AU2021104370A4

AU2021104370A4 - Kit for quantitatively detecting cytokines using fluorescent microarray

Info

Publication number: AU2021104370A4
Application number: AU2021104370A
Authority: AU
Inventors: Shanshan Chen; Shidan LI; Yi Liu; Huahao SHEN; Shandong Wu; Shaochang WU; Zhoujie Wu; Xukai Yang; Mingzhi Zhu
Original assignee: Hangzhou Zheda Dixun Biological Gene Engineering Co Ltd
Current assignee: Hangzhou Zheda Dixun Biological Gene Engineering Co Ltd
Priority date: 2021-07-21
Filing date: 2021-07-21
Publication date: 2021-09-16
Anticipated expiration: 2029-07-21

Abstract

OF THE DISCLOSURE The present disclosure relates to a kit for quantitatively detecting cytokines using a fluorescent microarray, and belongs to the technical field of protein detection. The kit of the present disclosure includes a detecting plate and detection antibodies coupled with fluorescent microspheres, wherein the detecting plate is provided with a plurality of reaction chambers, the reaction chamber is provided with an opening, and an inner bottom surface of the reaction chamber is provided with a plurality of detection sites that are arranged side by side along a length direction of the reaction chamber at an interval. The kit of the present disclosure may detect cytokines with high sensitivity, which may quickly and quantitatively detect the cytokine levels in human serum or plasma and may detect a dozen or more cytokines at one time. ABSTRACT DRAWING - Fig 3 17894483_1 (GHMatters) P116788.AU 2/3 1 FIG3 FIG4

Description

2/3 1

FIG3

FIG4

KIT FOR QUANTITATIVELY DETECTING CYTOKINES USING FLUORESCENT MICROARRAY TECHNICAL FIELD

[01] The present disclosure relates to the technical field of protein detection, and in particular to a highly-sensitive kit for quantitatively detecting various cytokine levels in human serum or plasma using a fluorescent microarray.

BACKGROUNDART

[02] The term "cytokine storm" (hypercytokineemia) was first proposed in 1993 as the pathogenesis of graft-versus-host disease (GVHD). This term was first used in infectious disease research in early 2000, which was used in reports of cytomegalovirus (CMV), hemophagocytic lymphohistiocytosis (HLH), influenza virus, severe acute respiratory syndrome coronavirus (SARS-CoV), etc. Cytokine storm is an important cause of acute respiratory distress syndrome (ARDS) and multiple organ failure (MOF), and the concentration of cytokine is related to the severity and prognosis of the diseases.

[03] Cytokines are low-molecular-weight (LMW) soluble proteins produced by a variety of cells under the induction of stimulants such as immunogens or mitogens, which have various functions such as innate immune and adaptive immune regulation, hematopoiesis, cell growth, and reparation of wounded tissue. Cytokines can include interleukin (IL), interferon (IFN), tumor necrosis factor (TNF), colony-stimulating factor (CSF), chemokine, growth factor, etc. Numerous cytokines mutually promote or restrict in the body, forming an extremely-complex immunoregulatory network of cytokines. Specific cytokines exert their biological functions in the three ways of autocrine, paracrine, and endocrine, which have multiple characteristics such as pleiotropic, overlapping, antagonistic, and synergistic characteristics. Cytokines function as a "double-edged sword" like other immune molecules, which can not only play an immunoregulatory role, but also participate in the occurrence of various diseases under some conditions, and even trigger cytokine storm and cytokine storm syndrome, leading to damage and dysfunction of multiple organs and death.

[04] Cytokine storm, related to a variety of infectious and non-infectious diseases, is a systemic inflammatory response induced by infections, drugs, and other factors. Cytokine storm-related inflammation begins in local tissues and spreads throughout the body through the circulation, with specific manifestations of increased blood flow, increased local temperature (fever), myalgia/arthralgia, nausea, rash, lassitude, and other mild flu-like acute inflammation

17894483_1 (GHMatters) P116788.AU symptoms, which mobilizes the body's immune system to resist pathogen infection. Acute inflammation is also characterized by the release of proinflammatory cytokines or chemokines. A compensatory repair process begins shortly after the inflammation begins, and in many cases, the compensatory repair process may completely restore the functions of tissues and organs.

[05] In an infection status, pathogens try to disrupt a sophisticated immunoregulatory system to evade immune responses and evolve a variety of evasion strategies to achieve mass reproduction. In some cases, pathogens can escape an immune response and thus will not induce an effective immune reaction. In other cases, some pathogens can excessively stimulate the immune system. When a local tissue structure is destroyed, dysregulated proinflammatory cytokines/chemokines may overflow into the circulatory system, causing a large-scale inflammatory cascade. When cytokine storm occurs, single-organ or multi-organ systemic inflammation is over-presented, such as pulmonary symptoms (hypoxemia, pulmonary edema caused by vascular leakage, and even ARDS), cardiovascular symptoms (hypotension, arrhythmia, myocardial damage, and shock), hematological symptoms (continuous reduction of blood cell, coagulation disorder, disseminated intravascular coagulation (DIC)), acute kidney injury (AKI), MOF, and even death. This uncontrolled systemic inflammation is caused by the release of extreme inflammatory mediators due to excessive activation and expansion of primary immune cells.

[06] Cytokines are highly related to inflammation, so cytokines can be detected to regulate inflammation as soon as possible, clinically guide the use of antibiotics, and assist in the diagnosis of viral infections. Current methods for detecting cytokines include flow fluorescence and enzyme-linked immunosorbent assay (ELISA). The flow fluorescence requires a relatively-high cost and a high instrument cost. ELISA can only achieve single-item detection and involves large serum consumption. Cytokines are items with many detection indexes, and available methods currently on the market have problems such as large instrument, high detection cost, long test time, and large sample size.

SUMMARY

[07] The present disclosure is intended to provide a kit for quantitatively detecting cytokines using a fluorescent microarray. The kit of the present disclosure has high throughput, low reaction cost, high accuracy, and prominent repeatability, and can detect cytokines with high sensitivity.

[08] The present disclosure provides a kit for quantitatively detecting cytokines using a fluorescent microarray, including a detecting plate and detection antibodies coupled with

2 17894483_1 (GHMatters) P116788.AU fluorescent microspheres, wherein the detecting plate is provided with a plurality of reaction chambers; the reaction chamber is provided with an opening, and an inner bottom surface of the reaction chamber is provided with a plurality of detection sites that are arranged side by side along a length direction of the reaction chamber at an interval; the detection sites on the detecting plate are fixed with cytokine-specific monoclonal antibodies (mAbs) that acted as capture antibodies; and the detection antibodies coupled with fluorescent microspheres are the paired antibodies of cytokine-specific monoclonal antibodies.

[09] In one embodiment, a method for fixing the test cytokine-specific monoclonal antibodies include: coating each detection site with streptavidin, coupling the streptavidin to biotin-labeled test cytokine-specific monoclonal antibodies and coupling biotin-labeled test cytokine-specific monoclonal antibodies to different detection sites on the detecting plate respectively.

[10] In one embodiment, a material of the detecting plate include polystyrene (PS); an upper end of the reaction chamber may be provided with an opening; and the detection site may be a groove or a raised column.

[11] In one embodiment, the test cytokine may include IL-Ibeta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12P70, IL-17A, TNF-a, IFN-y, and IFN-a.

[12] The present disclosure also provides a method for high-throughput detection of cytokines using the kit according to the above technical solution, including the following steps:

[13] (1) horizontally fixing and placing the detecting plate fixed with the test cytokine-specific monoclonal antibodies at room temperature;

[14] (2) adding test serum or plasma into the reaction chambers of the detecting plate, thoroughly mixing, and incubating at room temperature for 30 min to 60 min;

[15] (3) rinsing the reaction chambers with a washing liquid;

[16] (4) adding the test cytokine-specific paired antibodies coupled with fluorescent microspheres into the reaction chambers, thoroughly mixing, and incubating at room temperature for 30 min to 60 min;

[17] (5) rinsing the reaction chambers with a washing liquid; and

[18] (6) drying, and reading results with a reader.

[19] The present disclosure provides a kit for quantitatively detecting cytokines using a fluorescent microarray. A dual signal amplification system of the fluorescent microsphere method and the biotin-streptavidin biological method was used in the present disclosure to rapidly and quantitatively detect the cytokine levels in human serum or plasma with high sensitivity, which may rapidly and accurately screen a dozen or more cytokines at a time with

3 17894483_1 (GHMatters) P116788.AU high sensitivity and is suitable for high-throughput detection.

BRIEF DESCRIPTION OF DRAWINGS

[20] FIG. 1 is a schematic structural diagram of the reaction chamber provided by the present disclosure;

[21] FIG. 2 is a schematic structural diagram of an outer bottom surface of the reaction chamber provided by the present disclosure;

[22] FIG. 3 is a schematic structural diagram of the detecting plate provided by the present disclosure;

[23] FIG. 4 is a schematic structural diagram of a fixing frame in the detecting plate provided by the present disclosure; and

[24] FIG. 5 is a schematic diagram of a back structure of the fixing frame in the detecting plate provided by the present disclosure.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[25] The present disclosure provides a kit for quantitatively detecting cytokines using a fluorescent microarray, including a detecting plate and detection antibodies coupled with fluorescent microspheres, where the detecting plate is provided with a plurality of reaction chambers, the reaction chamber is provided with an opening, and an inner bottom surface of the reaction chamber is provided with a plurality of detection sites that are arranged side by side along a length direction of the reaction chamber at an interval. In the kit of the present disclosure, fluorescent microspheres for amplifying a signal and a detecting plate (biotin-streptavidin biological method) are used to obtain a dual signal amplification system, so the kit may simultaneously detect multiple indexes and may also quantify by fluorescence, which greatly improves the detection efficiency and detection sensitivity. In one embodiment, the detecting plate and the detection antibodies coupled with fluorescent microspheres are placed separately.

[26] In one embodiment, a material of the detecting plate may preferably include PS; an upper end of the reaction chamber may be provided with an opening, and an inner bottom surface of the reaction chamber is provided with a plurality of detection sites that are arranged side by side along a length direction of the reaction chamber at an interval; and the detection site may preferably be a groove or a raised column.

[27] In one embodiment, the reaction chamber may preferably be made of a transparent material.

4 17894483_1 (GHMatters) P116788.AU

[28] In one embodiment, an outer bottom surface of the reaction chamber is recessed inwardly at least at locations corresponding to the plurality of detection sites.

[29] In one embodiment, a first handle and a second handle may preferably be fixedly disposed on side walls of two ends of the reaction chamber along the length direction, respectively, and the first handle and the second handle may be embedded in a fixing frame to fix the reaction chamber on the fixing frame, thus the detecting plate of the present disclosure is obtained. In one embodiment, the fixing frame may preferably be a chamber body with an open upper end, and the plurality of reaction chambers may be fixedly arranged side by side in the chamber body along a width direction of the fixing frame.

[30] In one embodiment, the first handle may preferably have a shape different from that of the second handle.

[31] In one embodiment, a first handle and a second handle may preferably be fixedly disposed on side walls of two ends of the reaction chamber along the length direction, respectively; a plurality of first handle-embedding grooves and a plurality of second handle-embedding grooves are disposed on upper end surfaces of two opposite side walls of the fixing frame along a width direction of the reaction chamber; a shape of the first handle-embedding groove matches a shape of the first handle, and a shape of the second handle-embedding groove matches a shape of the second handle; and the reaction chamber is fixedly connected to the fixing frame by embedding the first handle and the second handle into the first handle-embedding groove and the second handle-embedding groove, respectively. In one embodiment, a plurality of weight-reducing holes may preferably be disposed on a lower bottom surface of the chamber body, and a weight-reducing slot may preferably be disposed on a lower end surface of a side wall of thefixing frame.

[32] In one embodiment, a structure of the detecting plate may preferably be shown in FIG. 1 to FIG. 5, wherein FIG. 1 is a schematic structural diagram of the reaction chamber; FIG. 2 is a schematic structural diagram of the outer bottom surface of the reaction chamber; FIG. 3 is a schematic structural diagram of the detecting plate; FIG. 4 is a schematic structural diagram of the fixing frame in the detecting plate; and FIG. 5 is a schematic diagram of a back structure of the fixing frame in the detecting plate. In the figures: 1 represents a reaction chamber, 2 represents a reaction site, 3 represents an anti-skidding groove, 4 represents a first handle, 5 represents a second handle, 6 represents a fixing frame, 7 represents afirst handle-embedding groove, 8 represents a second handle-embedding groove, 9 represents a weight-reducing hole, and 10 represents a weight-reducing slot.

[33] In one embodiment, an upper end of the reaction chamber is open; an inner bottom

5 17894483_1 (GHMatters) P116788.AU surface of the reaction chamber is provided with a plurality of detection sites that are arranged side by side along a length direction of the reaction chamber at an interval; and the detection sites may carry proteins or antibodies, and the proteins or antibodies may be adsorbed on the detection sites. The present disclosure has no special limitations on a spacing among the plurality of detection sites.

[34] In one embodiment, the kit may preferably further include a diluent and a washing liquid. In one embodiment, the diluent may preferably be a PBS buffer. In one embodiment, the washing liquid may preferably be 0.01 M, pH 7.4 PBS with 0.05% (mass percentage content) of Tween 20.

[35] In one embodiment, test cytokine-specific monoclonal antibodies are fixed on the detection sites of the detecting plate, and the detection antibodies coupled with fluorescent microspheres are the paired antibodies of cytokine-specific monoclonal antibodies; and the test cytokine includes IL-Ibeta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12P70, IL-17A, TNF-a, IFN-y, and IFN-a.

[36] The present disclosure has no special limitations on sources of the test cytokine-specific monoclonal antibodies coupled with fluorescent microspheres, and conventional commercially-available products well known to those skilled in the art may be used, such as Anti-IL-i beta Antibody (SinoBiological, 10139-MM07); IL-2 (abeam, ab222639); Anti-IL-4 Antibody (SinoBiological, 11846-MM04); Anti-IL-5 Antibody (SinoBiological, 15673-ROO); Anti-IL-6 Antibody (SinoBiological, 10395-MM14); Anti-IL-8 Antibody (SinoBiological, 10098-MM05); Anti-IL-10 Antibody (SinoBiological, 10947-MM19); Anti-IL-12p70 Antibody (SinoBiological, CT11-ROO1); Anti-IL17 Antibody (SinoBiological, 12047-MM25); Anti-TNF-a Antibody (SinoBiological, 10602-MMO1); Anti-IFN-y Antibody (SinoBiological, 11725-R209); and Anti IFN-a Antibody (ProSpec-Tany, ANT-208).

[37] The present disclosure has no special limitations on sources of the test cytokine-specific paired antibodies coupled with fluorescent microspheres, and conventional commercially-available products well known to those skilled in the art may be used, such as Anti-IL-i beta Antibody (SinoBiological, 10139-MM097); IL-2 (abeam, ab222640); Anti-IL-4 Antibody (SinoBiological, 11846-MM05); Anti-IL-5 Antibody (SinoBiological, 15673-R013); Anti-IL-6 Antibody (SinoBiological, 10395-MM72); Anti-IL-8 Antibody (SinoBiological, 10098-MM18); Anti-IL-10 Antibody (SinoBiological, 10947-T16); Anti-IL-12p70 Antibody (SinoBiological, CTO11-R70); Anti-IL17 Antibody (SinoBiological, 12047-MM31); TNF alpha (SinoBiological, 10602-MM08); IFN-y (SinoBiological, 11725-R238); Anti IFN-a

6 17894483_1 (GHMatters) P116788.AU

Antibody (ProSpec-Tany, ANT-208).

[38] In one embodiment, a method for preparing the detecting plate fixed with the test cytokine-specific monoclonal antibodies, including the following steps:

[39] coating each detection site of the detecting plate with streptavidin to obtain a coated detecting plate; labeling test cytokine-specific monoclonal antibodies with biotin to obtain biotin-labeled test cytokine-specific monoclonal antibodies; and coupling the biotin-labeled test cytokine-specific monoclonal antibodies to different detection sites of the detecting plate to obtain the detecting plate fixed with the test cytokine-specific monoclonal antibodies.

[40] In one embodiment, each detection site of the detecting plate is coated with streptavidin to obtain a coated detecting plate. The present disclosure has no special limitations on a method of the coating, and a conventional streptavidin coating method well known to those skilled in the art may be used.

[41] In one embodiment, test cytokine-specific monoclonal antibodies are labeled with biotin to obtain biotin-labeled test cytokine-specific monoclonal antibodies. In one embodiment, before the biotin labeling, the test cytokine-specific monoclonal antibodies may preferably be dissolved in 0.01 M, pH 7.4 PBS. In one embodiment, a volume ratio of the test cytokine-specificmAb to the 0.01 M, pH 7.4 PBS may be preferably 1:(1-10) and more preferably 1:10 to achieve the optimal sensitivity and specificity.

[42] In one embodiment, the biotin-labeled test cytokine-specific monoclonal antibodies are coupled to different detection sites of the detecting plate to obtain the detecting plate fixed with the test cytokine-specific monoclonal antibodies. That is, in one embodiment, different test cytokine-specific monoclonal antibodies need to be fixed at different sites in the reaction chamber of the detecting plate. In one embodiment, the test cytokine-specific paired antibodies, before being coupled with fluorescent microspheres, may preferably be dissolved in 0.01 M, pH 7.4 PBS that includes 0.05% of Tween 20, 0.05% of Proclin-300, and 0.1% of BSA (mass percentage content) to achieve the optimal sensitivity and specificity. In one embodiment, a volume ratio of the test cytokine-specific paired antibodies to the 0.01 M, pH 7.4 PBS that includes 0.05% of Tween 20, 0.05% of Proclin-300, and 0.1% of BSA (mass percentage content) may be preferably 1:(10-1,000) and more preferably 1:100 to achieve the optimal sensitivity and specificity. In one embodiment, the fluorescent microspheres may preferably be purchased from Invitrogen, with model: F8807.

[43] The present disclosure also provides a method for high-throughput detection of cytokines using the kit, preferably including the following steps:

[44] (1) horizontally fixing and placing the detecting plate fixed with the test

7 17894483_1 (GHMatters) P116788.AU cytokine-specific monoclonal antibodies at room temperature;

[45] (2) adding test serum or plasma into the reaction chambers of the detecting plate, thoroughly mixing, and incubating at room temperature for 30 min to 60 min;

[46] (3) rinsing the reaction chambers with a washing liquid;

[47] (4) adding the test cytokine-specific paired antibodies coupled with fluorescent microspheres into the reaction chambers, thoroughly mixing, and incubating at room temperature for 30 min to 60 min;

[48] (5) rinsing the reaction chambers with a washing liquid; and

[49] (6) drying, and reading results with a reader.

[50] In one embodiment, the detecting plate fixed with the test cytokine-specific monoclonal antibodies is horizontally fixed and placed at room temperature (18°C to 26°C). In one embodiment, the detecting plate fixed with the test cytokine-specific monoclonal antibodies may preferably be horizontally fixed on a plate fixing holder.

[51] In one embodiment, test serum or plasma is added into the reaction chambers of the detecting plate, and a resulting mixture is thoroughly mixed and incubated at room temperature (18°C to 26°C) for 30 min to 60 min. In one embodiment, the mixing may preferably be conducted with a mixer, and the mixer may preferably include a shaker. The shaker of the present disclosure may preferably be a WD-9405A decolorizing shaker purchased from Ward Biomedical Instrument Company.

[52] In one embodiment, after the incubation, the reaction chambers are rinsed with a washing liquid. In one embodiment, the rinsing may be conducted preferably for 10 s to 30 s each time, and may be conducted preferably 3 to 5 times. The present disclosure has no special limitations on an amount of washing liquid added to the reaction chamber during washing, and a conventional washing liquid amount may be used, for example, the washing liquid may be added to cover all detection sites but not overflow during reaction.

[53] In one embodiment, after the rinsing, the test cytokine-specific paired antibodies coupled with fluorescent microspheres are added into the reaction chambers, and a resulting mixture is thoroughly mixed and incubated at room temperature (18°C to 26°C) for 30 min to min.

[54] In one embodiment, after the incubation, the reaction chambers are rinsed with a washing liquid. In one embodiment, the rinsing may be conducted preferably for 10 s to 30 s each time, and may be conducted preferably 3 to 5 times. The present disclosure has no special limitations on an amount of washing liquid added to the reaction chamber during washing, and a conventional washing liquid amount may be used, for example, the washing liquid may be

8 17894483_1 (GHMatters) P116788.AU added to cover all detection sites but not overflow during reaction.

[55] In one embodiment, after the rinsing, the detecting plate is dried, and results are read with a reader. In one embodiment, a method of the drying may preferably include pat-drying with a hand on a paper towel. The reader of the present disclosure may preferably be a reader with a data processing function, which can quantitatively detect the levels of various cytokines in human serum or plasma. In one embodiment, the reader may preferably be a fluorescence immunoassay analyzer (model: F1OPro) purchased from Tianjin Paipu Daye Company. The reader of the present disclosure can read a fluorescence value at a corresponding position, and then a concentration is calculated according to a standard curve.

[56] The kit of the present disclosure can rapidly and quantitatively detect the cytokine levels in human serum or plasma with high sensitivity, which can rapidly and accurately screen dozens of cytokines at a time with high sensitivity and is suitable for high-throughput detection.

[57] The kit for quantitatively detecting cytokines using a fluorescent microarray according to the present disclosure will be further described in detail below with reference to specific examples. The technical solutions of the present disclosure include but are not limited to the following examples.

[58] Example 1

[59] Preparation of the kit of the present disclosure

[60] 1. Fixation of cytokine-specific monoclonal antibodies on a PS detecting plate

[61] A. Commercially-available streptavidin was prepared into a solution with an appropriate concentration (such as 0.1 mg/mL to 2 mg/mL) using 0.01 M, pH 7.4 PBS. 0.5 L to 2 L of the solution was added to each detection site of the reaction chambers of the detecting plate to statically react overnight (more than 16 h) at 4°C.

[62] B. The reaction chamber of the detecting plate was washed with 0.5 mL to 1.5 mL of 0.01 M, pH 7.4 PBS with 0.05% of Tween 20 once and then pat-dried.

[63] C. 0.5 mL to 1.5 mL of 0.01 M, pH 7.4 PBS with 2% of BSA was added to each reaction chamber to statically react overnight (more than 16 h) at 4°C for blocking.

[64] D The detecting plate was washed with 0.5 mL to 1.5 mL of 0.01 M, pH 7.4 PBS with 0.05% of Tween 20 once and then pat-dried.

[65] E. Biotin-labeled cytokine-specific monoclonal antibodies were prepared into solutions with appropriate concentrations (for example, IL-Ibeta: 0.1 mg/mL to 7.0 mg/mL; IL-2: 2.0 mg/mL to 5.0 mg/mL; IL-4: 0.05 mg/mL to 3.0 mg/mL; IL-5: 1.0 mg/mL to 8.0 mg/mL; IL-6: 1.5 mg/mL to 10.0 mg/mL; IL-8: 0.1 mg/mL to 3.0 mg/mL; IL-10: 2.0 mg/mL to 5.0 mg/mL; IL-12P70:1.5 mg/mL to 10.0 mg/mL; IL-17A: 1.0 mg/mL to 8.0 mg/mL; TNFa: 2.0 mg/mL to

9 17894483_1 (GHMatters) P116788.AU

5.0 mg/mL; and IFN-: 1.5 mg/mL to 10.0 mg/mL) using 0.01 M, pH 7.4 PBS. 0.5 L to 2 L of each of the solutions was added to a well at a corresponding position to react at 37°C for 30 min.

[66] F. The detecting plate was washed with 0.5 mL to 1.5 mL of 0.01 M, pH 7.4 PBS with 0.05% of Tween 20 three times and then pat-dried for later use.

[67] 2. Mixed antibodies of cytokines coupled with fluorescent microspheres

[68] (1) IL-lbeta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12P70, IL-17A, TNF-a, IFN-7, and IFN-a monoclonal antibodies were mixed in a specified ratio (such as 1:1:1:1:1:1:1:1:1:1:1:1) and prepared into a solution (where each mAb had a concentration of 1 mg/mL) with PBS.

[69] (2) Microsphere coupling

[70] A. 835 pL of purified water and 50 L of a coupling buffer (a 500 mM 2-(N-morpholino)ethanesulfonic acid (MES) solution, pH 6.1) were sequentially added to a 2 mL centrifuge tube and thoroughly mixed.

[71] B. 100 L of 200 nm fluorescent microspheres (Invitrogen, F8807) (solid content: 2%) was added, and a resulting mixture was thoroughly mixed.

[72] C. 50 g of the mAb solution obtained in step (1) to the centrifuge tube, and a resulting mixture was thoroughly mixed and then reacted for 30 min at room temperature on a rotation reactor (gently and continuously rotating).

[73] D. After the reaction was completed, a 10 mg/mL EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) aqueous solution was prepared (which was prepared just before use and was used for activating carboxyl in labeling), pL of the EDC solution was immediately transferred to the centrifuge tube, and a resulting mixture was thoroughly mixed by rapid pipetting.

[74] E. Then the mixture was vortexed for thorough mixing, and then reacted for 2 h at room temperature on a rotation reactor.

[75] F. After the reaction was completed, a resulting system was centrifuged (15,000 rpm, 8 min), and a supernatant was removed. 1 mL of a washing buffer (0.01 M, pH 7.4 PBS with 0.05% of Tween 20) was added to the centrifuge tube, and a resulting mixture was thoroughly mixed by ultrasonic treatment (power: 15%; pulse duration: 3 s, interval: 3 s, and 1 min in total) such that a reaction product was completely dispersed.

[76] G. The reaction product was washed twice: a resulting system was centrifuged (15,000 rpm, 10 min), and a supernatant was removed; and 1 mL of the washing buffer was added, and a resulting mixture was thoroughly mixed by ultrasonic treatment (power: 10%; pulse duration: 3 s, interval: 3 s, and 1 min in total) such that the reaction product was completely dispersed.

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[77] H. 1 mL of a blocking buffer (0.01 M, pH 7.4 PBS with 0.05% of Tween 20 and 0.5% of BSA) was added to re-disperse the product by ultrasonic treatment, and a resulting reaction system reacted at room temperature for 1 h on a rotation reactor.

[78] I. After the reaction was completed, a resulting system was centrifuged (15,000 rpm, 6 min), and a supernatant was removed; and a reaction product was washed twice with 1 mL of a storage buffer (0.01 M, pH 7.4 PBS with 0.05% of Tween 20, 0.05% of Proclin-300, and 0.1% of BSA) and finally stored in 2 mL of a storage buffer.

[79] Example 2

[80] Dual signal amplification system using both the fluorescence method and the biological method (biotin-streptavidin method)

[81] The preparation of a kit can be seen in Example 1.

[82] A detection method was as follows:

[83] (1) The detecting plate fixed with different cytokine-specific monoclonal antibodies was horizontally placed on a special plate holder at room temperature for later use.

[84] (2) 200 L to 400 L of serum or plasma was added to each reaction chamber, and then the plate holder was placed on a mixer and incubated at room temperature for 30 min to 60 min.

[85] (3) The reaction chamber was rinsed 3 to 5 times using a washing liquid, with 10 s to s for each time.

[86] (4) 200 L to 400 L of the working solution of mixed paired antibodies coupled with fluorescent microspheres was diluted with PBS in a volume ratio of 1:(10-1,000) and then added to each reaction chamber, and then the detecting plate was placed on a mixer and incubated at room temperature for 30 min to 60 min.

[87] (5) The reaction chamber was rinsed 3 to 5 times using a washing liquid, with 10 s to s for each time; and

[88] (6) Then the detecting plate was pat-dried, a result was read with a reader to obtain a fluorescence value, and then a concentration was calculated according to a standard curve. Results were shown in Table 1.

[89] Example 3

[90] Dual signal amplification system using the conventional ELISA double-antibody sandwich method (the fluorescence method + the biological method of the present disclosure was not used):

[91] The detection method was as follows:

[92] (1) Cytokine-specific monoclonal antibodies were prepared into solutions with

11 17894483_1 (GHMatters) P116788.AU appropriate concentrations (for example, IL-lbeta: 0.1 mg/mL to 7.0 mg/mL; IL-2: 2.0 mg/mL to 5.0 mg/mL; IL-4: 0.05 mg/mL to 3.0 mg/mL; IL-5: 1.0 mg/mL to 8.0 mg/mL; IL-6: 1.5 mg/mL to 10.0 mg/mL; IL-8: 0.1 mg/mL to 3.0 mg/mL; IL-10: 2.0 mg/mL to 5.0 mg/mL; IL-12P70: 1.5 mg/mL to 10.0 mg/mL; IL-17A: 1.0 mg/mL to 8.0 mg/mL; TNF-a: 2.0 mg/mL to 5.0 mg/mL; IFN-y: 1.5 mg/mL to 10.0 mg/mL; and IFN-a: 1.5 mg/mL to 10.0 mg/mL) using 0.01 M, pH 7.4 PBS, and 50 L of each of the solutions was added to a well at a corresponding position of an reaction trough to statically react overnight (more than 16 h) at 4°C.

[93] (2) The reaction trough was washed with 0.01 M, pH 7.4 PBS with 0.05% of Tween 20 (150 L/well) once and then pat-dried.

[94] (3) 0.01 M, pH 7.4 PBS with 2% of BSA was added (100 L/well) to statically react overnight (more than 16 h) at 4°C for blocking.

[95] (4) The reaction trough was washed with 0.01 M, pH 7.4 PBS with 0.05% of Tween 20 (150 L/well) once and then pat-dried for later use.

[96] (5) The reaction trough coated with different cytokine-specific monoclonal antibodies was horizontally placed on a shaker at room temperature for later use.

[97] (6) 20 L to 100 L of serum or plasma was added to the reaction trough, and then the reaction trough was placed on a shaker and incubated at room temperature for 30 min to 60 min.

[98] (7) The wells of the reaction trough were rinsed 3 to 5 times using a washing liquid, with 10 s to 30 s for each time.

[99] (8) 20 L to 100 L of a working solution of HRP-coupled mixed paired antibodies (IL-lbeta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12P70, IL-17A, TNF-a, IFN-y, and IFN-a monoclonal antibodies were mixed in a specified ratio (such as1:1:1:1:1:1:1:1:1:1:1:1),with the same concentrations as Example 1) was diluted with PBS in a volume ratio of 1:(10-1,000) and then added to the reaction trough, and then the reaction trough was placed on a mixer and incubated at room temperature for 30 min to 60 min.

[100] (9) The reaction trough was rinsed 3 to 5 times using a washing liquid, with 10 s to 30 s for each time, and then pat-dried.

[101] (10) 20 L to 100 L of a TMB chromogenic solution was added, and the plate was placed on a mixer and incubated at room temperature for 5 min to 15 min.

[102] (11) Results were read with a microplate reader.

[103] Table 1 Comparison of detection results of cytokine levels in serum samples by the two systems

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IL-12P7 IL-17 Item IL-lbeta IL-2 IL-4 IL-5 IL-6 IL-8 IL-10 TNF-a IFN-y IFN-a 0 A

6 10 10 10 12 Exam 60 100 10 10 50 100 pg/m pg/m 5 pg/mL pg/m pg/m pg/m ple 2 pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL L L L L L

not not 13 not 11 not not Exam 105 10 12 55 112 detectab detect pg/m detectab pg/m detect detect ple 3 pg/mL pg/mL pg/mL pg/mL pg/mL le able L le L able able

[104] According to comparison of the detection results in Table 1, it can be known that the dual signal amplification system without streptavidin-biotin and fluorescent microspheres in Example 3 have a low detection sensitivity, such that low-concentration cytokines are not detectable.

[105] The above results show that the kit of the present disclosure, when used for detection, can achieve dual amplification of a signal, resulting in high detection sensitivity; and can simultaneously detect multiple indexes, with simple and convenient operations.

[106] The above descriptions are merely preferred embodiments of the present disclosure. It should be noted that a person of ordinary skill in the art may further make several improvements and modifications without departing from the principle of the present disclosure, but such improvements and modifications should be deemed as falling within the protection scope of the present disclosure.

[107] It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art, in Australia or any other country.

[108] In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

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Claims

WHAT IS CLAIMED IS:

1. A kit for quantitatively detecting cytokines using a fluorescent microarray, wherein the kit comprises a detecting plate and detection antibodies coupled with fluorescent microspheres, wherein the detecting plate is provided with a plurality of reaction chambers; the reaction chamber is provided with an opening, and an inner bottom surface of the reaction chamber is provided with a plurality of detection sites that are arranged side by side along a length direction of the reaction chamber at an interval; the detection sites on the detecting plate are fixed with cytokine-specific monoclonal antibodies (mAbs) that acted as capture antibodies; and the detection antibodies coupled with fluorescent microspheres are the paired antibodies of cytokine-specific monoclonal antibodies.

2. The kit according to claim 1, wherein a method for fixing the test cytokine-specific monoclonal antibodies comprises: coating each detection site with streptavidin, coupling the streptavidin to biotin-labeled test cytokine-specific monoclonal antibodies and coupling biotin-labeled test cytokine-specific monoclonal antibodies to different detection sites on the detecting plate respectively.

3. The kit according to claim 1, wherein a material of the detecting plate comprises polystyrene (PS); an upper end of the reaction chamber is provided with an opening; and the detection site is a groove or a raised column.

4. The kit according to claim 1 or 2, wherein the test cytokine comprises IL-lbeta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12P70, IL-7A, TNF-a, IFN-y, and IFN-a.

5. A method for high-throughput detection of cytokines using the kit according to any one of claims 1 to 4, comprising the following steps: (1) horizontally fixing and placing the detecting plate fixed with the test cytokine-specific monoclonal antibodies at room temperature; (2) adding test serum or plasma into the reaction chambers of the detecting plate, thoroughly mixing, and incubating at room temperature for 30 min to 60 min; (3) rinsing the reaction chambers with a washing liquid;

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(4) adding the test cytokine-specific paired antibodies coupled with fluorescent microspheres into the reaction chambers, thoroughly mixing, and incubating at room temperature for 30 min to 60 min; (5) rinsing the reaction chambers with a washing liquid; and (6) drying, and reading results with a reader.

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