Method for detecting beta-amyloid protein in urine through magnetic particle chemiluminescence based on acridinium ester

Abstract

The invention discloses a method for detecting beta-amyloid protein in urine by magnetic particle chemiluminescence based on acridinium ester. The method comprises the following steps: (1) coupling carboxyl magnetic beads with an A beta 42 N-terminal antibody; (2) labeling acridinium ester and a Lec antibody; and (3) respectively diluting the A beta calibrator, the urine sample to be detected, the magnetic bead compound and the acridinium ester-Lec antibody compound, and carrying out luminescence detection on a luminescence instrument.

Description

Technical Field

[0001] The invention belongs to the technical field of magnetic particle chemiluminescence immunoassay, and specifically relates to a method for detecting beta-amyloid protein in urine by magnetic particle chemiluminescence based on acridinium ester. Background Art

[0002] Alzheimer's disease (AD), commonly known as senile dementia, is called a devastating disease by the FDA. It is a chronic progressive disease caused by the degeneration and necrosis of neuronal cells, which mainly occurs in the elderly or pre-elderly age. Clinical manifestations include memory loss and cognitive impairment. The 2018 Global Report of the Alzheimer's Disease International (ADI) pointed out that one dementia patient appears every three seconds in the world, and one in every seven elderly people in my country has cognitive impairment of varying degrees or types. According to a national cross-sectional study, there are 15.07 million patients with cognitive impairment aged 60 and above in China, including nearly 10 million patients with Alzheimer's disease. With the aging of the population in my country, the prevalence of Alzheimer's disease is increasing year by year, and it is showing a trend of younger age.

[0003] β-amyloid protein (Aβ) is a product of the breakdown of β-amyloid precursor protein (APP) by β and γ secretases. It is composed of 39 to 43 amino acid residues and accumulates inside and outside nerve cells to form senile plaques of Alzheimer's disease. It can also cause toxic reactions leading to neuronal degeneration and death. It is often used as an early screening signal for Alzheimer's disease in clinical practice.

[0004] Increased expression of β-amyloid protein in cerebral blood vessels and excessive deposition is one of the pathological mechanisms of Alzheimer's disease (AD). In the early stages of Alzheimer's disease, the concentration of β-amyloid protein will increase significantly, and in the middle and late stages, as the precipitation increases, the concentration will decrease. Therefore, β-amyloid protein detection is of great value in the early screening of Alzheimer's disease, dynamic observation of progressive disease changes, and dynamic tracking and evaluation of treatment effects.

[0005] There are many biological markers in cerebrospinal fluid that have advantages such as high efficiency and specificity in the diagnosis of Alzheimer's disease (AD) patients. However, since cerebrospinal fluid testing is an invasive examination and it is inconvenient to obtain samples, patients are not easy to accept, so it is not conducive to clinical development. Plasma is one of the best choices when routine examinations cannot confirm the diagnosis of Alzheimer's disease (AD) because it is simple and convenient to collect. However, the collection of plasma is traumatic and increases the risk of infection, local hematoma and puncture site pain. Urine specimens are easy to obtain and non-invasive, and patients have a high degree of acceptance.

[0006] Chemiluminescent immunoassay (CLIA) is highly sensitive and can detect analytes at low concentrations, and magnetic particles have the advantages of simple operation, high separation efficiency, large specific surface area and good physical stability. Currently, the detection of Aβ in the market mainly uses cerebrospinal fluid and blood samples, while the magnetic particle chemiluminescence method based on acridinium ester is still blank for detecting Aβ in urine samples. Summary of the invention

[0007] The present invention aims to overcome the deficiencies of the prior art and provides a method for detecting beta-amyloid protein in urine by chemiluminescence of magnetic microparticles based on acridinium ester.

[0008] In order to achieve the above object, the technical solution provided by the present invention is:

[0009] The method for detecting β-amyloid protein in urine by chemiluminescence of magnetic microparticles based on acridinium ester is characterized in that the method for detecting β-amyloid protein in urine by chemiluminescence of magnetic microparticles based on acridinium ester comprises the following steps:

[0010] (1) coupling carboxyl magnetic beads with Aβ42 N-terminal antibody: after magnetic separation and cleaning of carboxyl magnetic beads, adding EDC solution to activate them, magnetic separation and cleaning and ultrasonic treatment, adding Aβ42 N-terminal antibody solution, reacting at room temperature for 2-3 hours, after magnetic separation, washing with magnetic bead cleaning buffer and ultrasonic treatment, blocking the magnetic bead complex at least twice to obtain a coupled magnetic bead complex, which is stored in a preservation solution at 4°C; the mass volume ratio of the carboxyl magnetic beads to the EDC solution is 10 mg: (40-100) μL, preferably 10 mg: 60 μL, the EDC concentration in the EDC solution is 10 mg / mL, and the concentration of the Aβ42 N-terminal antibody in the Aβ42 N-terminal antibody solution is 40-500 μg / mL, preferably 200 μg / mL;

[0011] (2) Labeling with acridinium ester and Lec antibody: prepare a Lec antibody solution with a Lec antibody concentration of 0.25 mg / mL, then add acridinium ester, mix well, react at room temperature in the dark for 1 hour, then add lysine to react for 0.5 hour, pass through a G25 dextran column to remove free acridinium ester, and obtain an acridinium ester-Lec antibody complex; the molar mass ratio of the Lec antibody to the acridinium ester is 1:5 to 1:50, preferably 1:30, and the initial mass percentage of the lysine in the reaction system is 1%;

[0012] (3) The Aβ calibrator, the urine sample to be tested, the magnetic bead complex obtained in step (1), and the acridinium ester-Lec antibody complex obtained in step (2) are diluted separately and then subjected to luminescence detection in a luminometer.

[0013] Preferably, in step (1), the EDC solution is prepared from a MES solution having an EMS concentration of 0.05-0.2 M and a pH value of 5.0-7.0, preferably 0.1 M and a pH value of 5.0, and the Aβ42 N-terminal antibody solution is prepared from a MES solution having an EMS concentration of 0.05-0.2 M and a pH value of 5.0-8.0, preferably 0.1 M and a pH value of 7.0.

[0014] Preferably, in step (1), the magnetic bead complex is subjected to two blocking treatments, each blocking treatment lasting 1 hour.

[0015] More preferably, the first blocking treatment is performed using an ethanolamine solution with an ethanolamine concentration of 0.025-0.25 M, preferably 0.25 M, and a pH value of 7.4, and the second blocking treatment is performed using 1% BSA.

[0016] Preferably, the components and concentrations of the preservation solution in step (1) are: 0.1M PBS, 1% BSA, 0.1% Triton, 2% trehalose, 0.05% PC-300, and the pH value of the 0.1M PBS is 7.4.

[0017] Preferably, in step (2), a Lec antibody solution having a Lec antibody concentration of 0.25 mg / mL is prepared using a NaHCO3 solution having a NaHCO3 concentration of 50 mM, and the pH value of the NaHCO3 solution is 8.3-9.6, preferably 9.0.

[0018] Preferably, in step (3), the Aβ calibrator is gradiently diluted with a sample diluent so that the concentration of the standard is 0, 5, 10, 20, 100, 500 pg / mL; the urine sample to be tested is gradiently diluted with a sample diluent so that the concentration of the urine sample to be tested is original urine, 1 / 2 original urine, 1 / 4 original urine, 1 / 8 original urine, and sample diluent; the magnetic bead complex is diluted with a magnetic bead diluent at a volume ratio of 1:30-1:60, preferably 1:30; the acridinium ester-Lec antibody complex is diluted with an acridinium ester marker diluent to a luminescence value of 400,000-6400,000, preferably 3200,000.

[0019] Preferably, the components and concentrations in the sample diluent are: 200mM PBS, 1% BSA, 0.1% Triton, 2% trehalose, 0.05% PC-300, and the pH value of the 200mM PBS is 6.5; the components and concentrations in the magnetic bead diluent are: 0.1M PBS, 1% BSA, 0.1% Triton, 2% trehalose, 0.05% PC-300, and the pH value of the 0.1M PBS is 7.4; the components and concentrations in the acridinium ester marker diluent are: 20mM PBS, 1% BSA, 0.1% Triton, 2% trehalose, 0.05% PC-300, and the pH value of the 20mM PBS is 7.4.

[0020] The Aβ42 N-terminal antibody in the present invention can be a monoclonal antibody WO-2 or the like.

[0021] The present invention provides a method for detecting β-amyloid protein in urine by acridinium ester-based magnetic microparticle chemiluminescence using Aβ42 N-terminal antibody and Lec antibody (lecanemab, an Aβ monoclonal antibody drug jointly developed by Biogen and Eisai, with a trade name of Leqembi). The reagent prepared by this method can be used for the detection of Aβ in urine samples with high detection sensitivity. DETAILED DESCRIPTION

[0022] Example 1: Reagent and its preparation process, and its use in the detection process of Aβ in urine samples

[0023] 1 Carboxyl magnetic beads are coupled with the AΒ42 N-terminal antibody

[0024] 1.1.1 Magnetic bead activation

[0025] 1) After vortexing the carboxyl magnetic bead solution, take 10 mg of the carboxyl magnetic bead solution into a 1.5 mL centrifuge tube, magnetically absorb for 1 min, and remove the supernatant.

[0026] 2) Add 1000 μL 0.1 M MES (pH 5.0) to the magnetic beads, vortex to disperse, magnetically absorb for 1 min, remove the supernatant, and wash 3 times;

[0027] 3) Add 1000 μL 0.1 M MES (pH 5.0) to the magnetic beads, vortex to disperse and mix, and add 60 μL EDC solution (10 mg / mL);

[0028] (Note: EDC is prepared with 0.1M MES (pH 5.0) solution and needs to be prepared and used immediately)

[0029] 4) After vortexing and mixing the magnetic beads, place them on a rotator and allow the magnetic beads to react at room temperature for 30 minutes;

[0030] 5) After the reaction is completed, remove the supernatant by magnetic separation;

[0031] 6) Add 1000 μL of 0.1 M MES (pH 7.0) solution to the magnetic beads, vortex to disperse, magnetically absorb for 1 min, remove the supernatant, and wash 3 times;

[0032] 7) The activated magnetic beads were ultrasonically disrupted on ice for 1 second on, 1 second off, for a total of 3 minutes;

[0033] 1.1.2 Coupling of magnetic beads with Aβ42 N-terminal antibody

[0034] 1) Add 1000 μL of 0.1 M MES (pH 7.0) solution (containing the antibody at a final concentration of 200 μg / mL) to the magnetic beads and vortex to mix (vortex gently to avoid creating bubbles);

[0035] 2) Place the magnetic bead complex on a rotator and react at room temperature for 2-3 hours;

[0036] 3) After the reaction is completed, remove the supernatant by magnetic separation;

[0037] 4) Add 1000 μL of magnetic bead washing buffer (25 mM Tris-HCl (tris(hydroxymethylaminomethane) hydrochloride, pH 7.4), 0.15 M Nacl (sodium chloride), 0.05% Tween 20)) to the magnetic bead complex, vortex to disperse, remove the supernatant by magnetic separation, and wash 3 times;

[0038] 5) The coupled magnetic bead complex was subjected to ultrasonic treatment on ice, with an ultrasonic pulse of 1 second and a stop pulse of 1 second for a total of 3 minutes;

[0039] 1.1.3 Closure process

[0040] 1) The magnetic bead complex was blocked with 0.25 M ethanolamine (diluted with 25 mM Tris-HCl (pH 7.4)) and placed on a rotator for reaction at room temperature for 1 h. After blocking, the supernatant was removed by magnetic separation;

[0041] 2) Add 1000 μL of magnetic bead washing buffer (25 mM Tris-HCl (pH 7.4), 0.15 M Nacl, 0.05% Tween 20) to the magnetic bead complex, vortex to disperse, remove the supernatant by magnetic separation, and wash three times;

[0042] 3) The magnetic bead complex was blocked with 1% BSA and placed on a rotator for reaction at room temperature for 1 hour. After blocking, the supernatant was removed by magnetic separation;

[0043] 4) Add 1000 μL of magnetic bead washing buffer (25 mM Tris-HCl (pH 7.4), 0.15 M Nacl, 0.05% Tween 20) to the magnetic bead complex, vortex to disperse, remove the supernatant by magnetic separation, and wash three times;

[0044] 5) Add 1000 μL magnetic bead storage solution (0.1M PBS (pH 7.4), 1% BSA, 0.1% Triton, 2% trehalose, 0.05% PC-300) to the magnetic bead complex

[0045] 6) After overnight storage, the magnetic bead complex was ultrasonically disrupted on ice, with an ultrasound cycle of 1 second for 3 minutes and 2 rounds of ultrasound.

[0046] 1.2 Labeling with acridinium ester and Lec antibody

[0047] 1) Take 0.2 mg of Lec antibody and dilute the stock solution with 50 mM NaHCO3 (pH 9.0) solution to make the working solution concentration 0.25 mg / mL.

[0048] 2) The concentration of the acridinium ester stock solution is 3 mg / mL.

[0049] 3) mixing the protein and the acridinium ester in a ratio of 1:30 by molar mass;

[0050] 4) The molecular weight of Lec is 150 kD, and the molecular weight of acridinium ester is 681.73. To label 0.2 mg of Lec antibody, 0.027 mg of acridinium ester is required.

[0051] Specific implementation: Take 2 μL of Lec antibody with a mother solution concentration of 100 mg / mL, add 798 μL of 50 mM NaHCO3 (pH 9.0) solution to prepare 800 μL of Lec antibody solution with a working concentration of 0.25 mg / mL, vortex mix, add 9 μL of acridinium ester with a working concentration of 3 mg / mL, and vortex mix.

[0052] 4) Place on a rotator and react for about 1 hour at room temperature and away from light;

[0053] 5) Add lysine at a final concentration of 1% to terminate the reaction and react for 0.5 h;

[0054] 6) Use a G25 dextran column to remove free acridinium esters, wash the column with 2 column volumes of water, equilibrate the column with 2 column volumes of 10 mM PBS (pH 7.4), load the sample, elute with 3 column volumes of 10 mM PBS (pH 7.4), collect the first 10 tubes, 1 mL per tube, then wash the column with 3 column volumes of water and store with 1 column volume of 20% ethanol.

[0055] 7) The collected eluate was tested for protein concentration using A280 and luminescence value using a luminometer;

[0056] 8) Add an equal volume of glycerol to the target acridinium ester-Lec and store it at 4°C.

[0057] 1.3 Detection of calibrants and urine by coupling the magnetic bead complex with labeled acridinium ester-Lec (Table 1)

[0058] 1) The calibrator was used as a sample for detection. The stock solution (1 mg / mL) was diluted with sample diluent (200 mM PBS (pH 6.5), 1% BSA, 0.1% Triton, 2% trehalose, 0.05% PC-300) in proportion to make the concentrations 0, 5, 10, 20, 100, and 500 pg / mL.

[0059] 2) Urine was used as a sample for testing, and the original urine was diluted with sample diluent (200 mM PBS (pH 6.5), 1% BSA, 0.1% Triton, 2% trehalose, 0.05% PC-300) at a ratio of 1:1, so that the concentration gradient was original urine, 1 / 2 original urine, 1 / 4 original urine, 1 / 8 original urine, and sample diluent.

[0060] 3) diluting the coupled magnetic bead complex with magnetic bead diluent (0.1M PBS (pH 7.4), 1% BSA, 0.1% Triton, 2% trehalose, 0.05% PC-300) at a ratio of 1:30;

[0061] 1) Dilute the labeled acridinium ester-Lec with acridinium ester labeling diluent (20 mM PBS (pH 7.4), 1% BSA, 0.1% Triton, 2% trehalose, 0.05% PC-300) in proportion so that its luminescence value is controlled at about 32 million;

[0062] 2) The treated samples, magnetic bead complex and acridinium ester-Lec are detected on a luminometer.

[0063] Table 1

[0064]

[0065] Example 2: Optimization of reagents and preparation methods

[0066] 1) Optimization of coupling pH between magnetic beads and Aβ42 N-terminal antibody

[0067] 10 mg of carboxyl magnetic beads were coupled with Aβ42 N-terminal antibody. The pH in the coupling system was 5.0, 5.5, 6.0, 6.5, 7.0, and 7.5, respectively. The experimental results are shown in Table 2: With the increase of coupling pH, there was no obvious change in the background, but there were certain differences in the signal response. At pH 7.0, the signal response value was the strongest, which had certain advantages.

[0068] Table 2. Different coupling pH values ​​when magnetic beads are coupled to antibodies

[0069] Magnetic beads coupled with QK-03 pH 5.0 5.5 6.0 6.5 7.0 7.5 C3=500pg / mL 3716 3910 3263 2616 2888 2471 C2=100pg / mL 704 793 664 628 651 541 C1=0pg / mL 221 254 234 183 150 203 C3 / C1 16.8 15.4 13.9 14.3 19.3 12.2

[0070] 2) Optimization of the dilution ratio of magnetic bead complex

[0071] The magnetic bead complex was diluted with different dilution ratios. The experimental results are shown in Table 3. As the dilution ratio of the magnetic bead complex increased, the background did not change significantly, but the signal response gradually increased. When diluted at 1:30, the signal response value was the strongest, which had certain advantages.

[0072] Table 3. Optimization of dilution ratio of magnetic bead complex

[0073] Dilution ratio of the compound 1:60 1:50 1:40 1:30 C3=500pg / mL 1280 1341 1454 1847 C2=100pg / mL 517 676 443 528 C1=0pg / mL 138 132 127 150 C3 / C1 9.3 10.1 11.4 12.3

[0074] 3) Optimization of magnetic bead complex release

[0075] By optimizing the pH value and ion concentration in the magnetic bead diluent system, the experimental results are shown in Table 4. The magnetic bead diluent 0.1M PBS (pH7.4), 1% BSA, 0.1% Triton, 2% trehalose, and 0.05% PC300 has obvious advantages.

[0076] Table 4. Optimization of magnetic bead complex dilution

[0077]

[0078] 4) pH value of acridinium ester and Lec

[0079] 0.2 mg of Lec antibody was labeled with acridinium ester at a molar mass ratio of 1:30 under different pH systems. The experimental results are shown in Table 5: For the AE-Lec marker, pH 9.0 in the labeling system and blocking for half an hour have obvious advantages, with low background and high response value.

[0080] Table 5. Labeled pH values ​​of acridinium esters and Lec

[0081]

[0082]

[0083] 5) Optimization of AE-Lec marker concentration

[0084] Different AE-Lec marker concentrations were used for detection. The experimental results are shown in Table 6. As the concentration of AE-Lec marker increases, its signal response gradually increases. When the luminescence value is controlled at around 32 million, it has a clear advantage.

[0085] Table 6. Optimization of AE-Lec marker concentrations

[0086] Luminous value 1 million 2 million 4 million 8 million 16 million 32 million 64 million C6=500pg / mL 15302 22275 15291 26727 46193 102109 158606 C5=100pg / mL 3817 5841 3552 6345 11085 25227 36501 C4=20pg / mL 1342 1881 1038 1505 2443 5360 7196 C3=10pg / mL 937 1310 663 871 1701 2875 4442 C2=5pg / mL 810 1131 564 683 1187 1861 2322 C1=0pg / mL 623 758 340 385 606 1072 3054 C6 / C1 24.6 29.4 45.0 69.4 76.2 95.3 51.9

[0087] 6) Optimization of AE marker dilution

[0088] By optimizing the pH and ion concentration of the AE marker diluent, the experimental results are shown in Table 7. With the increase of ion concentration and the decrease of pH value, the background is significantly increased and the sensitivity is greatly reduced. The diluents 0.02M PBS (pH7.4), 1% BSA, 0.1% Triton, 2% trehalose, and 0.05% PC300 have obvious advantages.

[0089] Table 7. Optimization of pH and ion concentration of AE marker dilution solution

[0090]

[0091] 7) Calibrator dilution optimization

[0092] By optimizing the ion concentration and pH value of the calibration diluent, the experimental results are shown in Table 8, and it is found that the diluent 0.2M PBS (pH 6.5), 1% BSA, 0.1% Triton, 2% trehalose, and 0.05% PC300 has certain advantages.

[0093] Table 8. Optimization of ion concentration and pH of calibrator dilutions

[0094]

Claims

1. A method for detecting β-amyloid protein in urine by chemiluminescence of magnetic microparticles based on acridinium ester, characterized in that: The method comprises the following steps: (1) coupling carboxyl magnetic beads with Aβ42 N-terminal antibody: after magnetic separation and cleaning of carboxyl magnetic beads, adding EDC solution to activate them, magnetic separation and cleaning and ultrasonic treatment, adding Aβ42 N-terminal antibody solution, reacting at room temperature for 2-3 hours, after magnetic separation, washing with magnetic bead cleaning buffer and ultrasonic treatment, blocking the magnetic bead complex at least twice to obtain a coupled magnetic bead complex, which is stored in a preservation solution at 4°C; the mass volume ratio of the carboxyl magnetic beads to the EDC solution is 10 mg: (40-100) μL, the EDC concentration in the EDC solution is 10 mg / mL, and the concentration of the Aβ42 N-terminal antibody in the Aβ42 N-terminal antibody solution is 40-500 μg / mL; (2) Labeling with acridinium ester and Lec antibody: prepare a Lec antibody solution with a Lec antibody concentration of 0.25 mg / mL, then add acridinium ester, mix well, react at room temperature in the dark for 1 hour, then add lysine to react for 0.5 hour, pass through a G25 dextran column to remove free acridinium ester, and obtain an acridinium ester-Lec antibody complex; the molar mass ratio of the Lec antibody to the acridinium ester is 1:5 to 1:50, and the initial mass percentage of the lysine in the reaction system is 1% at the final concentration; (3) The Aβ calibrator, the urine sample to be tested, the magnetic bead complex obtained in step (1), and the acridinium ester-Lec antibody complex obtained in step (2) are diluted separately and then subjected to luminescence detection on a luminometer.

2. The method for detecting β-amyloid protein in urine by chemiluminescence of magnetic microparticles based on acridinium ester according to claim 1, characterized in that: In step (1), the EDC solution is prepared from a MES solution having an EMS concentration of 0.05-0.2 M and a pH value of 5.0-7.0, and the Aβ42 N-terminal antibody solution is prepared from a MES solution having an EMS concentration of 0.05-0.2 M and a pH value of 5.0-8.

0.

3. The acridinium ester-based magnetic microparticle chemiluminescence detection method according to claim 1, characterized in that: In step (1), the magnetic bead complex is blocked twice, each blocking treatment lasting 1 hour.

4. The method for detecting β-amyloid protein in urine by chemiluminescence of magnetic microparticles based on acridinium ester according to claim 3, characterized in that: The first blocking treatment was performed using an ethanolamine solution with an ethanolamine concentration of 0.025-0.25 M, and the second blocking treatment was performed using 1% BSA.

5. The method for detecting β-amyloid protein in urine by chemiluminescence of magnetic microparticles based on acridinium ester according to claim 1, characterized in that: The components and concentrations of the preservation solution in step (1) are: 0.1M PBS, 1% BSA, 0.1% Triton, 2% trehalose, and 0.05% PC-300. The pH value of the 0.1M PBS is 7.

4.

6. The method for detecting β-amyloid protein in urine by chemiluminescence of magnetic microparticles based on acridinium ester according to claim 1, characterized in that: In step (2), a Lec antibody solution with a Lec antibody concentration of 0.25 mg / mL is prepared using a NaHCO3 solution with a NaHCO3 concentration of 50 mM, and the pH value of the NaHCO3 solution is 8.3-9.

6.

7. The method for detecting β-amyloid protein in urine by chemiluminescence of magnetic microparticles based on acridinium ester according to claim 1, characterized in that: In step (3), the Aβ calibrator is diluted with the sample diluent in a gradient manner so that the concentration of the standard is 0, 5, 10, 20, 100, and 500 pg / mL; the urine sample to be tested is diluted with the sample diluent in a gradient manner so that the concentration of the urine sample to be tested is original urine, 1 / 2 original urine, 1 / 4 original urine, 1 / 8 original urine, and sample diluent; The magnetic bead complex was diluted with magnetic bead diluent at a volume ratio of 1:30-1:60, and the acridinium ester-Lec antibody complex was diluted with acridinium ester marker diluent to a luminescence value of 400,000-6400,000.

8. The method for detecting β-amyloid protein in urine by chemiluminescence of magnetic microparticles based on acridinium ester according to claim 7, characterized in that: The components and concentrations in the sample diluent are: 200mM PBS, 1% BSA, 0.1% Triton, 2% trehalose, 0.05% PC-300, and the pH value of the 200mM PBS is 6.5; the components and concentrations in the magnetic bead diluent are: 0.1M PBS, 1% BSA, 0.1% Triton, 2% trehalose, 0.05% PC-300, and the pH value of the 0.1M PBS is 7.4; the components and concentrations in the acridinium ester marker diluent are: 20mM PBS, 1% BSA, 0.1% Triton, 2% trehalose, 0.05% PC-300, and the pH value of the 20mM PBS is 7.

4.

9. A magnetic microparticle chemiluminescent detection reagent based on acridinium ester, characterized in that: The acridinium ester-based magnetic microparticle chemiluminescent detection reagent is a magnetic bead complex obtained by coupling carboxyl magnetic beads with Aβ42 N-terminal antibodies, and the magnetic bead complex is prepared according to step (1) of the method for detecting β-amyloid protein in urine by acridinium ester-based magnetic microparticle chemiluminescent detection according to claim 1.

10. A magnetic microparticle chemiluminescent detection reagent based on acridinium ester, characterized in that: The acridinium ester-based magnetic microparticle chemiluminescent detection reagent is an acridinium ester-Lec antibody complex, and the acridinium ester-Lec antibody complex is prepared according to step (2) of the method for detecting β-amyloid protein in urine by acridinium ester-based magnetic microparticle chemiluminescent detection according to claim 1.

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