Kit for auxiliary diagnosis of Alzheimer's disease and preparation method and application thereof

Abstract

The invention discloses a kit for auxiliary diagnosis of Alzheimer's disease and a preparation method and application thereof, and relates to the field of medical detection. The kit for auxiliary diagnosis of the Alzheimer's disease comprises a magnetic particle solution coated with a capture antibody, an enzyme-labeled antibody compound, a cleaning solution and a luminous substrate, the magnetic particle solution coated with the capture antibody comprises magnetic particles connected with a first antibody, and the first antibody is specifically combined with a biomarker; the enzyme-labeled antibody compound comprises a second antibody coupled with an enzyme, and the second antibody and the first antibody are specifically combined with the same biomarker; the magnetic particles comprise carboxyl magnetic beads, the carboxyl magnetic beads are coated with Protein A, and the Protein A is connected with a first antibody. By optimizing the coupling process of the antibody, the problems of poor biomarker detection sensitivity and low efficiency of the traditional detection technology are solved, and redundant inspection items for early diagnosis of the Alzheimer's disease and other neurodegenerative diseases are reduced.

Description

Technical Field

[0001] The present invention relates to the field of medical detection, and in particular to a kit for assisting in the diagnosis of Alzheimer's disease, and a preparation method and application thereof. Background Art

[0002] In the field of neurodegenerative disease research and diagnosis, Alzheimer's disease is the most common type of dementia, and its early identification is crucial for effective intervention. Traditionally, the diagnosis of Alzheimer's disease (AD) relies on clinical evaluation, biomarker changes in cerebrospinal fluid (CSF) analysis, and expensive positron emission tomography (PET) imaging technology. However, these methods are either invasive or costly, limiting their application in large-scale screening and routine clinical practice.

[0003] In recent years, with the in-depth study of protein biomarkers, especially the phosphorylated forms of Tau protein, such as Tau217, NfL, Tau181, Aβ1-40, Aβ1-42, Tau231, GFAP and other biomarkers, its key role in the disease process has been revealed. Although the concentration of biomarkers in blood is much lower than that in cerebrospinal fluid, its potential as an early biomarker of AD has attracted widespread attention. Early studies have shown that biomarkers in plasma can distinguish AD patients from healthy controls with high accuracy, and even detect abnormal changes before cognitive function declines.

[0004] The technical challenge is to develop a sufficiently sensitive detection platform to capture tiny changes in biomarker concentrations in the blood. Traditional detection methods may not be able to achieve the necessary sensitivity, but with the advancement of single molecule analysis technology, such as single molecule array (Simoa) technology, quantification of biomarkers in the blood has become possible, providing a new direction for non-invasive AD diagnosis.

[0005] However, despite significant progress, the existing technology still needs to be further optimized to improve the specificity and repeatability of the test, while reducing costs to ensure its widespread application in clinical practice. Therefore, the development of more efficient, sensitive and economical AD blood testing technology is crucial to promote the early diagnosis and treatment strategies of Alzheimer's disease and other neurodegenerative diseases. Summary of the invention

[0006] The technical problem to be solved by the present invention is that since the content of biomarkers such as Tau217, NfL, Tau181, Aβ1-40, Aβ1-42, Tau231, GFAP in the blood is low, and the existing technology has low detection sensitivity, poor efficiency and is not economical enough for biomarkers, the purpose is to provide a kit for auxiliary diagnosis of Alzheimer's disease and its preparation method and application, which solves the problem that the existing technology has low detection sensitivity, poor efficiency and is not economical enough for AD.

[0007] The present invention is achieved through the following technical solutions: In a first aspect, the present invention provides a kit for assisting in the diagnosis of Alzheimer's disease, comprising a capture antibody-coated magnetic particle solution, an enzyme-labeled antibody complex, a cleaning solution, and a luminescent substrate. wherein the capture antibody-coated magnetic particle solution includes magnetic particles linked to a first antibody, the first antibody specifically binding to a Tau217, NfL, Tau181, Aβ1-40, Aβ1-42, Tau231 or GFAP biomarker; The enzyme-labeled antibody complex includes a second antibody coupled to an enzyme, and the second antibody and the first antibody specifically bind to the same biomarker; The magnetic particles include carboxyl magnetic beads, which are coated with Protein A, and the Protein A is linked to a first antibody.

[0008] As a possible design, the capture antibody-coated magnetic particle solution further includes a first buffer, which is a Tris buffer, and is used to maintain the solution at a specific pH value, which is about 7-9.

[0009] As a possible design, the enzyme-labeled antibody complex includes a second buffer, which includes a 0.1 M MgCl2 buffer and a 0.01 M ZnCl2 buffer. The second buffer is used to maintain the stability of the solution.

[0010] Preferably, the capture antibody-coated magnetic particle solution comprises the following raw materials: carboxyl magnetic beads, protein A, monoclonal primary antibody, 1% bovine serum albumin (BSA), 0.1M Tris, PC300 (preservative ProClin300), tween20 (polyoxyethylene sorbitan monolaurate) and PC950 (preservative ProClin950).

[0011] In the capture antibody-coated magnetic microparticle solution, Protein A is a protein that can specifically bind to the Fc fragment of most immunoglobulin G (IgG) class antibodies. Here it acts as a bridge, allowing the antibody to indirectly bind to the magnetic beads.

[0012] Preferably, the capture antibody-coated magnetic particle solution is prepared by the following method: 1. Activation of carboxyl magnetic beads: 10 mg / ml EDC and 20 mg / ml N-hydroxysuccinimide (NHS) were used to activate carboxyl magnetic beads (concentration 1.0 mg / ml) at room temperature for 30 min, where the mass ratio of EDC, NHS and magnetic beads was 1:2:5; 2. Carboxyl magnetic beads coated with protein A: At room temperature, mix the activated carboxyl magnetic beads and protein A, add 20 μg protein A per mg of magnetic beads, react for 1 hour, and obtain protein A magnetic beads; 3. Protein A magnetic beads linked to monoclonal primary antibodies: add 20 μg of biomarker (Tau217, NfL, Tau181, Aβ1-40, Aβ1-42, Tau231 or GFAP) monoclonal primary antibodies per mg of protein A magnetic beads and react at room temperature for 1 hour; 4. Magnetic bead blocking: Add 20% BSA to the antibody-coated magnetic beads, add 50 μl 20% BSA per mg of magnetic beads, mix well at room temperature, and incubate for 1 hour; 5. Wash the magnetic beads and adjust the volume. Washing can be done with buffer.

[0013] Preferably, the enzyme-labeled antibody complex comprises the following raw materials: biomarker monoclonal secondary antibody, alkaline phosphatase (ALP), 1% bovine serum albumin, 0.1M Tris, PC300, tween20, PC950, 0.1M MgCl2 buffer and 0.01M ZnCl2 buffer.

[0014] Preferably, the enzyme-labeled antibody complex is prepared by the following steps: 1. ALP activation: add 30 μL of 50 mM Sulfo-SMCC to 10 mg ALP (10 mg / mL) solution, mix well at pH 7.4 and room temperature, react for 30 min, and dialyze overnight; 2. Activated ALP marker biomarker monoclonal antibody Secondary antibody: (1) Add 2.4 μL 2-IT (2-iminothiolane, 50 mM) to 0.2 mg Tau217 monoclonal secondary antibody, mix and react for 30 min at room temperature at pH 8.5, and dialyze overnight. (2) 0.2 mg of 2-IT-conjugated biomarker monoclonal antibody 2 was added to 48 μl of activated ALP, and the reaction was carried out at pH 7.4 and room temperature for 2 hours; 3. Dilute the enzyme working solution to a fixed volume.

[0015] As a possible design, the first antibody is a monoclonal antibody.

[0016] As a possible design, the second antibody is a monoclonal antibody.

[0017] As a possible design, the magnetic particles include carboxyl magnetic beads.

[0018] As a possible design, the enzyme coupled to the second antibody includes alkaline phosphatase.

[0019] As a possible design, the luminescent substrate includes an APS-5 substrate.

[0020] Preferably, the cleaning solution comprises the following raw materials in weight percentage: 1.8% NaCl, 0.1% ProClin 950, 0.05% Tween 20, and the balance is 50 mM tris.

[0021] In a second aspect, the present invention provides a method for preparing a kit for detecting Tau217, comprising the following steps: Preparing a capture antibody-coated magnetic microparticle solution: activating magnetic beads, and then connecting with protein A to obtain protein A magnetic beads; combining the protein A magnetic beads with a first antibody specifically binding to Tau217, NfL, Tau181, Aβ1-40, Aβ1-42, Tau231 or GFAP biomarkers, and then blocking to obtain a capture antibody-coated magnetic microparticle solution; Preparation of enzyme-labeled antibody complex: coupling the enzyme with cross-linker 1 for activation; coupling the second antibody that specifically binds to the same biomarker as the first antibody with cross-linker 2 to obtain a labeled second antibody; coupling the enzyme-coupled cross-linker 1 with the labeled second antibody to obtain an enzyme-labeled antibody complex, wherein the cross-linker 1 is Sulfo-SMCC and the cross-linker 2 is 2-IT.

[0022] Preferably, the above activation is specifically to activate the magnetic beads by EDC (water-soluble carbodiimide) and NHS (N-hydroxysuccinimide). Two times the amount of NHS is added to the magnetic beads, and in the presence of NHS, EDC is added, and the carboxyl groups of EDC and the magnetic beads react to form active esters that are more easily reactive with the amino groups of proteins, so that the proteins can be more stably connected to the magnetic beads.

[0023] Preferably, the blocking is specifically done with 20% BSA. Blocking after connecting with Protein A can block the remaining active groups on the surface of the magnetic beads that are not occupied by antibodies, reduce nonspecific binding, and improve the specificity and sensitivity of the detection.

[0024] Preferably, the crosslinking agent 1 is Sulfo-SMCC, and the crosslinking agent 2 is 2-IT.

[0025] Sulfo-SMCC (Sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) is a water-soluble bifunctional crosslinker. It contains a sulfonated N-hydroxysuccinimide (NHS) ester at one end, which can react rapidly with primary amine groups in proteins or other molecules at physiological pH to form stable amide bonds. The other end has a maleimide group, which reacts specifically with sulfhydryl (-SH) groups in proteins or peptides at similar pH values ​​to form thioether bonds. 2-IT is 2-iminothiolane.

[0026] As a possible design, zinc chloride and magnesium chloride are also added to the above enzyme-labeled antibody complex.

[0027] The addition of zinc chloride and magnesium chloride can increase ALP activity and enhance the structural stability of the enzyme.

[0028] In a third aspect, the present invention provides a kit for assisting in the diagnosis of Alzheimer's disease, wherein the kit is used in the preparation of a detection product for detecting Alzheimer's disease, and the method comprises the following steps: Add the sample to be tested into the reaction cup, add the magnetic particle solution coated with the capture antibody, and then add the enzyme-labeled antibody complex. After reaction and adsorption, separate the waste liquid, wash with cleaning solution, add the luminescent substrate, incubate, and read the readings through the instrument.

[0029] Preferably, the above application comprises the following steps: Add 50ul of the sample to be tested to the reaction cup, add 30ul of the capture antibody-coated magnetic microparticle solution, add 100ul of the enzyme-labeled antibody complex, react at 37℃ for 15min, discard the waste liquid after adsorption, add 300ul of the cleaning solution, discard the waste liquid after cleaning (repeat the cleaning 3 times), add 200ul of APS-5 luminescent substrate, incubate for 5min, and then measure the readings on the instrument.

[0030] Compared with the prior art, the present invention has the following advantages and beneficial effects: The present invention solves the problems of poor sensitivity and low efficiency of traditional detection technology for Tau217, NfL, Tau181, Aβ1-40, Aβ1-42, Tau231 or GFAP biomarkers by optimizing the antibody coupling process, reduces redundant examination items for early diagnosis of Alzheimer's disease and other neurodegenerative diseases, reduces comprehensive detection costs, and improves economic value.

[0031] In addition, the present invention coats activated magnetic beads with Protein A, combines Protein A with the Fc end of the monoclonal antibody, increases the chain length of the linker, and reduces steric hindrance; and Protein A can purify the target linker protein, and other impurities in the raw material can be washed away by the adsorption of the magnetic beads. At the same time, one Protein A can combine with at least two IgG molecules to improve the labeling sensitivity; Protein A is connected to the Fc end of the monoclonal antibody, which can make the Fab end of the monoclonal antibody face the outside in a specific direction, and more efficiently combine with the antigen to be detected in the sample, thereby improving the detection sensitivity. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with Examples. The illustrative embodiments of the present invention and their description are only used to explain the present invention and are not intended to limit the present invention. If specific conditions are not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0033] The "range" disclosed in the present invention is defined in the form of a lower limit and an upper limit, and a given range is defined by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundaries of the particular range. The range defined in this way can be inclusive or exclusive of the end values, and can be arbitrarily combined, that is, any lower limit can be combined with any upper limit to form a range. For example, if a range of 60 to 120 and 80 to 110 is listed for a particular parameter, it is understood that a range of 60 to 110 and 80 to 120 is also expected. In addition, if the minimum range values ​​1 and 2 are listed, and if the maximum range values ​​3, 4 and 5 are listed, the following ranges can all be expected: 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4 and 2 to 5. In the present invention, unless otherwise specified, the numerical range "a to b" represents an abbreviation of any real number combination between a and b, wherein a and b are both real numbers. For example, the numerical range "0-5" means that all real numbers between "0-5" are listed in this document, and "0-5" is just an abbreviation of these numerical combinations. In addition, when a parameter is expressed as an integer ≥ 2, it is equivalent to disclosing that the parameter is, for example, an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0034] If not otherwise specified, all embodiments and optional embodiments of the present invention can be combined with each other to form a new technical solution. If not otherwise specified, all technical features and optional technical features of the present invention can be combined with each other to form a new technical solution. If not otherwise specified, all steps of the present invention can be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), which means that the method may include steps (a) and (b) performed sequentially, or may include steps (b) and (a) performed sequentially. For example, it is mentioned that the method may also include step (c), which means that step (c) can be added to the method in any order. For example, the method may include steps (a), (b) and (c), or may include steps (a), (c) and (b), or may include steps (c), (a) and (b), etc.

[0035] If there is no special explanation, the "include" and "comprising" mentioned in the present invention represent open-ended or closed-ended expressions. For example, the "include" and "comprising" may represent that other components not listed may also be included or only the listed components may be included or only the listed components may be included.

[0036] If not specifically stated, in the present invention, the term "or" is inclusive. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, any of the following conditions satisfies the condition "A or B": A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0037] The magnetic beads used in this embodiment and the comparative example were purchased from JSR manufacturer, micron-grade MS300 carboxyl magnetic beads.

[0038] Example 1

[0039] A kit for assisting in the diagnosis of Alzheimer's disease, including: Capture antibody-coated magnetic microparticle solution: a carboxyl magnetic bead solution coated with a Tau217 antibody, wherein the Tau217 antibody is a Tau217 monoclonal antibody specific to the Tau217 antigen in the sample; Enzyme-labeled antibody complex: Tau217 monoclonal antibody solution specific to Tau217 antigen in the sample coupled with alkaline phosphatase; Washing solution: contains 50mM tris, 1.8% NaCl, 0.1% ProClin 950, 0.05% Tween 20; Luminescent substrate: APS-5, source from Hanhai New Enzyme.

[0040] The concentrations of Tau217 in the tested samples were 0 ng / mL, 0.6 ng / mL, 1.5 ng / mL, 3 ng / mL, 10 ng / mL, and 20 ng / mL, respectively.

[0041] Preparation of capture antibody-coated magnetic microparticle solution: 1. Activation of carboxyl magnetic beads: Activate carboxyl magnetic beads (concentration 1.0 mg / ml) at room temperature for 30 min using 10 mg / ml EDC and 20 mg / ml NHS. The mass ratio of EDC, NHS and magnetic beads is 1:2:5. 2. Carboxyl magnetic beads coated with protein A: At room temperature, mix the activated carboxyl magnetic beads and protein A, add 20 μg protein A per mg of magnetic beads, and react for 1 hour; 3. Protein A magnetic beads linked to Tau217 monoclonal primary antibody: add 20 μg Tau217 monoclonal primary antibody per mg of protein A magnetic beads and react at room temperature for 1 hour; 4. Magnetic bead blocking: Add 20% BSA to the antibody-coated magnetic beads, add 50 μl 20% BSA per mg of magnetic beads, mix well at room temperature, and incubate for 1 hour; 5. Wash the magnetic beads and adjust the volume.

[0042] Preparation of enzyme-labeled antibody complex: 1. ALP activation: add 30 μL of 50 mM Sulfo-SMCC to 10 mg ALP (10 mg / mL) solution, mix well at pH 7.4 and room temperature, react for 30 min, and dialyze overnight; 2. Activated ALP labeled Tau217 monoclonal antibody 2: (1) Add 2.4 μL 2-IT (50 mM) to 0.2 mg Tau217 monoclonal secondary antibody, mix and react for 30 min at room temperature at pH 8.5, and dialyze overnight; (2) 0.2 mg of 2-IT-conjugated Tau217 monoclonal secondary antibody was added to 48 μl of activated ALP and reacted at pH 7.4 and room temperature for 2 hours; 3. Dilute the enzyme working solution to a fixed volume to obtain the enzyme-labeled antibody complex.

[0043] Comparative Example 1 This comparative example is basically the same as Example 1, except that: the magnetic beads are carboxyl magnetic beads coated with antibodies under the existing process; the enzyme-labeled antibody complex, the cleaning solution and the luminescent substrate are the same as those in Example 1.

[0044] A kit for assisting in the diagnosis of Alzheimer's disease, including: Capture antibody-coated magnetic microparticle solution: antibody-coated carboxyl magnetic beads under current process; Enzyme-labeled antibody complex: Tau217 monoclonal antibody solution specific to Tau217 antigen in the sample coupled with alkaline phosphatase; Washing solution: contains 50mM tris, 1.8% NaCl, 0.1% ProClin 950, 0.05% Tween 20; Luminescent substrate: APS-5, source from Hanhai New Enzyme.

[0045] The concentrations of Tau217 in the tested samples were 0 ng / mL, 0.6 ng / mL, 1.5 ng / mL, 3 ng / mL, 10 ng / mL, and 20 ng / mL, respectively.

[0046] Preparation of capture antibody-coated magnetic microparticle solution: 1. Activation of carboxyl magnetic beads: 10 mg / m EDC and 20 mg / ml NHS were used to activate carboxyl magnetic beads (concentration 1.0 mg / ml) at room temperature for 30 min, with the mass ratio of EDC, NHS and magnetic beads being 1:2:5; 2. Carboxyl magnetic beads coated with Tau217 monoclonal primary antibody: 1 magnetic beads plus 20 μg Tau217 monoclonal primary antibody, react at room temperature for 1 hour; 3. Magnetic bead blocking: add 20% BSA to the magnetic beads coated with antibodies, add 50 μl 20% BSA to 1 volume of magnetic beads, mix well at room temperature, and incubate for 1 hour; 4. Wash the magnetic beads and adjust the volume.

[0047] Preparation of enzyme-labeled antibody complex: 1. ALP activation: add 30 μL of 50 mM Sulfo-SMCC to 10 mg ALP (10 mg / mL) solution, mix well at pH 7.4 and room temperature, react for 30 min, and dialyze overnight; 2. Activated ALP labeled Tau217 monoclonal antibody 2: (1) Add 2.4 μL 2-IT (50 mM) to 0.2 mg Tau217 monoclonal secondary antibody, mix and react for 30 min at room temperature at pH 8.5, and dialyze overnight; (2) 0.2 mg of 2-IT-conjugated Tau217 monoclonal secondary antibody was added to 48 μl of activated ALP and reacted at pH 7.4 and room temperature for 2 hours; 3. Dilute the enzyme working solution to a fixed volume to obtain the enzyme-labeled antibody complex.

[0048] The Alzheimer's disease project was tested in Example 1 and Comparative Example 1 respectively, and blood samples of Alzheimer's disease patients were obtained. The blood samples were processed and then tested with a kit, and the data were recorded.

[0049] Testing process: Operate on the fully automatic chemiluminescence immunoassay analyzer, add 50uL of the plasma sample to be tested into the reaction cup with the sample aspirator, and after cleaning the sample aspirator, draw 30ul of immunomagnetic beads coated with the Tau217 monoclonal primary antibody and 100ul of enzyme working solution labeled with the Tau217 monoclonal secondary antibody into the reaction cup, react at 37 degrees for 15min, then discard the waste liquid after the reactant is adsorbed, add 300ul of cleaning solution, and discard the waste liquid after cleaning (repeat cleaning 3 times), finally add 200ul of APS-5 to the reaction cup, incubate for 2min, and then detect the reading on the instrument, substitute the luminescence value into the standard curve of the kit, and the instrument automatically fits and calculates the concentration of the test object.

[0050] The data analysis results are as follows: The kits of Example 1 and Comparative Example 1 were used to obtain Tau217 calibration curves, respectively. The results are shown in Table 1.

[0051] Table 1 Tau217 calibration curve Tau217 concentration (ng / ml) Comparative Example 1 (RLU) Front-to-back ratio Example 1 (RLU) Front-to-back ratio 0 2464 - 2944 - 0.6 41865 16.99 65297 22.18 1.5 101475 2.42 153575 2.35 3 218577 2.15 356534 2.32 10 624385 2.86 1075764 3.02 20 1085699 1.74 1974574 1.84 The kits of Example 1 and Comparative Example 1 were used to detect different Tau217 clinical samples, respectively, to obtain Tau217 concentration detection results, as shown in Table 2. Wherein, N represents a clinically judged negative sample, and P represents a clinically judged positive sample.

[0052] Table 2 Clinical samples (Tau217) sample Comparative Example 1 (pg / ml) Example 1 (pg / ml) N1 0.30 0.14 N2 0.25 0.21 N3 0.48 0.25 N4 1.39 0.48 N5 2.77 0.61 N6 0.34 0.19 N7 0.30 0.21 N8 0.38 0.23 N9 0.31 0.15 N10 1.92 0.72 N11 1.16 0.53 N12 1.28 0.45 P1 2.75 2.91 P2 0.94 1.82 P3 4.32 3.26 P4 1.46 1.94 P5 0.93 1.32 P6 1.16 2.01 P7 1.28 1.96 P8 9.02 10.45 Reference interval: <1.8 pg / mL According to Tables 1 and 2, compared with Comparative Example 1, the sensitivity of the reagent in Example 1 is effectively improved and the test results of the samples are more consistent with the clinical positive and negative judgment.

[0053] Example 2 A kit for assisting in the diagnosis of Alzheimer's disease, including: Capture antibody-coated magnetic microparticle solution: NfL antibody-coated carboxyl magnetic bead solution, where the NfL antibody is a monoclonal antibody specific to the NfL antigen in the sample; Enzyme-labeled antibody complex: a solution of NfL monoclonal antibodies specific to the NfL antigen in the sample coupled with alkaline phosphatase; Washing solution: contains 50mM tris, 1.8% NaCl, 0.1% ProClin 950, 0.05% Tween 20; Luminescent substrate: APS-5, source from Hanhai New Enzyme.

[0054] The NfL concentrations in the tested samples were 0 ng / mL, 15 ng / mL, 50 ng / mL, 250 ng / mL, 1000 ng / mL, and 2000 ng / mL, respectively.

[0055] Preparation of capture antibody-coated magnetic microparticle solution: 1. Activation of carboxyl magnetic beads: Activate carboxyl magnetic beads (concentration 1.0 mg / ml) at room temperature for 30 min using 10 mg / ml EDC and 20 mg / ml NHS. The mass ratio of EDC, NHS and magnetic beads is 1:2:5. 2. Carboxyl magnetic beads coated with protein A: At room temperature, mix the activated carboxyl magnetic beads and protein A, add 20 μg protein A per mg of magnetic beads, and react for 1 hour; 3. Protein A magnetic beads linked to NfL monoclonal primary antibody: add 20 μg NfL monoclonal primary antibody per mg of protein A magnetic beads and react at room temperature for 1 hour; 4. Magnetic bead blocking: Add 20% BSA to the antibody-coated magnetic beads, add 50 μl 20% BSA per mg of magnetic beads, mix well at room temperature, and incubate for 1 hour; 5. Wash the magnetic beads and adjust the volume.

[0056] Preparation of enzyme-labeled antibody complex: 1. ALP activation: add 30 μL of 50 mM Sulfo-SMCC to 10 mg ALP (10 mg / mL) solution, mix well at pH 7.4 and room temperature, react for 30 min, and dialyze overnight; 2. Activated ALP labeled NfL monoclonal antibody 2: (1) Add 2.4 μL 2-IT (50 mM) to 0.2 mg NfL monoclonal secondary antibody, mix and react for 30 min at room temperature at pH 8.5, and dialyze overnight; (2) 0.2 mg of 2-IT-conjugated NfL monoclonal secondary antibody was added to 48 μl of activated ALP, and the reaction was carried out at pH 7.4 and room temperature for 2 hours; 3. Dilute the enzyme working solution to a fixed volume to obtain the enzyme-labeled antibody complex.

[0057] Comparative Example 2 This comparative example is basically the same as Example 2, except that: the magnetic beads are carboxyl magnetic beads coated with antibodies under the existing process; the enzyme-labeled antibody complex, the cleaning solution and the luminescent substrate are the same as those in Example 2.

[0058] The Alzheimer's disease project was tested in Example 2 and Comparative Example 2 respectively, and blood samples of Alzheimer's disease patients were obtained. The blood samples were processed and tested with a kit, and the data were recorded.

[0059] Detection process: The detection process is the same as that of Example 1 and Comparative Example 1.

[0060] The data analysis results are as follows: The NfL calibration curves were obtained using the kits of Example 2 and Comparative Example 2, respectively. The results are shown in Table 3.

[0061] Table 3 NfL calibration curve ng / ml Comparative Example 2 (RLU) Front-to-back ratio Example 2 (RLU) Front-to-back ratio 0 2401 1976 15 28861 12.0 30345 15.4 50 128052 4.4 144562 4.8 250 630854 4.9 802743 5.6 1000 2436521 3.9 3258212 4.1 2000 4536305 1.9 5485431 1.7 The kits of Example 2 and Comparative Example 2 were used to detect different NfL clinical samples, respectively, to obtain NfL concentration detection results, as shown in Table 4. Wherein, N represents a sample clinically judged to be negative, and P represents a sample clinically judged to be positive.

[0062] Table 4 Clinical samples (NfL) sample Comparative Example 2 (RLU) Comparative Example 2 (pg / ml) Example 2 (RLU) Example 2 (pg / ml) N1 10041 4.9 13423 6.9 N2 10328 5.1 14254 7.3 N3 22744 12.0 16235 8.3 N4 12997 6.7 10874 5.5 N5 13151 6.8 18563 9.5 N6 31338 16.2 18073 9.3 N7 14119 7.3 11846 6.0 N8 14357 7.5 12832 6.5 N9 14824 7.7 13863 7.1 N10 18539 9.8 11387 5.7 N11 20883 11.0 14287 7.3 N12 31354 16.2 26735 13.4 P1 62534 29.1 78711 32.4 P2 51499 24.9 66927 28.7 P3 55409 26.4 89362 35.6 P4 30952 16.0 41374 19.5 P5 26369 13.8 38371 18.4 P6 31747 16.4 49763 22.7 P7 30354 15.7 38275 18.3 P8 93553 39.7 117394 43.3 Reference interval: <17 pg / mL According to Tables 3 and 4, compared with Comparative Example 2, the sensitivity of the reagent in Example 2 is effectively improved and the test results of the samples are more consistent with the clinical positive and negative judgment.

[0063] Example 3

[0064] A kit for assisting in the diagnosis of Alzheimer's disease, including: Capture antibody-coated magnetic microparticle solution: a carboxyl magnetic bead solution coated with Tau181 antibody, wherein the Tau181 antibody is a Tau181 monoclonal antibody specific to the Tau181 antigen in the sample; Enzyme-labeled antibody complex: Tau181 monoclonal antibody solution specific to Tau181 antigen in the sample coupled with alkaline phosphatase; Washing solution: contains 50mM tris, 1.8% NaCl, 0.1% ProClin 950, 0.05% Tween 20; Luminescent substrate: APS-5, source from Hanhai New Enzyme.

[0065] The concentrations of Tau181 in the tested samples were 0 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 60 ng / mL, and 100 ng / mL, respectively.

[0066] Preparation of capture antibody-coated magnetic microparticle solution: 1. Activation of carboxyl magnetic beads: Activate carboxyl magnetic beads (concentration 1.0 mg / ml) at room temperature for 30 min using 10 mg / ml EDC and 20 mg / ml NHS. The mass ratio of EDC, NHS and magnetic beads is 1:2:5. 2. Carboxyl magnetic beads coated with protein A: At room temperature, mix the activated carboxyl magnetic beads and protein A, add 20 μg protein A per mg of magnetic beads, and react for 1 hour; 3. Protein A magnetic beads linked to Tau181 monoclonal primary antibody: add 20 μg Tau181 monoclonal primary antibody per mg of protein A magnetic beads and react at room temperature for 1 hour; 4. Magnetic bead blocking: Add 20% BSA to the antibody-coated magnetic beads, add 50 μl 20% BSA per mg of magnetic beads, mix well at room temperature, and incubate for 1 hour; 5. Wash the magnetic beads and adjust the volume.

[0067] Preparation of enzyme-labeled antibody complex: 1. ALP activation: add 30 μL of 50 mM Sulfo-SMCC to 10 mg ALP (10 mg / mL) solution, mix well at pH 7.4 and room temperature, react for 30 min, and dialyze overnight; 2. Activated ALP labeled Tau181 monoclonal antibody 2: (1) Add 2.4 μL 2-IT (50 mM) to 0.2 mg Tau181 monoclonal secondary antibody, mix and react for 30 min at room temperature at pH 8.5, and dialyze overnight. (2) 0.2 mg of 2-IT-conjugated Tau181 monoclonal secondary antibody was added to 48 μl of activated ALP and reacted at pH 7.4 and room temperature for 2 hours; 3. Dilute the enzyme working solution to a fixed volume to obtain the enzyme-labeled antibody complex.

[0068] Comparative Example 3 This comparative example is basically the same as Example 3, except that: the magnetic beads are carboxyl magnetic beads coated with antibodies under the existing process; the enzyme-labeled antibody complex, the cleaning solution and the luminescent substrate are the same as those in Example 3.

[0069] Detection process: The detection process is the same as that of Example 1 and Comparative Example 1.

[0070] The data analysis results are as follows: The Tau181 calibration curves were obtained using the kits of Example 3 and Comparative Example 3, respectively. The results are shown in Table 5.

[0071] Table 5 Tau181 calibration curve ng / ml Comparative Example 3 (RLU) Front-to-back ratio Example 3 (RLU) Front-to-back ratio 0 2143 　- 678 -　 10 6847 3.2 8247 12.2 20 14247 2.1 22423 2.7 30 21543 1.5 34908 1.6 60 63886 3.0 71335 2.0 100 117816 1.8 124650 1.7 The kits of Example 3 and Comparative Example 3 were used to detect different Tau181 clinical samples, respectively, to obtain Tau181 concentration detection results, as shown in Table 6. Wherein, N represents a sample clinically judged to be negative, and P represents a sample clinically judged to be positive.

[0072] Table 6 Clinical samples (Tau181) sample Comparative Example 3 (RLU) Comparative Example 3 (pg / ml) Example 3 (RLU) Example 3 (pg / ml) N1 13733 19.4 18716 17.8 N2 23653 32.1 21048 19.2 N3 32165 39.3 20354 18.8 N4 41846 46.2 36733 31.7 N5 12907 18.4 14098 14.7 N6 11059 16.1 10354 11.8 N7 37853 43.5 22733 19.4 N8 13553 19.2 13553 14.3 N9 15482 22.2 25482 21.9 P1 51122 52.1 76051 63.0 P2 79546 69.8 103377 84.1 P3 71414 64.6 81940 67.6 P4 76101 67.6 88295 72.5 P5 78287 69.0 76852 63.6 P6 70986 64.4 109600 89.0 P7 54494 54.2 56448 47.7 P8 47856 50.1 97324 79.5 Reference interval: <30 pg / mL According to Tables 5 and 6, compared with Comparative Example 3, the sensitivity of the reagent in Example 3 is significantly improved and the test results of the samples are more consistent with the clinical positive and negative judgment; the negative verification of Comparative Example 3 cannot be carried out and the sensitivity is low.

[0073] Example 4 A kit for assisting in the diagnosis of Alzheimer's disease, including: Capture antibody-coated magnetic microparticle solution: Aβ1-40 antibody-coated carboxyl magnetic bead solution, wherein the Aβ1-40 antibody is an Aβ1-40 monoclonal antibody specific to the Aβ1-40 antigen in the sample; Enzyme-labeled antibody complex: Aβ1-40 monoclonal antibody solution specific to the Aβ1-40 antigen in the sample coupled with alkaline phosphatase; Washing solution: contains 50mM tris, 1.8% NaCl, 0.1% ProClin 950, 0.05% Tween 20; Luminescent substrate: APS-5, source from Hanhai New Enzyme.

[0074] The concentrations of Aβ1-40 in the tested samples were 0 ng / mL, 20 ng / mL, 50 ng / mL, 105 ng / mL, 200ng / mL, and 300 ng / mL, respectively.

[0075] Preparation of capture antibody-coated magnetic microparticle solution: 1. Activation of carboxyl magnetic beads: Activate carboxyl magnetic beads (concentration 1.0 mg / ml) at room temperature for 30 min using 10 mg / ml EDC and 20 mg / ml NHS. The mass ratio of EDC, NHS and magnetic beads is 1:2:5. 2. Carboxyl magnetic beads coated with protein A: At room temperature, mix the activated carboxyl magnetic beads and protein A, add 20 μg protein A per mg of magnetic beads, and react for 1 hour; 3. Protein A magnetic beads linked to Aβ1-40 monoclonal primary antibody: add 20 μg Aβ1-40 monoclonal primary antibody per mg of protein A magnetic beads and react at room temperature for 1 hour; 4. Magnetic bead blocking: Add 20% BSA to the antibody-coated magnetic beads, add 50 μl 20% BSA per mg of magnetic beads, mix well at room temperature, and incubate for 1 hour; 5. Wash the magnetic beads and adjust the volume.

[0076] Preparation of enzyme-labeled antibody complex: 1. ALP activation: add 30 μL of 50 mM Sulfo-SMCC to 10 mg ALP (10 mg / mL) solution, mix well at pH 7.4 and room temperature, react for 30 min, and dialyze overnight; 2. Activated ALP marker Aβ1-40 monoclonal antibody 2: (1) Add 2.4 μL 2-IT (50 mM) to 0.2 mg Aβ1-40 monoclonal secondary antibody, mix and react for 30 min at room temperature at pH 8.5, and dialyze overnight; (2) 0.2 mg of 2-IT-coupled Aβ1-40 monoclonal secondary antibody was added to 48 μl of activated ALP and reacted at pH 7.4 and room temperature for 2 hours; 3. Dilute the enzyme working solution to a fixed volume to obtain the enzyme-labeled antibody complex.

[0077] Comparative Example 4 This comparative example is basically the same as Example 4, except that: the magnetic beads are carboxyl magnetic beads coated with antibodies under the existing process; the enzyme-labeled antibody complex, the cleaning solution and the luminescent substrate are the same as those in Example 4.

[0078] Detection process: The detection process is the same as that of Example 1 and Comparative Example 1.

[0079] The data analysis results are as follows: The kits of Example 4 and Comparative Example 4 were used to obtain Aβ1-40 calibration curves, respectively. The results are shown in Table 7.

[0080] Table 7 Aβ1-40 calibration curve ng / ml Comparative Example 4 (RLU) Front-to-back ratio Example 4 (RLU) Front-to-back ratio 0 46983 - 16983 - 20 155850 3.3 105850 6.2 50 255190 1.6 255190 2.4 105 677568 2.7 607568 2.4 200 1269000 1.9 1659000 2.7 300 2440459 1.9 3030459 1.8 The kits of Example 4 and Comparative Example 4 were used to test different Aβ1-40 clinical samples, respectively, to obtain the Aβ1-40 concentration test results, as shown in Table 8. Wherein, N represents a clinically judged negative sample, and P represents a clinically judged positive sample.

[0081] Table 8 Clinical samples (Aβ1-40) sample Comparative Example 4 (RLU) Comparative Example 4 (pg / ml) Example 4 (RLU) Example 4 (pg / ml) N1 536418 85.8 333001 66.0 N2 637686 101.4 361914 71.0 N3 635809 101.1 779418 123.9 N4 946491 144.5 1071573 151.8 N5 643035 102.2 685989 114.0 N6 530003 84.8 560642 99.3 N7 438947 69.8 330261 65.5 P1 723436 113.9 1284728 170.2 P2 925338 141.7 10418770 450.0 P3 1055743 158.7 1296999 171.2 P4 825972 128.3 1333682 174.2 P5 924293 141.6 2225402 242.1 P6 745365 117.1 1694384 202.7 Reference interval: <160 pg / mL According to Tables 7 and 8, compared with Comparative Example 4, the sensitivity of the reagent in Example 4 is effectively improved and the positive and negative test results of the samples are clearly distinguished.

[0082] Example 5

[0083] A kit for assisting in the diagnosis of Alzheimer's disease, including: Capture antibody-coated magnetic microparticle solution: Aβ1-42 antibody-coated carboxyl magnetic bead solution, wherein the Aβ1-42 antibody is an Aβ1-42 monoclonal antibody specific to the Aβ1-42 antigen in the sample; Enzyme-labeled antibody complex: Aβ1-42 monoclonal antibody solution specific to the Aβ1-42 antigen in the sample coupled with alkaline phosphatase; Washing solution: contains 50mM tris, 1.8% NaCl, 0.1% ProClin 950, 0.05% Tween 20; Luminescent substrate: APS-5, source from Hanhai New Enzyme.

[0084] The concentrations of Aβ1-42 in the tested samples were 0 ng / mL, 20 ng / mL, 50 ng / mL, 105 ng / mL, 200 ng / mL, and 300 ng / mL, respectively.

[0085] Preparation of capture antibody-coated magnetic microparticle solution: 1. Activation of carboxyl magnetic beads: Activate carboxyl magnetic beads (concentration 1.0 mg / ml) at room temperature for 30 min using 10 mg / ml EDC and 20 mg / ml NHS. The mass ratio of EDC, NHS and magnetic beads is 1:2:5. 2. Carboxyl magnetic beads coated with protein A: At room temperature, mix the activated carboxyl magnetic beads and protein A, add 20 μg protein A per mg of magnetic beads, and react for 1 hour; 3. Protein A magnetic beads linked to Aβ1-42 monoclonal primary antibody: add 20 μg Aβ1-42 monoclonal primary antibody per mg of protein A magnetic beads and react at room temperature for 1 hour; 4. Magnetic bead blocking: Add 20% BSA to the antibody-coated magnetic beads, add 50 μl 20% BSA per mg of magnetic beads, mix well at room temperature, and incubate for 1 hour; 5. Wash the magnetic beads and adjust the volume.

[0086] Preparation of enzyme-labeled antibody complex: 1. ALP activation: add 30 μL of 50 mM Sulfo-SMCC to 10 mg ALP (10 mg / mL) solution, mix well at pH 7.4 and room temperature, react for 30 min, and dialyze overnight; 2. Activated ALP marker Aβ1-42 monoclonal antibody 2: (1) Add 2.4 μL 2-IT (50 mM) to 0.2 mg Aβ1-42 monoclonal secondary antibody, mix and react for 30 min at room temperature at pH 8.5, and dialyze overnight; (2) 0.2 mg of 2-IT-conjugated Aβ1-42 monoclonal secondary antibody was added to 48 μl of activated ALP and reacted at pH 7.4 and room temperature for 2 hours; 3. Dilute the enzyme working solution to a fixed volume to obtain the enzyme-labeled antibody complex.

[0087] Comparative Example 5 This comparative example is basically the same as Example 5, except that: the magnetic beads are carboxyl magnetic beads coated with antibodies under the existing process; the enzyme-labeled antibody complex, the cleaning solution and the luminescent substrate are the same as those in Example 5.

[0088] Detection process: The detection process is the same as that of Example 1 and Comparative Example 1.

[0089] The data analysis results are as follows: The kits of Example 5 and Comparative Example 5 were used to obtain Aβ1-42 calibration curves, respectively. The results are shown in Table 9.

[0090] Table 9 Aβ1-42 calibration curve ng / ml Comparative Example 5 (RLU) Front-to-back ratio Example 5 (RLU) Front-to-back ratio 0.0 5464 - 1274 - 0.6 24354 4.5 35081 27.5 1.5 53452 2.2 90716 2.6 3.0 84353 1.6 192048 2.1 10.0 327532 3.9 797532 4.2 20.0 712433 2.2 1652433 2.1 According to Table 9, compared with Comparative Example 5, the sensitivity of the reagent in Example 5 is effectively improved.

[0091] Example 6

[0092] A kit for assisting in the diagnosis of Alzheimer's disease, including: Capture antibody-coated magnetic microparticle solution: a carboxyl magnetic bead solution coated with Tau231 antibody, wherein the Tau231 antibody is a Tau231 monoclonal antibody specific to the Tau231 antigen in the sample; Enzyme-labeled antibody complex: Tau231 monoclonal antibody solution specific to Tau231 antigen in the sample coupled with alkaline phosphatase; Washing solution: contains 50mM tris, 1.8% NaCl, 0.1% ProClin 950, 0.05% Tween 20; Luminescent substrate: APS-5, source from Hanhai New Enzyme.

[0093] The Tau231 concentrations in the tested samples were 0 ng / mL, 0.6 ng / mL, 1.5 ng / mL, 3 ng / mL, 10 ng / mL, and 20 ng / mL, respectively.

[0094] Preparation of capture antibody-coated magnetic microparticle solution: 1. Activation of carboxyl magnetic beads: Activate carboxyl magnetic beads (concentration 1.0 mg / ml) at room temperature for 30 min using 10 mg / ml EDC and 20 mg / ml NHS. The mass ratio of EDC, NHS and magnetic beads is 1:2:5. 2. Carboxyl magnetic beads coated with protein A: At room temperature, mix the activated carboxyl magnetic beads and protein A, add 20 μg protein A per mg of magnetic beads, and react for 1 hour; 3. Protein A magnetic beads linked to Tau231 monoclonal primary antibody: add 20 μg Tau231 monoclonal primary antibody per mg of protein A magnetic beads and react at room temperature for 1 hour; 4. Magnetic bead blocking: Add 20% BSA to the antibody-coated magnetic beads, add 50 μl 20% BSA per mg of magnetic beads, mix well at room temperature, and incubate for 1 hour; 5. Wash the magnetic beads and adjust the volume.

[0095] Preparation of enzyme-labeled antibody complex: 1. ALP activation: add 30 μL of 50 mM Sulfo-SMCC to 10 mg ALP (10 mg / mL) solution, mix well at pH 7.4 and room temperature, react for 30 min, and dialyze overnight; 2. Activated ALP labeled Tau231 monoclonal antibody 2: (1) Add 2.4 μL 2-IT (50 mM) to 0.2 mg Tau231 monoclonal secondary antibody, mix and react for 30 min at room temperature at pH 8.5, and dialyze overnight; (2) 0.2 mg of 2-IT-conjugated Tau231 monoclonal secondary antibody was added to 48 μl of activated ALP and reacted at pH 7.4 and room temperature for 2 hours; 3. Dilute the enzyme working solution to a fixed volume to obtain the enzyme-labeled antibody complex.

[0096] Comparative Example 6 This comparative example is basically the same as Example 6, except that: the magnetic beads are carboxyl magnetic beads coated with antibodies under the existing process; the enzyme-labeled antibody complex, the cleaning solution and the luminescent substrate are the same as those in Example 6.

[0097] Detection process: The detection process is the same as that of Example 1 and Comparative Example 1.

[0098] The data analysis results are as follows: The kits of Example 6 and Comparative Example 6 were used to obtain Tau231 calibration curves, respectively. The results are shown in Table 10.

[0099] Table 10 Tau231 calibration curve ng / ml Comparative Example 6 (RLU) Front-to-back ratio Example 6 (RLU) Front-to-back ratio 0.0 5464 - 1274 - 0.6 24354 4.5 35081 27.5 1.5 53452 2.2 90716 2.6 3.0 84353 1.6 192048 2.1 10.0 327532 3.9 797532 4.2 20.0 712433 2.2 1652433 2.1 According to Table 10, compared with Comparative Example 6, the sensitivity of the reagent in Example 6 is effectively improved.

[0100] Example 7 A kit for assisting in the diagnosis of Alzheimer's disease, including: Capture antibody-coated magnetic microparticle solution: GFAP antibody-coated carboxyl magnetic bead solution, the GFAP antibody is a GFAP monoclonal antibody specific to the GFAP antigen in the sample; Enzyme-labeled antibody complex: a GFAP monoclonal antibody solution specific to the GFAP antigen in the sample coupled with alkaline phosphatase; Washing solution: contains 50mM tris, 1.8% NaCl, 0.1% ProClin 950, 0.05% Tween 20; Luminescent substrate: APS-5, source from Hanhai New Enzyme.

[0101] The GFAP concentrations in the tested samples were 0 ng / mL, 100 ng / mL, 500 ng / mL, 2500 ng / mL, 10000 ng / mL, and 30000 ng / mL, respectively.

[0102] Preparation of capture antibody-coated magnetic microparticle solution: 1. Activation of carboxyl magnetic beads: Activate carboxyl magnetic beads (concentration 1.0 mg / ml) at room temperature for 30 min using 10 mg / ml EDC and 20 mg / ml NHS. The mass ratio of EDC, NHS and magnetic beads is 1:2:5. 2. Carboxyl magnetic beads coated with protein A: At room temperature, mix the activated carboxyl magnetic beads and protein A, add 20 μg protein A per mg of magnetic beads, and react for 1 hour; 3. Protein A magnetic beads linked to GFAP monoclonal primary antibody: add 20 μg GFAP monoclonal primary antibody per mg of protein A magnetic beads and react at room temperature for 1 hour; 4. Magnetic bead blocking: Add 20% BSA to the antibody-coated magnetic beads, add 50 μl 20% BSA per mg of magnetic beads, mix well at room temperature, and incubate for 1 hour; 5. Wash the magnetic beads and adjust the volume.

[0103] Preparation of enzyme-labeled antibody complex: 1. ALP activation: add 30 μL of 50 mM Sulfo-SMCC to 10 mg ALP (10 mg / mL) solution, mix well at pH 7.4 and room temperature, react for 30 min, and dialyze overnight; 2. Activated ALP labeled GFAP monoclonal antibody 2: (1) Add 2.4 μL 2-IT (50 mM) to 0.2 mg GFAP monoclonal secondary antibody, mix and react for 30 min at room temperature at pH 8.5, and dialyze overnight. (2) 0.2 mg of 2-IT-conjugated GFAP monoclonal secondary antibody was added to 48 μl of activated ALP and reacted at pH 7.4 and room temperature for 2 hours; 3. Dilute the enzyme working solution to a fixed volume to obtain the enzyme-labeled antibody complex.

[0104] Comparative Example 7 This comparative example is basically the same as Example 7, except that: the magnetic beads are carboxyl magnetic beads coated with antibodies under the existing process; the enzyme-labeled antibody complex, the cleaning solution and the luminescent substrate are the same as those in Example 7.

[0105] Detection process: The detection process is the same as that of Example 1 and Comparative Example 1.

[0106] The data analysis results are as follows: The GFAP calibration curves were obtained using the kits of Example 7 and Comparative Example 7, respectively. The results are shown in Table 11.

[0107] Table 11 GFAP calibration curve ng / ml Comparative Example 7 (RLU) Front-to-back ratio Example 7 (RLU) Front-to-back ratio 0.0 1314 - 1053 - 100.0 8037 6.1 12346 11.7 500.0 43468 5.4 63257 5.1 2500.0 209890 4.8 340123 5.4 10000.0 1031736 4.9 1424508 4.2 30000.0 2396791 2.3 3154873 2.2 The kits of Example 7 and Comparative Example 7 were used to detect different GFAP clinical samples, respectively, to obtain GFAP concentration detection results, as shown in Table 12.

[0108] Table 12 Clinical samples (GFAP) sample Comparative Example 7 (RLU) Comparative Example 7 (pg / ml) Example 7 (RLU) Example 7 (pg / ml) Random physical examination samples 3023 26.7 1851 7.2 Random physical examination samples 2746 22.4 1795 6.7 Random physical examination samples 2145 13.1 1795 6.7 Random physical examination samples 2643 20.8 1946 8.1 Random physical examination samples 3867 39.5 1812 6.9 Random physical examination samples 2964 25.8 2043 9.0 Random physical examination samples 2351 16.3 1983 8.4 Random physical examination samples 2835 23.8 2154 10.0 Brain injury samples 11200 143.1 15099 123.6 Brain injury samples 18940 240.7 20571 169.8 Brain injury samples 34205 409.0 58140 462.8 Brain injury samples 213468 2535.8 277520 2079.0 Brain injury samples 675304 6722.6 783833 5423.7 Brain injury samples 12426 159.3 19901 164.2 Brain injury samples 825975 8075.2 1511106 10681.5 Brain injury samples 34300 410.0 42180 343.0 Brain injury samples 12252 157.0 15535 127.4 Reference interval: <27 pg / mL According to Tables 11 to 12, compared with Comparative Example 7, the sensitivity of the reagent in Example 7 is effectively improved and the test results of the samples are more specific.

[0109] Example 8

[0110] A kit for assisting in the diagnosis of Alzheimer's disease, including: Capture antibody-coated magnetic microparticle solution: a carboxyl magnetic bead solution coated with an S100β antibody, wherein the S100β antibody is a S100β monoclonal antibody specific to the S100β antigen in the sample; Enzyme-labeled antibody complex: a solution of S100β monoclonal antibody specific to the S100β antigen in the sample coupled with alkaline phosphatase; Washing solution: contains 50mM tris, 1.8% NaCl, 0.1% ProClin 950, 0.05% Tween 20; Luminescent substrate: APS-5, source from Hanhai New Enzyme.

[0111] The concentrations of S100β in the tested samples were 0 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 2.5 ng / mL, 10 ng / mL, and 30 ng / mL, respectively.

[0112] Preparation of capture antibody-coated magnetic microparticle solution: 1. Activation of carboxyl magnetic beads: Activate carboxyl magnetic beads (concentration 1.0 mg / ml) at room temperature for 30 min using 10 mg / ml EDC and 20 mg / ml NHS. The mass ratio of EDC, NHS and magnetic beads is 1:2:5. 2. Carboxyl magnetic beads coated with protein A: At room temperature, mix the activated carboxyl magnetic beads and protein A, add 20 μg protein A per mg of magnetic beads, and react for 1 hour; 3. Protein A magnetic beads linked to S100β monoclonal primary antibody: add 20μg S100β monoclonal primary antibody per mg of protein A magnetic beads and react at room temperature for 1 hour; 4. Magnetic bead blocking: Add 20% BSA to the antibody-coated magnetic beads, add 50 μl 20% BSA per mg of magnetic beads, mix well at room temperature, and incubate for 1 hour; 5. Wash the magnetic beads and adjust the volume.

[0113] Preparation of enzyme-labeled antibody complex: 1. ALP activation: add 30 μL of 50 mM Sulfo-SMCC to 10 mg ALP (10 mg / mL) solution, mix well at pH 7.4 and room temperature, react for 30 min, and dialyze overnight; 2. Activated ALP-labeled S100β monoclonal antibody 2: (1) Add 2.4 μL 2-IT (50 mM) to 0.2 mg S100β monoclonal secondary antibody, mix and react for 30 min at room temperature at pH 8.5, and dialyze overnight. (2) 0.2 mg of 2-IT-conjugated S100β monoclonal secondary antibody was added to 48 μl of activated ALP and reacted at pH 7.4 and room temperature for 2 hours; 3. Dilute the enzyme working solution to a fixed volume to obtain the enzyme-labeled antibody complex.

[0114] Comparative Example 8 This comparative example is basically the same as Example 8, except that: the magnetic beads are carboxyl magnetic beads coated with antibodies under the existing process; the enzyme-labeled antibody complex, the cleaning solution and the luminescent substrate are the same as those in Example 8.

[0115] Detection process: The detection process is the same as that of Example 1 and Comparative Example 1.

[0116] The data analysis results are as follows: The S100β calibration curves were obtained using the kits of Example 8 and Comparative Example 8, respectively. The results are shown in Table 13.

[0117] Table 13 S100β calibration curve ng / ml Comparative Example 8 (RLU) Front-to-back ratio Example 8 (RLU) Front-to-back ratio 0.000 2035 　- 1390 -　 0.100 4970 2.4 6527 4.7 0.500 17749 3.6 27820 4.3 2.500 84924 4.8 119838 4.3 10.000 477634 5.6 617267 5.2 30.000 1217850 2.5 2078699 3.4 The kits of Example 8 and Comparative Example 8 were used to detect different S100β clinical samples, respectively, to obtain S100β concentration detection results, as shown in Table 14.

[0118] Table 14 Clinical samples (S100β) sample Comparative Example 8 (RLU) Comparative Example 8 (pg / ml) Example 8 (RLU) Example 8 (pg / ml) Hospital CT value Random physical examination samples 4067 0.07 2860 0.03 / Random physical examination samples 3993 0.07 2925 0.03 / Random physical examination samples 3735 0.06 1696 0.01 / Random physical examination samples 4112 0.00 2313 0.02 / Random physical examination samples 2597 0.02 1848 0.01 / Random physical examination samples 3612 0.05 2654 0.03 / Random physical examination samples 2708 0.02 2250 0.02 / Random physical examination samples 2323 0.01 1734 0.01 / Brain injury samples 20897 0.64 42874 0.90 / 　 Brain injury samples 36686 1.22 58574 1.27 / 　 Brain injury samples 24750 0.80 52423 1.13 / 　 Brain injury samples 33743 1.12 68234 1.48 / 　 Brain injury samples 15087 0.42 32532 0.63 / 　 Brain injury samples 58347 1.84 84446 1.82 / 　 Brain injury samples 23906 0.77 55433 1.20 / 　 Brain injury samples 28881 0.95 46445 0.99 / 　 Brain injury samples 38521 1.27 66335 1.44 / 　 Reference interval: <0.1 ug / mL According to Tables 13 to 14, compared with Comparative Example 8, the sensitivity of the reagent is effectively improved in Example 8, and both labeling methods have good positive and negative detection.

[0119] The present invention coats activated magnetic beads with Protein A, combines Protein A with the Fc end of a monoclonal antibody, increases the chain length of the connection, and reduces steric hindrance; Protein A can purify the target connection protein, and other impurities in the raw material can be washed away by the adsorption of the magnetic beads. At the same time, one Protein A can combine with at least two IgG molecules, thereby improving the labeling sensitivity; Protein A is connected to the Fc end of the monoclonal antibody, so that the Fab end of the monoclonal antibody can be directed toward the outside in a specific direction, and can be more efficiently combined with the antigen to be detected in the sample, thereby improving the detection sensitivity.

[0120] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A kit for assisting in the diagnosis of Alzheimer's disease, characterized in that: It includes a capture antibody-coated magnetic particle solution, an enzyme-labeled antibody complex, a washing solution, and a luminescent substrate. wherein the capture antibody-coated magnetic particle solution comprises magnetic particles linked to a first antibody, wherein the first antibody specifically binds to a Tau217, NfL, Tau181, Aβ1-40, Aβ1-42, Tau231 or GFAP biomarker; The enzyme-labeled antibody complex includes a second antibody coupled to an enzyme, wherein the second antibody and the first antibody specifically bind to the same biomarker; The magnetic particles include carboxyl magnetic beads, the carboxyl magnetic beads are coated with Protein A, and the Protein A is linked to a first antibody.

2. The kit for assisting diagnosis of Alzheimer's disease according to claim 1, characterized in that: The capture antibody-coated magnetic particle solution further comprises a first buffer, which is a tris(hydroxymethyl)aminomethane buffer.

3. The kit for assisting diagnosis of Alzheimer's disease according to claim 1, characterized in that: The first antibody is a monoclonal antibody.

4. The kit for assisting diagnosis of Alzheimer's disease according to claim 1, characterized in that: The second antibody is a monoclonal antibody.

5. The kit for assisting diagnosis of Alzheimer's disease according to claim 1, characterized in that: The enzyme-labeled antibody complex further comprises a second buffer, which comprises a 0.1 M MgCl2 buffer and a 0.01 M ZnCl2 buffer.

6. The kit for assisting diagnosis of Alzheimer's disease according to claim 1, characterized in that: The enzyme conjugated to the second antibody includes alkaline phosphatase.

7. The kit for assisting diagnosis of Alzheimer's disease according to claim 1, characterized in that: The luminescent substrate includes an APS-5 substrate.

8. The method for preparing a kit for assisting diagnosis of Alzheimer's disease according to any one of claims 1 to 7, characterized in that: The steps include: Preparing a capture antibody-coated magnetic microparticle solution: activating magnetic beads and then connecting them with Protein A to obtain Protein A magnetic beads; combining the Protein A magnetic beads with a first antibody specifically binding to Tau217, NfL, Tau181, Aβ1-40, Aβ1-42, Tau231 or GFAP biomarkers, and then blocking to obtain a capture antibody-coated magnetic microparticle solution; Preparation of enzyme-labeled antibody complex: coupling an enzyme with a cross-linker 1 for activation; coupling a second antibody that specifically binds to the same biomarker as the first antibody with a cross-linker 2 to obtain a labeled second antibody; coupling the enzyme-coupled cross-linker 1 with the labeled second antibody to obtain an enzyme-labeled antibody complex, wherein the cross-linker 1 is Sulfo-SMCC and the cross-linker 2 is 2-IT.

9. The method for preparing a kit for assisting diagnosis of Alzheimer's disease according to claim 8, characterized in that: Zinc chloride and magnesium chloride are also added to the enzyme-labeled antibody complex.

10. Use of the kit for assisting diagnosis of Alzheimer's disease according to any one of claims 1 to 7 in preparing a detection product for detecting Alzheimer's disease, characterized in that: The steps include: Add the sample to be tested into the reaction cup, add the magnetic particle solution coated with the capture antibody, and then add the enzyme-labeled antibody complex. After reaction and adsorption, separate the waste liquid, wash with cleaning solution, add the luminescent substrate, incubate, and read the readings through the instrument.