Application of a buffer solution in preparing a kit for detecting cytokines based on a flow cytometer

Abstract

The present invention discloses an application of a buffer solution in the preparation of a kit for detecting cytokines based on a flow cytometer, belonging to the technical field of flow cytometry analysis. The buffer solution is a Tris buffer solution, and the Tris buffer solution further 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, and the first surfactant is domiphen bromide. When using the buffer solution as a microsphere diluent for detecting cytokines based on a flow cytometer, it still has good linearity for low-concentration samples, has high sensitivity so as to be able to detect low-concentration samples, finally has high detection accuracy, and can eliminate random sample anomalies, and has very important clinical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of flow cytometry analysis. Specifically, it relates to the application of a buffer in the preparation of a kit for detecting cytokines based on a flow cytometer. Background Art

[0002] Cytokines are a class of small molecular proteins with a wide range of biological activities, which are synthesized and secreted by immune cells and certain non-immune cells upon stimulation. Cytokines can be classified 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, etc. The detection of cytokines provides a scientific auxiliary basis for the prevention, diagnosis, and treatment of diseases clinically.

[0003] Helper T cells (Th) are one of the subsets of T cells, usually referring to a subset of T cells with the ability to assist humoral and cellular immune responses. According to the different cytokines secreted, Th cells can be divided into cell subsets such as Th1 and Th2. Currently, the cells that secrete cytokines and are studied more are Th1 and Th2 cells. Th1 cells mainly secrete pro-inflammatory 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 antibodies combined with complement (IgM, IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ), and 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, autoantibodies in the human body (such as rheumatoid factor, anti-mitochondrial antibody, HAMA, etc.) often interfere with the test results, producing non-specific immune responses, that is, false positives, resulting in the test results may be different from the actual situation, thus affecting the clinical judgment of diseases.

[0004] However, in the prior art, the accuracy of 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. is not high, the sensitivity is low, and many positive cases cannot be detected, which is not conducive to the detection of rare samples. Summary of the Invention

[0005] To solve at least one of the above technical problems, the inventors conducted a series of explorations on detection reagents and detection conditions and unexpectedly found that when domiphen bromide is added to the microsphere diluent, the method can solve microsphere aggregation, improve the sensitivity and accuracy of simultaneously detecting 12 cytokines, and improve the stability of the reagent, thus completing the present invention. On this basis, the technical solution adopted by the present invention is as follows:

[0006] The first aspect of the present invention provides an application of a buffer solution in the preparation of a kit for detecting 12 cytokines based on a flow cytometer. The buffer solution is a Tris buffer solution, and the Tris buffer solution further 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 domiphen bromide, 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.

[0007] Domiphen bromide is a quaternary ammonium cationic surfactant and is commonly used as a chemical preservative or antibacterial agent. In the present invention, when domiphen bromide is added to the buffer solution and used as a microsphere diluent, microsphere aggregation can be avoided.

[0008] In some embodiments of the present invention, the concentration of the Tris buffer solution is 80-150 mmol / L.

[0009] In some embodiments of the present invention, the inorganic salt is NaCl, KCl, MgCl2, and / or ZnCl2.

[0010] In some embodiments of the present invention, the preservative is at least one of the KroVin series preservatives.

[0011] In some specific embodiments of the present invention, the KroVin series preservatives include KroVin 100, KroVin 400, KroVin 500, and KroVin 750.

[0012] In some embodiments of the present invention, the preservative is at least one of the ProClin series preservatives.

[0013] 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.

[0014] In the present invention, there are no special requirements 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.

[0015] In some embodiments of the present invention, the kit includes an antibody solution conjugated with fluorescent microspheres, a fluorescently labeled antibody solution, and a microsphere buffer solution, wherein,

[0016] The antibody solution conjugated with fluorescent microspheres is prepared by adding the first antibodies of IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, IL-17A, IL-1β, IL-5, IL-8, IL-12P70, and IFN-α conjugated with fluorescent microspheres to the Tris buffer solution. The fluorescence intensity of the fluorescent microspheres conjugated with each first antibody is different;

[0017] The fluorescently labeled antibody solution includes fluorescently labeled second antibodies of IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, IL-17A, IL-1β, IL-5, IL-8, IL-12P70, and IFN-α;

[0018] The microsphere buffer solution includes 0.2% - 0.3% m / v KH2PO4, 3% - 4% m / v Na2HPO4·12H2O, 0.5% - 1.0% m / v NaCl, 0.1% - 0.5% m / v KCl, 2% - 4% m / v BSA, 0.1% - 0.2% m / v ProClin300, 0.05% - 0.1% v / v Tween-20, and 0.1% - 0.3% m / v PVP-k30.

[0019] In the present invention, the microsphere buffer solution is used to replace the Tris buffer solution of the antibody solution conjugated with fluorescent microspheres when detecting 12 cytokines in a liquid biological sample using the kit. The specific steps are as follows: Centrifuge the antibody solution conjugated with fluorescent microspheres to remove the supernatant, and add the microsphere buffer solution. Simultaneously or sequentially add the liquid biological sample and the fluorescently labeled antibody solution, and incubate in the dark at room temperature to obtain a solution to be detected.

[0020] In a second aspect of the present invention, a kit for detecting 12 cytokines based on a flow cytometer is provided, which includes an antibody solution conjugated with fluorescent microspheres, a fluorescently labeled antibody solution, and a microsphere buffer. Among them,

[0021] The antibody solution conjugated with fluorescent microspheres is prepared by adding the first antibodies of IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, IL-17A, IL-1β, IL-5, IL-8, IL-12P70, and IFN-α conjugated with fluorescent microspheres into the Tris buffer described in the first aspect of the present invention. The fluorescent intensities of the fluorescent microspheres conjugated with each first antibody are different;

[0022] The fluorescently labeled antibody solution includes fluorescently labeled second antibodies of IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, IL-17A, IL-1β, IL-5, IL-8, IL-12P70, and IFN-α;

[0023] The microsphere buffer includes 0.2% - 0.3% m / v KH2PO4, 3% - 4% m / v Na2HPO4·12H2O, 0.5% - 1.0% m / v NaCl, 0.1% - 0.5% m / v KCl, 2% - 4% m / v BSA, 0.1% - 0.2% m / v ProClin300, 0.05% - 0.1% v / v Tween-20, and 0.1% - 0.3% m / v PVP-k30.

[0024] In the present invention, the buffer in the antibody solution conjugated with fluorescent microspheres is replaced with the microsphere buffer before detection. Specifically: centrifuge the antibody solution conjugated with fluorescent microspheres to remove the supernatant, and then add the same volume of the microsphere buffer.

[0025] In a third aspect of the present invention, a method for detecting 12 cytokines in a liquid biological sample using the kit described in the second aspect of the present invention is provided, including the following steps:

[0026] S1, centrifuge the antibody solution conjugated with fluorescent microspheres to remove the supernatant, add the microsphere buffer, the liquid biological sample, and the fluorescently labeled antibody solution, and incubate in the dark at room temperature to obtain a solution to be detected;

[0027] S2, detect the fluorescence intensity of the fluorescence label in the solution to be detected to calculate the content of different cytokines in the sample to be detected.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] When detecting cytokines based on a flow cytometer using the buffer of the present invention, it still has good linearity for low-concentration samples, high sensitivity so that it can detect low-concentration samples, and finally has high detection accuracy, and can eliminate random sample anomalies, having very important clinical application value. It can solve microsphere aggregation, thereby improving the accuracy and sensitivity of the reagent. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Shows the linear relationship between the MFI measurement value and the concentration of cytokine TNF-α and IFN-γ standards in the microsphere diluent of formulation #1 in Example 1 of the present invention.

[0031] Figure 2 Shows the microsphere distribution diagram when detecting using the microsphere diluent of formulation #1 in Example 9 of the present invention. 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, P9 are the distribution diagrams of P1 in A respectively, and the percentage ratio is between 1.52% - 51.38%; C: P10, P11, P12, P13, P14, P15, P16 are the distribution diagrams of P2 in A respectively, and the percentage ratio is between 0.19% - 23.76%.

[0032] Figure 3 Shows the microsphere distribution diagram when detecting using the microsphere diluent of formulation #9 in Example 9 of the present invention. 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, P9 are the distribution diagrams of P1 in A respectively, and the percentage ratio is between 10.86% - 17.24%; C: P10, P11, P12, P13, P14, P15, P16 are the distribution diagrams of P2 in A respectively, and the percentage ratio is between 0.68% - 4.11%.

[0033] Figure 4 Shows the MFI measurement value deviation of different concentrations of cytokine standards detected after the microsphere diluent of formulation #9 in Example 12 of the present invention is stored at two storage temperatures.

[0034] Figure 5 Shows the detection concentration deviation of 15 clinical samples obtained after the microsphere diluent of formulation #9 in Example 12 of the present invention is stored at two storage temperatures. DETAILED DESCRIPTION OF THE INVENTION

[0035] Unless otherwise specified, implied from the context, or conventional in the art, all parts and percentages in this application are based on weight, and the test and characterization methods used are contemporaneous with the filing date of this application. To the extent applicable, any patents, patent applications, or publications referred to in this application are incorporated herein by reference in their entirety, and their equivalent family patents are also incorporated by reference, particularly 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.

[0036] The numerical ranges in this application are approximate values, and thus, unless otherwise specified, may include values outside the range. The numerical range includes all values increasing in increments of one unit from the lower limit value to the upper limit value, provided that there is an interval of at least two units between any lower value and any higher value. For ranges that include values less than 1 or include fractions greater than 1 (such as 1.1, 1.5, etc.), one unit is appropriately considered to be 0.0001, 0.001, 0.01, or 0.1. For ranges that include single digits less than 10 (such as 1 to 5), one unit is generally considered to be 0.1. These are merely 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 recited in this application.

[0037] The terms "comprising", "including", "having", and their derivatives do not exclude the presence of any other components, steps, or processes, and are independent of whether or not these other components, steps, or processes are disclosed in this application. To eliminate any doubt, unless explicitly stated, all compositions in this application using the terms "comprising", "including", or "having" may contain any additional additives, excipients, or compounds. In contrast, the term "consisting essentially of" excludes any other components, steps, or processes from the scope of any description following such term, except for those necessary for the operating properties. The term "consisting of" does not include any components, steps, or processes not specifically described or listed. Unless explicitly stated, the term "or" refers to the individual members listed or any combination thereof.

[0038] In order to make the technical problems, technical solutions, and beneficial effects solved by the present invention more clearly understood, the present invention will be further described in detail below in conjunction with embodiments.

[0039] The following examples are used herein to demonstrate the preferred embodiments of the present invention. Those skilled in the art will understand that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present invention and can thus be regarded as preferred embodiments for implementing the present invention. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein and still obtain the same or similar results without departing from the spirit or scope of the present invention.

[0040] Unless otherwise defined, 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. All publications, patent applications, patents, and other references mentioned herein and the materials they cited are hereby incorporated by reference.

[0041] Those skilled in the art will recognize or, through routine experimentation, will be able to understand many equivalent techniques to the specific embodiments of the invention described herein. These equivalents will be included in the claims.

[0042] Unless otherwise specified, the experimental methods in the following examples are all conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the test materials used in the following examples are all obtained from regular biochemical reagent stores.

[0043] Example 1 Detection of 12 Cytokines in Plasma Samples

[0044] 1. Samples to be Tested

[0045] In the present invention, 15 clinical plasma samples from the infectious disease department were obtained for the detection of 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. 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, with the unit of pg / mL.

[0046] The concentrations of each cytokine detected in the 15 plasma samples using a cytokine multiplex detection kit (immunofluorescence method) (Jiangxi Saiji Biotechnology Co., Ltd., Jiangxi Medical Device Approval No. 20192400359) are shown in Table 1:

[0047] Table 1 Concentrations of 12 Cytokines in Clinical Plasma Samples (pg / mL)

[0048]

[0049] 2. Microsphere Diluent Formulation

[0050] The conventional microsphere diluent formulation for detection (Formulation #1) is as follows:

[0051] Based on 1000 mL, it includes 6.052 g of Trizma® base, with a pH of 8.2 ± 0.1.

[0052] 3. Detection Reagents

[0053] Standards: There are a total of 11 standards. Each standard contains the above 12 cytokines and the concentrations of each cytokine are the same. The concentrations of each cytokine in each standard are 2.5 pg / mL, 5 pg / mL, 10 pg / mL, 20 pg / mL, 40 pg / mL, 80 pg / mL, 156 pg / mL, 312 pg / mL, 625 pg / mL, 1250 pg / mL, and 2500 pg / mL respectively.

[0054] 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 Patent Publication No. CN117368493A.

[0055] Antibody solution conjugated with fluorescent microspheres: Take polystyrene fluorescent microspheres with 12 fluorescence intensities (manufacturer Polysciences, model BLI239C-20, BLI250C-10) respectively. The concentration of each type of microsphere is 5×10 7cells / mL. Take 0.1 mL of the first fluorescent microspheres (L1) for each type, wash them twice with the microsphere diluent of formulation #1, add 0.5 mL of the microsphere diluent of formulation #1 to the washed first fluorescent microspheres, add 100 μg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 50 μg of N-hydroxysulfosuccinimide (NHS), and let stand for 30 min to activate the fluorescent microspheres. Add 100 μg of IL-1β antibody 1 and react by rotation at room temperature for 5 h. Wash the first fluorescent microspheres with the microsphere diluent of formulation #1 to remove the excess antibody, then add 5% skim milk powder by mass to block for 30 min. After removing the skim milk powder, add 0.5 mL of the microsphere diluent of formulation #1 to obtain a fluorescent microsphere solution coated with IL-1β antibody 1. Prepare 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 respectively in the same way. As shown in Table 2, different types of polystyrene microspheres can be distinguished by the fluorescence signal value in the allophycocyanin channel (APC-A) and the fluorescence signal value in the allophycocyanin-conjugated Cy7 dye channel (APC-Cy7--A) on the BriCyte E6 flow cytometer produced by Mindray. Here, 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.

[0056] Table 2 Microspheres Containing Different Antibodies 1

[0057]

[0058] Take 188 μL of the microsphere diluent, and add 1 μL of each of the fluorescent microsphere solutions 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 respectively to prepare an antibody solution conjugated with fluorescent microspheres.

[0059] Fluorescently labeled antibody solution: Take 2 of IL-1β antibody, 2 of IL-2 antibody, 2 of IL-4 antibody, 2 of IL-5 antibody, 2 of IL-6 antibody, 2 of IL-8 antibody, 2 of IL-10 antibody, 2 of IL-12p70 antibody, 2 of IL-17A antibody, 2 of IFN-γ antibody, 2 of TNF-α antibody, 2 of IFN-α antibody. Add phycoerythrin fluorescein to each antibody at a molar ratio of antibody 2 to phycoerythrin fluorescein of 1:30. After oscillating and incubating at room temperature for 5 h, wash 3 times with a 0.01 mol / L Tris buffer using a 50K ultrafiltration tube to remove excess unbound phycoerythrin fluorescein, obtaining 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. Dilute the 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 with 0.01 mol / L Tris buffer to 2 μg / mL respectively.

[0060] Take 1 μL each of the diluted 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 with a concentration of 2 μg / mL, and add to 36 μL of 0.01 mol / L Tris buffer and mix to prepare a fluorescently labeled antibody solution.

[0061] 1× Wash buffer: Take 3.0275 g of Tris and 9.0 g of NaCl, dissolve in 800 mL of pure water, add 0.8 mL of ProClin300 preservative, add 0.6 mL of Tween 20, add 20 g of BSA, adjust the pH value to 7.4 to obtain a wash buffer (10×) for standby. Let the wash buffer (10×) reach room temperature, wait for all salts to dissolve, take 10 mL of the wash buffer (10×) and add to 90 mL of pure water to obtain a wash buffer (1×).

[0062] Microsphere buffer: Dissolve 2.4 g of KH2PO4, 36.32 g of Na2HPO4·12H2O, 8 g of NaCl, and 2 g of KCl in 1000 mL of pure water. Add 25 g of BSA, 1.2 mL of ProClin300, 0.6 mL of Tween 20, and 2 mL of PVP-k30 (purchased from aladdin, English name RNase-free PVP k30 Solution, CAS No. 9003-39-8) for standby.

[0063] 4. Detection method

[0064] Incubation: Take 25 μL of the antibody solution conjugated with fluorescent microspheres, centrifuge to remove the supernatant, add 25 μL of microsphere buffer, then add 25 μL of the standard or plasma sample, and finally add 25 μL of the fluorescently labeled antibody solution. Mix well and let stand in the dark at room temperature for 2.5 h (one-step incubation);

[0065] Washing: Add 1000 μL of 1× washing buffer, resuspend the conjugated microspheres by vortexing, mix well, shake on an oscillator for more than 30 s, and centrifuge to remove the supernatant;

[0066] Detection: Add 100 μL of 1× washing buffer and detect the fluorescence type and fluorescence signal intensity (MFI) on a BriCyte E6 flow cytometer produced by Mindray.

[0067] The detection results of each standard are shown in Table 3.

[0068] Table 3 MFI values of each standard detected using the microsphere diluent of formulation #1

[0069]

[0070] For the detection MFI value of a certain concentration standard, the detection MFI value of the adjacent lower concentration standard is used as the baseline noise. After comparison, the signal-to-noise ratio (S / N) is obtained to evaluate the different concentration resolution capabilities of the curve. For example, for the IL-2 cytokine, when the concentration is 10 pg / mL, the MFI value is 721.20, and the MFI value of the adjacent lower concentration standard of this cytokine at 5 pg / mL is 474.00. Then the signal-to-noise ratio of the IL-2 cytokine standard at a concentration of 10 pg / mL is 721.20 / 474.00 = 1.52.

[0071] When S / N ≤ 1.2, it indicates that it is impossible to distinguish it from the adjacent low concentration, thus affecting the detection sensitivity. As can be seen from Table 2, when using the microsphere diluent of Formulation #1 for detection, when the concentration of each cytokine is 5 pg / mL, the S / N values 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 using the microsphere diluent of Formulation #1 for detection, the standard curve is not applicable to low-concentration samples. The double logarithmic curves of the detection results of TNF-α and IFN-γ are as Figure 1 shown. It can be seen that for the cytokine TNF-α, the standard curve has a high fitting degree, and the calculated concentration of the detection result will be very accurate. 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 calculated sample concentration is inaccurate, showing a higher value, and false positive results may occur.

[0072] The concentrations of 15 clinical plasma samples detected using the microsphere diluent of Formulation #1 are shown in Table 4.

[0073] Table 4 Concentrations of clinical plasma samples detected using the microsphere diluent of Formulation #1

[0074]

[0075] For a certain cytokine, deviation = (A - B) / B × 100%, where A represents the concentration of this cytokine detected using the microsphere diluent of Formulation #1; B represents the concentration of this cytokine detected using the cytokine combined detection kit (immunofluorescence method) (Jiangxi Saiji Biotechnology Co., Ltd., Jiangxi Medical Device Approval No. 20192400359).

[0076] As can be seen from Table 4, when using the microsphere diluent of Formulation #1 for detection, the concentrations of some cytokines in some samples deviate greatly from the concentrations detected using the cytokine combined detection kit (immunofluorescence method) (Jiangxi Saiji Biotechnology Co., Ltd., Jiangxi Medical Device Approval No. 20192400359), and some exceed 1000% or even 10000%.

[0077] Furthermore, the detection accuracies of each cytokine are shown in Table 5.

[0078] Table 5 Detection accuracies of each cytokine detected using the microsphere diluent of Formulation #1

[0079]

[0080] As can be seen from Table 5, when using the microsphere diluent of Formulation #1 for detection, the detection accuracy of each cytokine is not ideal. For some cytokines such as IL-4 and IFN-γ, the accuracy is only 53%, which is quite untrustworthy.

[0081] The overall accuracy of each clinical sample is shown in Table 6:

[0082] Table 6 Overall accuracy of clinical plasma samples detected using the microsphere diluent of Formulation #1

[0083]

[0084] As can be seen from Table 6, among the 15 samples, only 2 samples have an overall accuracy reaching 100%, that is, the detection results of all cytokines are accurate. The overall accuracy of the remaining samples is not ideal. For the clinical plasma sample, such as the plasma sample numbered 8, the overall accuracy is only 25%, that is, there are abnormal measured values in the random samples.

[0085] In order to further improve the detection accuracy of each cytokine and the consistency and stability of the detection results of different samples, the inventor tried to adjust the microsphere diluent.

[0086] Example 2 Optimization of the microsphere diluent formula - adding formula components

[0087] The inventor improved on the basis of the microsphere diluent of Formulation #1 in Example 1 to obtain a new microsphere diluent formula (Formulation #2), as follows:

[0088] Based on 1000 mL, it includes 6.052 g of Trizma® base, 1.2 mL of Krovin 500, 0.6 mL of Tween 20, 8 g of NaCl, 25 g of BSA, and the pH is 8.2 ± 0.1.

[0089] Using the same method as in Example 1 to detect each standard product, the results are shown in Table 7.

[0090] Table 7 MFI measured values of each standard product detected using the microsphere diluent of Formulation #2

[0091]

[0092] As can be seen from Table 7, when using the microsphere diluent of Formulation #2 for detection, when the concentration of each cytokine is 5 pg / mL, the S / N of cytokine IL-5 is 1.18, which is less than 1.2, indicating that when using the microsphere diluent of Formulation #2 for detection, the standard curve of cytokine IL-5 is not applicable to low-concentration samples.

[0093] The concentrations of 15 clinical plasma samples detected using the microsphere diluent of Formulation #2 are shown in Table 8.

[0094] Table 8 Concentrations of clinical plasma samples detected using the microsphere diluent of Formulation #2

[0095]

[0096] As can be seen from Table 8, when detecting using the microsphere diluent of Formulation #2, the absolute value of the concentration deviation of some cytokines in some samples is relatively large compared with the concentration detected using the cytokine combined detection kit (immunofluorescence method) (Jiangxi Saiji Biotechnology Co., Ltd., Jiangxi Medical Device Registration No. 20192400359), and some exceed 200% or even 400%.

[0097] Furthermore, the detection accuracy of each cytokine is shown in Table 9.

[0098] Table 9 Detection accuracy of each cytokine using the microsphere diluent of Formulation #2

[0099]

[0100] As can be seen from Table 9, when detecting using the microsphere diluent of Formulation #2, the detection accuracy of only some cytokines (IL-6, TNF-α, IL-17A, IL-1β, and IL-12p70) reaches 100%, and the detection accuracy of the remaining cytokines is still not ideal.

[0101] The overall accuracy of each clinical sample is shown in Table 10:

[0102] Table 10 Overall accuracy of clinical plasma samples detected using the microsphere diluent of Formulation #2

[0103]

[0104] As can be seen from Table 10, compared with the microsphere diluent of Formulation #1, when using the microsphere diluent of Formulation #2, the number of cases with an overall accuracy reaching 100% increases from 2 to 7, but there are still 4 plasma samples with an overall accuracy of only 83%, which is difficult to meet the clinical detection requirements.

[0105] Example 3 Optimization of the microsphere diluent formulation - replacement of the preservative

[0106] The inventor replaced the preservative on the basis of Formulation #2 in Example 2 to obtain a new buffer formulation (Formulation #3) as follows:

[0107] Based on 1000 mL, it includes 6.052 g of Trizma® base, 1.2 mL of Proclin 300, 0.6 mL of Tween 20, 8 g of NaCl, 25 g of BSA, and the pH is 8.2 ± 0.1.

[0108] The various reference standards were detected in the same manner as in Example 1, and the results are shown in Table 11.

[0109] Table 11 MFI measurement values of various reference standards detected using the microsphere diluent of Formulation #3

[0110]

[0111] As can be seen from Table 11, when the concentrations of the various cytokines were 5 pg / mL and detected using the microsphere diluent of Formulation #3, the S / N values 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 detected using the microsphere diluent of Formulation #3, the standard curves of cytokines IL-2, IL-4, and IFN-γ were not applicable to low-concentration samples.

[0112] The concentrations of 15 clinical plasma samples detected using the microsphere diluent of Formulation #3 are shown in Table 12.

[0113] Table 12 Concentrations of clinical plasma samples detected using the microsphere diluent of Formulation #3

[0114]

[0115] As can be seen from Table 12, when detected using the microsphere diluent of Formulation #3, the absolute values of the concentration deviations of some cytokines in some samples were relatively large compared to those detected using the cytokine combined detection kit (immunofluorescence method) (Jiangxi Saiji Biotechnology Co., Ltd., Jiangxi Medical Device Registration No. 20192400359), and some exceeded 200% or even 500%.

[0116] Furthermore, the detection accuracies of the various cytokines are shown in Table 13.

[0117] Table 13 Detection accuracies of various cytokines detected using the microsphere diluent of Formulation #3

[0118]

[0119] As can be seen from Table 13, when detected using the microsphere diluent of Formulation #3, the detection accuracies of only some cytokines (IL-6, IL-17A, IL-5, IL-12p70, and IFN-α) reached 100%, and the detection accuracies of the remaining cytokines were still not ideal. The detection accuracy of cytokine IL-4 was only 47%, which was highly untrustworthy.

[0120] The overall accuracies of the various clinical samples are shown in Table 14:

[0121] Table 14 Overall accuracies of clinical plasma samples detected using the microsphere diluent of Formulation #3

[0122]

[0123] As can be seen from Table 14, compared with the microsphere diluent of Formulation #2, the number of cases where the overall detection accuracy of the microsphere diluent of Formulation #3 with the preservative replaced reaches 100% does not increase but decreases, from 7 cases to 3 cases, making it difficult to meet the clinical detection requirements.

[0124] Example 4 Formulation Optimization of Microsphere Diluent - Adding Sucrose

[0125] The inventor added sucrose with a volume fraction of 1% to the basis of Formulation #2 in Example 2 to obtain a new buffer formulation (Formulation #4) as follows:

[0126] Based on 1000 mL, it includes 6.052 g of Trizma® base, 1.2 mL of Krovin 500, 0.6 mL of Tween 20, 8 g of NaCl, 25 g of BSA, 10 g of sucrose, and the pH is 8.2 ± 0.1.

[0127] Using the same method as in Example 1 to detect each standard product, the results are shown in Table 15.

[0128] Table 15 MFI Measurement Values of Each Standard Product Detected Using the Microsphere Diluent of Formulation #4

[0129]

[0130] As can be seen from Table 15, when using the microsphere diluent of Formulation #4 for detection, when the concentration of each cytokine is 5 pg / mL, the S / N values of cytokines IL-2, IL-6, IL-10, TNF-α, IL-17A, IL-1β, IL-12p70, and IFN-α are 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 using the microsphere diluent of Formulation #4 for detection, the standard curves of cytokines IL-2, IL-6, IL-10, TNF-α, IL-17A, IL-1β, IL-12p70, and IFN-α are not applicable to low-concentration samples.

[0131] The concentrations of 15 clinical plasma samples detected using the microsphere diluent of Formulation #4 are shown in Table 16.

[0132] Table 16 Concentrations of Clinical Plasma Samples Detected Using the Microsphere Diluent of Formulation #4

[0133]

[0134] As can be seen from Table 16, when using the microsphere diluent of Formulation #4 for detection, the absolute value of the concentration deviation of some cytokines in some samples is relatively large compared to the concentration detected using the cytokine combined detection kit (immunofluorescence method) (Jiangxi Saiji Biotechnology Co., Ltd., Medical Device Registration No. 20192400359 in Jiangxi), and some exceed 200% or even 400%.

[0135] Furthermore, the detection accuracy of each cytokine is shown in Table 17.

[0136] Table 17 Detection accuracy of each cytokine using the microsphere diluent of Formulation #4

[0137]

[0138] As can be seen from Table 17, when using the microsphere diluent of Formulation #4 for detection, only the detection accuracy of some cytokines (TNF-α, IL-17A, and IL-5) reaches 100%, and the detection accuracy of the remaining cytokines is still not ideal.

[0139] The overall accuracy of each clinical sample is shown in Table 18:

[0140] Table 18 Overall accuracy of clinical plasma samples detected using the microsphere diluent of Formulation #4

[0141]

[0142] As can be seen from Table 18, compared with the microsphere diluent of Formulation #2, the number of samples with an overall detection accuracy of 100% for the microsphere diluent of Formulation #4 with added sucrose is still only 4 cases, which is difficult to meet the clinical detection requirements.

[0143] Example 5 Optimization of the microsphere diluent formulation - adding trehalose

[0144] Considering the detection results of the microsphere diluent of Formulation #4, the detection deviation of some cytokines in individual samples is relatively large. The inventor optimized on the basis of Formulation #2 in Example 2. Specifically, trehalose was added on the basis of Formulation #2 in Example 2 to obtain a new buffer formulation (Formulation #5), as follows:

[0145] Based on 1000 mL, it includes 6.052 g of Trizma® base, 1.2 mL of Krovin 500, 0.6 mL of Tween 20, 8 g of NaCl, 25 g of BSA, 10 g of trehalose, and the pH is 8.2 ± 0.1.

[0146] Using the same method as in Example 1 to detect each standard product, the results are shown in Table 19.

[0147] MFI values of each standard product detected using the microsphere diluent of formulation #5

[0148]

[0149] As can be seen from Table 19, when using the microsphere diluent of formulation #5 for detection, when the concentration of each cytokine was 5 pg / mL, the S / N values of cytokines IL-2 and IFN-α were 1.11 and 1.18 respectively, both less than 1.2, indicating that when using the microsphere diluent of formulation #5 for detection, the standard curves of cytokines IL-2 and IFN-α were not applicable to low-concentration samples.

[0150] The concentrations of 15 clinical plasma samples detected using the microsphere diluent of formulation #5 are shown in Table 20.

[0151] Table 20 Concentrations of clinical plasma samples detected using the microsphere diluent of formulation #5

[0152]

[0153] As can be seen from Table 20, when using the microsphere diluent of formulation #5 for detection, the absolute values of the concentration deviations of some cytokines in some samples were relatively large compared with those detected using the cytokine combined detection kit (immunofluorescence method) (Jiangxi Saiji Biotechnology Co., Ltd., Jiangxi Medical Device Approval No. 20192400359), and some exceeded 200% or even 400%.

[0154] Furthermore, the detection accuracy of each cytokine is shown in Table 21.

[0155] Table 21 Detection accuracy of each cytokine detected using the microsphere diluent of formulation #5

[0156]

[0157] As can be seen from Table 21, when using the microsphere diluent of formulation #5 for detection, except that the detection accuracy of cytokine IL-10 was 87%, the others all reached over 90%, showing a great improvement.

[0158] The overall accuracy of each clinical sample is shown in Table 22:

[0159] Table 22 Overall accuracy of clinical plasma samples detected using the microsphere diluent of formulation #5

[0160]

[0161] As can be seen from Table 22, compared with the microsphere diluent of formulation #2, the overall detection accuracy of the microsphere diluent of formulation #5 did not show a significant improvement.

[0162] Example 6 Optimization of the formulation of the microsphere diluent - addition of glycine

[0163] Based on the formulation #2 in Example 2, the inventors added glycine with a volume fraction of 0.25% to obtain a new formulation of the buffer solution (formulation #6) as follows:

[0164] Calculated based on 1000 mL, it includes 6.052 g of Trizma® base, 1.2 mL of Krovin 500, 0.6 mL of Tween 20, 8 g of NaCl, 25 g of BSA, 2.5 g of glycine, and the pH is 8.2 ± 0.1.

[0165] Using the same method as in Example 1 to detect each standard product, the results are shown in Table 23.

[0166] Table 23 MFI measurement values of each standard product detected using the microsphere diluent of formulation #6

[0167]

[0168] As can be seen from Table 23, when using the microsphere diluent of formulation #6 for detection, when the concentration of each cytokine is 5 pg / mL, the S / N of cytokine IL-6 is 1.12, less than 1.2, indicating that when using the microsphere diluent of formulation #6 for detection, the standard curve of cytokine IL-6 is not applicable to low-concentration samples.

[0169] The concentrations of 15 clinical plasma samples detected using the microsphere diluent of formulation #6 are shown in Table 24.

[0170] Table 24 Concentrations of clinical plasma samples detected using the microsphere diluent of formulation #6

[0171]

[0172] As can be seen from Table 24, when using the microsphere diluent of formulation #6 for detection, the absolute value of the concentration deviation of some cytokines in some samples is relatively large compared with the concentration detected using the cytokine combined detection kit (immunofluorescence method) (Jiangxi Saiji Biotechnology Co., Ltd., Jiangxi Medical Device Approval No. 20192400359), and some exceed 200% or even 400%.

[0173] Furthermore, the detection accuracy of each cytokine is shown in Table 25.

[0174] Table 25 Detection accuracy of each cytokine using the microsphere diluent of formulation #6

[0175]

[0176] As can be seen from Table 25, when using the microsphere diluent of formulation #6 for detection, the detection accuracy of 5 cytokines still cannot reach 100%.

[0177] The overall accuracy of each clinical sample is shown in Table 26:

[0178] Table 26 Overall accuracy of clinical plasma samples detected using the microsphere diluent of formulation #6

[0179]

[0180] As can be seen from Table 26, compared with the microsphere diluent of formulation #2, the overall detection accuracy of the microsphere diluent of formulation #6 with glycine added has not been significantly improved.

[0181] Example 7 Formulation optimization of microsphere diluent - adding benzalkonium chloride dodecyl sulfate

[0182] The inventor optimized on the basis of formulation #2 in Example 2. Specifically, on the basis of formulation #2 in Example 2, benzalkonium chloride dodecyl sulfate with a volume fraction of 0.2% was added to obtain a new buffer formulation (formulation #7), as follows:

[0183] Based on 1000 mL, it includes 6.052 g of Trizma® base, 1.2 mL of Krovin 500, 0.6 mL of Tween 20, 8 g of NaCl, 25 g of BSA, 2 mL of benzalkonium chloride dodecyl sulfate, and the pH is 8.2 ± 0.1.

[0184] Using the same method as in Example 1 to detect each standard product, the results are shown in Table 27.

[0185] Table 27 MFI measurement values of each standard product detected using the microsphere diluent of formulation #7

[0186]

[0187] As can be seen from Table 27, when using the microsphere diluent of formulation #7 for detection, the S / N of each cytokine at different concentrations is greater than 1.2, indicating that when using the microsphere diluent of formulation #7 for detection, the detection sensitivity of each cytokine is significantly relatively high.

[0188] The concentrations of 15 clinical plasma samples detected using the microsphere diluent of formulation #7 are shown in Table 28.

[0189] Table 28 Concentrations of clinical plasma samples detected using the microsphere diluent of formulation #7

[0190]

[0191] As can be seen from Table 28, when using the microsphere diluent of formulation #7 for detection, the absolute value of the concentration deviation of some cytokines in some samples is still relatively large compared to the concentration detected using the cytokine combined detection kit (immunofluorescence method) (Jiangxi Saiji Biotechnology Co., Ltd., Jiangxi Medical Device Approval No. 20192400359), and some exceed 100% or even 200%.

[0192] Furthermore, the detection accuracy of each cytokine is shown in Table 29.

[0193] Table 29 Detection accuracy of each cytokine using the microsphere diluent of formulation #7

[0194]

[0195] As can be seen from Table 29, when using the microsphere diluent of formulation #7 for detection, the detection accuracy of some cytokines (IL-6) still does not reach 100%.

[0196] The overall accuracy of each clinical sample is shown in Table 30:

[0197] Table 30 Overall accuracy of clinical plasma samples detected using the microsphere diluent of formulation #7

[0198]

[0199] As can be seen from Table 30, compared with the microsphere diluent of formulation #2, for the microsphere diluent of formulation #7 added with benzalkonium chloride, since the detection accuracy of some cytokines (IL-6) still does not reach 100%, the overall accuracy of individual clinical samples also does not reach 100%.

[0200] Example 8 Optimization of the formulation of the microsphere diluent - addition of benzethonium chloride

[0201] The inventor optimized on the basis of formulation #2 in Example 2. Specifically, on the basis of formulation #2 in Example 2, benzethonium chloride with a mass-volume fraction of 0.2% was added to obtain a new buffer formulation (formulation #8) as follows:

[0202] Calculated based on 1000 mL, it includes 6.052 g of Trizma® base, 1.2 mL of Krovin 500, 0.6 mL of Tween 20, 8 g of NaCl, 25 g of BSA, 2 mL of benzethonium chloride, and the pH is 8.2 ± 0.1.

[0203] Using the same method as in Example 1 to detect each standard product, the results are shown in Table 31.

[0204] Table 31 MFI measurement values of each standard product detected using the microsphere diluent of formulation #8

[0205]

[0206] As can be seen from Table 31, when using the microsphere diluent of formulation #8 for detection, the S / N of each cytokine at different concentrations is greater than 1.2, indicating that when using the microsphere diluent of formulation #8 for detection, the detection sensitivity of each cytokine is significantly relatively high.

[0207] The concentrations of 15 clinical plasma samples detected using the microsphere diluent of formulation #8 are shown in Table 32.

[0208] Table 32 Concentrations of clinical plasma samples detected using the microsphere diluent of formulation #8

[0209]

[0210] As can be seen from Table 32, when using the microsphere diluent of formulation #8 for detection, the absolute value of the concentration deviation of some cytokines in some samples is still relatively large compared with the concentration detected using the cytokine combined detection kit (immunofluorescence method) (Jiangxi Saiji Biotechnology Co., Ltd., Jiangxi Medical Device Registration No. 20192400359), and some exceed 50% or even 100%.

[0211] Furthermore, the detection accuracy of each cytokine is shown in Table 33.

[0212] Table 33 Detection accuracy of each cytokine using the microsphere diluent of formulation #8

[0213]

[0214] As can be seen from Table 33, when using the microsphere diluent of formulation #8 for detection, the detection accuracy of some cytokines (IL-6 and IL-10) still does not reach 100%.

[0215] The overall accuracy of each clinical sample is shown in Table 34:

[0216] Table 34 Overall accuracy of clinical plasma samples detected using the microsphere diluent of formulation #8

[0217]

[0218] As can be seen from Table 34, compared with the microsphere diluent of formulation #2, for the microsphere diluent of formulation #8 with benzethonium chloride added, since the detection accuracy of some cytokines (IL-6 and IL-10) still does not reach 100%, the overall accuracy of 3 clinical samples does not reach 100%.

[0219] Example 9 Formulation optimization of microsphere diluent - adding domiphen

[0220] The inventor optimized based on Formulation #2 in Example 2. Specifically, Domiphen Bromide with a mass-volume fraction of 0.2% was added to the basis of Formulation #2 in Example 2 to obtain a new buffer formulation (Formulation #9) as follows:

[0221] Based on 1000 mL, it includes 6.052 g of Trizma® base, 1.2 mL of Krovin 500, 0.6 mL of Tween 20, 8 g of NaCl, 25 g of BSA, 2 mL of Domiphen Bromide, and the pH is 8.2 ± 0.1.

[0222] Each standard product was detected in the same method as in Example 1, and the results are shown in Table 35.

[0223] Table 35 MFI measurement values of each standard product detected using the microsphere diluent of Formulation #9

[0224]

[0225] As can be seen from Table 35, when detected using the microsphere diluent of Formulation #9, the S / N of each cytokine at different concentrations is greater than 1.2, indicating that when detected using the microsphere diluent of Formulation #9, the detection sensitivity of each cytokine is significantly relatively high.

[0226] The concentrations of 15 clinical plasma samples detected using the microsphere diluent of Formulation #9 are shown in Table 36.

[0227] Table 36 Concentrations of clinical plasma samples detected using the microsphere diluent of Formulation #9

[0228]

[0229] As can be seen from Table 36, when detected using the microsphere diluent of Formulation #9, the absolute value of the concentration deviation of all cytokines in each sample from the concentration detected using the cytokine combined detection kit (immunofluorescence method) (Jiangxi Saiji Biotechnology Co., Ltd., Jiangxi Medical Device Registration No. 20192400359) does not exceed 10%, and most are even almost exactly the same.

[0230] Furthermore, the detection accuracy of each cytokine is shown in Table 37.

[0231] Table 37 Detection accuracy of each cytokine detected using the microsphere diluent of Formulation #9

[0232]

[0233] As can be seen from Table 37, when detected using the microsphere diluent of Formulation #9, the detection accuracy of all cytokines reaches 100%.

[0234] The overall accuracy of each clinical sample is shown in Table 38:

[0235] Table 38 Overall accuracy of clinical plasma samples detected using the microsphere diluent of Formulation #9

[0236]

[0237] As can be seen from Table 38, compared with the microsphere diluent of Formulation #2, the microsphere diluent of Formulation #9 with domiphen bromide added has an overall accuracy of 100% for all clinical samples.

[0238] The inventor further analyzed the test results and unexpectedly found that the reason for the above phenomenon was that when detecting using the microsphere diluent of Formulation #1, microsphere aggregation occurred, as Figure 2 shown, resulting in inaccurate test results. When detecting using the microsphere diluents of Formulations #2 - #8, the microsphere aggregation phenomenon did not alleviate and even became more severe. When detecting using the microsphere diluent of Formulation #9, only slight microsphere aggregation occurred, as Figure 3 shown.

[0239] Example 10 Without replacement using a buffer

[0240] Different from the method of Example 1, take 25 μL of the antibody solution conjugated with fluorescent microspheres, directly add 25 μL of the standard product or plasma sample, then add 25 μL of the fluorescently labeled antibody solution, mix well, and let it stand in the dark at room temperature for 2.5 h (one-step incubation); detect each standard product, and the results are shown in Table 39.

[0241] Table 39 MFI measurement values of each standard product detected in the microsphere diluent of Formulation #9

[0242]

[0243] As can be seen from Table 39, when detecting using the microsphere diluent of Formulation #9, the S / N of each cytokine at different concentrations is greater than 1.2, indicating that when detecting in the microsphere diluent of Formulation #9, the detection sensitivity of each cytokine is also relatively high.

[0244] The concentrations of 15 clinical plasma samples detected in the microsphere diluent of Formulation #9 are shown in Table 40.

[0245] Table 40 Concentrations of clinical plasma samples detected in the microsphere diluent of Formulation #9

[0246]

[0247] As can be seen from Table 40, when directly detecting using the microsphere diluent of formulation #9, the absolute value of the concentration deviation of all cytokines in each sample from the concentration detected using the cytokine combined detection kit (immunofluorescence method) (Jiangxi Saiji Biotechnology Co., Ltd., Jiangxi Medical Device Approval No. 20192400359) is very large.

[0248] Furthermore, the detection accuracy of each cytokine is shown in Table 41.

[0249] Table 41 Detection accuracy of each cytokine directly detected in the microsphere diluent of formulation #9

[0250]

[0251] As can be seen from Table 41, when directly detecting using the microsphere diluent of formulation #9, the detection accuracy of cytokines is poor.

[0252] The overall accuracy of each clinical sample is shown in Table 42:

[0253] Table 42 Overall accuracy of clinical plasma samples directly detected in the microsphere diluent of formulation #9

[0254]

[0255] As can be seen from Table 42, when directly detecting in the microsphere diluent of formulation #9 instead of after converting to microsphere buffer, the overall accuracy of all clinical samples fails to reach 100%.

[0256] Example 11 Determination of the limit of blank and the limit of detection

[0257] Detection method for the limit of blank: Use the zero-concentration standard product as the sample for detection, repeat the determination 20 times, obtain the concentration values of the 20 measurement results according to the curve equation of the standard product used in the kit, calculate their average value (M) and standard deviation (SD), and obtain M + 2SD, which is the blank limit value.

[0258] Detection method for the limit of detection: Detect 5 low-value samples with concentrations approximately at the limit of detection (the approximate limit of detection is estimated according to the obtained blank limit value, slightly higher than the limit of blank), detect each sample 5 times, sort the detection results by size, and the number of detection results lower than the blank limit value should be less than or equal to 3.

[0259] The limits of blank and the limits of detection of formulations #6 to #9 are determined according to the above method. Taking the cytokine combined detection kit (immunofluorescence method) (Jiangxi Saiji Biotechnology Co., Ltd., Jiangxi Medical Device Approval No. 20192400359) as the control, the results are shown in Table 43.

[0260] Table 43 Blank limits and detection limits of microsphere diluents of Formulation #6 to Formulation #9 for detection

[0261]

[0262] As can be seen from Table 43, when using the microsphere diluent of Formulation #9 for detection, the blank limits and detection limits for each cytokine are significantly lower than those of other formulations, indicating that the microsphere diluent of Formulation #9 has the best performance.

[0263] In summary, when using the diluent of Formulation #9 to detect cytokine proteins, the detection signal value is high, and theoretically, the detection sensitivity can be improved. To verify this, the inventor used a cytokine combined detection kit (immunofluorescence method) (Jiangxi Saiji Biotechnology Co., Ltd., Jiangxi Medical Device Registration No. 20192400359) as a control to detect cytokine protein samples.

[0264] Among them, the cytokine protein samples were prepared using international calibrators (all purchased from NIBSC), and the detection results are shown in Table 44.

[0265] Table 44 Measured values of cytokine proteins detected by different methods

[0266]

[0267] Overall, when the concentration of 12 international calibrators of cytokine proteins was 0.400 pg / mL, the kit from Jiangxi Saiji could not produce results, while the measured values of the microsphere diluent of Formulation #9 were very close to the true values.

[0268] Example 12 Stability test of the microsphere diluent of Formulation #9

[0269] The microsphere diluent of Formulation #9 was stored for 3 days under refrigerated conditions (2 - 8°C) or heat treatment conditions (37°C) respectively. The MFI measured values of each cytokine standard were measured and further used for the detection of 15 clinical samples.

[0270] The MFI measured values of different concentrations of each cytokine standard obtained from the microsphere diluent of Formulation #9 under the two storage conditions were used to obtain the MFI measured value deviation. The results are as Figure 4 shown. As can be seen from Figure 4 , when the microsphere diluent of Formulation #9 was stored under refrigerated conditions or heat treatment conditions, the MFI measured value deviation of different concentrations of each cytokine standard did not exceed 10%, indicating stable performance. The detection concentration deviation of the microsphere diluent of Formulation #9 after storage under different conditions for the detection of clinical samples is as Figure 5 shown. As can be seen from Figure 5It can be seen that the microsphere diluent of formulation #9 has little deviation in the test results of clinical samples when stored under refrigerated conditions or heat treatment conditions, indicating little difference, and further indicating that the microsphere diluent of formulation #9 has high stability.

[0271] All documents mentioned in the present invention are cited herein by reference as if each individual document was cited by reference. 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 appended claims of the present application.

Claims

1. A method for detecting 12 cytokines in a liquid biological sample, characterized in that: The following steps are involved: S1, centrifuging the antibody solution coupled to fluorescent microspheres to remove the supernatant, adding 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, Among them, the 12 cytokines are 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. 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 into Tris buffer, and the fluorescence intensity of the fluorescent microspheres coupled with each first antibody is different. 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, the first surfactant is domiphene, and the preservative is Krovin 500; 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.

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

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

4. The method 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.

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