A reagent and kit for detecting glial fibrillary acidic protein and its preparation method and application

Abstract

The present invention discloses a reagent for detecting glial fibrillary acidic protein, a kit, and a preparation method and application thereof. The present invention provides a reagent for detecting glial fibrillary acidic protein, the reagent containing magnetic beads coated with capture antibodies and / or detection antibodies labeled with fluorescent microspheres; the magnetic beads coated with capture antibodies contain glial fibrillary acidic protein capture antibodies and magnetic beads, and the diameter of the magnetic beads is 1.0-3.0 μm; the detection antibodies labeled with fluorescent microspheres contain glial fibrillary acidic protein detection antibodies and fluorescent microspheres, and the diameter of the fluorescent microspheres is 200-400 nm. It can quickly detect GFAP in cerebrospinal fluid and serum samples, with high sensitivity and good repeatability, and the detection limit of GFAP can reach 0.13 pg / mL. The detection repeatability CV value can reach 3%. No need for live puncture to perform detection, the use process is simple and convenient, and the result can be detected in 1 hour.

Description

Technical Field

[0001] The invention relates to the technical field of single molecule immunoassay, and in particular to a reagent and a kit for detecting glial fibrillary acid protein, and a preparation method and application thereof. Background Art

[0002] Astrocytes are the most numerous cell type in the human central nervous system (CNS), accounting for 50% of the non-neuronal cells in the brain. The pathology of Alzheimer's disease (AD) is associated with the morphological, molecular and functional remodeling of astrocytes, a process known as astrogliosis. The cytoskeleton of astrocytes is composed of characteristic intermediate filaments (IFs) and is called glial filaments, among which glial fibrillary acid protein (GFAP) is the main component of intermediate filament proteins. GFAP is a key IFs protein. Like other members of IFs, it has vimentin, desmin and peripherin, and has head, rod and tail domains. Its structure is highly conserved in IFs. GFAP is an acidic protein composed of 432 encoded amino acids, which are encoded by chromosome 17q21 genes. GFAP is a unique component of the cytoskeleton protein of astrocytes in the brain and plays an important role in many complex cellular activities. Astrocyte proliferation is an important case change in AD, and astrocyte proliferation is accompanied by increased expression of GFAP. Therefore, GFAP can be used as a biomarker for astrocyte proliferation in central nervous system damage.

[0003] So far, most of the reports on GFAP are clinical studies, lacking further research on the mechanism of GFAP such as animal model experiments, in vitro cell culture and biopsy. In addition, there is no unified international diagnostic standard for GFAP, and clinical diagnosis mainly relies on clinical manifestations, imaging manifestations and GFAP antibody detection. At present, the main detection methods for GFAP include immunohistochemistry, fluorescent immunochromatography and magnetic particle chemiluminescence detection. Enzyme-linked immunosorbent assay is a relatively popular detection method. Its main experimental principle is to use double antibody one-step sandwich enzyme-linked immunosorbent assay: add specimens, calibrators and detection antibodies to the coated microwells pre-coated with GFAP capture antibodies in turn, incubate and wash thoroughly. The substrate TMB (3,3',5,5'-tetramethylbenzidine) is used for color development. TMB is converted into blue under the catalysis of peroxidase and converted into the final yellow under the action of acid. The depth of color is positively correlated with the GFAP content in the sample. The absorbance (OD value) is measured at a wavelength of 450nm using an enzyme marker to calculate the sample concentration. However, these detection methods have complicated detection processes, poor repeatability and large batch differences, and are not suitable for clinical promotion. Summary of the invention

[0004] The embodiments of the present invention provide a reagent and a kit for detecting glial fibrillary acid protein, and a preparation method and application thereof, so as to at least solve one of the problems existing in the related art.

[0005] One of the purposes of the present invention is to provide a reagent for detecting glial fibrillary acid protein.

[0006] The second object of the present invention is to provide the use of the reagent in preparing a product for detecting glial fibrillary acid protein.

[0007] The third object of the present invention is to provide a kit for detecting glial fibrillary acid protein.

[0008] A fourth object of the present invention is to provide an application of the kit in the preparation of a product for detecting Alzheimer's disease.

[0009] A fifth object of the present invention is to provide a method for preparing the reagent.

[0010] To achieve the object, the present invention is implemented through the following technical solutions.

[0011] The invention provides a reagent for detecting glial fibrillary acid protein, the reagent contains magnetic beads coated with capture antibodies and / or detection antibodies labeled with fluorescent microspheres; the magnetic beads coated with capture antibodies contain glial fibrillary acid protein capture antibodies and magnetic beads, the diameter of the magnetic beads is 1.0-3.0 μm; the detection antibodies labeled with fluorescent microspheres contain glial fibrillary acid protein detection antibodies and fluorescent microspheres, the diameter of the fluorescent microspheres is 200-400 nm. The sensitivity and coefficient of variation are good.

[0012] Preferably, the diameter of the magnetic beads is 3.0 μm, the diameter of the fluorescent microspheres is 300 nm, the reagent reaction background is greatly reduced, the detection sensitivity can reach 0.13 pg / mL, and the coefficient of variation is low, which is 3.0%.

[0013] Further, according to the classification of antibody specific characteristics, the capture antibody and / or the detection antibody can be at least one of a polyclonal antibody and a monoclonal antibody, and according to the classification of sources, the capture antibody can be at least one of a mouse antibody, a rabbit antibody and a sheep antibody.

[0014] The magnetic beads coated with capture antibodies and the detection antibodies labeled with fluorescent microspheres can bind to the GFAP antigen. The amino acid sequence of the GFAP antigen is: MERRRITSAARRSYVSSGEMMVGGLAPGRRLGPGTRLSLARMPPPLPTRV DFSLAGALNAGFKETRASERAEMMELNDRFASYIEKVRFLEQQNKALAAELNQLRAKEPTKLADVYQAELRELRLRLDQLTANSARLEVERDNLAQDLATVRQKLQDETNLRLEAENNLAAYRQEADEATLARLDLERKIESLEEEIRFLRKIHEEEVRELQEQLARQQVHVELDVAKPDLTAALKEIRTQ YEAMASSNMHEAEEWYRSKFADLTDAAARNAELLRQAKHEANDYRRQLQSLTCNLESLRGTNESLERQMREQEERHVREAASYQEALARLEEEGQSLKDEMARHLQEYQDLLNVKLALDIEIATYRKLLEGEENRITIPVQTFSNLQIRETSLDTKSVSEGHLKRNIVVKTVEMRDGEVIKESKQEHKDVM.

[0015] Furthermore, the surface of the magnetic beads is modified with at least one of carboxyl, amino and toluenesulfonyl groups, preferably with carboxyl-modified magnetic beads. The carboxyl concentration is 10 to 35 μeq / g. If the carboxyl concentration is greater than 35 μeq / g, the low concentration will be too high, and if it is less than 10 μeq / g, the antibody will not be fully connected to the magnetic beads, resulting in non-specific connection in the kit.

[0016] Furthermore, the material of the fluorescent microspheres is one or more of silicon dioxide, polyacrylamide or polystyrene. Silicon dioxide is not easy to swell, the dye cannot be wrapped inside it, and the fluorescence intensity is unstable. The preparation cost of polyacrylamide is high, and the dispersibility and stability are poor, and the fluorescence signal and background noise are strong. We prefer polystyrene as a material mainly considering its good monodispersity, high luminescence rate, strong adsorption and good stability. The surface of the fluorescent microspheres is modified with one or more of carboxyl, amino or toluenesulfonyl groups, preferably fluorescent microspheres modified with carboxyl groups. Modifying carboxyl groups on fluorescent microspheres helps to further enhance the signal and further improve the sensitivity.

[0017] Furthermore, the fluorescent microspheres were prepared by an internal dye embedding method, and the obtained fluorescent microspheres emitted more stable light.

[0018] Further, the dye encapsulated in the fluorescent microsphere is fluorescein, quantum dots, rare earth elements, rare earth chelates, fluorescent proteins or up-conversion nanoparticles. The fluorescence intensity of the fluorescent microsphere using fluorescein as the dye is strong, can be separated from the background signal, is conducive to signal collection during the final result analysis, and can significantly improve the precision of the detection. Preferably, the dye encapsulated in the fluorescent microsphere is fluorescein isothiocyanate (FITC), mainly considering its high absorption rate and good water solubility, can be efficiently combined with protein, and the fluorescent color is bright and easy to observe, which is better for the sensitivity of the kit.

[0019] The invention provides application of the reagent in preparing a product for detecting glial fibrillary acid protein.

[0020] The invention provides a kit for detecting glial fibrillary acid protein, and the kit contains the reagent.

[0021] In a specific embodiment of the present invention, the kit may further contain PBST buffer.

[0022] In a specific embodiment of the present invention, the kit further contains quality control products and calibrators.

[0023] In a specific embodiment of the present invention, the calibrator is a GFAP antigen solution, and the concentration of the GFAP antigen solution is 0-100 pg / mL, preferably 0, 40 pg / mL and 80 pg / mL.

[0024] In a specific embodiment of the present invention, the quality control product is a GFAP antigen solution, and the concentration of the GFAP antigen solution is 40-80 pg / mL, preferably 40 pg / mL and 80 pg / mL.

[0025] The present invention provides application of the kit in preparing a product for detecting Alzheimer's disease.

[0026] The present invention provides a method for preparing the reagent, wherein the preparation of magnetic beads coated with capture antibodies comprises the following steps:

[0027] S11: activating the magnetic beads for 30 to 50 minutes to obtain activated magnetic beads;

[0028] S12: mixing the magnetic beads activated in step S11 with the glial fibrillary acid protein capture antibody, and incubating for 1 to 2 hours to obtain a reaction product of the magnetic beads and the glial fibrillary acid protein capture antibody;

[0029] S13: The magnetic beads of step S12 are mixed with the reaction product of the glial fibrillary acid protein capture antibody and the blocking solution, and magnetic separation and blocking are performed at 40-50 rpm / min for 1-2 hours to obtain magnetic beads coated with the capture antibody.

[0030] Furthermore, in step S11, the concentration of the magnetic beads is 0.25-1.00 mg / mL, and the sensitivity and coefficient of variation are good.

[0031] Preferably, the concentration of the magnetic beads is 0.25 mg / mL, the detection sensitivity can reach 0.13 pg / mL, the coefficient of variation is low, which is 3.0%, and a low production cost can be guaranteed.

[0032] Furthermore, in step S11, when the activation time of the magnetic beads is greater than 50 min, the activation time is too long, which will cause the intermediate formed by activation to be unstable and reduce the coupling efficiency of the antibody. When the activation time of the magnetic beads is less than 30 min, the activation time is too short, which will cause insufficient activation of the carboxyl group and affect the subsequent antibody coupling. The preferred activation time of the magnetic beads is 30 min.

[0033] Furthermore, in steps S11 and S12, an activator is required for coupling the magnetic beads with the capture antibody. When the modification group on the surface of the magnetic beads is a carboxyl group, the activator can be at least one of N-hydroxysuccinimide (NHS), SulfoNHS and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC). The present invention controls the type of activator used for coupling the magnetic beads with the capture antibody, and finds that when the activator is EDC, the antibody and the magnetic beads are firmly covalently bound.

[0034] Furthermore, in step S12, when the incubation time is greater than 2h, the experimental time is too long, and long-term coupling does not increase the coupling efficiency, but will cause magnetic beads to agglomerate. When the incubation time is less than 1h, the incubation time is too short, which will cause the antibody and magnetic beads to not be completely combined, the antibody coupling is insufficient, and the coupling efficiency is reduced. The preferred incubation time is 2h.

[0035] The present invention provides a method for preparing the reagent, wherein preparing a detection antibody labeled with fluorescent microspheres comprises the following steps:

[0036] S21: activating the fluorescent microspheres for 30 to 50 minutes to obtain activated fluorescent microspheres;

[0037] S22: mixing the fluorescent microspheres activated in step S21 with the glial fibrillary acidic protein detection antibody, and incubating for 1 to 2 hours to obtain a reaction product of the fluorescent microspheres and the glial fibrillary acidic protein detection antibody;

[0038] S32: Mix the reaction product of the fluorescent microspheres in step S22 with the glial fibrillary acid protein detection antibody and the blocking solution, and block at 40-50 rpm / min for 1-2 hours to obtain the detection antibody labeled with the fluorescent microspheres.

[0039] Furthermore, in steps S21 and S22, an activator is required to couple the fluorescent microspheres with the detection antibody. When the modification group on the surface of the fluorescent microspheres is a carboxyl group, the activator can be at least one of NHS, SulfoNHS and EDC. EDC and NHS allow the detection antibody to be firmly covalently bonded to the fluorescent microspheres.

[0040] Furthermore, in step S21, when the activation time of the fluorescent microspheres is greater than 50 min, the activation time is too long, which will cause the intermediate formed by activation to be unstable and reduce the coupling efficiency of the antibody. When the activation time of the fluorescent microspheres is less than 30 min, the activation time is too short, which will cause insufficient activation of the carboxyl group and affect the subsequent antibody coupling. Preferably, the activation time of the fluorescent microspheres is 30 min.

[0041] Furthermore, in step S22, when the incubation time is greater than 2 hours, the fluorescent microspheres will agglomerate, and when the incubation time is less than 1 hour, if the incubation time is too short, the antibody and the fluorescent microspheres will not be completely combined, thereby reducing the coupling efficiency. The preferred incubation time is 2 hours.

[0042] Further, in step S22, the rotation speed is greater than 50 rpm / min. If the rotation speed is too fast, the magnetic beads (or fluorescent microspheres) cannot be evenly dispersed and cannot fully combine with the antibody, and the coupling efficiency is reduced. The rotation speed is less than 40 rpm / min. If the rotation speed is too slow, the magnetic beads (or fluorescent microspheres) will agglomerate and the coupling efficiency will be reduced. The preferred rotation speed is 50 rpm / min, which can make the magnetic beads (or fluorescent microspheres) evenly dispersed and fully combined with the antibody.

[0043] Furthermore, in step S23, the rotation speed is greater than 50 rpm / min. If the rotation speed is too fast, the magnetic beads (or fluorescent microspheres) will not be evenly dispersed, and the blocking solution cannot bind to the sites of the magnetic beads (or fluorescent microspheres) that are not bound to the antibody, the blocking efficiency is reduced, and non-specific reactions will occur in later detections, affecting the sensitivity; the rotation speed is less than 40 rpm / min. If the rotation speed is too slow, the magnetic beads (or fluorescent microspheres) will agglomerate and the blocking efficiency will be reduced. The preferred rotation speed after adding the blocking solution is 50 rpm / min, which can make the magnetic beads (or fluorescent microspheres) evenly dispersed and fully combined with the antibody.

[0044] Furthermore, the blocking solution contains at least one of CE210, CE510 and ethanolamine.

[0045] Furthermore, the labeling buffer is usually one or more of Tris buffer, phosphate buffer, MES buffer, MOPS buffer, HEPES buffer, and glycine buffer, with a concentration of 10 to 200 mM.

[0046] The embodiments of the present invention have the following beneficial effects:

[0047] In order to solve the above problems, this paper uses single molecule detection technology combined with high-throughput testing technology based on the enzyme-linked immunosorbent assay of the double antibody sandwich method to detect low-abundance protein markers with a detection limit of fg / mL. Single molecule immunoassay technology can be used to measure proteins in various matrices (such as blood, cerebrospinal fluid, cell extracts, etc.), and its sensitivity is up to 1000 times higher than that of traditional enzyme-linked immunosorbent assays. For these low-abundance proteins, the single molecule complexes formed in the single molecule array can also be accurately detected.

[0048] During the development of the GFAP test kit, I explored the particle size of the magnetic beads, the particle size of the fluorescent microspheres, and the types of detection and capture antibodies, and experimented with the types of various buffers including pH in the coupling process, so that the correlation coefficient of the calibration curve of the kit reached 0.99 and the detection limit reached the fg / mL level.

[0049] The invention solves the problem that the traditional GFAP test method is cumbersome and the test sensitivity cannot meet the requirements. The sensitivity of the traditional ELISA kit can only reach 100pg / mL level, and the test results are affected by factors such as laboratory environment and laboratory operator operation. The test results have poor repeatability and the operation process is cumbersome.

[0050] The kit for detecting glial fibrillary acid protein of the present invention can quickly detect GFAP in cerebrospinal fluid and serum samples with high sensitivity and good repeatability. In some embodiments, the detection limit of GFAP can reach 0.17pg / mL, and in other embodiments, the detection limit of GFAP can reach 0.19pg / mL. In other embodiments, the detection limit of GFAP can reach 0.13pg / mL. The detection repeatability CV value can reach 3% in some embodiments. The present invention can use cerebrospinal fluid or serum as a sample for detection. The source of serum sample is simple and easy to obtain, and detection can be performed without live puncture. The finished kit is ready for use after disassembly, and the use process is simple and convenient, and the result can be detected in 1 hour. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0052] Figure 1 This is the detection range of glial fibrillary acid protein detected by the kit in Example 1 of the present invention.

[0053] Figure 2 The kit of Example 1 of the present invention is used to detect the glial fibrillary acid protein content in the serum of AD patients and healthy subjects. DETAILED DESCRIPTION

[0054] To make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings. However, it will be appreciated by those skilled in the art that in the embodiments of the present invention, many technical details are proposed in order to enable the reader to better understand the present application. However, even without these technical details and various changes and modifications based on the following embodiments, the technical scheme claimed in the present application can be implemented. The division of the following embodiments is for the convenience of description, and should not constitute any limitation on the specific implementation of the present invention. The various embodiments can be combined and referenced with each other without contradiction.

[0055] Examples 1 to 9

[0056] 1. Experimental Methods

[0057] 1. Prepare reagents

[0058] Labeling dispersion solution: BSA (bovine serum albumin) is dissolved in 20mM PBS (phosphate buffered saline), pH = 7.4, and the final volume concentration of BSA is 0.1% (v / v); labeling preservation solution: NaCl and FBS (fetal bovine serum) are dissolved in 50mM PBS, pH = 7.4, and the final volume concentration of NaCl is 0.9% (w / v), and the final volume concentration of FBS is 0.2% (v / v).

[0059] Magnetic bead washing solution: 5×PBST buffer (phosphate-Tween buffer); magnetic bead coupling solution: 0.1M MES buffer (4-morpholineethanesulfonic acid); magnetic bead storage solution: NaCl and FBS dissolved in 50mM PBS, pH=7.4, NaCl final volume concentration is 0.9% (w / v), FBS final concentration is 0.2% (v / v).

[0060] Calibrator diluent or quality control diluent: BSA, casein, trehalose, NaCl, betaine, mannitol, Tween-20 and antibacterial agent Proclin 300 (manufacturer: Sigma, catalog number: 48912-U) were dissolved in PBS buffer (50 mM, pH = 7.4), and filtered with a 0.22 μm microporous filter membrane to obtain a calibrator diluent or quality control diluent, wherein the final concentration of BSA is 1% (v / v), the final concentration of Casein is 0.1% (w / v), the final concentration of trehalose is 1% (w / v), the final concentration of NaCl is 0.9% (w / v), the final concentration of betaine is 0.1% (w / v), the final concentration of mannitol is 0.1% (v / v), the final concentration of Tween-20 is 0.1% (v / v), and the final concentration of antibacterial agent Proclin 300 is 0.1% (v / v).

[0061] 2. Preparation of magnetic beads coated with GFAP capture antibody

[0062] (1) Take 100 μL of 3 mg / mL carboxyl-modified magnetic beads (manufacturer: JSR, catalog number: MS300CA / Carboxyl), resuspend the magnetic beads in 1 mL of MES buffer (50 mM, pH = 6.0), mix well on a vortexer, place on a magnetic stand for magnetic separation, and wash the beads three times with magnetic bead washing solution.

[0063] (2) Weigh 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), prepare an activation solution with a concentration of 10 mg / mL using a magnetic bead coupling solution, add it to the magnetic beads treated in step (1), and activate it on a roller incubator at 25° C. for 30 minutes at a speed of 50 rpm / min to obtain activated magnetic beads. The magnetic bead coupling solution is used to make the carboxyl groups on the magnetic beads in a state that is easy to modify.

[0064] (3) Take 20 μg of GFAP capture antibody (manufacturer: Merck, catalog number: AB5541), add it to the activated magnetic beads in step (2), mix well, and incubate on a roller incubator at 25° C. for 1 to 2 h at a speed of 50 rpm / min.

[0065] (4) Add magnetic bead cleaning solution to the magnetic beads treated in step (3), mix well on a vortexer, place on a magnetic rack, perform magnetic separation, and clean three times with magnetic bead cleaning solution.

[0066] (5) CE210 and CE510 (manufacturer: JSR, item number: Blockmaster) were added to the magnetic beads treated in step (4) in sequence. TM CE210 / CE510) and ethanolamine blocking solution, incubate on a roller incubator for 1 hour, rotation speed: 50 rpm / min, remove the supernatant after magnetic separation, and obtain magnetic beads coated with GFAP capture antibody.

[0067] (6) Add magnetic bead storage solution to the magnetic beads coated with GFAP capture antibody in step (5) and store at 4°C. This is reagent 1.

[0068] 3. Preparation of GFAP detection antibody labeled with fluorescent microspheres

[0069] (1) Take 200 μL of carboxyl-modified fluorescent microspheres (manufacturer: Weidu, catalog number: FG0300CA), wash them three times with the labeling dispersion, and centrifuge at 12000 rpm for 15 min.

[0070] (2) Weigh a certain amount of EDC and N-hydroxysuccinimide (NHS), dissolve them in MES buffer (50 mM, pH = 6.0) to a concentration of 10 mg / mL, add them to the fluorescent microspheres treated in step (1), and activate them on a roller incubator at 25°C for 30 min at a speed of 50 rpm / min.

[0071] (3) Take 20 μg of GFAP detection antibody (manufacturer: Merck, catalog number: MAB5628), add it to the fluorescent microspheres treated in step (2), mix well, and incubate on a roller incubator at 25° C. for 2 h at a speed of 50 rpm / min.

[0072] (4) CE210, CE510 and ethanolamine blocking solution were added to the fluorescent microspheres treated in step (3) in sequence, and incubated on a roller incubator for 1 hour at a speed of 50 rpm / min.

[0073] (5) The fluorescent microspheres blocked in step (4) were centrifuged at 12,000 rpm for 15 min, added with labeling preservation solution and stored at 4°C. The final concentration of the fluorescent microspheres was 1.0 mg / mL. This is reagent 2.

[0074] 4. Configure calibration products and quality control products

[0075] The GFAP antigen (manufacturer: Thermo Fisher, catalog number: GFAP-FITC) was diluted to 0 pg / mL, 20 pg / mL and 100 pg / mL with calibrator diluent, and used as calibrators 1, 2 and 3, respectively; the GFAP antigen was diluted to 40 pg / mL and 80 pg / mL with control diluent, and used as controls 1 and 2, respectively.

[0076] 5. Composition of the kit

[0077] Reagent 1, reagent 2 and 5×PBST buffer, calibrators 1 to 3 and quality controls 1 and 2.

[0078] 6. How to use the kit

[0079] (1) Take 50 μL of Reagent 1, add it to a reaction cup, place it on a magnetic separation rack, wash it three times with 5× PBST buffer, and resuspend it to obtain a magnetic bead resuspension solution coated with GFAP capture antibody. The concentration of the magnetic beads is the same as that of the magnetic beads in Reagent 1.

[0080] (2) Take 50 μL of the serum to be tested and add it to the reaction cup treated in step (1), and incubate it on a roller incubator at 25°C for 30 min.

[0081] (3) On a magnetic separation rack, add 5×PBST to the serum treated in step (2), wash three times, and resuspend. Add 50 μL of reagent 2 to each tube, mix thoroughly with a mixer, and incubate on a roller incubator at 25°C for 30 min.

[0082] (4) On a magnetic separation rack, add 5×PBST buffer to the serum treated in step (3), wash three times, and then resuspend to obtain a test solution. The test solution in step (4) is transferred to a detection position multi-well plate, and single-molecule imaging is performed using a fluorescence microscope, and the fluorescence intensity and count are recorded; the magnetic field is released to rearrange the magnetic beads in the multi-well plate, and single-molecule imaging is performed using a fluorescence microscope, and the fluorescence intensity and count are recorded to detect the GFAP concentration in the serum.

[0083] The diameter of the magnetic beads used in Examples 1 to 9, the concentration of magnetic beads in reagent 1, the diameter of magnetic beads, and the concentration and diameter of fluorescent microspheres in reagent 2 are shown in Table 1. Among them, the manufacturer of the magnetic beads in Examples 1, 5 to 9 is: Weidu, the item number is: CMP1003CA; the manufacturer of the magnetic beads in Example 2 is: JSR, the item number is: MS300CA; the manufacturer of the magnetic beads in Example 3 is: JSR, the item number is: MS160CA; the manufacturer of the magnetic beads in Example 4 is: Weidu, the item number is: CMP1001CA; the manufacturer of the fluorescent microspheres in Examples 1 to 7 is: Weidu, the item number is: FG0300CA; the manufacturer of Example 8 is: Weidu, the item number is: FG0200CA, and the manufacturer of Example 9 is: Weidu, the item number is: FG0400CA.

[0084] Table 1

[0085]

[0086] Example 10 Detection limit and reagent CV value

[0087] 1. Experimental Methods

[0088] The concentration of GFAP was measured using the kit prepared in Examples 1 to 9.

[0089] 1. Dilute the concentration of GFAP antigen (manufacturer: Thermo Fisher, catalog number: GFAP-FITC) with the calibration diluent to 0, 0.05, 0.1, 0.5, 1, 5, 10, 25, 50, 100 and 200 pg / mL, respectively, to obtain samples of different concentrations.

[0090] 2. Take 50 μL of the sample to be tested in step 1 for each concentration, add it to different reaction cups, and perform the test according to the method of use of the kit of Examples 1 to 9.

[0091] 2. Experimental Methods

[0092] The test detection limit and reagent CV values ​​are shown in Table 2, and the slope of the calibration curve is shown in Figure 1 The CV value mainly reflects the degree of dispersion of the data, the smaller the better, and 3% and below is the best. It can be seen that the kit prepared in Example 1 has the best sensitivity, the smaller the CV value, and the better comprehensive detection effect.

[0093] Table 2

[0094] Sensitivity (pg / mL) CV value (%) Example 1 0.13 3.0 Example 2 0.17 6.1 Example 3 0.32 4.3 Example 4 0.38 5.2 Example 5 0.19 8.3 Example 6 0.90 10.2 Example 7 1.32 9.0 Example 8 0.35 8.5 Example 9 0.73 7.3

[0095] Example 11 Detection of GFAP concentration in serum samples using the kit prepared in Example 1

[0096] 1. Experimental Methods

[0097] Serum samples from 5 healthy subjects and 5 AD patients were collected respectively, and the GFAP concentration in the serum samples was detected using the kit prepared in Example 1.

[0098] 2. Experimental Results

[0099] like Figure 2 As shown, the GFAP content in the serum of AD patients is significantly higher than that in the serum of healthy people. The kit of Example 1 of the present invention can effectively distinguish AD patients from healthy people.

[0100] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A reagent for detecting glial fibrillary acidic protein, characterized in that: The reagent contains magnetic beads coated with capture antibodies and / or detection antibodies labeled with fluorescent microspheres; the magnetic beads coated with capture antibodies contain glial fibrillary acid protein capture antibodies and magnetic beads, and the diameter of the magnetic beads is 1.0-3.0 μm; the detection antibodies labeled with fluorescent microspheres contain glial fibrillary acid protein detection antibodies and fluorescent microspheres, and the diameter of the fluorescent microspheres is 200-400 nm.

2. Use of the reagent according to claim 1 in the preparation of a product for detecting glial fibrillary acidic protein.

3. A kit for detecting glial fibrillary acidic protein, characterized in that: The kit contains the reagent according to claim 1.

4. The kit according to claim 3, characterized in that The kit also contains PBST buffer.

5. Use of the kit according to claim 3 or 4 in the preparation of a product for detecting Alzheimer's disease.

6. The method for preparing the reagent according to claim 1, characterized in that: Preparation of magnetic beads coated with capture antibodies comprises the following steps: S11: activating the magnetic beads for 15 to 30 minutes to obtain activated magnetic beads; S12: mixing the magnetic beads activated in step S11 with the glial fibrillary acid protein capture antibody, and incubating for 1 to 2 hours to obtain a reaction product of the magnetic beads and the glial fibrillary acid protein capture antibody; S13: The magnetic beads of step S12 are mixed with the reaction product of the glial fibrillary acid protein capture antibody and the blocking solution, and magnetic separation and blocking are performed at 40-50 rpm / min for 1-2 hours to obtain magnetic beads coated with the capture antibody.

7. The preparation method according to claim 6, characterized in that: The concentration of the magnetic beads is 0.25-1.00 mg / mL.

8. The method for preparing the reagent according to claim 1, characterized in that: The preparation of the detection antibody labeled with fluorescent microspheres comprises the following steps: S21: activating the fluorescent microspheres for 15 to 30 minutes to obtain activated fluorescent microspheres; S22: mixing the fluorescent microspheres activated in step S21 with the glial fibrillary acidic protein detection antibody, and incubating for 1 to 2 hours to obtain a reaction product of the fluorescent microspheres and the glial fibrillary acidic protein detection antibody; S32: Mix the reaction product of the fluorescent microspheres in step S22 with the glial fibrillary acid protein detection antibody and the blocking solution, and block at 40-50 rpm / min for 1-2 hours to obtain the detection antibody labeled with the fluorescent microspheres.

9. The preparation method according to any one of claims 6 to 8, characterized in that: The blocking solution contains at least one of CE210, CE510 and ethanolamine buffer.

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