A kit for detecting interleukin 6, a detection method and application

Abstract

The application discloses a kit for detecting interleukin 6 and preparation and application. The kit comprises magnetic microspheres coated first antibodies and tracer marker labeled second antibodies. The first antibodies are 12H6 with a catalog number of DA011, IL-6-55E9 with a catalog number of P1810035 of Jin Sui, or IL-6-01 with a catalog number of 20180915 of Gelin. The second antibodies are 15H5 with a catalog number of DA012, IL-6-55C6 with a catalog number of P1904008 of Jin Sui, or IL-6-02 with a catalog number of 20180919 of Gelin. The application has the advantages of simple operation, low cost, short detection time, high sensitivity and wide linear range, and can realize full-automatic, rapid, sensitive and quantitative detection of the concentration of IL-6 in samples.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of immunological medical detection, and particularly relates to a kit for detecting interleukin 6, a detection method and application. BACKGROUND

[0002] In 1985, Kishimoto et al. first obtained the cDNA clone of interleukin 6 (IL-6) from human T cells. The human IL-6 gene is located on chromosome 7, and the molecular weight is 26 kDa. In 1986, IL-6 was uniformly named as Interleukin-6 (IL-6), which was previously named as hybridoma / plasmacytoma growth factor (HPGF), B cell stimulating factor-2 (BSF-2) and hepatocyte stimulating factor (HSF) according to different experimental systems and functions.

[0003] Interleukin 6 (IL-6) is produced by fibroblasts, T lymphocytes, B lymphocytes, mononuclear macrophages and various tumor cells. Interleukin 6 (IL-6) is a pleiotropic cytokine, which can not only induce B cells to differentiate to produce immunoglobulins, promote the proliferation and growth of T cells, promote the proliferation of bone marrow hematopoietic stem cells, enhance the differentiation of blood cells and their anti-tumor effects, etc., but also is a key component in the inflammatory mediator network, and plays an important role in inflammatory response.

[0004] IL-6 is involved in the occurrence and development of many diseases, and its plasma level is closely related to inflammation, viral infection and autoimmune diseases. Its change is earlier than CRP. At present, it is considered that IL-6 is an early sensitive marker of inflammation, sepsis and a quantitative marker of chronic inflammation, which can effectively guide the use of antibiotics; and can also effectively evaluate the prognosis of sepsis and septic shock.

[0005] At present, the immunodetection methods of interleukin 6 mainly include fluorescence immunoassay (FICA) and chemiluminescence method (CLIA).

[0006] The fluorescence immunoassay (FICA) is a new type of membrane detection technology based on the specific immune reaction of antigen and antibody. The strip fibrous chromatography material fixed with detection line (coated antibody or coated antigen) and quality control line (anti-antibody) is used as the stationary phase, the test liquid is used as the mobile phase, and the fluorescently labeled antibody or antigen is fixed on the connecting pad. Through capillary action, the analyte moves on the chromatographic strip. Taking the double antibody sandwich method as an example, the analyte is first combined with the fluorescently labeled antibody under the action of the mobile phase, and then combined with the coated antibody to form a double antibody sandwich "sandwich" type when reaching the detection line. The fluorescence immunoassay (FICA) has the shortcomings of low degree of automation and small detection flux.

[0007] Chemiluminescence immunoassay (CLIA) is a kind of immunoassay technology that has gradually emerged in recent years. It is a product of the close combination of the immune response system and the chemiluminescence technology. The chemiluminescence technology is a detection method that uses luminescent substances such as acridinium ester, which is oxidized by an oxidizing agent and catalyzed by a catalyst to form an excited-state intermediate, and then uses a corresponding measuring instrument to measure the light quantum yield produced when the intermediate returns to the ground state. The immune response system is to label the antigen or antibody with a luminescent marker, and then detect it through the formation of an antigen-antibody complex. This technology has the advantages of strong specificity, high sensitivity, good precision, wide linear range, high throughput and easy automation.

[0008] Chemiluminescence is divided into direct chemiluminescence immunoassay, enzyme-catalyzed chemiluminescence immunoassay and electrochemiluminescence immunoassay according to the different markers.

[0009] Direct chemiluminescence immunoassay refers to the use of acridinium ester to directly label antibodies (antigens), which will react with the corresponding antigens (antibodies) in the sample to be tested to form a solid-phase coated antibody-test antigen-acridinium ester-labeled antibody complex. Under the action of oxidizing agent (H2O2) and NaOH, acridinium ester can be decomposed and emit light without the need for a catalyst. The strong direct light is completed in one second, which is a rapid flash light. The light intensity is proportional to the amount of the antigen to be tested, and the content of the antigen to be tested can be calculated from the standard curve. Chinese patent application CN107817354A also uses acridinium ester labeling, the amount of magnetic suspension and acridinium ester marker added is 150 μl, the incubation time is 15 min, and the analysis sensitivity is 3.8 pg / mL.

[0010] Enzyme-catalyzed chemiluminescence method is horseradish peroxidase system and alkaline phosphatase system. Chinese patent application CN103048465A uses horseradish peroxidase system, which needs 60 min for reaction incubation time, and 5 min for substrate liquid A and B to react in the dark. Chinese patent application CN104330551A uses alkaline phosphatase system, which has a minimum detection limit of 1.3 pg / mL and a precision of 6% or less. Chinese patent application CN108519487A uses avidin-biotin system, which has many reaction steps and a long reaction time of about 8-30 min.

[0011] Electrochemiluminescence method is that the antigen-antibody complex labeled with trispyridine ruthenium emits visible light after oxidation-reduction reaction under the action of tripropylamine. Its excitation process is complex and time-consuming, and each light emission speed is about 25 seconds. However, the electrochemiluminescence method has high instrument cost and complex technology, which is difficult to implement. SUMMARY

[0012] In view of the defects of low sensitivity and narrow linear range of the chemiluminescence kit for detecting interleukin 6 (IL-6) in the prior art, the present application provides a chemiluminescence kit for detecting interleukin 6, a detection method and application. The kit of the present application for detecting IL-6 is simple to operate, low in cost, short in detection time, high in sensitivity, wide in linear range (3-5000 pg / ml), good in precision (coefficient of variation CV less than 5%), and good in clinical coincidence rate (clinical sample correlation R value 0.9962).

[0013] The present inventors have unexpectedly found, through a large number of experiments, that based on direct chemiluminescence method, two antibodies coated with magnetic beads and tracer labels respectively and having different antigen binding sites are used in combination with a chemiluminescence immunoassay instrument, so that interleukin 6 can be conveniently and quickly detected, and high sensitivity and wide linear range are achieved. Further, the present inventors have also screened the detection conditions in the detection process, further improving the sensitivity and linear range.

[0014] To solve the above technical problems, the first aspect of the present application provides a kit for detecting interleukin 6 (IL-6), which comprises: a magnetic microsphere coated first antibody and a tracer labeled second antibody; the first antibody and the second antibody are IL-6 monoclonal antibodies and recognize different antigen epitopes.

[0015] Preferably, the first antibody is a 12H6 monoclonal antibody with catalog number DA011 of nearshore (nearshore protein technology co., ltd.), and the second antibody is a 15H5 monoclonal antibody with catalog number DA012 of nearshore.

[0016] Preferably, the first antibody is an IL-6-55E9 monoclonal antibody with catalog number P1810035 of jinsirui (Nanjing jinsirui biological technology co., ltd.), and the second antibody is an IL-6-55C6 monoclonal antibody with catalog number P1904008 of jinsirui.

[0017] Preferably, the first antibody is an IL-6-01 monoclonal antibody with catalog number 20180915 of gurilin (Guangzhou gurilin biological technology co., ltd.), and the second antibody is an IL-6-02 monoclonal antibody with catalog number 20180919 of gurilin.

[0018] The present application adopts double antibody sandwich method to detect IL-6, uses two different specific monoclonal antibodies, wherein the binding sites of the antibody labeled with a tracer marker are different from the binding sites of the antibody coated on the magnetic microspheres. The selection of these binding sites is not only conducive to the labeling of the tracer or the coating of the magnetic microspheres, but also does not hinder the combination of the antibody and the antigen to form a sandwich complex, thus improving the specificity and sensitivity of the reaction.

[0019] And the double reagent reaction system formed by the double antibody sandwich method saves cost and reaction time compared with the three reagent reaction system, thereby increasing the test throughput.

[0020] The magnetic microspheres can be conventional in the art; preferably, the magnetic microspheres are micron-sized solid-phase microspheres with paramagnetism and extremely large protein adsorption capacity formed by compounding nanoscale Fe2O3 and Fe3O4 magnetic microparticles with high molecular materials, which can improve the coating efficiency.

[0021] The magnetic microspheres can be rapidly magnetized under the action of an external magnetic field, and the residual magnetism is zero after the magnetic field disappears. The type of high molecular material used for compounding is not limited.

[0022] The particle size of the magnetic microspheres can be 1-5 μm, and the magnetic microspheres can be added with multiple active groups through surface modification, including but not limited to -OH and -COOH.

[0023] The particle size of the magnetic microspheres is 1-5 μm; preferably 3 μm.

[0024] The concentration of the magnetic microspheres is 0.1-1 mg / ml; preferably 0.1-0.5 mg / ml; more preferably 0.15-0.25 mg / ml.

[0025] In a preferred embodiment of the present application, the average particle size of the magnetic microspheres is 3 μm, purchased from JSR Company, and the solid content is about 10%.

[0026] The preparation of the first antibody coated on the magnetic microspheres comprises the step of blocking the first antibody coated on the magnetic microspheres with a BSA solution with a concentration of 200 mg / mL.

[0027] In a preferred embodiment of the present application, the preparation of the first antibody coated on the magnetic microspheres further comprises the step of coating the magnetic microspheres with the first antibody in a coating buffer before the blocking.

[0028] During coating, the mass ratio of the magnetic microspheres to the first antibody is 100:0.5-100:1.25; preferably 100:0.75-100:1; more preferably 100:1.

[0029] The coupling time of the magnetic microspheres and the first antibody is 0.5 h to 2 h; preferably 0.5 h.

[0030] The coupling buffer during the coating is 0.05 to 0.2 M MES buffer, preferably 0.1 M MES buffer; the pH of the MES buffer is 5.0 to 6.0; preferably 5.2 to 5.8, more preferably 5.4 to 5.6; for example 5.5.

[0031] The working concentration of the first antibody is 0.1 to 10 μg / ml; preferably 0.1 to 5 μg / ml; more preferably 1.5 to 2.5 μg / ml.

[0032] The coated first antibody of the magnetic microspheres is stored, cleaned and diluted using a luminescence recovery solution.

[0033] In a preferred embodiment of the present application, the luminescence recovery solution comprises PBS with a pH of 7.4, 2% bovine serum albumin and 1 to 2‰ sucrose; the % and ‰ are mass percentage and mass ten thousandth, respectively.

[0034] The tracer marker can be conventional in the art, preferably luminol, oxalate ester, acridinium ester or adamantane, for example acridinium ester.

[0035] When the tracer marker is acridinium ester, the small molecular weight of acridinium ester can further reduce the steric hindrance effect of the reaction, which helps to improve the sensitivity of the overall system.

[0036] The molar ratio of the tracer marker to the second antibody can be conventional in the art (it can be within the conventional range of the present application), preferably greater than or equal to 15:1.

[0037] The preparation of the second antibody labeled with the tracer marker comprises the step of labeling the second antibody with the tracer marker in a labeling buffer.

[0038] In a preferred embodiment of the present application, the labeling buffer is 0.05 to 0.1 M HEPES buffer, preferably 0.05 M HEPES buffer; the pH of the HEPES buffer is 6.8 to 8.2; preferably 7.4 to 8.2; more preferably 7.9 to 8.1; for example 8.0.

[0039] The labeling time is 1 to 2 h; preferably 1 h.

[0040] The working concentration of the second antibody is 0.1 to 2.5 μg / ml; preferably 0.75 to 1.25 μg / ml.

[0041] The concentration of the tracer marker is 0.1-2.5 μg / ml; preferably 0.1-1.25 μg / ml; more preferably 0.375-0.625 μg / ml.

[0042] In a preferred embodiment of the present application, the detection kit further comprises an IL-6 calibrator, a washing solution and a chemiluminescent substrate solution (i.e. chemiluminescent excitation solution) for reacting with the tracer marker to generate a detection signal.

[0043] In a preferred embodiment of the present application, the IL-6 calibrator is diluted by a 0.02M PBS solution containing 0.1% Proclin 300 and 1% BSA.

[0044] The percentage of 0.1% Proclin 300 is mass percentage, and the percentage of 1% BSA is mass volume percentage.

[0045] In a preferred embodiment of the present application, a preservative is further added to the IL-6 calibrator. The calibrator can be stored stably at 4℃.

[0046] The washing solution is a PBS buffer containing a surfactant.

[0047] In a preferred embodiment of the present application, a preservative is further added to the washing solution.

[0048] The chemiluminescent substrate solution comprises hydrogen peroxide and nitric acid solution.

[0049] In a preferred embodiment of the present application, the chemiluminescent substrate solution (i.e. chemiluminescent excitation solution) comprises substrate solution A (i.e. chemiluminescent pre-excitation solution A) and substrate solution B (i.e. chemiluminescent pre-excitation solution B); the substrate solution A is a mixture of H2O2 and HNO3, wherein the mass fraction of H2O2 is 0.01-5.0%, and the concentration of HNO3 is 0.01-1.0 mol / L; the substrate solution B is a mixture of Triton X-100 and NaOH, wherein the mass fraction of Triton X-100 is 0.01-2.0%, and the concentration of NaOH is 0.05-1 mol / L.

[0050] The preservative can be one or two of the following conventional preservatives in the art: potassium sorbate, sodium benzoate, sodium nitrite, sodium azide, proclin-300 (the main active ingredients are 2-methyl-4-isothiazolin-3-ketone and 5-chloro-2-methyl-4-isothiazolin-3-ketone, which is a safer preservative and one of the most commonly used preservatives in immunodiagnosis) and antibiotics.

[0051] The surface active agent can be conventional in the art, preferably one or two of Triton X-100, Triton X-405, Tween 20 and Tween 80. The surface active agent is added in an amount of 0.1-2‰, which is more conducive to the dispersion of the magnetic microspheres.

[0052] In a preferred embodiment of the application, the cleaning solution is a PBST solution with a pH of 7.0-9.0 and a concentration of 0.02 mol / L, wherein the mass fraction of Tween-20 is 0.5%.

[0053] To solve the above technical problems, the second aspect of the application provides a method for detecting IL-6 using the above kit, comprising the following steps:

[0054] (1) mixing and incubating the working concentration of magnetic microsphere-coated first antibody, tracer-labeled second antibody and sample to be tested;

[0055] (2) placing the mixture incubated in (1) under a magnetic condition, cleaning, adding chemiluminescent substrate solution, and detecting the photon value.

[0056] Preferably, the volumes of the working concentration of magnetic microsphere-coated first antibody, tracer-labeled second antibody and sample to be tested in (1) are the same.

[0057] Preferably, the incubation is at 37°C, and the incubation time is 6 min.

[0058] Preferably, the cleaning in (2) is cleaning using a cleaning solution.

[0059] The sample to be tested can be directly obtained serum, plasma and whole blood.

[0060] The method for detecting IL-6 according to the application can realize fully automatic, rapid, high-sensitivity and quantitative detection of the concentration of IL-6 in the sample, which is conducive to early monitoring of the immune state and inflammatory response of the body, and can effectively guide the use of antibiotics, and can also be used as a reference for evaluating the prognosis of sepsis and septic shock. Meanwhile, those skilled in the art should know that the method can also be used for non-diagnostic purposes, such as laboratory detection of animal blood samples for subsequent physiological and biochemical analysis and the like.

[0061] To solve the above technical problems, the third aspect of the application provides a use of the kit according to the first aspect of the application in the detection of IL-6.

[0062] On the basis of common sense in the art, the above preferred conditions can be combined arbitrarily, i.e. to obtain each preferred example of the application.

[0063] The reagents and raw materials used in the present application are commercially available.

[0064] The positive progress effect of the present application is that:

[0065] (1) The kit of the present application for detecting IL-6 is simple to operate, low in cost, short in detection time, and high in sensitivity. In a preferred embodiment, the sensitivity of the kit of the present application for detecting IL-6 can reach 1.29 pg / ml, which is much higher than 3.8 pg / ml in the prior art. In a preferred embodiment, the reagent liquid volume of the kit of the present application is only 50 μl, the incubation time is 6 min, and the whole detection can be completed within 10 min.

[0066] (2) The linear range of the kit of the present application is wide, which can reach 3~5000 pg / ml (while the linear range in the prior art is not more than 1000 pg / ml); the precision is good, the coefficient of variation CV is less than 5%; the accuracy is good, the recovery rate can reach 101.16%; the clinical coincidence rate is good, and the correlation R value of the clinical sample can reach 0.9962. BRIEF DESCRIPTION OF DRAWINGS

[0067] Figure 1 The schematic diagram of the detection principle in the examples is shown in

[0068] Figure 2 The linear correlation diagram of Example 9 is shown in

[0069] Figure 3 The sample coincidence rate diagram of Example 9 is shown in

[0070] Figure 4 The hook effect diagram of Example 9 is shown in DETAILED DESCRIPTION

[0071] The present application will be further described by way of examples, but the present application is not limited in the scope of the examples. The experimental methods in the following examples without specific conditions are selected according to the conventional methods and conditions, or according to the product instructions.

[0072] The schematic diagram of the detection principle in the examples is shown in Figure 1 .

[0073] Example 1

[0074] Preparation 1: Magnetic microsphere suspension coated with first antibody

[0075] (1) 10 mg of magnetic microspheres (average particle size 3 μm, JSR Corporation, solid content 10%) were measured and suspended in 1 mL of 0.1 M MES buffer solution at pH 5.5, and the magnetic microspheres were adsorbed with a magnet for 5 to 10 min to perform cleaning, and after the supernatant was discarded, the aforementioned cleaning step was repeated three times, 1 mL of 0.1 M MES was added, and the mixture was vortexed to mix.

[0076] (2) 100 μg of the first antibody (Kangshi, Lot: DA011) was added to make the mass ratio of magnetic microspheres:antibody 100:1, and after the mixture was vortexed to mix, it was incubated at 37°C for 30 min.

[0077] (3) 10 μL of 10 mg / ml of 1-(3-dimethylaminopropyl)-3-ethyl diimide hydrochloride (EDC) was added, the mixture was vortexed to mix, and the mixture was incubated at 37°C for 1.5 h.

[0078] (5) 200 μL of a 200 mg / mL BSA solution was added to the coated antibody solution, the mixture was vortexed to mix at room temperature overnight (not less than 16 h) to perform the second-stage blocking, the magnetic microspheres were adsorbed with a magnet or a magnetic stand, and the supernatant was discarded;

[0079] (6) 1 mL of a luminescence recovery solution was added to the blocked magnetic microsphere-antibody suspension, the mixture was adsorbed with a magnet to perform cleaning, and after the supernatant was discarded, the aforementioned cleaning step was repeated three times to complete the preparation of the magnetic microsphere-antibody suspension coated with the first antibody.

[0080] The luminescence recovery solution comprises: pH 7.4 PBS, 2% bovine serum albumin, and 1-2‰ sucrose; and the % and ‰ are mass percentage and mass parts per thousand, respectively.

[0081] (7) The prepared magnetic microsphere-antibody suspension was placed in 1 mL of the luminescence recovery solution and stored at 2 to 8°C.

[0082] Preparation 2: Preparation of acridinium ester-labeled second antibody solution

[0083] (1) 1 mg of the second antibody (Kangshi, Lot: DA012) was placed in an ultrafiltration centrifuge tube, and the antibody was dialyzed by centrifugation at 7500 g for 15 min using not less than 1 mL of 0.05 M HEPES buffer solution (pH 8.0), and the dialysis was repeated twice to make the concentration of the antibody greater than 5 mg / mL.

[0084] (2) 100 μL of a 5 mg / mL NSP-SA-NHS DMF solution was added to the dialyzed second antibody to make the molar ratio of NSP-SA-NHS to the second antibody not less than 15:1, and after the mixture was mixed, the mixture was reacted at 4°C (avoiding light) for 1 h to 2 h to perform labeling.

[0085] (3) Add 100 μL of 100 mg / mL lysine solution, react for 30 min, and terminate the reaction. Then add no less than 2 mL of PBS buffer (10 M, pH 7.4) and ultrafiltrate at 7500 g for 20 min, with 3 times of intermediate liquid change, to separate free NSP-SA-NHS, and complete the preparation of acridinium ester-labeled secondary antibody solution.

[0086] (4) Collect the separated label, add 5% BSA solution to make the final concentration of BSA 1%, then add an equal volume of glycerol, and store at -20°C.

[0087] Preparation 3: Preparation of calibrators

[0088] Take IL-6 working solution (Zhongkean body, LOT: AY10001-Ag), dilute to 50 pg / mL and 250 pg / mL using antigen diluent, and prepare high and low point standard solution.

[0089] The antigen diluent is a 0.02 M PBS solution containing 1% bovine serum albumin.

[0090] Preparation 4: Preparation of chemiluminescent substrate solution

[0091] Substrate solution A is a mixture of H2O2 and HNO3, with a mass fraction of H2O2 of 0.01-5.0% and a concentration of HNO3 of 0.01-1.0 mol / L.

[0092] Substrate solution B is a mixture of Triton X-100 and NaOH, with a mass fraction of Triton X-100 of 0.01-2.0% and a concentration of NaOH of 0.05-1 mol / L.

[0093] Preparation 5: Preparation of washing solution

[0094] The washing solution is a PBST solution with a pH of 7.0-9.0 and a concentration of 0.02 mol / L, with a mass fraction of Tween-20 of 0.5%.

[0095] Example 2

[0096] The kit composed of the components prepared in Example 1 is used in conjunction with a self-made full-automatic chemiluminescence analyzer to detect the IL-6 concentration in the sample to be tested.

[0097] The specific steps are as follows:

[0098] 1. The coated first antibody magnetic microspheres suspension obtained from Preparation 1 in Example 1 was diluted with the light emission recovery solution to dilute the concentration of the first antibody to 2.5 μg / mL (reagent 1) at the working concentration of the magnetic microspheres, and the concentration of the acridinium ester-labeled second antibody obtained from Preparation 2 in Example 1 was diluted with a 1% BSA solution to 1.25 μg / mL (reagent 2);

[0099] 2. 50 μL of reagent 1 and 50 μL of reagent 2 were sequentially added to the reaction cup;

[0100] 3. 50 μL of the sample to be tested or 50 μL of the calibrator was added to the reaction cup, and the entire sample addition process took 0.5 minutes;

[0101] 4. After the solution in the reaction cup was thoroughly mixed, it was incubated at 37°C for 6 minutes;

[0102] 5. The incubated reaction cup was placed under magnetic conditions, and the washing solution was used to wash 3 times, and the entire process took 2 minutes;

[0103] 6. 100 μL of the chemiluminescent substrate solution was added to the reaction cup, and the photon value was detected;

[0104] 7. According to the light intensity detected in the reaction cup, the instrument automatically calculated the concentration of IL-6 in the sample to be tested, and the entire testing and calculation process took 1 minute.

[0105] The entire process took about 9 minutes.

[0106] Example 3 Screening of the paired combination of the first antibody and the second antibody

[0107] (1) Antibody paired combination (pairing) screening

[0108] There are 8 candidate IL-6 monoclonal antibodies:

[0109] Antibody 1: IL-6-2F7 (Chongqing Taosheng, Lot: 20190521);

[0110] Antibody 2: IL-6-7E5 (Chongqing Taosheng, Lot: 20190520);

[0111] Antibody 3: 15H5 (Jin'an, Lot: DA012);

[0112] Antibody 4: 12H6 (Jin'an, Lot: DA011);

[0113] Antibody 5: IL-6-55C6 (Jinshui, Lot: P1904008);

[0114] Antibody 6: IL-6-55E9 (Jinshui, Lot: P1810035);

[0115] Antibody 7: IL-6-01 (Growth hormone, Lot: 20180915)

[0116] Antibody 8: IL-6-02 (Growth hormone, Lot: 20180919)

[0117] Antibodies 1, 2, 3, 4, 5, 6, 7, and 8 were coated on magnetic microspheres to form antibody 1-magnetic microspheres, antibody 2-magnetic microspheres, antibody 3-magnetic microspheres, antibody 4-magnetic microspheres, antibody 5-magnetic microspheres, antibody 6-magnetic microspheres, antibody 7-magnetic microspheres, and antibody 8-magnetic microspheres, respectively; meanwhile, antibodies 1, 2, 3, 4, 5, 6, 7, and 8 were labeled with acridinium ester to form antibody 1-acridinium ester, antibody 2-acridinium ester, antibody 3-acridinium ester, antibody 4-acridinium ester, antibody 5-acridinium ester, antibody 6-acridinium ester, antibody 7-acridinium ester, and antibody 8-acridinium ester. The above antibodies were combined and paired, and serum samples from different patients collected from a hospital were detected using the experimental procedures in Examples 1 and 2 to screen the optimal combination, and the relative luminescence value (RLU) results are shown in Tables 1-1, 1-2, 1-3, 1-4, and 1-5.

[0118] Table 1-1 Antibody pairing combination screening results

[0119]

[0120] Table 1-2 Antibody pairing combination screening results

[0121]

[0122] Table 1-3 Antibody pairing combination screening results

[0123]

[0124] Table 1-4 Antibody pairing combination screening results

[0125]

[0126] Table 1-5 Antibody pairing combination screening results

[0127]

[0128] As shown in the table, in combination 1, combination 2, combination 3, combination 13, combination 36, the pairing of antibody 1, antibody 2, antibody 3, antibody 5, antibody 8 and antibody 2, antibody 1, antibody 4, antibody 6, antibody 7, the overall signal value of interleukin 6 is low, and the discrimination is low; the antibody pairing of other combinations has no obvious reaction. In combination 4, combination 20 and combination 29, the pairing of antibody 4 and antibody 3, the pairing of antibody 6 and antibody 5 and the pairing of antibody 7 and antibody 8 have better results (higher signal value and high discrimination), and the further detection sample coincidence rate is as shown in Table 2 and Table 3.

[0129] Table 2 Serum sample coincidence rate detection results

[0130]

[0131]

[0132] Table 3 Blood homologous sample coincidence rate detection results

[0133]

[0134] Referring to the results of Table 2 and Table 3, the sample coincidence rate and homologous sample detection coincidence rate of the combination of antibody 4 and antibody 3 are better than those of other combinations.

[0135] Example 4 Antigen diluent screening

[0136] The antigen diluent of the dilution calibrator working solution is screened by using 1% casein containing 0.1% Proclin 300, 1% BSA 0.02M PBS solution and newborn calf serum as the antigen diluent.

[0137] Table 4 Antigen diluent thermal acceleration stability results

[0138]

[0139] As shown in Table 4, after 7 days of acceleration at 37°C, the stability of the antigen diluted with newborn calf serum decreased by more than 70%; after 7 days of acceleration, the stability of the antigen diluted with 1% casein decreased by more than 15%; and after the antigen was diluted with 1% BSA, the stability decreased by less than 10% compared with 4°C storage. Therefore, 1% BSA containing 0.1% Proclin 300 is selected as the calibrator diluent to improve the stability of the calibrator.

[0140] Example 5 Optimization of magnetic microsphere-antibody coating process

[0141] The coupling buffer optimization screening results of magnetic microspheres-antibodies are shown in Table 5.

[0142] Table 5 Optimization of magnetic microsphere-antibody conjugation buffer

[0143]

[0144] Based on the comprehensive comparison of the data of each group, 0.1 M pH 5.5 MES buffer was selected as the conjugation buffer.

[0145] The ratio of magnetic microspheres to antibody was optimized, and the results are shown in Table 6.

[0146] Table 6 Results of magnetic microsphere-antibody ratio screening

[0147]

[0148] As can be seen from the above table, as the amount of antibody added increases, the antigen detection signal value also increases, and there is no obvious effect on the antigen coincidence rate; when the mass ratio of magnetic microspheres to antibody reaches 100:1.25, the overall luminescence value does not increase significantly, therefore, 100:1 is selected as the preferred ratio of magnetic microspheres to antibody.

[0149] The results of the screening of the blocking solution are shown in Table 7.

[0150] Table 7 Blocking solution screening

[0151]

[0152] As can be seen from Table 7, the three blocking solutions do not have a significant difference in the luminescence value and coincidence rate of the detection calibrators. The three magnetic microspheres blocked with the three different blocking solutions were used to detect samples together with acridinium ester, and the coincidence rates were detected, and the results are shown in Table 8.

[0153] Table 8 Detection of samples with different blocking solutions

[0154]

[0155] As shown in Table 8, the coincidence rate of the sample detected by the magnetic microspheres blocked with 20% BSA and acridinium ester was the best, which was 0.9674, and no detection abnormalities occurred during the detection process. The coincidence rate of the sample detected by the magnetic microspheres blocked with 1M Gly and 5% BSA and acridinium ester was poorer, and some samples showed detection abnormalities, with the phenomenon of excessively high luminescence value, therefore, 20% BSA was still selected as the blocking solution.

[0156] Example 6 Process optimization of acridinium ester-labeled antibody

[0157] The conjugation buffer for acridinium ester labeling was optimized, and the results are shown in Table 9.

[0158] Table 9 Coupling buffer optimization of acridinium ester labeled antibody

[0159]

[0160] From Table 9, it can be seen that the three coupling buffers have no obvious effect on the coincidence rate of antigen diluent detection, the HEPES sample detection signal value is overall higher, and the low value range is larger, and HEPES is preferred as the acridinium ester labeled antibody coupling buffer.

[0161] The coupling time of acridinium ester labeling was optimized, and the results are shown in Table 10.

[0162] Table 10 Optimization of acridinium ester labeled antibody coupling time

[0163]

[0164] From Table 10, it can be seen that the antibody is coupled with acridinium ester for 1 h, and the reaction reaches the platform period.

[0165] The use ratio of acridinium ester: antibody was screened, and the results are shown in Table 11.

[0166] Table 11 Screening of acridinium ester: antibody use ratio

[0167]

[0168] According to the experimental data in the above table, the acridinium ester: antibody should not be less than 15: 1.

[0169] Example 7 Antibody working concentration screening

[0170] The working concentration of the first antibody and the second antibody was screened, and the results are shown in Table 12-1 and Table 12-2.

[0171] Table 12-1 Antibody working concentration screening results

[0172]

[0173] Table 12-2 Antibody working concentration screening results

[0174]

[0175] As shown in Table 12-1 and Table 12-2, when the working concentration of the first antibody is 2.5 μg / ml and the working concentration of the second antibody is 1.25 μg / ml, the coincidence rate is the best, and the signal-to-noise ratio and the discrimination are also better.

[0176] Example 8 Stability study

[0177] The reagent 1, reagent 2 and calibrators in the kit components of Example 2 were placed in an oven at 35-37℃ for 7 days, and the relative deviation of the 7-day accelerated stability and the 4℃ reagent was tested, and the results are shown in Table 13.

[0178] Table 13 Stability test results

[0179]

[0180] As shown in Table 13, the signal values of all components of the kit after 7 days of acceleration at 35-37℃ were within ±15% of the relative deviation of the reagent placed at 4℃, and the stability of the kit was very good.

[0181] Example 9 Performance test

[0182] The kit prepared in Example 1 was subjected to performance testing.

[0183] (1) Detection of blank limit

[0184] The detection method refers to the test method of YY / T 1233-2014 "Myocardial Troponin-I Quantitative Determination Reagent (Kit) (Chemiluminescence Immunoassay)" issued by the State Drug Administration.

[0185] The blank sample (sample diluent) was detected 20 times repeatedly, and then according to the detection results, the blank limit was calculated by formula, and the results are shown in Tables 14-1 and 14-2.

[0186] Table 14-1 Detection results of initial calibration curve

[0187]

[0188] Table 14-2 Detection results of blank limit

[0189]

[0190] According to the concentration-chemiluminescence (RLU) value results between the zero concentration calibrator (S0) and the adjacent calibrator (S1), a one-time equation was obtained by two-point regression fitting, and then the RLU value corresponding to Mean+2SD obtained by detecting 20-hole blank samples (S0) was substituted into the above equation. The concentration value obtained is the analytical sensitivity, and the blank limit of the kit can reach 1.29 pg / mL, which has great significance for early diagnosis of inflammation.

[0191] (2) Detection of within-day precision

[0192] The high and low point calibrators were detected 10 times repeatedly, and then according to the detection results, the CV was calculated, and the results are shown in Table 14-3.

[0193] Table 14-3 Intra-day precision test results

[0194]

[0195] The intra-day precision of the kit can reach 4.82% and 4.81%, which is of great significance for the accuracy of inflammation diagnosis.

[0196] (3) Detection of linear correlation

[0197] The linear sample was detected by multiple holes, and the linear correlation coefficient of the theoretical value and the measured value of each point was calculated, and the results are shown in Table 14-4 and Figure 2 .

[0198] Table 14-4 Linear correlation test results

[0199]

[0200] The linear correlation R of the kit is greater than 0.990. The linear sample (7 points) was detected by multiple holes, and the linear correlation coefficient of the theoretical value and the measured value of each point was calculated. Among them, the theoretical concentration is the concentration obtained without using the calibration curve, and the measured value is the sample value after detection by the Roche comparison machine. Figure 2 It shows that the linear correlation is good.

[0201] (4) Detection of accuracy

[0202] The detection method refers to the test method of YY / T 1233-2014 "Myocardial Troponin-I Quantitative Determination Reagent (Kit) (Chemiluminescence Immunoassay)" issued by the State Drug Administration, and the serum is diluted by 1:9 for detection.

[0203] The accuracy of the kit was detected by recovery test, and the detection results are shown in Table 14-5.

[0204] Table 14-5 Recovery test results

[0205]

[0206] The recovery test result can reach 101.16%, and the accuracy is good.

[0207] The hook effect of the kit was detected, and the results are shown in Table 14-6 and Figure 4 .

[0208] Table 14-6 Hook effect

[0209]

[0210] The kit of Example 1 was used to detect high concentration antigen samples. When the sample concentration reached 200 ng / ml, the hook effect began to appear, but the detection value was still greater than 5,000 pg / ml, exceeding the upper limit of detection, thus indicating that the kit can ignore the influence of the hook effect on sample detection.

[0211] (6) Methodology comparison

[0212] After some serum or plasma samples were detected by using the interleukin 6 detection kit (electrochemiluminescence method) produced by Roche Diagnostics, the above samples were detected by using the kit of the present application, and the detection results are shown in Table 1. Figure 3 The kit of the present application was used to detect 101 Roche samples, and the concentration values (pg / ml) detected by the Roche kit and the detection signal values of the kit of the present application were plotted as a scatter plot. It was found that the coincidence rate was good, and the overall coincidence rate R value could reach 0.9962. It can be seen that the kit of the present application has good correlation with the internationally recognized Roche interleukin 6 kit, and can accurately screen out diseased individuals from healthy people.

Claims

1. A method of detecting IL-6, characterized by, The IL-6 is detected by using a kit, the kit comprising: a magnetic microsphere coated first antibody, a tracer marker labeled second antibody, an interleukin 6 calibrator, a cleaning solution and a chemiluminescent substrate solution reacting with the tracer marker to generate a detection signal; the method is for non-diagnostic purposes; the method comprises the following steps: (1) using the kit to prepare a working concentration of magnetic microsphere coated first antibody, tracer marker labeled second antibody, mixing and incubating the first antibody, second antibody and the sample to be tested; the incubation time is 6 min; (2) placing the incubated mixture in (1) under magnetic conditions, cleaning and adding the chemiluminescent substrate solution to detect the photon value; The first antibody is 12H6 monoclonal antibody with catalog number DA011 from R&D Systems; the second antibody is 15H5 monoclonal antibody with catalog number DA012 from R&D Systems; or, The first antibody is IL-6-55E9 monoclonal antibody with catalog number P1810035 from KPL; the second antibody is IL-6-55C6 monoclonal antibody with catalog number P1904008 from KPL; or, The first antibody is IL-6-01 monoclonal antibody with catalog number 20180915 from Grubbi; the second antibody is IL-6-02 monoclonal antibody with catalog number 20180919 from Grubbi; When preparing the magnetic microsphere coated first antibody, a step of blocking the magnetic microsphere coated first antibody is further included, and the blocking solution used in the blocking is a 20% BSA solution; the % is mass percentage; The working concentration of the first antibody is 2.5 μg / ml; the working concentration of the second antibody is 1.25 μg / ml; The interleukin 6 calibrator is diluted by using a 0.02M PBS solution containing 0.1% Proclin 300 and 1% BSA; the percentage of 0.1% Proclin 300 is mass percentage, and the percentage of 1% BSA is mass percentage.

2. The method of claim 1, wherein, When coating the first antibody with the magnetic microspheres, a coupling buffer of MES buffer is used; And / or, when coating the first antibody with the magnetic microspheres, the mass ratio of the magnetic microspheres to the first antibody is 100:0.5-100:1.25; And / or, when coating the first antibody with the magnetic microspheres, the coupling time of the magnetic microspheres to the first antibody is 0.5h-2h.

3. The method of claim 2, wherein, The concentration of the MES buffer is 0.05-0.2M; And / or, the pH of the MES buffer is 5.0-6.0; And / or, the mass ratio of the magnetic microspheres to the first antibody is 100:0.75-100:1; And / or, the coupling time of the magnetic microspheres to the first antibody is 0.5h.

4. The method of claim 3, wherein, The concentration of the MES buffer is 0.1M; And / or, the pH of the MES buffer is 5.2-5.

8.

5. The method of claim 4, wherein, The pH of the MES buffer is 5.4-5.

6.

6. The method of claim 5, wherein, The pH of the MES buffer is 5.

5.

7. The method according to any one of claims 1 to 6, wherein The tracer marker is luminol, acridinum ester or adamantane; And / or, when the tracer label marks the second antibody, the concentration of the HEPES buffer used is 0.05-0.1M; And / or, when the tracer label marks the second antibody, the molar ratio of the tracer label to the second antibody is ≥15:1; And / or, when the tracer label marks the second antibody, the marking time is 1-2h.

8. The method of claim 7, wherein, The tracer label is acridinium ester; And / or, the concentration of the HEPES buffer is 0.05M; And / or, the pH of the HEPES buffer is 6.9-8.

2.

9. The method of claim 7, wherein, The pH of the HEPES buffer is 7.4-8.

2.

10. The method of claim 9, wherein, The pH of the HEPES buffer is 7.9-8.

1.

11. The method of claim 10, wherein, The pH of the HEPES buffer is 8.

0.

12. The method of claim 1, wherein, The particle size of the magnetic microspheres is 1-5μm; And / or, the concentration of the tracer label is 0.1-2.5μg / ml.

13. The method of claim 12, wherein, The particle size of the magnetic microspheres is 3μm; And / or, the concentration of the tracer label is 0.1-1.25μg / ml.

14. The method of claim 13, wherein, The concentration of the tracer label is 0.375-0.625μg / ml.

15. The method of claim 1, wherein, The first antibody coated with the magnetic microspheres is stored, cleaned and diluted using a luminescence recovery solution.

16. The method of claim 15, wherein, The luminescence recovery solution comprises PBS with pH 7.4, 2% bovine serum albumin and 1-2‰ sucrose; the % and ‰ are mass percentage and mass ten thousandth, respectively.

17. The method of claim 1, wherein, The chemiluminescence substrate solution comprises substrate solution A and substrate solution B; the substrate solution A is a mixture of H2O2 and HNO3, wherein the mass fraction of H2O2 is 0.01-5.0%, and the concentration of HNO3 is 0.01-1.0mol / L; the substrate solution B is a mixture of Triton X-100 and NaOH, wherein the mass fraction of Triton X-100 is 0.01-2.0%, and the concentration of NaOH is 0.05-1mol / L.

18. The method of claim 17, wherein, The interleukin 6 calibrator comprises a calibrator buffer; and / or, the cleaning solution comprises a preservative and a buffer containing a surfactant.

19. The method of claim 18, wherein, The cleaning solution is a PBST solution with pH 7.0-9.0, 0.02M and containing 0.5% Tween-20 in mass fraction.

20. The method of claim 19, wherein, The addition amount of the surfactant is 0.1-2‰ of the total volume of the cleaning solution, and the ‰ is mass ten thousandth.

21. The method of claim 1, wherein, The volumes of the first antibody coated with the magnetic microspheres, the second antibody labeled with the tracer label and the sample to be detected in the working concentration in (1) are the same; The incubation is incubation at 37℃; The cleaning in (2) is cleaning using a cleaning solution.

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