Chemiluminescence-based FGF21 detection kit and preparation method thereof

Abstract

The invention belongs to the technical field of in-vitro diagnosis, and discloses a chemiluminescence-based FGF21 detection kit and a preparation method thereof, and the kit comprises magnetic microspheres of which the surfaces are coupled with FGF21 specific antibodies, the FGF21 specific antibodies containing acridinium ester labels, a sample diluent, a luminescence pre-excitation liquid, an excitation liquid and FGF21 calibrators with different concentrations. According to the FGF21 detection kit disclosed by the invention, a method for preparing magnetic bead microspheres, carrying out surface modification and carrying out surface coupling on an FGF21 specific antibody is deeply explored, and the FGF21 specific antibody containing an acridinium ester label and a luminescence excitation system are matched, so that the prepared FGF21 detection kit can be used for accurately detecting FGF21 protein; and the detection kit has the advantages of high sensitivity, good accuracy, good repeatability and high automation degree of test operation, can treat a large batch of samples, further provides effective detection products and methods for clinical detection, and has strong practicability.

Description

Technical Field

[0001] The present invention belongs to the technical field of in vitro diagnosis, and in particular relates to a chemiluminescence-based FGF21 detection kit and a preparation method thereof. Background Art

[0002] The fibroblast growth factor family (FGF) has many physiological functions, such as promoting body development, cell proliferation, wound repair, and angiogenesis. Currently, there are 22 known members of the FGF family, which participate in the regulation of physiological functions in the form of paracrine. Among them, fibroblast growth factor-21 (FGF21) is a newly discovered endocrine hormone, which is mainly produced by the liver and also expressed in adipose tissue, pancreas and skeletal muscle. It can be secreted into the blood and participate in the regulation of the body's metabolism. The physiological effects of FGF21 are mainly in lowering blood sugar and improving blood lipids. According to research findings, FGF21 participates in regulating the body's blood sugar metabolism by improving insulin resistance, increasing gluconeogenesis and ketone body production. Many studies have shown that FGF21 levels increase in patients with type 2 diabetes, obesity, hyperlipidemia, metabolic syndrome, and renal insufficiency who require dialysis. Recent studies have found that increased FGF21 levels are also associated with fatty liver and coronary heart disease. Therefore, the detection of FGF21 content provides a reference indicator for the research, diagnosis and treatment of metabolic diseases, and has important practical value.

[0003] At present, the detection of FGF21 mainly adopts enzyme-linked immunosorbent assay (ELISA), which has the characteristics of high sensitivity and suitability for large-scale sample detection operations. However, the detection results of ELISA mainly depend on the coating matrix. The coating matrix of the solid phase carrier is difficult to achieve consistency between batches, which leads to poor repeatability and low accuracy of ELISA detection of FGF21. Therefore, how to solve the stability and uniformity of the cross-linking relationship between the coating matrix and the FGF21-specific antibody is one of the important ways to improve the accuracy and repeatability of FGF21 detection results, and is one of the problems that need to be solved in the current FGF21 detection. Summary of the invention

[0004] In view of the shortcomings and deficiencies of the above prior art, the primary purpose of the present invention is to provide a chemiluminescence-based FGF21 detection kit and a preparation method thereof.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] A chemiluminescence-based FGF21 detection kit, the kit comprising: magnetic microspheres with FGF21-specific antibodies coupled to the surface, FGF21-specific antibodies labeled with acridinium ester, sample diluent, luminescent pre-excitation solution, excitation solution and FGF21 calibrators of different concentrations;

[0007] The sample diluent comprises the following raw materials: 100-120 mM PBS buffer, 3-6 mM tris(2-hydroxyethyl)phosphine TCEP, 0.05-0.15 wt% Tween 20, 0.05-0.15 wt% triton X-100, 0.1-0.12 wt% dodecyl glucoside and 0.5-1.5 w t% BSA.

[0008] Furthermore, the method for preparing the magnetic microspheres with FGF21-specific antibodies coupled to the surface comprises the following steps:

[0009] S1. Take 10 mg of magnetic microspheres, separate them by magnetic separation for 1 min, remove the supernatant, and wash them several times with 0.02 mol / L Tris-NaCl buffer (pH 7.4) by shaking;

[0010] S2, prepare 10 mg / mL EDC and 12 mg / mL NHS solution with 0.05 mol / L pH 6.0 MES buffer, and take 100 μL of each into the magnetic microspheres washed in step S1, and shake for 20-40 minutes;

[0011] S3, separate the magnetic microspheres using an external magnetic field, discard the supernatant, add 0.05 mol / L MES buffer at pH 6.0 and FGF21-specific antibody solution, and shake at room temperature for 1-3 hours;

[0012] S4. Separate the magnetic microspheres with an external magnetic field, discard the supernatant, add 300 μL of blocking solution A, mix and oscillate, then perform magnetic separation and discard the supernatant, repeat several times, add 3 mL of blocking solution A, and oscillate at room temperature for 0.5-1 h to obtain the magnetic microspheres with FGF21-specific antibodies coupled to the surface.

[0013] Furthermore, the magnetic microspheres are of a core-shell structure formed by ferrosoferric oxide as a core layer and polylactic acid polymer as a shell layer.

[0014] Furthermore, in step S4, the blocking solution A comprises the following components: 0.04-0.06 mol / L Tris-NaCl buffer at pH 7.4, 0.5%-2% BSA, 0.01-0.02% ProClin-300, 4-6% sucrose and 0.1-0.8% ascorbic acid.

[0015] In the present invention, the magnetic microspheres are blocked by the blocking solution A composed of the above-mentioned specific components, and in the subsequent use process, the binding of the magnetic microspheres with non-specific proteins can be inhibited, thereby improving the sensitivity and repeatability of FGF21 detection, and maintaining the luminescent signal value at a high level and stabilizing the luminescence.

[0016] Furthermore, the preparation method of the magnetic microspheres comprises the following steps:

[0017] A1, dissolving sodium hydroxide in diethylene glycol, heating and stirring to form solution A, then adding polyacrylic acid and ferric chloride to diethylene glycol, mixing and heating to form solution B, rapidly adding solution A to solution B under stirring, continuing to keep warm for 1-1.5h, filtering, washing and drying to obtain ferroferric oxide core layer particles;

[0018] A2. Take the iron oxide core layer particles and polylactic acid polymer precursor obtained in step A1, add them into chloroform to fully dissolve, and ultrasonically prepare them into solution C. The solution C is transferred to the outside of the SPG membrane emulsifier as a dispersed phase. The external nitrogen pressurization acts on the outside of the SPG membrane emulsifier to form droplets, and reacts with the SDS solution to form a uniformly dispersed emulsion. The obtained emulsion is stirred at room temperature overnight, and then centrifuged, washed and dried to obtain the magnetic microspheres.

[0019] Furthermore, in step A1, the molar ratio of sodium hydroxide, polyacrylic acid and ferric chloride is 12-13:1:0.1.

[0020] Furthermore, in step A2, the preparation method of the polylactic acid polymer precursor includes the following steps: fully dissolving polylactic acid, maleic anhydride, and benzoyl peroxide in dichloromethane, and placing them at room temperature for 20-24 hours, then transferring them to a 110-120°C oven and heating them for 16-20 hours, dissolving the obtained solid mixture by chloroform, precipitating with ether, purifying multiple times, and obtaining the polylactic acid precursor by centrifugation and drying.

[0021] Furthermore, the mass ratio of the polylactic acid, maleic anhydride and benzoyl peroxide is 5:0.5:0.01.

[0022] Furthermore, the method for preparing magnetic microspheres further comprises step A3, wherein the magnetic microspheres prepared in step A2 are uniformly dispersed in an acid solution, stirred at room temperature overnight, centrifuged and washed multiple times, and dried to obtain surface carboxylated magnetic microspheres.

[0023] Furthermore, in step A3, the acid solution is at least one of hydrochloric acid, nitric acid, acetic acid, lactic acid, citric acid or sulfuric acid, and the pH of the acid solution is 1. In the present invention, preferably, the acid solution is compounded by citric acid and hydrochloric acid in a mass ratio of 3:1.

[0024] Furthermore, in step A3, the mass volume ratio of the magnetic microspheres to the acid solution is 1:2.

[0025] In the present invention, the ferroferric oxide core layer particles are first prepared, and then the polylactic acid material is coated on the surface thereof to form magnetic microspheres with uniform particle size and uniform dispersion and core-shell structure, so that the magnetic microspheres have more carboxyl groups and high stability. On this basis, the surface of the magnetic microspheres is carboxylated by step A3, so that the surface of the magnetic microspheres has more carboxyl groups, which helps to enhance the specific binding of the magnetic microspheres to the FGF21 protein, thereby improving the detection sensitivity and accuracy of the FGF21 detection kit.

[0026] Furthermore, the luminescent pre-excitation solution is a 0.2 mol / L nitric acid solution containing 0.5% hydrogen peroxide, and the exciting solution is a 0.5 mol / L sodium hydroxide solution containing 1% Tween 20.

[0027] Furthermore, the preparation method of the FGF21-specific antibody containing an acridinium ester label comprises the following steps: fully dissolving the acridinium ester in DMSO, adding PBS buffer containing the FGF21-specific antibody thereto, shaking at room temperature for 1-3 hours, and then dialyzing with 0.01 mol / L PBS buffer with a pH of 7.2 to remove excess acridinium ester in the solution; then adding blocking solution B thereto, shaking at room temperature for 0.5-1 hour, and obtaining the FGF21-specific antibody containing an acridinium ester label.

[0028] Furthermore, the blocking solution B includes the following raw materials: 8-10% glycine, 0.5-2% BSA, 0.04-0.06 mol / L Tris-NaCl buffer solution with pH 7.4 and 0.05% Proclin-300.

[0029] Furthermore, the concentrations of FGF21 calibrators were 0 pg / ml, 30 pg / ml, 60 pg / ml, 120 pg / ml, 240 pg / ml, 480 pg / ml, 960 pg / ml and 1 920 pg / ml, respectively.

[0030] The kit prepared by the preparation method of the FGF21 detection kit based on chemiluminescence is applied to FGF21 detection. The method of using the kit comprises the following steps:

[0031] (1) adding magnetic microspheres with FGF21-specific antibodies coupled to the surface to dilute the sample to a concentration of 30-60 mg / L, and then adding the sample to be tested to mix and incubate;

[0032] (2) removing the supernatant in the sample solution to be tested in step (1), and then adding a cleaning solution to perform multiple shaking cleaning;

[0033] (3) adding the FGF21-specific antibody labeled with acridinium ester to the sample diluent for dilution to a concentration of 0.1-0.3 mg / L, and adding the diluted solution to the cleaned sample to be tested;

[0034] (4) Under the action of an external magnetic field, the magnetic microspheres are separated from the sample solution to be tested, and the luminescent pre-excitation solution and the excitation solution are added while shaking, and the luminescent signal value is immediately read.

[0035] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention conducts in-depth research on the preparation, surface modification and surface coupling of FGF21-specific antibodies of magnetic bead microspheres, and cooperates with the application of FGF21-specific antibodies labeled with acridinium ester and a luminescent excitation system to prepare an FGF21 detection kit that can accurately detect FGF21 protein, and the detection kit has high sensitivity, good accuracy and good repeatability, a high degree of automation in the test operation, and can process large quantities of samples, thereby providing effective detection products and methods for clinical testing, and having strong practicality. DETAILED DESCRIPTION

[0036] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

[0037] Example 1

[0038] This embodiment provides a chemiluminescence-based FGF21 detection kit, comprising magnetic microspheres with FGF21-specific antibodies coupled to the surface, FGF21-specific antibodies labeled with acridinium ester, sample diluent, luminescent pre-excitation solution, excitation solution, and FGF21 calibrators of different concentrations;

[0039] The sample diluent includes the following raw materials: 100 mM PBS buffer, 4 mM tris(2-hydroxyethyl)phosphine TCEP, 0.1 wt % Tween 20, 0.1 wt % triton X-100, 0.1 wt % dodecyl glucoside and 0.5 wt % BSA.

[0040] Furthermore, the method for preparing the magnetic microspheres with FGF21-specific antibodies coupled to the surface comprises the following steps:

[0041] S1. Take 10 mg of magnetic microspheres, separate them by magnetic separation for 1 min, remove the supernatant, and wash them three times with 0.02 mol / L Tris-NaCl buffer (pH 7.4) by shaking;

[0042] S2, prepare 10 mg / mL EDC and 12 mg / mL NHS solutions with 0.05 mol / L pH 6.0 MES buffer, and take 100 μL of each into the magnetic microspheres washed in step S1, and shake for 30 minutes;

[0043] S3, separate the magnetic microspheres with a magnet, discard the supernatant, add 200 μL of 0.05 mol / L pH 6.0 MES buffer and 3 μL of FGF21-specific antibody solution, and shake at room temperature for 1 hour;

[0044] S4. Separate the magnetic microspheres with a magnet, discard the supernatant, add 300 μL of blocking solution A, mix and oscillate, and then discard the supernatant by magnetic separation. Repeat three times, then add 3 mL of blocking solution A, and oscillate at room temperature for 0.5 h to obtain the magnetic microspheres with FGF21-specific antibodies coupled to the surface.

[0045] Furthermore, the magnetic microspheres are of a core-shell structure formed by ferrosoferric oxide as a core layer and polylactic acid polymer as a shell layer.

[0046] Furthermore, in step S4, the blocking solution A includes the following components: 0.05 mol / L Tris-NaCl buffer at pH 7.4, 1% BSA, 0.01% ProClin-300, 5% sucrose and 0.3% ascorbic acid.

[0047] Furthermore, the preparation method of the magnetic microspheres comprises the following steps:

[0048] A1. Dissolve sodium hydroxide in 2 mL of diethylene glycol, heat and stir to form solution A, then add polyacrylic acid and ferric chloride to 4 mL of diethylene glycol, mix and form solution B at 220°C, stir for 0.5 h, and quickly add solution A to solution B under stirring, continue to keep warm for 1 h, filter, wash and dry to obtain ferroferric oxide core layer particles;

[0049] A2. The ferroferric oxide core layer particles and the polylactic acid polymer precursor obtained in step A1 are added to 4 mL of chloroform at a mass ratio of 1.25:1 to fully dissolve, and ultrasonically prepared into solution C. Solution C is transferred to the outside of the SPG membrane emulsifier as a dispersed phase, and is pressurized by external nitrogen (nitrogen pressure is 0.008 MPa) to act on the outside of the SPG membrane emulsifier to form droplets, and stirred with 0.5 wt% SDS solution at a stirring speed of 500 rpm to form a uniformly dispersed emulsion. The obtained emulsion is stirred at room temperature overnight, and then centrifuged, washed and dried to obtain the magnetic microspheres.

[0050] Furthermore, in step A1, the molar ratio of sodium hydroxide, polyacrylic acid and ferric chloride is 4.5:1:0.1.

[0051] Furthermore, in step A2, the preparation method of the polylactic acid polymer precursor includes the following steps: polylactic acid, maleic anhydride, and benzoyl peroxide are fully dissolved in 50 mL of dichloromethane, and placed at room temperature for 24 hours, and then transferred to a 110°C oven and heated for 18 hours. The obtained solid mixture is dissolved by chloroform, precipitated with ether, purified three times, and the polylactic acid precursor is obtained by centrifugation and drying.

[0052] Furthermore, the mass ratio of the polylactic acid, maleic anhydride and benzoyl peroxide is 5:0.5:0.01.

[0053] Furthermore, the luminescent pre-excitation solution is a 0.2 mol / L nitric acid solution containing 0.5% hydrogen peroxide, and the exciting solution is a 0.5 mol / L sodium hydroxide solution containing 1% Tween 20.

[0054] Furthermore, the preparation method of the FGF21-specific antibody containing an acridinium ester label comprises the following steps: 0.5 mg of acridinium ester is fully dissolved in 500 μL DMSO, and then an equal volume of 1 mg of PBS buffer (pH 7.2, concentration of 0.05 mol / L) containing the FGF21-specific antibody is added thereto, and the mixture is shaken at room temperature for 2 hours, and then dialyzed with 0.01 mol / L PBS buffer with a pH of 7.2 to remove excess acridinium ester in the solution; then, blocking solution B is added thereto, and the mixture is shaken at room temperature for 0.5 hours to obtain the FGF21-specific antibody containing an acridinium ester label.

[0055] Furthermore, the acridinium ester is M e -DMAE-NHS.

[0056] Furthermore, the blocking solution B comprises the following raw materials: 8% glycine, 1% BSA, 0.05 mol / L Tris(II) at pH 7.4 s -NaCl buffer and 0.05% Proclin-300.

[0057] Furthermore, the concentrations of FGF21 calibrators were 0 pg / ml, 30 pg / ml, 60 pg / ml, 120 pg / ml, 240 pg / ml, 480 pg / ml, 960 pg / ml and 1920 pg / ml, respectively.

[0058] The method for using the FGF21 detection kit prepared in this embodiment comprises the following steps:

[0059] (1) adding magnetic microspheres with FGF21-specific antibodies coupled to their surfaces into a sample diluent for dilution to a concentration of 50 mg / L, and then adding an equal volume of the sample to be tested for mixing and incubation;

[0060] (2) removing the supernatant in the sample solution to be tested in step (1), and then adding a cleaning solution to perform multiple shaking cleaning;

[0061] (3) adding the FGF21-specific antibody labeled with acridinium ester to the sample diluent for dilution to a concentration of 0.2 mg / L, and adding the diluted solution to the cleaned sample to be tested;

[0062] (4) Under the action of an external magnetic field, the magnetic microspheres are separated from the sample solution to be tested, and 100 μL of the luminescent pre-excitation solution and the excitation solution are added while shaking, and the luminescent signal value is immediately read (the detection kits in the present invention are all measured using the Feipeng open automatic biochemical analyzer IncreCare Shinei 1910).

[0063] Example 2

[0064] The difference between this embodiment and embodiment 1 is that:

[0065] In this embodiment, the method for preparing magnetic microspheres further includes step A3, uniformly dispersing the magnetic microspheres prepared in step A2 in an acid solution, stirring at room temperature overnight, centrifuging and washing multiple times, and drying to obtain surface carboxylated magnetic microspheres.

[0066] Furthermore, in step A3, the pH of the acid solution is 1, the acid solution is compounded by citric acid and hydrochloric acid in a mass ratio of 3:1, and the mass volume ratio of the magnetic microspheres to the acid solution is 1:2.

[0067] Example 3

[0068] The difference between this embodiment and embodiment 2 is that:

[0069] In this embodiment, the method for preparing magnetic microspheres further includes step A3, uniformly dispersing the magnetic microspheres prepared in step A2 in an acid solution, stirring at room temperature overnight, centrifuging and washing multiple times, and drying to obtain surface carboxylated magnetic microspheres.

[0070] Furthermore, in step A3, the pH of the acid solution is 1, the acid solution is a hydrochloric acid solution, and the mass volume ratio of the magnetic microspheres to the acid solution is 1:2.

[0071] Comparative Example 1

[0072] The difference between this comparative example and Example 1 is:

[0073] The preparation method of the magnetic microspheres comprises the following steps:

[0074] A1. Sodium hydroxide is dissolved in 2 mL of diethylene glycol, heated and stirred to form solution A. Polyacrylic acid and ferric chloride are then added to 4 mL of diethylene glycol and mixed at 220° C. to form solution B. The mixture is stirred for 0.5 h. Solution A is quickly added to solution B under stirring. The mixture is kept warm for 1 h. The magnetic microspheres are obtained by filtering, washing and drying.

[0075] The rest of the contents of this comparative example are the same as those of Example 1 and will not be described again here.

[0076] Comparative Example 2

[0077] The difference between this comparative example and Example 1 is:

[0078] In the method for preparing magnetic microspheres with FGF21-specific antibodies coupled to the surface of the magnetic microspheres in this comparative example, the blocking solution A in Example 1 is replaced by the 0.05 mol / L Tris-NaCl buffer solution with a pH of 7.4 in step S4.

[0079] Comparative Example 3

[0080] The difference between this comparative example and Example 1 is:

[0081] In this embodiment, the method for preparing the magnetic microspheres comprises the following steps:

[0082] A1. Dissolve sodium hydroxide in 2 mL of diethylene glycol, heat and stir to form solution A, then add polyacrylic acid and ferric chloride to 4 mL of diethylene glycol, mix and form solution B at 220°C, stir for 0.5 h, and quickly add solution A to solution B under stirring, continue to keep warm for 1 h, filter, wash and dry to obtain ferroferric oxide core layer particles;

[0083] A2. The ferroferric oxide core layer particles and the polylactic acid polymer precursor obtained in step A1 are added to 4 mL of chloroform at a mass ratio of 0.5:1 to fully dissolve, and ultrasonically prepared into solution C. Solution C is transferred to the outside of the SPG membrane emulsifier as a dispersed phase, and is pressurized by external nitrogen (nitrogen pressure is 0.005 MPa) to act on the outside of the SPG membrane emulsifier to form droplets, and stirred with 0.5wt% SDS solution at a stirring speed of 800 rpm to form a uniformly dispersed emulsion. The obtained emulsion is stirred at room temperature overnight, and then centrifuged, washed and dried to obtain the magnetic microspheres.

[0084] Furthermore, in step A1, the molar ratio of sodium hydroxide, polyacrylic acid and ferric chloride is 5.5:1:0.1.

[0085] Furthermore, in step A2, the preparation method of the polylactic acid polymer precursor includes the following steps: polylactic acid, maleic anhydride, and benzoyl peroxide are fully dissolved in 50 mL of dichloromethane, and placed at room temperature for 24 hours, and then transferred to a 110°C oven and heated for 18 hours. The obtained solid mixture is dissolved by chloroform, precipitated with ether, purified three times, and the polylactic acid precursor is obtained by centrifugation and drying.

[0086] Furthermore, the mass ratio of the polylactic acid, maleic anhydride and benzoyl peroxide is 5:0.3:0.01.

[0087] The present invention performs a detection performance test on the FGF21 detection kits prepared in Examples 1-3 and Comparative Examples 1-3 by the following test method:

[0088] (1) The luminescent signal values ​​of FGF21 calibrators of different concentrations in FGF21 detection in Examples 1-3 and Comparative Examples 1-3 were determined using the Feipeng open automatic biochemical analyzer IncreCare Shine i1910. The results are shown in the following table;

[0089]

[0090]

[0091] It can be seen from the data in the above table that the FGF21 reagent prepared by Examples 1-3 of the present invention has a low blank background, a high luminescent signal value of the calibration curve, and a high sensitivity of the reagent. Moreover, as the concentration of the calibrator increases, the luminescent signal value of the corresponding reagent increases, and the sensitivity is also improved.

[0092] (2) Magnetic separation efficiency test:

[0093] According to the luminescence signal value detection results of Feipeng open fully automatic biochemical analyzer, a corresponding curve between reaction time and luminescence signal value was drawn. The time when the luminescence signal value reached the highest and stable was recorded as the reaction completion time. The shorter the reaction completion time, the higher the magnetic separation efficiency. The results are shown in the following table.

[0094]

[0095] It can be seen from the data in the above table that the reaction completion time of the FGF21 detection kit prepared in Example 1-3 is significantly shorter than that of Comparative Example 1-3, indicating that the magnetic separation efficiency of the FGF21 detection kit prepared in Example 1-3 is relatively high. This is mainly because Example 1-3 prepares magnetic microbeads with a core-shell structure that have strong specificity for binding to FGF21.

[0096] (3) Repeatability test

[0097] Take the FGF21 detection kit prepared in Example 1-3 and Comparative Example 1-3, detect 120pg / ml FGF21 calibrator according to the usage method, test 20 times, calculate the CV value, and the calculation method of the CV value is: CV=SD / M×100%, wherein CV is the coefficient of variation, SD is the standard deviation of the 20 test results, and M is the average value of the 20 test results.

[0098] According to the test results, the CV values ​​of the FGF21 detection kits prepared in Examples 1, 2 and 3 were 4%, 2% and 3%, respectively, while the CV values ​​of the FGF21 detection kits prepared in Comparative Examples 1, 2 and 3 were 8%, 6% and 11%, respectively. This shows that the detection results of FGF21 detected by the FGF21 detection kits prepared in Examples 1-3 of the present invention are accurate and reliable, with high repeatability.

[0099] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A chemiluminescence-based FGF21 detection kit, characterized in that: The kit comprises: magnetic microspheres with FGF21-specific antibodies coupled to the surface, FGF21-specific antibodies labeled with acridinium ester, sample diluent, luminescent pre-excitation solution, excitation solution and FGF21 calibrators of different concentrations; The sample diluent comprises the following raw materials: 100-120 mM PBS buffer, 3-6 mM tris(2-hydroxyethyl)phosphine, 0.05-0.15 wt % Tween 20, 0.05-0.15 wt % triton X-100, 0.1-0.12 wt % dodecyl glucoside and 0.5-1.5 wt % BSA.

2. The method for preparing a chemiluminescence-based FGF21 detection kit according to claim 1, characterized in that: The method for preparing the magnetic microspheres with FGF21-specific antibodies coupled to the surface comprises the following steps: S1. Take 10 mg of magnetic microspheres, separate them by magnetic separation for 1 min, remove the supernatant, and wash them several times with 0.02 mol / L Tris-NaCl buffer (pH 7.4) by shaking; S2, prepare 10 mg / mL EDC and 12 mg / mL NHS solution with 0.05 mol / L pH 6.0 MES buffer, and take 100 μL of each into the magnetic microspheres washed in step S1, and shake for 20-40 minutes; S3, separate the magnetic microspheres using an external magnetic field, discard the supernatant, add 0.05 mol / L MES buffer at pH 6.0 and FGF21-specific antibody solution, and shake at room temperature for 1-3 hours; S4. Separate the magnetic microspheres with an external magnetic field, discard the supernatant, add 300 μL of blocking solution A, mix and oscillate, then perform magnetic separation and discard the supernatant, repeat several times, add 3 mL of blocking solution A, and oscillate at room temperature for 0.5-1 h to obtain the magnetic microspheres with FGF21-specific antibodies coupled to the surface.

3. The method for preparing a chemiluminescence-based FGF21 detection kit according to claim 2, characterized in that: The magnetic microspheres have a core-shell structure with ferroferric oxide as a core layer and polylactic acid polymer as a shell layer.

4. The method for preparing the chemiluminescence-based FGF21 detection kit according to claim 2, characterized in that: In step S4, the blocking solution A includes the following components: 0.04-0.06 mol / L Tris-NaCl buffer at pH 7.4, 0.5%-2% BSA, 0.01-0.02% ProClin-300, 4-6% sucrose and 0.1-0.8% ascorbic acid.

5. The method for preparing a chemiluminescence-based FGF21 detection kit according to claim 3, characterized in that: The preparation method of the magnetic microspheres comprises the following steps: A1, dissolving sodium hydroxide in diethylene glycol, heating and stirring to form solution A, then adding polyacrylic acid and ferric chloride to diethylene glycol, mixing and heating to form solution B, rapidly adding solution A to solution B under stirring, continuing to keep warm for 1-1.5h, filtering, washing and drying to obtain ferroferric oxide core layer particles; A2. Take the iron oxide core layer particles and polylactic acid polymer precursor obtained in step A1, add them into chloroform to fully dissolve, and ultrasonically prepare them into solution C. The solution C is transferred to the outside of the SPG membrane emulsifier as a dispersed phase. The external nitrogen pressurization acts on the outside of the SPG membrane emulsifier to form droplets, and reacts with the SDS solution to form a uniformly dispersed emulsion. The obtained emulsion is stirred at room temperature overnight, and then centrifuged, washed and dried to obtain the magnetic microspheres.

6. The method for preparing a chemiluminescence-based FGF21 detection kit according to claim 5, characterized in that: In step A1, the molar ratio of sodium hydroxide, polyacrylic acid and ferric chloride is 12-13:1:0.

1.

7. The method for preparing the chemiluminescence-based FGF21 detection kit according to claim 5, characterized in that: In step A2, the preparation method of the polylactic acid polymer precursor includes the following steps: fully dissolving polylactic acid, maleic anhydride, and benzoyl peroxide in dichloromethane, and placing them at room temperature for 20-24 hours, and then transferring them to a 110-120°C oven and heating them for 16-20 hours. The obtained solid mixture is dissolved by chloroform, precipitated with ether, purified multiple times, and centrifuged and dried to obtain the polylactic acid precursor.

8. The method for preparing a chemiluminescence-based FGF21 detection kit according to claim 1, characterized in that: The concentrations of FGF21 calibrators were 0pg / ml, 30pg / ml, 60pg / ml, 120pg / ml, 240pg / ml, 480pg / ml, 960pg / ml and 1920pg / ml, respectively.

9. Use of the kit prepared by the method for preparing a chemiluminescence-based FGF21 detection kit according to any one of claims 2 to 8 in detecting FGF21.

10. Use of the chemiluminescence-based FGF21 detection kit according to claim 9 in detecting FGF21, characterized in that: The method for using the FGF21 detection kit comprises the following steps: (1) adding magnetic microspheres with FGF21-specific antibodies coupled to the surface to dilute the sample to a concentration of 30-60 mg / L, and then adding the sample to be tested to mix and incubate; (2) removing the supernatant in the sample solution to be tested in step (1), and then adding a cleaning solution to perform multiple shaking cleaning; (3) adding the FGF21-specific antibody labeled with acridinium ester to the sample diluent for dilution to a concentration of 0.1-0.3 mg / L, and adding the diluted solution to the cleaned sample to be tested; (4) Under the action of an external magnetic field, the magnetic microspheres are separated from the sample solution to be tested, and the luminescent pre-excitation solution and the excitation solution are added while shaking, and the luminescent signal value is immediately read.

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