Kit for detecting Abeta1-40 protein as well as preparation method and application thereof

Abstract

The invention discloses a kit for detecting Abeta1-40 protein as well as a preparation method and application of the kit. The invention provides a reagent for detecting Abeta1-40 protein. The reagent contains magnetic beads coupled with a capture antibody and / or a detection antibody labeled with fluorescent microspheres, the magnetic bead coupled with the capture antibody contains an A beta 1-40 protein capture antibody and a magnetic bead, and the diameter of the magnetic bead is 1.0-3.0 [mu] m; the detection antibody marked with the fluorescent microspheres contains an A beta 1-40 protein detection antibody and the fluorescent microspheres, and the diameter of the fluorescent microspheres is 190-400 nm. According to the present invention, the Abeta1-40 in the serum sample can be rapidly detected, the sensitivity is high, the repeatability is good, the sensitivity can achieve 0.12 pg / mL, and the data dispersion degree CV value can achieve 3.2%; the serum can be used as a sample for detection, the sample source is simple and easy to obtain, detection can be performed without living body puncture, and the use process is simple and convenient.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro diagnosis, and in particular to a kit for detecting Aβ1-40 protein, and a preparation method and application thereof. Background Art

[0002] Alzheimer's disease (AD) is a chronic disease of the central nervous system that develops slowly. Clinically, AD patients have symptoms such as memory loss, cognitive dysfunction, language disorders, decreased executive ability, and personality changes.

[0003] The etiology of AD is complex, and its pathogenesis is still unclear. At present, the widely accepted pathogenic hypotheses include: Aβ aggregation hypothesis, Tau protein abnormality hypothesis, cholinergic hypothesis and neuroinflammation hypothesis. The Aβ aggregation hypothesis is one of the most classic theories in the pathogenesis of AD. This theory explains that Alzheimer's disease is caused by the large deposition of Aβ in the cerebral cortex and hippocampus, and the subsequent intracellular neurofibrillary tangles mediated by phosphorylated tau protein, which in turn leads to neuronal apoptosis and damage that affects the patient's cognitive function. According to the latest ATN diagnostic criteria of the National Institute on Aging and the Alzheimer's Association (NIA-AA), changes in the concentrations of Aβ, total tau and phosphorylated tau protein are clearly defined as important markers for diagnosing AD. Due to the complex etiology of AD and the unclear pathogenesis, the research on its therapeutic drugs has progressed slowly, and there is still a lack of drugs or methods to cure AD in the world.

[0004] AD is an irreversible process. Therefore, early screening and diagnosis are particularly important for intervening in AD and delaying the progression of the disease in clinical practice. However, there is no accurate method for early prediction or diagnosis of AD. At present, the diagnostic method of AD is mainly combined diagnosis, which mainly includes: neuropsychological assessment, cognitive impairment test, brain senile plaque and Tau protein PET scan, brain magnetic resonance imaging (MRI) and cerebrospinal fluid (CSF) marker detection, β-amyloid protein detection and phosphorylated Tau protein detection. Although cerebrospinal fluid can better reflect the metabolic situation in the brain, invasive lumbar puncture is required to obtain cerebrospinal fluid. Its invasiveness and high cost make it impossible to sample on a large scale and repeatedly, so long-term monitoring is impossible. Compared with cerebrospinal fluid, blood samples are easy to obtain and less invasive. Blood can be collected multiple times in a short period of time, and patients have a high degree of acceptance. However, AD markers in the blood are difficult to detect due to their extremely low content and low specificity.

[0005] Therefore, how to provide an early screening method for AD that is easy to operate, low in cost and safer has become an urgent problem to be solved. β-Amyloid (1-40), also known as Aβ1-40, is a polypeptide produced by cleavage of amyloid precursor protein (APP) by β- and γ-secretase. It is the main protein fragment found in brain plaques of patients with Alzheimer's disease, and its amino acid sequence is DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV. At present, there are few Aβ1-40 detection kits on the market, and most of them use traditional detection methods (enzyme-linked immunosorbent assay, fluorescence immunoassay, chemiluminescence). The detection principle is to compare the overall optical signal of the solution with the standard curve to achieve quantitative detection. Due to the background noise of the detection equipment itself, the deviation is large at ultra-low concentrations. Due to the limitations of the underlying technology, high-sensitivity detection and analysis cannot be achieved, and the detection requirements of this project cannot be met. Summary of the invention

[0006] The embodiments of the present invention provide a kit for detecting Aβ1-40 protein and a preparation method and application thereof, so as to at least solve one of the problems existing in the related art.

[0007] One of the purposes of the present invention is to provide a reagent for detecting Aβ1-40 protein.

[0008] The second object of the present invention is to provide the use of the reagent in preparing a product for detecting Aβ1-40 protein.

[0009] The third object of the present invention is to provide a kit for detecting Aβ1-40 protein.

[0010] 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.

[0011] A fifth object of the present invention is to provide a method for preparing the reagent.

[0012] To achieve the object, the present invention is implemented through the following technical solutions.

[0013] The invention provides a reagent for detecting Aβ1-40 protein, the reagent contains magnetic beads coupled with capture antibodies and / or detection antibodies labeled with fluorescent microspheres; the magnetic beads coupled with capture antibodies contain Aβ1-40 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 Aβ1-40 protein detection antibodies and fluorescent microspheres, the diameter of the fluorescent microspheres is 190-400 nm. The sensitivity and coefficient of variation are good.

[0014] The magnetic beads coupled with the capture antibody and the detection antibody labeled with the fluorescent microspheres can bind to the Aβ1-40 antigen. The amino acid sequence of the Aβ1-40 antigen is: DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV.

[0015] Preferably, the diameter of the magnetic beads is 3.0 μm, the diameter of the fluorescent microspheres is 190 nm, the reagent reaction background is greatly reduced, the detection sensitivity can reach 0.12 pg / mL, and the coefficient of variation is low, which is 4.2%.

[0016] 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. According to the classification of source, the capture antibody can be at least one of a mouse antibody, a rabbit antibody and a sheep antibody, preferably a mouse monoclonal antibody.

[0017] 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 background 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.

[0018] Further, the fluorescent microspheres are made of one or more of silicon dioxide, polyacrylamide or polystyrene, preferably polystyrene. Silicon dioxide is not easy to swell, and the dye cannot be wrapped therein, and the fluorescence intensity is unstable. 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.

[0019] Furthermore, the fluorescent microspheres were prepared by an internal dye embedding method, and the obtained fluorescent microspheres emitted more stable light.

[0020] Furthermore, the dye encapsulated in the fluorescent microspheres is fluorescein, quantum dots, rare earth elements, rare earth chelates, fluorescent proteins or up-conversion nanoparticles. The fluorescent microspheres using fluorescein as the dye have a strong fluorescence intensity and can be separated from the background signal, which is beneficial for signal collection in the final result analysis and can significantly improve the precision of the detection. The dye encapsulated in the fluorescent microspheres is preferably Alexa Fluor. 488, the sensitivity of the kit is better.

[0021] The present invention provides application of the reagent in preparing a product for detecting Aβ1-40 protein.

[0022] The present invention provides a kit for detecting Aβ1-40 protein, and the kit contains the reagent.

[0023] In a specific embodiment of the present invention, the kit may further contain PBS buffer.

[0024] In a specific embodiment of the present invention, the kit further contains quality control products and calibrators.

[0025] In a specific embodiment of the present invention, the calibrator is an Aβ1-40 antigen solution, and the concentration of the Aβ1-40 antigen solution is 25 to 200 pg / mL, preferably 25 and 200 pg / mL.

[0026] In a specific embodiment of the present invention, the quality control product is an Aβ1-40 antigen solution, and the concentration of the Aβ1-40 antigen solution is 50 pg / mL.

[0027] The present invention provides application of the kit in preparing a product for detecting Alzheimer's disease.

[0028] The present invention provides a method for preparing the reagent, wherein the preparation of magnetic beads coupled to capture antibodies comprises the following steps:

[0029] S11: activating the magnetic beads for 15 to 30 minutes to obtain activated magnetic beads;

[0030] S12: mixing the magnetic beads activated in step S12 with the Aβ1-40 protein capture antibody, and incubating for 1 to 3 hours to obtain a reaction product of the magnetic beads and the Aβ1-40 protein capture antibody;

[0031] S13: The magnetic beads of step S12 are mixed with the reaction product of the Aβ1-40 protein capture antibody and the blocking solution, and magnetic separation and blocking are performed at 50-80 rpm / min for 1-3 hours to obtain magnetic beads coupled with the capture antibody.

[0032] 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.

[0033] Preferably, the concentration of the magnetic beads is 0.25 mg / mL, the detection sensitivity can reach 0.12 pg / mL, the coefficient of variation is low, which is 4.2%, and a low production cost can be guaranteed.

[0034] Furthermore, in step S11, when the activation time of the magnetic beads is greater than 30 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 15 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 25 min.

[0035] 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.

[0036] Furthermore, in step S12, when the incubation time is greater than 3h, 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.

[0037] The present invention provides a method for preparing the reagent, wherein preparing a detection antibody labeled with fluorescent microspheres comprises the following steps:

[0038] S21: activating the fluorescent microspheres for 15 to 30 minutes to obtain activated fluorescent microspheres;

[0039] S22: mixing the fluorescent microspheres activated in step S21 with the Aβ1-40 protein detection antibody, and incubating for 1 to 3 hours to obtain a reaction product of the fluorescent microspheres and the Aβ1-40 protein detection antibody;

[0040] S32: Mix the fluorescent microspheres in step S22 with the reaction product of the Aβ1-40 protein detection antibody and the blocking solution, and block at 50-80 rpm / min for 1-3 hours to obtain the detection antibody labeled with the fluorescent microspheres.

[0041] 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.

[0042] Furthermore, in step S21, when the activation time of the fluorescent microspheres is greater than 30 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 15 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 fluorescent microspheres is 25 min.

[0043] Furthermore, in step S22, when the incubation time is greater than 3 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 3 hours.

[0044] Further, in step S22, the rotation speed is greater than 80rpm / 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 50rpm / 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 80rpm / min, which can make the magnetic beads (or fluorescent microspheres) evenly dispersed and fully combined with the antibody.

[0045] Furthermore, in step S23, the rotation speed is greater than 80 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 50 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 80 rpm / min, which can make the magnetic beads (or fluorescent microspheres) evenly dispersed and fully combined with the antibody.

[0046] Furthermore, the blocking solution contains at least one of bovine serum albumin, Tween 20 and Tris-HCl buffer.

[0047] The embodiments of the present invention have the following beneficial effects:

[0048] The biomarker Aβ1-40 detection kit of the present invention can quickly detect Aβ1-40 in serum samples with high sensitivity and good repeatability. In some embodiments, the sensitivity can reach 0.12pg / mL, and the discrete degree CV value of the data can reach 3.2%. The present invention can use serum as a sample for detection, and the sample source is simple and easy to obtain, and the detection can be performed without live puncture. The finished kit is ready to use, and the use process is simple and convenient, and the results can be detected in 1 hour.

[0049] The single molecule immunoassay technology in the present invention directly signals the substance of the molecule to be tested, and achieves quantitative detection at the single molecule level by counting the number of single molecule signals and correcting them using a standard curve. The detection limit of this method can reach fg / mL, and it has obvious advantages in detecting low-abundance protein targets, and can measure targets in various matrices (serum, plasma, cerebrospinal fluid, urine, cell extracts, etc.).

[0050] The magnetic beads of the present invention are distributed in a porous plate, and the porous distribution can be calibrated with better precision; the microfluidic chip uses less material and saves cost. The present invention extracts magnetic bead signals from a single hole therein, lays them flat on a plane for signal counting, and calculates the number of real signals according to the ratio. 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 Aβ1-40 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 Aβ1-40 content in the serum of Alzheimer's 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] Example 1 Aβ1-40 single molecule immunoassay kit

[0056] 1. Preparation method

[0057] 1. Experimental components:

[0058] (1) Carboxyl fluorescent microspheres (manufacturer: Bangs, catalog number: FCDG003), carboxyl magnetic beads (manufacturer: JSR, catalog number: MS300 / Carboxyl), Aβ1-40 capture antibody (manufacturer: abcam, catalog number: ab20068), Aβ1-40 detection antibody (manufacturer: Merck, catalog number: MABN878).

[0059] (2) Other reagents: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS);

[0060] Microsphere labeling solution: BSA (bovine serum albumin) was dissolved in PBS buffer (phosphate buffer, 20 mM, pH = 7.4), and the final concentration of BSA was 0.1% (v / v);

[0061] Microsphere preservation solution: NaCl, BSA, sucrose, betaine and protein stabilizer StabilCoat Plus Microarray Stabilizer (manufacturer: Surmodics, catalog number: SC02-1000) were dissolved in PBS buffer (50 mM, pH = 7.4), with a final concentration of NaCl of 0.9% (w / v), a final concentration of BSA of 0.2% (v / v), a final concentration of sucrose of 0.5% (w / v), a final concentration of betaine of 0.2% (w / v), and a final concentration of StabilCoat Plus Microarray Stabilizer of 1% (v / v);

[0062] Blocking solution: BSA and Tween-20 were dissolved in Tris-HCl buffer (20 mM, pH = 8.2), with a final concentration of BSA of 5% (v / v) and a final concentration of Tween-20 of 0.2% (v / v);

[0063] Magnetic bead washing solution: PBST solution (phosphate-tween buffer);

[0064] Magnetic bead coupling solution: PB buffer (phosphate buffer, 0.1 M, pH = 7.4);

[0065] Magnetic bead storage solution: NaCl and PVP were dissolved in PBS buffer (50 mM, pH = 7.4), with a final concentration of NaCl of 0.9% (w / v) and a final concentration of PVP of 0.2% (w / v);

[0066] Calibrator diluent or quality control diluent: Dissolve BSA, casein, trehalose, NaCl, betaine, mannitol, Tween-20 and antibacterial agent Proclin 300 (manufacturer: Sigma-Aldrich, catalog number: 48914-U) in PBS buffer (50mM, pH=7.4), filter with a 0.22μm 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), the antibacterial agent Proclin 300 The final concentration was 0.1% (v / v).

[0067] 2. Preparation of reagent R1: coupling of capture antibody and magnetic beads

[0068] (1) Take 100 μL of 3 mg / mL carboxyl magnetic beads, wash them four times with 900 μL of magnetic bead washing solution, and resuspend them in 900 μL of magnetic bead coupling solution to obtain a magnetic bead resuspension solution.

[0069] (2) Weigh a certain amount of EDC and dissolve it in 50 mM 2-(N-morpholino)ethanesulfonic acid (MES) solution (pH 6.0) to prepare an EDC solution with a concentration of 20 mg / mL. Take 10 μL of the EDC solution and add it to the magnetic bead resuspension prepared in step (1). Under the condition of 25°C, activate it on a roller incubator at 80 rpm / min for 25 min to obtain an EDC-activated magnetic bead solution.

[0070] (3) Dilute hemocyanin to 25 mg / mL with PB buffer (phosphate buffer, 0.1 M, pH = 7.4) to obtain a hemocyanin dilution solution; take 20 μL of the hemocyanin dilution solution and 0.04 mg of Aβ1-40 capture antibody, add them to the EDC-activated magnetic bead solution prepared in step (2) (the final concentration of hemocyanin is 0.05% w / v), mix well, and incubate at 25°C and 80 rpm / min on a roller incubator for 2 h.

[0071] (4) Wash the capture antibody and magnetic beads treated in step (3) with 200 μL of blocking solution, mix well on a vortexer, place on a magnetic stand, perform magnetic separation, and wash three times.

[0072] (5) Add 200 μL of blocking solution to the capture antibody and magnetic beads treated in step (4), incubate on a roller incubator at 80 rpm / min at 25°C for 1 h, perform magnetic separation, remove the supernatant, and obtain magnetic beads coupled with capture antibodies.

[0073] (6) Add 1200 μL of magnetic bead storage solution to the magnetic beads coupled to the capture antibody in step (5) and store at 4° C. to obtain reagent R1. The final concentration of the magnetic beads is 0.25 mg / mL.

[0074] 3. Preparation of reagent R2: coupling of detection antibody and fluorescent microspheres

[0075] (1) Take 100 μL of 10 mg / mL carboxyl fluorescent microspheres, add 900 μL of microsphere labeling solution, and centrifuge at 12,000 rpm for 15 min to wash three times.

[0076] (2) Weigh a certain amount of EDC and NHS, dissolve them in 50mM pH 6.0 2-(N-morpholino)ethanesulfonic acid (MES) solution to prepare a 20mg / mL EDC solution and a 20mg / mL NHS solution, take 15μL of EDC solution and 15μL of NHS solution and add them to the fluorescent microspheres treated in step (1). Under 37°C, react on a roller incubator at 80rpm / min for 25min, centrifuge at 12000rpm for 15min, remove the supernatant, and resuspend with 300μL of microsphere labeling solution to obtain a resuspension of fluorescent microspheres reacted with EDC and NHS.

[0077] (3) Take 0.05 mg of Aβ1-40 detection antibody and add it to the fluorescent microsphere resuspension reacted with EDC and NHS in step (2), mix well, and incubate at 37°C and 80 rpm / min on a roller incubator for 3 h.

[0078] (4) Add 200 μL of blocking solution to the fluorescent microspheres containing the detection antibody treated in step (3), and incubate at 37° C. and 80 rpm / min on a roller incubator for 1 h.

[0079] (5) Centrifuge the fluorescent microspheres with the detection antibody blocked in step (4) at 12000 rpm for 15 min, remove the supernatant, obtain the detection antibody labeled with the fluorescent microspheres, add 1000 μL of microsphere preservation solution, and store at 4°C to obtain reagent R2, with the final concentration of the fluorescent microspheres being 1 mg / mL.

[0080] 4. Preparation of calibrators and quality control products

[0081] Aβ1-40 antigen (manufacturer: abcam, catalog number ab120992) was diluted to 25 pg / mL and 200 pg / mL with calibrator diluent as calibrator; Aβ1-40 antigen was diluted to 50 pg / mL with quality control diluent as quality control.

[0082] 2. Composition

[0083] Reagent R1, reagent R2, PBS buffer (20 mM, pH=7.4), calibrators and quality controls.

[0084] 3. Usage

[0085] 1. Add 50 μL of reagent R1 into a reaction cup, place it on a magnetic separation rack, wash it three times with 20 mM PBS buffer and resuspend it to obtain the reagent R1 resuspension.

[0086] 2. Take 50 μL of the serum to be tested and add it to the resuspension of reagent R1. Incubate on a roller incubator at 25°C for 30 minutes.

[0087] 3. On the magnetic separation stand, add 20 mM PBS buffer to the serum treated in step 2, wash three times and resuspend, add 50 μL reagent R2, mix thoroughly with a mixer, and incubate on a roller incubator at 25°C for 30 min.

[0088] 4. On the magnetic separation stand, add 20mM PBS buffer to the serum treated in step 3, wash three times, and resuspend to obtain the test solution. The test solution in step 4 is transferred to the 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 Aβ1-40 concentration in the serum.

[0089] Example 2

[0090] The diameter of the carboxyl magnetic beads was set to 2.7 μm (manufacturer: Agilent Technologies, item number: Lode Stars 2.7 Carboxyl), the final concentration of the magnetic beads was set to 0.25 mg / mL, and the other preparation methods were the same as in Example 1, to obtain Kit 2.

[0091] Example 3

[0092] The diameter of the carboxyl magnetic beads was set to 1.6 μm (manufacturer: JSR, item number: MS160 / Carboxyl), the final concentration of the magnetic beads was set to 0.25 mg / mL, and the other preparation methods were the same as in Example 1, to obtain Kit 3.

[0093] Example 4

[0094] The diameter of the carboxyl magnetic beads was set to 1 μm (manufacturer: Weidu Biotechnology, product number: CMP1001CA), and the final concentration of the magnetic beads was set to 0.25 mg / mL. The other preparation methods were the same as in Example 1, and kit 4 was obtained.

[0095] Example 5

[0096] The diameter of the carboxyl magnetic beads was set to 3 μm (manufacturer: JSR, item number: MS300 / Carboxyl), the final concentration of the magnetic beads was set to 0.5 mg / mL, and the other preparation methods were the same as in Example 1, to obtain kit 5.

[0097] Example 6

[0098] The diameter of the carboxyl magnetic beads was set to 3 μm (manufacturer: JSR, item number: MS300 / Carboxyl), the final concentration of the magnetic beads was set to 0.75 mg / mL, and the other preparation methods were the same as in Example 1, to obtain kit 6.

[0099] Example 7

[0100] The diameter of the carboxyl magnetic beads was set to 3 μm (manufacturer: JSR, item number: MS300 / Carboxyl), the final concentration of the magnetic beads was set to 1.0 mg / mL, and the other preparation methods were the same as in Example 1, to obtain kit 7.

[0101] Example 8

[0102] The diameter of the fluorescent microspheres is 200 nm (manufacturer: Weidu Biological, item number: FG0200CA), the final concentration of the magnetic beads is set to 0.25 mg / mL, and the other preparation methods are the same as in Example 1, to obtain kit 8.

[0103] Example 9

[0104] The diameter of the fluorescent microspheres is 400 nm (manufacturer: Bangs, item number: FCDG004), the final concentration of the magnetic beads is set to 0.25 mg / mL, and the other preparation methods are the same as in Example 1, to obtain Kit 9.

[0105] Example 10 Test kit detection range

[0106] 1. Dilute Aβ1-40 antigen (manufacturer: abcam, catalog number ab120992) with calibrator diluent to concentrations of 0, 0.05, 0.1, 0.5, 1, 5, 10, 25, 50, 100, 200 and 400 pg / mL respectively.

[0107] 2. Take 50 μL of each concentration of antigen dilution into different reaction cups, use the kit of Example 1 to perform detection, test each concentration 5 times, and draw a standard curve.

[0108] like Figure 1 As shown, the detection range of the Aβ1-40 standard curve is 0.05 pg / mL to 400 pg / mL, and within this range, the number of single molecule signals has a good linear relationship with the sample concentration.

[0109] Example 11 Sensitivity and coefficient of variation (CV value) of different kits

[0110] The antigen dilution of Example 10 was detected by the kits of Examples 1 to 9 respectively, and the sensitivity and coefficient of variation (CV value) of the kit were determined. The results are shown in Table 1. The CV value mainly reflects the degree of dispersion of the data, and the smaller the better. It can be seen that the sensitivity of the kit of Example 1 is the best, the CV is smaller, and the comprehensive detection effect is better.

[0111] Table 1

[0112]

[0113] Example 12 Actual sample detection

[0114] The kit of Example 1 was used to detect sera from 5 healthy subjects and sera from 5 patients with Alzheimer's disease.

[0115] like Figure 2As shown, the Aβ1-40 content in the serum of Alzheimer's patients is significantly higher than that in the serum of healthy people. The kit of Example 1 of the present invention can effectively distinguish Alzheimer's patients from healthy people.

[0116] Example 13 Effect of different magnetic bead coupling solutions

[0117] 1. Experimental Methods

[0118] 1. The magnetic bead coupling liquids were set to Tris-HCl buffer (pH: 7.0, 8.0 and 9.0, respectively), MES buffer (pH: 5.5 and 6.5, respectively), PB buffer (pH: 6.0, 7.0 and 8.0, respectively), boric acid borax buffer (pH: 7.4, 8.0 and 9.0, respectively), MOPS buffer (3-morpholinepropanesulfonic acid, pH: 6.5 and 7.4, respectively) and HEPES buffer (4-hydroxyethylpiperazineethanesulfonic acid, pH: 7.0 and 8.0, respectively), all with a concentration of 50 mM, and the antigen dilution was detected according to the kit of Example 1.

[0119] 2. The magnetic bead coupling solutions were set to be: Tris-HCl buffer, PB buffer, borate borax buffer, MOPS buffer and HEPES buffer at pH 7.4, all at a concentration of 50 mM, and the antigen dilution was detected according to the kit of Example 1.

[0120] 3. The magnetic bead coupling solutions were set to PB buffer solutions with concentrations of 20 mM, 50 mM and 0.1 M, with a pH of 7.4. The antigen dilution solution was detected according to the kit of Example 1.

[0121] 2. Experimental Results

[0122] The PB buffer with a concentration of 0.1 M and a pH of 7.4 had the best coupling effect.

[0123] 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 Aβ1-40 protein, characterized in that: The reagent contains magnetic beads coupled with capture antibodies and / or detection antibodies labeled with fluorescent microspheres; the magnetic beads coupled with capture antibodies contain Aβ1-40 protein capture antibodies and magnetic beads, and the diameter of the magnetic beads is 1.0 to 3.0 μm; the detection antibodies labeled with fluorescent microspheres contain Aβ1-40 protein detection antibodies and fluorescent microspheres, and the diameter of the fluorescent microspheres is 190 to 400 nm.

2. Use of the reagent according to claim 1 in the preparation of a product for detecting Aβ1-40 protein.

3. A kit for detecting Aβ1-40 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 PBS 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 coupled to 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 S12 with the Aβ1-40 protein capture antibody, and incubating for 1 to 3 hours to obtain a reaction product of the magnetic beads and the Aβ1-40 protein capture antibody; S13: The magnetic beads of step S12 are mixed with the reaction product of the Aβ1-40 protein capture antibody and the blocking solution, and magnetic separation and blocking are performed at 50-80 rpm / min for 1-3 hours to obtain magnetic beads coupled 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 Aβ1-40 protein detection antibody, and incubating for 1 to 3 hours to obtain a reaction product of the fluorescent microspheres and the Aβ1-40 protein detection antibody; S32: Mix the fluorescent microspheres in step S22 with the reaction product of the Aβ1-40 protein detection antibody and the blocking solution, and block at 50-80 rpm / min for 1-3 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 bovine serum albumin, Tween 20 and Tris-HCl buffer.

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