Phosphorylated tau181 protein assay kit and application

Abstract

This invention belongs to the field of detection technology. Specifically, it discloses a phosphorylated Tau181 protein assay kit and its application. In this application, the biotin in the p-Tau181 antibody in anti-reagent A and anti-reagent B reacts with alkaline phosphatase to form an antibody-antigen-antibody complex. Through the reaction of biotin with streptavidin, the complex binds to magnetic microparticles. The chemiluminescent substrate AMPPD is added, and alkaline phosphatase catalyzes AMPPD to generate a chemiluminescent signal. The luminescence intensity is measured by an optical detection system, and the instrument automatically calculates the detection result through a working curve. This application, through the design of anti-reagent A and anti-reagent B and the magnetic microparticle reagent in the kit, can accurately capture antibodies and amplify signals, effectively reducing the detection limit and improving the overall performance of the kit.

Description

Technical Field

[0001] This invention relates to the field of detection technology, specifically to a kit for the assay of phosphorylated Tau181 protein and its application. Background Technology

[0002] Alzheimer's disease, often abbreviated as AD, is a neurodegenerative disease with insidious onset and progressive development. It is the most common type of dementia, accounting for 60%-80% of all dementia cases. The disease is characterized by progressive cognitive decline, ultimately severely impacting patients' daily living abilities, behavior, and social function. The exact cause of Alzheimer's disease is not fully understood, but it is generally believed to be the result of multiple factors, including genetics, environment, and lifestyle.

[0003] TAU protein is a key protein for maintaining microtubule stability within neurons. In Alzheimer's disease (AD), TAU protein undergoes chemical alteration (hyperphosphorylation), causing it to detach from microtubules and aggregate into insoluble neurofibrillary tangles. This leads to the collapse of the neuronal transport system, cytoskeleton disintegration, and ultimately neuronal death. Hyperphosphorylation of Tau protein reduces its affinity for microtubules. Soluble hyperphosphorylated Tau protein aggregates into pathological aggregates, which are major components of neurofibrillary tangles and intracellular inclusion bodies in frontotemporal dementia cells in Alzheimer's disease. Phosphorylated Tau at threonine 181 (p-Tau181) is a highly specific biomarker for Alzheimer's disease pathology. p-Tau 181 detection is expected to be used to assess cognitive impairment caused by Alzheimer's disease (AD) as well as cognitive impairment caused by other factors. Results above the reference range are in good agreement with Tau protein positron emission tomography (Tau PET). Currently, a kit for the determination of phosphorylated Tau181 based on the magnetic microparticle chemiluminescence sandwich method has been put into clinical use, but the existing kits have the problem of insufficient detection sensitivity. Summary of the Invention

[0004] This invention provides a phosphorylated Tau181 protein assay kit and its application. By designing the anti-reagent A and anti-reagent B and the magnetic microparticle reagent in the kit, this application can accurately capture antibodies and amplify signals, effectively reduce the detection limit, and improve the overall performance of the kit.

[0005] The present invention solves its technical problem by adopting the following technical solution: A kit for the assay of phosphorylated Tau181 protein, comprising anti-reagent A, anti-reagent B, and magnetic microparticle reagent; The preparation method of the anti-reagent A is as follows: 100 μg of p-Tau217 antibody, 30-50 μg of biotin and 250-400 μg of phosphate buffer are mixed evenly, then 10-15 μg of 1-hydroxybenzotriazole, 5-10 μg of N,N-diisopropylethylamine and 5-10 μg of sodium borohydride are added and stirred evenly, then 100-200 μg of glycine is added and stirred evenly, and diluted with a stabilizer to obtain anti-reagent A; The method for preparing the anti-reagent B is as follows: 80-200 μg of p-Tau181 antibody solution, 50-100 μg of alkaline phosphatase solution and 40-80 μg of gold nanoparticle solution are mixed evenly, 50-120 μg of glycine is added, the mixture is stirred evenly, and then diluted with a stabilizer to obtain anti-reagent B.

[0006] As an embodiment of this application, the preparation method of the antibody solution is as follows: 100 μg of p-Tau181 antibody is added to 800~2000 μg of maleic acid buffer, stirred evenly, and then 20~50 μg of dithiothreitol is added. The mixture is stirred at 45~60℃ for 0.5~2h to obtain the antibody solution.

[0007] As an embodiment of this application, the molar concentration of the maleic acid buffer is 0.1~0.5 mol / L.

[0008] As an embodiment of this application, the alkaline phosphatase solution is prepared by adding 20-50 μg of alkaline phosphatase to 80-200 μg of succinimide-4-(N-maleimide)cyclohexane-1-hydroxy ester solution and stirring until homogeneous to obtain the alkaline phosphatase solution.

[0009] As an embodiment of this application, the preparation method of the nano-gold solution is as follows: 40-80 μg of polyethylene glycol, 40-80 μg of maleimide, and 40-80 μg of dioleoylphosphatidylethanolamine are added to 800-2000 μg of dimethyl sulfoxide, and then 80-120 μg of nano-gold particles are added and stirred evenly to obtain the nano-gold solution.

[0010] As an embodiment of this application, the molar concentration of the succinimide-4-(N-maleimide)cyclohexane-1-hydroxy ester solution is 0.01~0.1 mol / L.

[0011] As an embodiment of this application, the preparation method of the magnetic microparticle reagent is as follows: 10 mg of magnetic microparticles are resuspended in MES buffer, and then 100-200 μL of 0.05-0.2 mol / L 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide solution and 0.2-0.5 mg of N-hydroxysuccinimide are added. The mixture is stirred evenly, and 0.5-2 mg of streptavidin is added. The mixture is reacted at room temperature for 1-3 h. After magnetic separation of the supernatant, the mixture is washed, and then 100-200 μg of glycine is added. The volume is adjusted to 500 mL with a stabilizer to obtain the magnetic microparticle reagent.

[0012] As an embodiment of this application, the stabilizer comprises the following components in weight percentages: 0.1-0.2% streptomycin, 0.2-0.4% fish peptone, 0.2-0.5% sucrose, 0.2-0.5% sodium lauroyl glutamate, 0.8-1.5% bovine serum albumin, 0.8-1.4% chitosan, 0.05-0.12% polylysine, 0.01-0.04% preservative, and the balance being phosphate buffer.

[0013] As an embodiment of this application, the preservative is sodium azide.

[0014] This application also provides the application of a phosphorylated Tau181 protein assay kit in the preparation of reagents for detecting Alzheimer's disease.

[0015] The beneficial effects of this invention are as follows: The biotin in the p-Tau181 antibody in anti-reagent A and anti-reagent B of this application reacts with alkaline phosphatase to form an antibody-antigen-antibody complex. Through the reaction of biotin with streptavidin, the complex binds to magnetic microparticles. The chemiluminescent substrate AMPPD is added, and alkaline phosphatase catalyzes AMPPD to generate a chemiluminescent signal. The luminescence intensity is measured by an optical detection system, and the instrument automatically calculates the detection result through the working curve. This application, through the design of anti-reagent A and anti-reagent B and magnetic microparticle reagents in the kit, can accurately capture antibodies and amplify signals, effectively reduce the detection limit, and improve the overall performance of the kit. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0018] In this invention, numerical ranges are involved. Unless otherwise specified, the numerical ranges are considered continuous and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe features or characteristics, the ranges may be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0019] In this invention, there are no particular limitations on the specific dispersion and stirring methods.

[0020] Unless otherwise specified, all reagents or instruments used in this invention are commercially available conventional products. Unless otherwise specified, the raw materials used in each comparative example and the parallel experiments of each embodiment are the same commercially available products.

[0021] This application provides a phosphorylated Tau181 protein assay kit, including anti-reagent A, anti-reagent B, and magnetic microparticle reagent; The preparation method of the anti-reagent A is as follows: 100 μg of p-Tau217 antibody, 30-50 μg of biotin and 250-400 μg of phosphate buffer are mixed evenly, then 10-15 μg of 1-hydroxybenzotriazole, 5-10 μg of N,N-diisopropylethylamine and 5-10 μg of sodium borohydride are added and stirred evenly, then 100-200 μg of glycine is added and stirred evenly, and diluted with a stabilizer to obtain anti-reagent A; The method for preparing the anti-reagent B is as follows: 80-200 μg of p-Tau181 antibody solution, 50-100 μg of alkaline phosphatase solution and 40-80 μg of gold nanoparticle solution are mixed evenly, 50-120 μg of glycine is added, the mixture is stirred evenly, and then diluted with a stabilizer to obtain anti-reagent B.

[0022] The phosphorylated Tau181 protein assay kit described in this application The biotin in the p-Tau181 antibody in anti-reagent A and anti-reagent B described in this application reacts with alkaline phosphatase to form an antibody-antigen-antibody complex. This complex then binds to magnetic microparticles via a reaction between biotin and streptavidin. The addition of the chemiluminescent substrate AMPPD causes alkaline phosphatase to catalyze the AMPPD, generating a chemiluminescent signal. The luminescence intensity is measured using an optical detection system, and the instrument automatically calculates the detection result based on the working curve. This application, through the design of anti-reagent A and anti-reagent B and the magnetic microparticle reagent in the kit, can accurately capture antibodies and amplify signals, effectively reducing the detection limit and improving the overall performance of the kit.

[0023] The kit described in this application has a detection limit of ≤5.00 pg / mL, a linear correlation coefficient r≥0.9900 in the range of 1.00 pg / mL to 200.00 pg / mL, repeatability ≤8%, intermediate precision ≤15%, and good accuracy.

[0024] In some embodiments, the antibody solution is prepared by adding 100 μg of p-Tau181 antibody to 800-2000 μg of maleic acid buffer, stirring until homogeneous, then adding 20-50 μg of dithiothreitol, and stirring at 45-60°C for 0.5-2 h to obtain the antibody solution.

[0025] In some embodiments, the molar concentration of the maleic acid buffer is 0.1~0.5 mol / L.

[0026] In some embodiments, the alkaline phosphatase solution is prepared by adding 20-50 μg of alkaline phosphatase to 80-200 μg of succinimide-4-(N-maleimide)cyclohexane-1-hydroxy ester solution and stirring until homogeneous to obtain the alkaline phosphatase solution.

[0027] In some embodiments, the preparation method of the gold nanoparticle solution is as follows: 40-80 μg of polyethylene glycol, 40-80 μg of maleimide, and 40-80 μg of dioleoylphosphatidylethanolamine are added to 800-2000 μg of dimethyl sulfoxide, and then 80-120 μg of gold nanoparticles are added and stirred evenly to obtain the gold nanoparticle solution.

[0028] This application uses a special p-Tau181 antibody solution, alkaline phosphatase solution, and gold nanoparticle solution, which can improve the binding efficiency and stability with biotin, effectively enhance the signal amplification effect, effectively increase the luminescence intensity, and reduce the detection limit.

[0029] In some embodiments, the molar concentration of the succinimide-4-(N-maleimide)cyclohexane-1-hydroxy ester solution is 0.01~0.1 mol / L.

[0030] In some embodiments, the magnetic microparticle reagent is prepared as follows: 10 mg of magnetic microparticles are resuspended in MES buffer, then 100-200 μL of 0.05-0.2 mol / L 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide solution and 0.2-0.5 mg of N-hydroxysuccinimide are added, stirred evenly, 0.5-2 mg of streptavidin is added, and the mixture is reacted at room temperature for 1-3 h. After magnetic separation of the supernatant, the mixture is washed, and then 100-200 μg of glycine is added. The volume is adjusted to 500 mL with a stabilizer to obtain the magnetic microparticle reagent.

[0031] In some embodiments, the stabilizer comprises the following components in weight percentages: 0.1-0.2% streptomycin, 0.2-0.4% fish peptone, 0.2-0.5% sucrose, 0.2-0.5% sodium lauroyl urate, 0.8-1.5% bovine serum albumin, 0.8-1.4% chitosan, 0.05-0.12% polylysine, 0.01-0.04% preservative, and the balance being phosphate buffer.

[0032] The stabilizer described in this application has good compatibility with the anti-reagent A, anti-reagent B, and magnetic microparticle reagent, which can improve the stability of each reagent, enhance the anti-interference effect and binding efficiency, and effectively maintain activity.

[0033] In some embodiments, the preservative is sodium azide.

[0034] The assay kits and instruments described in this application have good compatibility and broad applicability. For example, they are compatible with the POClia chemiluminescence analyzer manufactured by Taizhou Zecheng Biotechnology Co., Ltd., including models POClia 8, POClia minus, POClia plus, and POClia auto; or the CIA 600, CIA 1200, CIA 1200M, CIA 1800, and CIA 2800 manufactured by Taizhou Zecheng Biotechnology Co., Ltd.; and the Shinei1900 and Shinei2000 manufactured by Shenzhen Yingkai Biotechnology Co., Ltd.

[0035] In some embodiments, the kit also includes calibrators and quality control samples.

[0036] In some embodiments, the calibrators include a first calibrator, a second calibrator, and a third calibrator, wherein the p-Tau 181 antigen in the first calibrator is diluted with buffer to a concentration of 0 ng / mL; the p-Tau 181 antigen in the second calibrator is diluted with buffer to a concentration of 0.5~5 ng / mL; and the p-Tau 181 antigen in the third calibrator is diluted with buffer to a concentration of 8~15 ng / mL.

[0037] More specifically, the quality control materials include a first quality control material and a second quality control material, wherein the p-Tau 181 antigen in the first quality control material is diluted with buffer to a concentration of 2-6 ng / mL; and the p-Tau 181 antigen in the second quality control material is diluted with buffer to a concentration of 20-60 ng / mL.

[0038] In some embodiments, a substrate solution is also included, wherein the substrate solution is an enzyme-catalyzed luminescent substrate.

[0039] In some embodiments, the enzyme-catalyzed luminescent substrate is the disodium salt of 3-(2-spirodalane)-4-methoxy-4-(3-phosphoryl)-phenyl-1,2-dioxane.

[0040] In some embodiments, the substrate solution is a 0.1-1 mol / L solution of 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoryl)-phenyl-1,2-dioxane disodium salt.

[0041] In some embodiments, 0.1 to 0.5 μL of substrate solution is also included.

[0042] In some embodiments, 1 to 5 mL of Tris buffer is also included.

[0043] This application also provides a phosphorylated Tau217 protein assay kit for the detection of the concentration of phosphorylated Tau217 protein in human serum and / or human plasma.

[0044] In some embodiments, the kit described in this application is suitable for serum and plasma samples, which should be equilibrated to room temperature and thoroughly mixed before testing. To ensure experimental requirements, the sample volume should be no less than 200 μL. Serum and plasma samples can be stored at 2–8°C for 3 days; or at -20±5°C for 3 months, with no more than 3 freeze-thaw cycles.

[0045] This application also provides an application of a phosphorylated Tau181 protein assay kit in the preparation of reagents for detecting Alzheimer's disease.

[0046] The present application is further illustrated below with specific embodiments:

[0047] Example 1 A phosphorylated Tau217 protein assay kit includes anti-reagent A, anti-reagent B, magnetic microparticle reagent, high-value calibrator, low-value calibrator, zero-point calibrator, high-value quality control, low-value quality control, substrate solution, and washing solution.

[0048] The preparation method of the anti-reagent A is as follows: 100 μg of p-Tau217 antibody, 40 μg of biotin and 350 μg of phosphate buffer (molar concentration of 0.05 mol / L) are mixed evenly, then 12 μg of 1-hydroxybenzotriazole, 8 μg of N,N-diisopropylethylamine and 8 μg of sodium borohydride are added and stirred evenly, then 120 μg of glycine is added and stirred evenly, and diluted 100 times with a stabilizer to obtain anti-reagent A.

[0049] The preparation method of the anti-reagent B is as follows: (1) 100 μg of p-Tau181 antibody is added to 1000 μg of maleic acid buffer (molar concentration of 0.2 mol / L), stirred evenly, and then 40 μg of dithiothreitol is added. The mixture is stirred at 50°C for 1 h to obtain p-Tau181 antibody solution.

[0050] Add 40 μg of alkaline phosphatase to 150 μg of succinimide-4-(N-maleimide)cyclohexane-1-hydroxy ester solution (0.05 mol / L), stir well to obtain alkaline phosphatase solution.

[0051] Add 50 μg of polyethylene glycol (PEG1000), 50 μg of maleimide, and 50 μg of dioleoylphosphatidylethanolamine to 1000 μg of dimethyl sulfoxide, then add 100 μg of gold nanoparticles (average particle size of 20 nm), and stir until homogeneous to obtain a gold nanoparticle solution.

[0052] (2) Mix 100 μg of p-Tau181 antibody solution, 80 μg of alkaline phosphatase solution and 50 μg of gold nanoparticle solution evenly, add 100 μg of glycine, stir evenly, and dilute 100 times with stabilizer to obtain anti-reagent B.

[0053] The magnetic microparticle reagent is prepared as follows: 10 mg of magnetic microparticles are resuspended in 100 mL of MES buffer (pH 5), then 150 μL of 0.1 mol / L 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide solution and 0.4 mg of N-hydroxysuccinimide are added, stirred evenly, 1 mg of streptavidin is added, and the mixture is reacted at room temperature for 2 h. After magnetic separation of the supernatant, the mixture is washed, and then 150 μg of glycine is added. The volume is adjusted to 500 mL with a stabilizer to obtain the magnetic microparticle reagent.

[0054] The stabilizer comprises the following components in weight percentage: 0.12% streptomycin, 0.35% fish peptone, 0.3% sucrose, 0.4% sodium lauroyl phosphate, 1% bovine serum albumin, 1.2% chitosan, 0.1% polylysine, 0.02% sodium azide, and the balance being phosphate buffer (molar concentration of 0.05 mol / L).

[0055] The p-Tau 181 antigen in the high-value calibrator was diluted with buffer to a concentration of 12 ng / mL; the p-Tau 181 antigen in the low-value calibrator was diluted with buffer to a concentration of 1.2 ng / mL; and the zero-value calibrator was diluted with buffer to a concentration of 0 ng / mL. The p-Tau 181 antigen in the high-value quality control was diluted with buffer to a concentration of 40 ng / mL; and the p-Tau 181 antigen in the low-value quality control was diluted with buffer to a concentration of 40 ng / mL. The buffer solution was a phosphate buffer of 0.5% BSA and 0.05% Prolin 300.

[0056] The substrate solution is a 0.5 mol / L solution of disodium 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoryl)-phenyl-1,2-dioxane.

[0057] The cleaning solution is Tris buffer.

[0058] The reagents can be packaged according to the following specifications: type A kits are 24 samples / box, and type B kits are 100 samples / box.

[0059] Table 1

[0060]

Test Methods

[0061] 1. Preparation before the experiment 1.1 All reagents must be brought to room temperature before the experiment, and the instrument must be turned on and preheated for at least 30 minutes; 1.2 Follow the system operation instructions to load the reaction tubes (cups), add or replace the reagents, clean the waste liquid and waste tubes, and fill the liquid lines. (This operation is only required for Type B reagents); 1.3 Calibrators: Once opened, calibrators can be stored sealed at 2–8°C for 28 days.

[0062] 2. Reagent and sample loading 2.1 Before loading the reagents, mix the reagents to be used thoroughly. Visually inspect the reagent solution components. They should be clear, free of foreign matter, precipitates and flocculent matter. The magnetic particle reagent should be a homogeneous suspension without obvious agglomeration. 2.2 Follow the system operation instructions to complete the reagent loading; 2.3 After mixing the calibrators and quality control samples, add them to the corresponding sample wells and place them in the appropriate adapter instrument. After scanning the samples, add them to the corresponding sample well positions and place them in the appropriate adapter instrument.

[0063] 3. Testing Steps For optimal detection performance, please follow the instructions in this manual and operate according to the corresponding instrument's operating manual.

[0064] Note: The detection reaction process and related parameters have been predefined in the instrument operating software.

[0065] 4. Calibration curve and calibration The calibration curve is obtained by scanning the master curve card directly, and then the fitted curve is corrected by three calibration points.

[0066] Note ①: Due to differences between instruments and system differences caused by different operators, operating environments and supporting general reagents, it is recommended to obtain the working calibration curve by three-point direct calibration.

[0067] Note ②: Due to reagent activity drift and batch changes of universal reagents, the calibrated working curve needs to be recalibrated after a certain period of use. Recalibration should be performed when the following situations occur: ① One month (28 days) after using the same batch of reagents; ② Use a new batch of reagent kits or substrates; ③ When the quality control value is outside the quality control range; ④ After each maintenance of the measuring instrument.

[0068] 5. Results Output The instrument automatically calculates the p-Tau181 concentration for each sample using the working curve, and the result is expressed in pg / mL.

[0069] 6. Quality Control 6.1 Quality control samples should be measured daily along with the sample testing, and treated as patient samples. 6.2 For the quality control scope, usage method and quality control cycle of the quality control products of this kit, please refer to the kit quality control sheet. If the quality control results do not meet the expectations, it indicates that the test results are unreliable and a test report should not be issued.

[0070] Example 2 The difference between Example 2 and Example 1 is that the composition of the stabilizer is different, but everything else is the same.

[0071] The stabilizer in this embodiment comprises the following components by weight percentage: 0.1% streptomycin, 0.4% fish peptone, 0.2% sucrose, 0.5% sodium lauroyl phosphate, 0.8% bovine serum albumin, 1.4% chitosan, 0.05% polylysine, 0.04% sodium azide, and the balance being phosphate buffer (molar concentration of 0.05 mol / L).

[0072] Example 3 The difference between Example 3 and Example 1 is that the composition of the stabilizer is different, but everything else is the same.

[0073] The stabilizer in this embodiment comprises the following components by weight percentage: 0.2% streptomycin, 0.2% fish peptone, 0.5% sucrose, 0.2% sodium lauroyl phosphate, 1.5% bovine serum albumin, 0.8% chitosan, 0.12% polylysine, 0.01% sodium azide, and the balance being phosphate buffer (molar concentration of 0.05 mol / L).

[0074] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that the preparation method of anti-reagent B is different, but everything else is the same.

[0075] The preparation method of anti-reagent B in this embodiment is as follows: (1) Add 100 μg of p-Tau181 antibody to 1000 μg of maleic acid buffer (molar concentration of 0.2 mol / L), stir well, then add 40 μg of dithiothreitol, stir at 50 °C for 1 h to obtain p-Tau181 antibody solution.

[0076] Add 40 μg of alkaline phosphatase to 150 μg of succinimide-4-(N-maleimide)cyclohexane-1-hydroxy ester solution (0.05 mol / L), stir well to obtain alkaline phosphatase solution.

[0077] (2) Mix 100 μg of p-Tau181 antibody solution and 80 μg of alkaline phosphatase solution evenly, add 100 μg of glycine, stir evenly, and dilute 100 times with stabilizer to obtain anti-reagent B.

[0078] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that the preparation method of anti-reagent B is different, but everything else is the same.

[0079] The preparation method of anti-reagent B in this embodiment is as follows: (1) Add 100 μg of p-Tau181 antibody to 1000 μg of maleic acid buffer (molar concentration of 0.2 mol / L), stir evenly, then add 20~50 μg of dithiothreitol, stir at 50℃ for 1 h to obtain p-Tau181 antibody solution.

[0080] Add 40 μg of alkaline phosphatase to 150 μg of succinimide-4-(N-maleimide)cyclohexane-1-hydroxy ester solution (0.05 mol / L), stir well to obtain alkaline phosphatase solution.

[0081] (2) Mix 100 μg of p-Tau181 antibody solution, 80 μg of alkaline phosphatase solution and 50 μg of gold nanoparticles (average particle size of 20 nm) evenly, add 100 μg of glycine, stir evenly, and dilute 100 times with stabilizer to obtain anti-reagent B.

[0082] Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that the preparation method of anti-reagent B is different, but everything else is the same.

[0083] The preparation method of the anti-reagent B is as follows: (1) Add 50 μg polyethylene glycol (PEG1000), 50 μg maleimide, and 50 μg dioleoylphosphatidylethanolamine to 1000 μg dimethyl sulfoxide, and then add 100 μg gold nanoparticles (average particle size of 20 nm), stir evenly, and obtain a gold nanoparticle solution.

[0084] (2) Mix 100 μg of p-Tau181 antibody, 80 μg of alkaline phosphatase and 50 μg of gold nanoparticle solution evenly, add 100 μg of glycine, stir evenly, and dilute 100 times with stabilizer to obtain anti-reagent B.

[0085] Comparative Example 4 The difference between Comparative Example 4 and Example 1 is that the stabilizer is different, but everything else is the same.

[0086] The stabilizer used in this comparative example was a phosphate buffer solution containing 0.5% BSA and 0.05% Prolin 300.

[0087] Comparative Example 5 The difference between Comparative Example 5 and Example 1 is that the stabilizer is different, but everything else is the same.

[0088] The stabilizer in this comparative example contains the following components by mass percentage: 0.12% streptomycin, 1% bovine serum albumin, 0.02% sodium azide, and the balance phosphate buffer (molar concentration of 0.05 mol / L).

[0089] 1. Limit of Detection Experiment: Using the stabilizer as a sample, the test was repeated 20 times. The RLU values ​​(relative luminescence values) of the 20 measurements were obtained, and their mean (M) and standard deviation (SD) were calculated to obtain M+2SD. A linear equation was obtained by performing a two-point regression fitting based on the calibration curve equation of the calibrators used in the kit or based on the concentration-RLU value results between zero concentration and adjacent calibrators. The RLU value corresponding to M+2SD was then substituted into the above equation to calculate the corresponding concentration, which is the limit of detection.

[0090] The detection limit (LOD) for Example 1 was 3.8 pg / mL. The LOD for Example 2 was 4.2 pg / mL; the LOD for Example 3 was 4.4 pg / mL; the LOD for Comparative Example 1 was 12.8 pg / mL; the LOD for Comparative Example 2 was 10.2 pg / mL; the LOD for Comparative Example 3 was 11.5 pg / mL; the LOD for Comparative Example 4 was 11.9 pg / mL; and the LOD for Comparative Example 5 was 10.6 pg / mL.

[0091] 2. Linearity Experiment: Dilute the high-value reference standard (close to the upper limit of the linear range) at a certain ratio to prepare five concentrations (1 pg / mL to 200 pg / mL), where the low-concentration samples must be close to the lower limit of the linear range. Follow the kit instructions, and repeat the test three times for each concentration. Calculate the average value, and fit the average result to the theoretical concentration using the least squares method to form a linear relationship. Calculate the linear correlation coefficient r.

[0092] ;

[0093] The results showed that within the range of 1 pg / mL to 200 pg / mL, the correlation coefficients (r) of Examples 1-3 and Comparative Example 3 were not less than 0.9900, which met the requirements. The correlation coefficients of Comparative Examples 1, 2, and 4-5 were less than 0.9900, which did not meet the requirements.

[0094] 3. Repeatability Test: Select repeatability reference standards with concentrations of 1 pg / mL and 200.00 pg / mL, perform the test, repeat the test 3 times, and calculate the relative deviation. The calculation formula is: ; In the formula: B -- Relative deviation; M -- Measurement result; T -- Calibration concentration.

[0095] The results showed that the repeatability of Examples 1-3 and Comparative Examples 3-4 was ≤8%, which met the requirements.

[0096] Finally, it should be noted that the above embodiments are used to illustrate the technical solutions of the present invention and not to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A kit for determining phosphorylated Tau181 protein, characterized in that, Including reagent A, reagent B, and magnetic microparticle reagent; The preparation method of the anti-reagent A is as follows: 100 μg of p-Tau217 antibody, 30-50 μg of biotin and 250-400 μg of phosphate buffer are mixed evenly, then 10-15 μg of 1-hydroxybenzotriazole, 5-10 μg of N,N-diisopropylethylamine and 5-10 μg of sodium borohydride are added and stirred evenly, then 100-200 μg of glycine is added and stirred evenly, and diluted with a stabilizer to obtain anti-reagent A; The method for preparing the anti-reagent B is as follows: 80-200 μg of p-Tau181 antibody solution, 50-100 μg of alkaline phosphatase solution and 40-80 μg of gold nanoparticle solution are mixed evenly, 50-120 μg of glycine is added, the mixture is stirred evenly, and then diluted with a stabilizer to obtain anti-reagent B.

2. The phosphorylated Tau181 protein assay kit according to claim 1, characterized in that, The preparation method of the p-Tau181 antibody solution is as follows: 100 μg of p-Tau181 antibody is added to 800~2000 μg of maleic acid buffer, stirred evenly, and then 20~50 μg of dithiothreitol is added. The mixture is stirred at 45~60℃ for 0.5~2h to obtain the antibody solution.

3. The phosphorylated Tau181 protein assay kit according to claim 2, characterized in that, The molar concentration of the maleic acid buffer solution is 0.1~0.5 mol / L.

4. The phosphorylated Tau181 protein assay kit according to claim 1, characterized in that, The alkaline phosphatase solution is prepared by adding 20-50 μg of alkaline phosphatase to 80-200 μg of succinimide-4-(N-maleimide)cyclohexane-1-hydroxy ester solution and stirring until homogeneous to obtain the alkaline phosphatase solution.

5. The phosphorylated Tau181 protein assay kit according to claim 1, characterized in that, The method for preparing the nano-gold solution is as follows: 40-80 μg of polyethylene glycol, 40-80 μg of maleimide, and 40-80 μg of dioleoylphosphatidylethanolamine are added to 800-2000 μg of dimethyl sulfoxide, and then 80-120 μg of nano-gold particles are added and stirred evenly to obtain the nano-gold solution.

6. The phosphorylated Tau181 protein assay kit according to claim 5, characterized in that, The molar concentration of the succinimide-4-(N-maleimide)cyclohexane-1-hydroxy ester solution is 0.01~0.1 mol / L.

7. The phosphorylated Tau181 protein assay kit according to claim 1, characterized in that, The magnetic microparticle reagent is prepared as follows: 10 mg of magnetic microparticles are resuspended in MES buffer, and then 100-200 μL of 0.05-0.2 mol / L 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide solution and 0.2-0.5 mg of N-hydroxysuccinimide are added. The mixture is stirred evenly, and 0.5-2 mg of streptavidin is added. The mixture is reacted at room temperature for 1-3 h. After magnetic separation of the supernatant, the mixture is washed, and then 100-200 μg of glycine is added. The volume is adjusted to 500 mL with a stabilizer to obtain the magnetic microparticle reagent.

8. The phosphorylated Tau181 protein assay kit according to claim 4, characterized in that, The stabilizer comprises the following components in weight percentages: 0.1-0.2% streptomycin, 0.2-0.4% fish peptone, 0.2-0.5% sucrose, 0.2-0.5% sodium lauroyl glutamate, 0.8-1.5% bovine serum albumin, 0.8-1.4% chitosan, 0.05-0.12% polylysine, 0.01-0.04% preservative, and the balance being phosphate buffer.

9. The phosphorylated Tau181 protein assay kit according to claim 1, characterized in that, The preservative is sodium azide.

10. The use of the phosphorylated Tau181 protein assay kit according to any one of claims 1 to 9 in the preparation of reagents for detecting Alzheimer's disease.

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