detection of 12 cytokines in plasma samples 1. Samples to be tested In the present invention, 15 clinical plasma samples from the infectious department were obtained for detecting 12 cytokines, the 12 cytokines were IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, IL-17A, IL-1β, IL-5, IL-12p70, IFN-α and IL-8, and the normal reference value ranges of each cytokine in the plasma samples were 0-5.71, 0-3, 0-5.3, 0-4.91, 0-4.6, 0-7.42, 0-20.6, 0-12.4, 0-3.1, 0-3.4, 0-8.5 and 0-20.6, respectively, in pg / mL.

[0034] The concentrations of various cytokines detected by 15 plasma samples using the cytokine combined detection kit (immunofluorescence method) (Jiangxi Saiji Biotechnology Co., Ltd., Gan Medical Device Registration No. 20192400359) are shown in Table 1: Table 1 Concentrations of 12 cytokines in clinical plasma samples (pg / mL)

[0035] 2. Microsphere diluent formulation The conventional microsphere diluent formulation for the assay (Formulation #1) is as follows: Based on 1000 mL, it includes 6.052 g Trizma® base, with a pH of 8.2 ± 0.1.

[0036] 3. Detection Reagents Standards: There are 11 standards in total, each of which includes the above 12 cytokines and the concentration of each cytokine is the same. The concentrations of each cytokine in each standard are 2.5pg / mL, 5pg / mL, 10pg / mL, 20pg / mL, 40pg / mL, 80pg / mL, 156pg / mL, 312pg / mL, 625pg / mL, 1250pg / mL, and 2500pg / mL, respectively.

[0037] Antibodies: IL-2 antibody 1, IL-2 antibody 2, IL-4 antibody 1, IL-4 antibody 2, IL-6 antibody 1, IL-6 antibody 2, IL-10 antibody 1, IL-10 antibody 2, TNF-α antibody 1, TNF-α antibody 2, IFN-γ antibody 1, IFN-γ antibody 2, IL-17A antibody 1, IL-17A antibody 2, IL-1β antibody 1, IL-1β antibody 2, IL-5 antibody 1, IL-5 antibody 2, IL-12p70 antibody 1, IL-12p70 antibody 2, IFN-α antibody 1, IFN-α antibody 2, IL-8 antibody 1 and IL-8 antibody 2 are the same as the antibodies disclosed in Chinese invention patent publication No. CN117368493A.

[0038] Antibody solution coupled to fluorescent microspheres: Take 12 different fluorescent intensities of polystyrene fluorescent microspheres (manufacturer Polysciences, model BLI239C-20, BLI250C-10), the concentration of each microsphere is 5×10 7Each of the first fluorescent microspheres (L1) was taken at 0.1 mL, washed twice with the microsphere diluent of formula #1, 0.5 mL of the microsphere diluent of formula #1 was added to the washed first fluorescent microspheres, 100 μg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 50 μg of N-hydroxysulfosuccinimide (NHS) were added and placed for 30 min to activate the fluorescent microspheres, 100 μg of IL-1β antibody 1 was added and rotated at room temperature for 5 h, the first fluorescent microspheres were washed with the microsphere diluent of formula #1 to remove excess antibodies, and then 5% skim milk powder was added to block for 30 min, and after removing the skim milk powder, 0.5 mL of the microsphere diluent of formula #1 was added to prepare a fluorescent microsphere solution coated with IL-1β antibody 1. According to the same method, 0.5 mL of fluorescent microsphere solutions coated with IL-2 antibody 1, IL-4 antibody 1, IL-5 antibody 1, IL-6 antibody 1, IL-8 antibody 1, IL-10 antibody 1, IL-12p70 antibody 1, IL-17A antibody 1, IFN-γ antibody 1, TNF-α antibody 1, and IFN-α antibody 1 were prepared respectively, as shown in Table 2. Different types of polystyrene microspheres can be distinguished by the fluorescence signal value of the allophycocyanin channel (APC-A) and the fluorescence signal value of the allophycocyanin-coupled Cy7 dye channel (APC-Cy7--A) on the BriCyte E6 flow cytometer produced by Mindray, where A refers to the area under the curve of the wavy signal generated when passing through the laser, representing the signal value; Cy7 refers to the name of the dye.

[0039] Table 2 Microspheres containing different antibodies 1

[0040] Take 188 μL of microsphere dilution solution and add 1 μL of fluorescent microsphere solution coated with IL-1β antibody 1, IL-2 antibody 1, IL-4 antibody 1, IL-5 antibody 1, IL-6 antibody 1, IL-8 antibody 1, IL-10 antibody 1, IL-12p70 antibody 1, IL-17A antibody 1, IFN-γ antibody 1, TNF-α antibody 1, and IFN-α antibody 1 to prepare an antibody solution coupled with fluorescent microspheres.

[0041] Fluorescently labeled antibody solution: Take IL-1β antibody 2, IL-2 antibody 2, IL-4 antibody 2, IL-5 antibody 2, IL-6 antibody 2, IL-8 antibody 2, IL-10 antibody 2, IL-12p70 antibody 2, IL-17A antibody 2, IFN-γ antibody 2, TNF-α antibody 2, and IFN-α antibody 2, add phycoerythrin fluorescein at a molar ratio of 1:30 to antibody 2, incubate at room temperature for 5 hours, and wash three times with a 50K ultrafiltration tube added with 0.01 mol / L Tris buffer to remove excess unbound phycoerythrin fluorescein to obtain phycoerythrin fluorescein-labeled IL-1β antibody 2, IL-2 antibody 2, IL-4 antibody 2, IL-5 antibody 2, IL-6 antibody 2, IL-8 antibody 2, IL-10 antibody 2, IL-12p70 antibody 2, IL-17A antibody 2, IFN-γ antibody 2, TNF-α antibody 2, IFN-α antibody 2, and phycoerythrin fluorescein-labeled IL-1β antibody 2, IL-2 antibody 2, IL-4 antibody 2, IL-5 antibody 2, IL-6 antibody 2, IL-8 antibody 2, IL-10 antibody 2, IL-12p70 antibody 2, IL-17A antibody 2, IFN-γ antibody 2, TNF-α antibody 2, and IFN-α antibody 2 were diluted to 2 μg / mL with 0.01 mol / L Tris buffer.

[0042] Take 1 μL each of phycoerythrin fluorescein-labeled IL-1β antibody 2, IL-2 antibody 2, IL-4 antibody 2, IL-5 antibody 2, IL-6 antibody 2, IL-8 antibody 2, IL-10 antibody 2, IL-12p70 antibody 2, IL-17A antibody 2, IFN-γ antibody 2, TNF-α antibody 2, and IFN-α antibody 2 at a diluted concentration of 2 μg / mL, add 36 μL of 0.01 mol / L Tris buffer and mix to prepare a fluorescently labeled antibody solution.

[0043] 1× Washing Buffer: Take 3.0275g Tris and 9.0g NaCl, dissolve in 800mL pure water, add 0.8mL ProClin300 preservative, add 0.6mL Tween 20, add 20g BSA, adjust the pH to 7.4, and obtain Washing Buffer (10×) for use. Stabilize the Washing Buffer (10×) to room temperature, wait until all salts are dissolved, take 10mL of Washing Buffer (10×) and add it to 90mL pure water to obtain Washing Buffer (1×).

[0044] Microsphere buffer: dissolve 2.4 g KH2PO4, 36.32 g Na2HPO4·12H2O, 8 g NaCl, and 2 g KCl in 1000 mL pure water, add 25 g BSA, 1.2 mL ProClin300, 0.6 mL Tween 20, and 2 mL PVP-k30 (purchased from aladdin, English name RNase-free PVP k30 Solution, CAS number 9003-39-8) for later use.

[0045] 4. Detection Methods Incubation: Take 25 μL of antibody solution coupled with fluorescent microspheres, centrifuge to remove the supernatant, add 25 μL of microsphere buffer, then add 25 μL of standard or plasma sample, and finally add 25 μL of fluorescent labeled antibody solution, mix well, and stand at room temperature in the dark for 2.5 hours (one-step incubation); Washing: Add 1000 μL of 1× washing buffer, resuspend the coupled microspheres by vortexing, mix thoroughly, shake on an oscillator for more than 30 seconds, and centrifuge to remove the supernatant; Detection: 100 μL of 1× washing buffer was added, and the fluorescence type and fluorescence signal intensity (MFI) were detected on a BriCyte E6 flow cytometer produced by Mindray.

[0046] The test results of each standard are shown in Table 3.

[0047] Table 3 MFI values ​​of each standard tested using microsphere diluent of formula #1

[0048] For the MFI value of a standard sample at a certain concentration, the MFI value of the adjacent low-concentration standard sample is used as the baseline noise, and the signal-to-noise ratio (S / N) is obtained after comparison, which is used to evaluate the resolution of different concentrations of the curve. For example, for the IL-2 cytokine, when the concentration is 10pg / mL, the MFI value is 721.20, and the MFI value of the adjacent low-concentration standard sample of 5pg / mL is 474.00. The signal-to-noise ratio of the IL-2 cytokine with a concentration of 10pg / mL is 721.20 / 474.00=1.52.

[0049] When S / N≤1.2, it indicates that it cannot be distinguished from the adjacent low concentration, thus affecting the detection sensitivity. As shown in Table 2, when the concentration of each cytokine is 5pg / mL, the S / N of cytokines IL-2, IL-4, IFN-γ, IL-1β, IFN-α and IL-8 are 1.13, 0.96, 1.03, 1.13, 1.11 and 1.04, respectively, all less than 1.2, indicating that when the microsphere diluent of formula #1 is used for detection, the standard curve cannot be applied to low-concentration samples. The double logarithmic curves of the detection results of TNF-α and IFN-γ are as follows: Figure 1 As shown, it can be seen that for the cytokine TNF-α, the standard curve has a high degree of fit, and the concentration estimated by the test results will be very accurate. However, for the cytokine IFN-γ, at low concentrations, there is a large deviation between the standard curve and the true value. Using this standard curve, the estimated sample concentration is inaccurate and will be too high, which may result in a false positive result.

[0050] The concentrations of 15 clinical plasma samples tested using microsphere diluent of formulation #1 are shown in Table 4.

[0051] Table 4 Concentrations of clinical plasma samples tested using microsphere diluent of formulation #1

[0052] For a certain cytokine, deviation = (AB) / B×100%, where A represents the concentration of the cytokine detected using the microsphere diluent of formula #1; B represents the concentration of the cytokine detected using the cytokine combined detection kit (immunofluorescence method) (Jiangxi Saiji Biotechnology Co., Ltd., Gan Medical Device Registration No. 20192400359).

[0053] As shown in Table 4, when the microsphere diluent of formula #1 was used for detection, the concentrations of some cytokines in some samples deviated greatly from the concentrations detected using the cytokine combined detection kit (immunofluorescence method) (Jiangxi Saiji Biotechnology Co., Ltd., Gan Medical Device Registration No. 20192400359), and some exceeded 1000% or even 10000%.

[0054] Furthermore, the detection accuracy of each cytokine is shown in Table 5.

[0055] Table 5 Accuracy of cytokine detection using microsphere diluent of formulation #1

[0056] As shown in Table 5, the detection accuracy of each cytokine using the microsphere diluent of formula #1 is not ideal. For some cytokines, such as IL-4 and IFN-γ, the accuracy is only 53%, which is very unreliable.

[0057] The overall accuracy of each clinical sample is shown in Table 6: Table 6 Overall accuracy of clinical plasma samples tested using microsphere diluent formulation #1

[0058] As shown in Table 6, among the 15 samples, only 2 samples had an overall accuracy of 100%, that is, the test results of all cytokines were accurate. The overall accuracy of the remaining samples was not ideal. For the clinical plasma samples, such as the plasma sample numbered 8, the overall accuracy was only 25%, that is, there were abnormal random sample measurements.

[0059] In order to further improve the accuracy of each cytokine detection and the consistency and stability of the detection results of different samples, the inventors tried to adjust the microsphere diluent.

[0060] Example 2 Microsphere diluent formula optimization - adding formula ingredients The inventors made improvements based on the microsphere diluent of formula #1 in Example 1 and obtained a new formula of microsphere diluent (formula #2), as follows: Based on 1000 mL, it includes 6.052 g Trizma® base, 1.2 mL Krovin 500, 0.6 mL Tween 20, 8 g NaCl, 25 g BSA, and the pH is 8.2±0.1.

[0061] The same method as in Example 1 was used to test each standard product. The results are shown in Table 7.

[0062] Table 7 MFI values ​​of each standard tested using microsphere diluent of formula #2

[0063] As shown in Table 7, when the microsphere diluent of formula #2 was used for detection, when the concentration of each cytokine was 5 pg / mL, the S / N of cytokine IL-5 was 1.18, which was less than 1.2, indicating that when the microsphere diluent of formula #2 was used for detection, the cytokine IL-5 standard curve was not applicable to low-concentration samples.

[0064] The concentrations of 15 clinical plasma samples tested using microsphere diluent of formulation #2 are shown in Table 8.

[0065] Table 8 Concentrations of clinical plasma samples tested using microsphere diluent of formulation #2

[0066] As can be seen from Table 8, when the microsphere diluent of formula #2 was used for detection, the concentrations of some cytokines in some samples were significantly different from those detected using the cytokine combined detection kit (immunofluorescence method) (Jiangxi Saiji Biotechnology Co., Ltd., Gan Medical Device Registration No. 20192400359), and some of them exceeded 200% or even 400%.

[0067] Furthermore, the detection accuracy of each cytokine is shown in Table 9.

[0068] Table 9 Accuracy of cytokine detection using microsphere diluent of formulation #2

[0069] As shown in Table 9, using the microsphere diluent of formula #2 for detection, only some cytokines (IL-6, TNF-α, IL-17A, IL-1β and IL-12p70) had a detection accuracy of 100%, while the detection accuracy of the remaining cytokines was still unsatisfactory.

[0070] The overall accuracy of each clinical sample is shown in Table 10: Table 10 Overall accuracy of clinical plasma samples tested using microsphere diluent formulation #2

[0071] As can be seen from Table 10, compared with the microsphere diluent of formula #1, the overall accuracy of 100% increased from 2 cases to 7 cases using the microsphere diluent of formula #2. However, there were still 4 plasma samples with an overall accuracy of only 83%, which was difficult to meet clinical testing needs.

[0072] Example 3 Optimization of the formulation of microsphere diluent - replacement of preservatives The inventors replaced the preservative on the basis of Formula #2 in Example 2 to obtain a new formula of the buffer solution (Formula #3), as follows: Based on 1000 mL, it includes 6.052 g Trizma® base, 1.2 mL Proclin 300, 0.6 mL Tween 20, 8 g NaCl, 25 g BSA, and a pH of 8.2 ± 0.1.

[0073] The same method as in Example 1 was used to test each standard product. The results are shown in Table 11.

[0074] Table 11 MFI values ​​of each standard tested using microsphere diluent of formula #3

[0075] As can be seen from Table 11, when the microsphere diluent of formula #3 was used for detection, when the concentration of each cytokine was 5 pg / mL, the S / N of cytokines IL-2, IL-4 and IFN-γ were 1.11, 1.16 and 1.14, respectively, all less than 1.2, indicating that when the microsphere diluent of formula #3 was used for detection, the standard curves of cytokines IL-2, IL-4 and IFN-γ were not applicable to low-concentration samples.

[0076] The concentrations of 15 clinical plasma samples tested using microsphere diluent of formulation #3 are shown in Table 12.

[0077] Table 12 Concentrations of clinical plasma samples tested using microsphere diluent of formulation #3

[0078] As can be seen from Table 12, when the microsphere diluent of formula #3 was used for detection, the concentrations of some cytokines in some samples were significantly different from those detected using the cytokine combined detection kit (immunofluorescence method) (Jiangxi Saiji Biotechnology Co., Ltd., Gan Medical Device Registration No. 20192400359), and some of them exceeded 200% or even 500%.

[0079] Furthermore, the detection accuracy of each cytokine is shown in Table 13.

[0080] Table 13 Accuracy of cytokine detection using microsphere diluent of formulation #3

[0081] As shown in Table 13, when using the microsphere diluent of formula #3 for detection, only some cytokines (IL-6, IL-17A, IL-5, IL-12p70 and IFN-α) have a detection accuracy of 100%, while the detection accuracy of the remaining cytokines is still not ideal. The detection accuracy of the cytokine IL-4 is only 47%, which is very unreliable.

[0082] The overall accuracy of each clinical sample is shown in Table 14: Table 14 Overall accuracy of clinical plasma samples tested using microsphere diluent formulation #3

[0083] As can be seen from Table 14, compared with the microsphere diluent of formula #2, the number of microsphere diluents with an overall accuracy of 100% in formula #3 with a change of preservatives decreased from 7 to 3, which is difficult to meet clinical testing needs.

[0084] Example 4 Optimization of the formulation of microsphere diluent - adding sucrose The inventors added 1% sucrose by volume to the formula #2 of Example 2 to obtain a new formula of the buffer solution (formula #4), as follows: Based on 1000 mL, it includes 6.052 g Trizma® base, 1.2 mL Krovin 500, 0.6 mL Tween 20, 8 g NaCl, 25 g BSA, 10 g sucrose, and the pH is 8.2±0.1.

[0085] The same method as in Example 1 was used to test each standard product. The results are shown in Table 15.

[0086] Table 15 MFI values ​​of each standard tested using microsphere diluent of formula #4

[0087] As can be seen from Table 15, when the concentration of each cytokine was 5 pg / mL, the S / N of cytokines IL-2, IL-6, IL-10, TNF-α, IL-17A, IL-1β, IL-12p70 and IFN-α were 1.11, 1.16, 1.03, 1.12, 1.14, 1.06, 1.06 and 1.13, respectively, all less than 1.2, indicating that when the microsphere diluent of formula #4 was used for detection, the standard curves of cytokines IL-2, IL-6, IL-10, TNF-α, IL-17A, IL-1β, IL-12p70 and IFN-α were not applicable to low-concentration samples.

[0088] The concentrations of 15 clinical plasma samples tested using microsphere diluent of formulation #4 are shown in Table 16.

[0089] Table 16 Concentrations of clinical plasma samples tested using microsphere diluent of formulation #4

[0090] As can be seen from Table 16, when the microsphere diluent of formula #4 was used for detection, the concentrations of some cytokines in some samples were significantly different from those detected using the cytokine combined detection kit (immunofluorescence method) (Jiangxi Saiji Biotechnology Co., Ltd., Gan Medical Device Registration No. 20192400359), and some exceeded 200% or even 400%.

[0091] Furthermore, the detection accuracy of each cytokine is shown in Table 17.

[0092] Table 17 Accuracy of cytokine detection using microsphere diluent of formula #4

[0093] As shown in Table 17, when using the microsphere diluent of formula #4 for detection, only some cytokines (TNF-α, IL-17A and IL-5) have a detection accuracy of 100%, and the detection accuracy of the remaining cytokines is still not ideal.

[0094] The overall accuracy of each clinical sample is shown in Table 18: Table 18 Overall accuracy of clinical plasma samples tested using microsphere diluent formulation #4

[0095] As can be seen from Table 18, compared with the microsphere diluent of formula #2, the microsphere diluent of formula #4 with added sucrose still had only 4 cases with an overall accuracy of 100%, which is difficult to meet clinical testing needs.

[0096] Example 5 Optimization of the formulation of microsphere diluent - adding trehalose Considering the test results of the microsphere dilution solution of formula #4, some cytokines have large detection deviations in individual samples. The inventors optimized the formula #2 in Example 2. Specifically, trehalose was added to the formula #2 in Example 2 to obtain a new buffer formula (formula #5), as follows: Based on 1000 mL, it includes 6.052 g Trizma® base, 1.2 mL Krovin 500, 0.6 mL Tween 20, 8 g NaCl, 25 g BSA, 10 g trehalose, and the pH is 8.2±0.1.

[0097] The same method as in Example 1 was used to test each standard product. The results are shown in Table 19.

[0098] Table 19 MFI values ​​of each standard tested using microsphere diluent of formula #5

[0099] As can be seen from Table 19, when the microsphere diluent of formula #5 was used for detection, when the concentration of each cytokine was 5pg / mL, the S / N of cytokines IL-2 and IFN-α were 1.11 and 1.18, respectively, both less than 1.2, indicating that when the microsphere diluent of formula #5 was used for detection, the standard curves of cytokines IL-2 and IFN-α were not applicable to low-concentration samples.

[0100] The concentrations of 15 clinical plasma samples tested using microsphere diluent of formulation #5 are shown in Table 20.

[0101] Table 20 Concentrations of clinical plasma samples tested using microsphere diluent of formulation #5

[0102] As can be seen from Table 20, when the microsphere diluent of formula #5 was used for detection, the concentrations of some cytokines in some samples were significantly different from those detected using the cytokine combined detection kit (immunofluorescence method) (Jiangxi Saiji Biotechnology Co., Ltd., Gan Medical Device Registration No. 20192400359), and some exceeded 200% or even 400%.

[0103] Furthermore, the detection accuracy of each cytokine is shown in Table 21.

[0104] Table 21 Accuracy of cytokine detection using microsphere diluent of formula #5

[0105] As can be seen from Table 21, when using the microsphere diluent of formula #5 for testing, except for the detection accuracy of cytokine IL-10, which is 87%, the others all reached more than 90%, which is a significant improvement.

[0106] The overall accuracy of each clinical sample is shown in Table 22: Table 22 Overall accuracy of clinical plasma samples tested using microsphere diluent formulation #5

[0107] As can be seen from Table 22, compared with the microsphere diluent of formula #2, the overall detection accuracy of the microsphere diluent of formula #5 was not significantly improved.

[0108] Example 6 Optimization of the formulation of microsphere diluent - addition of glycine The inventors added 0.25% glycine by volume to the formula #2 of Example 2 to obtain a new buffer formula (formula #6), as follows: Based on 1000 mL, it includes 6.052 g Trizma® base, 1.2 mL Krovin 500, 0.6 mL Tween 20, 8 g NaCl, 25 g BSA, 2.5 g glycine, and the pH is 8.2 ± 0.1.

[0109] The same method as in Example 1 was used to test each standard product. The results are shown in Table 23.

[0110] Table 23 MFI values ​​of each standard tested using microsphere diluent of formula #6

[0111] As can be seen from Table 23, when the microsphere diluent of formula #6 was used for detection, when the concentration of each cytokine was 5 pg / mL, the S / N of cytokine IL-6 was 1.12, which was less than 1.2, indicating that when the microsphere diluent of formula #6 was used for detection, the cytokine IL-6 standard curve was not applicable to low-concentration samples.

[0112] The concentrations of 15 clinical plasma samples tested using microsphere diluent of formulation #6 are shown in Table 24.

[0113] Table 24 Concentrations of clinical plasma samples tested using microsphere diluent of formulation #6

[0114] As can be seen from Table 24, when the microsphere diluent of formula #6 was used for detection, the concentrations of some cytokines in some samples were significantly different from those detected using the cytokine combined detection kit (immunofluorescence method) (Jiangxi Saiji Biotechnology Co., Ltd., Gan Medical Device Registration No. 20192400359), and some exceeded 200% or even 400%.

[0115] Furthermore, the detection accuracy of each cytokine is shown in Table 25.

[0116] Table 25 Accuracy of cytokine detection using microsphere diluent of formula #6

[0117] As shown in Table 25, when using the microsphere diluent of formula #6 for testing, the detection accuracy of 5 cytokines still cannot reach 100%.

[0118] The overall accuracy of each clinical sample is shown in Table 26: Table 26 Overall accuracy of clinical plasma samples tested using microsphere diluent formulation #6

[0119] As can be seen from Table 26, compared with the microsphere diluent of formula #2, the overall detection accuracy of the microsphere diluent of formula #6 with the addition of glycine was not significantly improved.

[0120] Example 7 Optimization of the formulation of microsphere diluent - adding sodium dodecyl sulfate benzyl ammonium chloride The inventors optimized the formula #2 in Example 2. Specifically, 0.2% of sodium dodecyl sulfate benzyl ammonium chloride by volume was added to the formula #2 in Example 2 to obtain a new formula of the buffer solution (formula #7), as follows: Based on 1000 mL, it includes 6.052 g Trizma® base, 1.2 mL Krovin 500, 0.6 mL Tween 20, 8 g NaCl, 25 g BSA, 2 mL sodium dodecyl sulfate benzyl ammonium chloride, and the pH is 8.2 ± 0.1.

[0121] The standard products were tested in the same manner as in Example 1. The results are shown in Table 27.

[0122] Table 27 MFI values ​​of each standard tested using microsphere diluent of formulation #7

[0123] As can be seen from Table 27, when the microsphere diluent of formula #7 was used for detection, the S / N of each cytokine at different concentrations was greater than 1.2, indicating that when the microsphere diluent of formula #7 was used for detection, the detection sensitivity of each cytokine was significantly higher.

[0124] The concentrations of 15 clinical plasma samples tested using microsphere diluent of formulation #7 are shown in Table 28.

[0125] Table 28 Concentrations of clinical plasma samples tested using microsphere diluent of formulation #7

[0126] As can be seen from Table 28, when the microsphere diluent of formula #7 was used for detection, the absolute values ​​of the concentrations of some cytokines in some samples were still significantly different from those detected using the cytokine combined detection kit (immunofluorescence method) (Jiangxi Saiji Biotechnology Co., Ltd., Gan Medical Device Registration No. 20192400359), and some exceeded 100% or even 200%.

[0127] Furthermore, the detection accuracy of each cytokine is shown in Table 29.

[0128] Table 29 Accuracy of cytokine detection using microsphere diluent of formulation #7

[0129] As shown in Table 29, when using the microsphere diluent of formula #7 for detection, the detection accuracy of some cytokines (IL-6) still did not reach 100%.

[0130] The overall accuracy of each clinical sample is shown in Table 30: Table 30 Overall accuracy of clinical plasma samples tested using microsphere diluent formulation #7

[0131] As shown in Table 30, compared with the microsphere diluent of formula #2, the microsphere diluent of formula #7 with the addition of sodium dodecyl sulfate benzyl ammonium chloride still did not reach 100% detection accuracy for some cytokines (IL-6), and the overall accuracy of individual clinical samples also did not reach 100%.

[0132] Example 8 Optimization of the formulation of microsphere diluent - adding benzethonium chloride The inventors optimized the formula #2 in Example 2. Specifically, 0.2% benzethonium chloride by mass volume fraction was added to the formula #2 in Example 2 to obtain a new formula of the buffer solution (formula #8), as follows: Based on 1000 mL, it includes 6.052 g Trizma® base, 1.2 mL Krovin 500, 0.6 mL Tween 20, 8 g NaCl, 25 g BSA, 2 mL benzethonium chloride, and the pH is 8.2±0.1.

[0133] The same method as in Example 1 was used to test each standard product. The results are shown in Table 31.

[0134] Table 31 MFI values ​​of each standard tested using microsphere diluent of formula #8

[0135] As can be seen from Table 31, when the microsphere diluent of formula #8 was used for detection, the S / N of each cytokine at different concentrations was greater than 1.2, indicating that when the microsphere diluent of formula #8 was used for detection, the detection sensitivity of each cytokine was significantly higher.

[0136] The concentrations of 15 clinical plasma samples tested using microsphere diluent of formulation #8 are shown in Table 32.

[0137] Table 32 Concentrations of clinical plasma samples tested using microsphere diluent of formulation #8

[0138] As can be seen from Table 32, when the microsphere diluent of formula #8 was used for detection, the absolute values ​​of the concentrations of some cytokines in some samples were still significantly different from those detected using the cytokine combined detection kit (immunofluorescence method) (Jiangxi Saiji Biotechnology Co., Ltd., Gan Medical Device Registration No. 20192400359), and some exceeded 50% or even 100%.

[0139] Furthermore, the detection accuracy of each cytokine is shown in Table 33.

[0140] Table 33 Accuracy of cytokine detection using microsphere diluent of formula #8

[0141] As shown in Table 33, when using the microsphere diluent of formula #8 for detection, the detection accuracy of some cytokines (IL-6 and IL-10) still did not reach 100%.

[0142] The overall accuracy of each clinical sample is shown in Table 34: Table 34 Overall accuracy of clinical plasma samples tested using microsphere diluent formulation #8

[0143] As can be seen from Table 34, compared with the microsphere diluent of formula #2, the microsphere diluent of formula #8 with added benzethonium chloride still had a detection accuracy of some cytokines (IL-6 and IL-10) that did not reach 100%, and the overall accuracy of 3 clinical samples did not reach 100%.

[0144] Example 9 Optimization of the formulation of microsphere diluent - adding domiphene The inventors optimized the formula #2 in Example 2. Specifically, 0.2% of domiphene by mass volume fraction was added to the formula #2 in Example 2 to obtain a new formula of the buffer solution (formula #9), as follows: Based on 1000 mL, it includes 6.052 g Trizma® base, 1.2 mL Krovin 500, 0.6 mL Tween 20, 8 g NaCl, 25 g BSA, 2 mL domiphene, and the pH is 8.2 ± 0.1.

[0145] The standard samples were tested in the same manner as in Example 1. The results are shown in Table 35.

[0146] Table 35 MFI values ​​of each standard tested using microsphere diluent of formula #9

[0147] As can be seen from Table 35, when the microsphere diluent of formula #9 was used for detection, the S / N of each cytokine at different concentrations was greater than 1.2, indicating that when the microsphere diluent of formula #9 was used for detection, the detection sensitivity of each cytokine was significantly higher.

[0148] The concentrations of 15 clinical plasma samples tested using microsphere diluent of formulation #9 are shown in Table 36.

[0149] Table 36 Concentrations of clinical plasma samples tested using microsphere diluent of formulation #9

[0150] As can be seen from Table 36, when the microsphere diluent of formula #9 was used for detection, the absolute value of the concentration deviation of all cytokines in each sample and the concentration detected by the cytokine combined detection kit (immunofluorescence method) (Jiangxi Saiji Biotechnology Co., Ltd., Gan Medical Device Registration No. 20192400359) did not exceed 10%, and most of them were almost completely consistent.

[0151] Furthermore, the detection accuracy of each cytokine is shown in Table 37.

[0152] Table 37 Accuracy of cytokine detection using microsphere diluent of formula #9

[0153] As shown in Table 37, the accuracy of all cytokine detection reached 100% when the microsphere diluent of formula #9 was used for detection.

[0154] The overall accuracy of each clinical sample is shown in Table 38: Table 38 Overall accuracy of clinical plasma samples tested using microsphere diluent formulation #9

[0155] As can be seen from Table 38, compared with the microsphere diluent of formula #2, the microsphere diluent of formula #9 with the addition of domiphene has an overall accuracy of 100% for all clinical samples.

[0156] The inventors further analyzed the test results and unexpectedly found that the reason for the above phenomenon was that when the microsphere diluent of formula #1 was used for testing, microspheres aggregated, such as Figure 2 As shown in the figure, the test results are inaccurate. When the microsphere diluents of formula #2 to #8 are used for testing, the microsphere aggregation phenomenon is not alleviated and even more serious aggregation occurs. When the microsphere diluent of formula #9 is used for testing, the microspheres only slightly aggregate, as shown in the figure. Figure 3 shown.

[0157] Example 10 Replacement without buffer Different from the method of Example 1, 25 μL of antibody solution coupled with fluorescent microspheres was directly added to 25 μL of standard or plasma sample, and then 25 μL of fluorescent-labeled antibody solution was added, mixed thoroughly, and allowed to stand at room temperature in the dark for 2.5 h (one-step incubation); each standard was tested, and the results are shown in Table 39.

[0158] Table 39 MFI values ​​of each standard in the microsphere diluent of formula #9

[0159] As can be seen from Table 39, when the microsphere diluent of formula #9 was used for detection, the S / N of each cytokine at different concentrations was greater than 1.2, indicating that the detection sensitivity of each cytokine was also high when the microsphere diluent of formula #9 was used for detection.

[0160] The concentrations of 15 clinical plasma samples tested in microsphere diluent of formulation #9 are shown in Table 40.

[0161] Table 40 Concentrations of clinical plasma samples tested in microsphere diluent of formulation #9

[0162] As can be seen from Table 40, when the microsphere diluent of formula #9 is used for direct detection, the absolute values ​​of the concentrations of all cytokines in each sample are very different from those detected using the cytokine combined detection kit (immunofluorescence method) (Jiangxi Saiji Biotechnology Co., Ltd., Gan Medical Device Registration No. 20192400359).

[0163] Furthermore, the detection accuracy of each cytokine is shown in Table 41.

[0164] Table 41 Accuracy of direct detection of various cytokines in microsphere diluent of formulation #9

[0165] As shown in Table 41, the accuracy of cytokine detection is poor when the microsphere diluent of formula #9 is used for direct detection.

[0166] The overall accuracy of each clinical sample is shown in Table 42: Table 42 Overall accuracy of clinical plasma samples tested directly in microsphere diluent of formulation #9

[0167] As shown in Table 42, when the microsphere diluent of formula #9 was directly tested instead of being converted into microsphere buffer, the overall accuracy of all clinical samples did not reach 100%.

[0168] Example 11 Determination of blank limit and detection limit Blank limit detection method: Use zero-concentration standard as the sample for detection, repeat the measurement 20 times, and obtain the concentration value of the 20 measurement results based on the curve equation of the standard used in the kit. Calculate the mean value (M) and standard deviation (SD) to obtain M+2SD, which is the blank limit value.

[0169] Detection limit detection method: Test 5 samples with low concentrations close to the detection limit (the approximate detection limit is estimated based on the obtained blank limit value, which is slightly higher than the blank limit). Test each sample 5 times and sort the test results by size. The number of test results below the blank limit value should be less than or equal to 3.

[0170] The blank limit and detection limit of formula #6~formula #9 were determined according to the above method, and the cytokine combined detection kit (immunofluorescence method) (Jiangxi Saiji Biotechnology Co., Ltd., Gan Medical Device Registration No. 20192400359) was used as a control. The results are shown in Table 43.

[0171] Table 43 Blank limits and detection limits of microsphere dilutions of formulation #6 to formulation #9

[0172] As can be seen from Table 43, when the microsphere diluent of formula #9 was used for testing, the blank limit and detection limit of each cytokine were significantly lower than those of other formulas, indicating that the microsphere diluent of formula #9 has the best performance.

[0173] In summary, the detection of cytokine protein using the diluent of formula #9 has a high detection signal value, which can theoretically improve the detection sensitivity. To verify this, the inventors used the cytokine combined detection kit (immunofluorescence method) (Jiangxi Saiji Biotechnology Co., Ltd., Gan Medical Device Registration No. 20192400359) as a control to detect cytokine protein samples.

[0174] Among them, cytokine protein samples were prepared using international calibrators (all purchased from NIBSC), and the test results are shown in Table 44.

[0175] Table 44 Measured values ​​of cytokine proteins detected by different methods

[0176] In summary, when the concentration of the international calibrators of the 12 cytokine proteins was 0.400 pg / mL, Jiangxi SecGene's test kit could not produce any test results, while the measured value of the microsphere dilution solution of formula #9 was very close to the true value.

[0177] Example 12 Stability test of microsphere diluent of formulation #9 The microsphere dilutions of formula #9 were stored under refrigerated conditions (2-8°C) or heat-treated conditions (37°C) for 3 days, and the MFI values ​​of each cytokine standard were measured and further used for the detection of 15 clinical samples.

[0178] The MFI values ​​of different concentrations of each cytokine standard obtained by the microsphere diluent of formula #9 under two storage conditions were obtained to obtain the MFI value deviation. The results are as follows Figure 4 As shown. Figure 4 It can be seen that the microsphere diluent of formula #9 is stored under refrigeration or heat treatment conditions, and the MFI measurement deviation of different concentrations of each cytokine standard is no more than 10%, indicating stable performance. The microsphere diluent of formula #9 stored under different conditions is used to test clinical samples, and the detection concentration deviation is as follows Figure 5 As shown. Figure 5 It can be seen that the microsphere diluent of formula #9 is stored under refrigeration conditions or heat treatment conditions, and the test results of clinical samples have little deviation, indicating that there is almost no difference, which further shows that the microsphere diluent of formula #9 has high stability.

[0179] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. Use of a buffer in preparing a kit for detecting 12 cytokines based on flow cytometry, characterized in that: The buffer is Tris buffer, which also contains 1~3‰ v / v preservative, 5~15‰ m / v inorganic salt, 1~5‰ m / v first surfactant, 0.3~1‰ v / v second surfactant and 20~50‰ m / v BSA, the first surfactant is domiphene, and the cytokines include IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, IL-17A, IL-1β, IL-5, IL-12p70, IFN-α and IL-8.

2. The use according to claim 1, characterized in that: The concentration of the Tris buffer is 80-150 mmol / L.

3. The use according to claim 1, characterized in that: The inorganic salt is NaCl, KCl, MgCl2 and / or ZnCl2.

4. The use according to claim 1, characterized in that: The preservative is at least one of the KroVin series preservatives.

5. The use according to claim 4, characterized in that The KroVin series preservatives include KroVin 100, KroVin 400, KroVin 500 and KroVin 750.

6. The use according to claim 1, characterized in that: The preservative is at least one of the ProClin series preservatives.

7. The use according to claim 6, characterized in that The ProClin series preservatives include ProClin50, ProClin 150, ProClin 200, ProClin 300, ProClin 950 and ProClin 5000.

8. The use according to claim 1, characterized in that: The second surfactant is selected from at least one of SDS, Triton X-100, Tween 80 and Tween 20.

9. The use according to any one of claims 1 to 8, characterized in that: The kit comprises an antibody solution coupled to fluorescent microspheres, a fluorescently labeled antibody solution and a microsphere buffer, wherein: The antibody solution coupled with fluorescent microspheres is prepared by adding IL-2 first antibody, IL-4 first antibody, IL-6 first antibody, IL-10 first antibody, TNF-α first antibody, IFN-γ first antibody, IL-17A first antibody, IL-1β first antibody, IL-5 first antibody, IL-8 first antibody, IL-12P70 first antibody and IFN-a first antibody coupled with fluorescent microspheres to the Tris buffer, and the fluorescent microspheres coupled with each first antibody have different fluorescence intensities; The fluorescently labeled antibody solution includes fluorescently labeled IL-2 second antibody, IL-4 second antibody, IL-6 second antibody, IL-10 second antibody, TNF-α second antibody, IFN-γ second antibody, IL-17A second antibody, IL-1β second antibody, IL-5 second antibody, IL-8 second antibody, IL-12P70 second antibody and IFN-a second antibody; The microsphere buffer includes 0.2% to 0.3% m / v KH2PO4, 3% to 4% m / v Na2HPO4·12H2O, 0.5% to 1.0% m / v NaCl, 0.1% to 0.5% m / v KCl, 2% to 4% m / v BSA, 0.1% to 0.2% m / v ProClin300, 0.05% to 0.1% v / v Tween-20 and 0.1% to 0.3% m / v PVP-k30.

10. The use according to claim 9, characterized in that: The microsphere buffer is used to replace the Tris buffer of the antibody solution coupled to the fluorescent microspheres when the kit is used to detect 12 cytokines in a liquid biological sample. The specific steps are: centrifuging the antibody solution coupled to the fluorescent microspheres to remove the supernatant and adding the microsphere buffer.

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