A reagent combination for detecting a beta-amyloid precursor fragment A beta in urine based on chemiluminescence

Abstract

The application discloses a reagent combination for detecting beta-amyloid precursor fragment A beta in urine based on a chemiluminescence method. It is composed of reagent R1, reagent R2 and reagent R3; the reagent R1 is prepared by diluting carboxyl magnetic beads coupled with beta-amyloid monoclonal antibodies with a magnetic bead diluent at a volume ratio of 1:30-1:60; the reagent R2 is prepared by diluting arycridine ester labeled humanized IgG1 monoclonal antibodies with an arycridine ester diluent to control the luminescence value to be more than 60 million; the reagent R3 is prepared by diluting a blocking agent with a blocking agent diluent to a concentration of the blocking agent being more than 8 ug / mL; and the blocking agent is a mouse IgG+HAMA and RF specific antibody.

Description

Technical Field

[0001] This invention belongs to the field of magnetic microparticle chemiluminescence immunoassay technology, specifically relating to a reagent combination for detecting the precursor fragment Aβ of β-amyloid protein in urine based on chemiluminescence method. Background Technology

[0002] Alzheimer's disease (AD), commonly known as senile dementia, is described by the FDA as a devastating disease. It is a chronic, progressive disease caused by the degeneration and death of neurons, primarily affecting the elderly or pre-senile. Clinically, it manifests as memory loss and cognitive impairment. The 2018 global report by Alzheimer's Disease International (ADI) indicates that one person worldwide is diagnosed with dementia every three seconds, and in my country, one in seven elderly people suffers from varying degrees or types of cognitive impairment. A national cross-sectional study shows that there are 15.07 million people aged 60 and above with cognitive impairment in China, of whom nearly 10 million have Alzheimer's disease. With the aging of my country's population, the prevalence of Alzheimer's disease is increasing year by year and showing a trend towards affecting younger people.

[0003] β-amyloid precursor is a transmembrane protein highly expressed in the brain. It can be rapidly metabolized by various proteases, including α, β, γ, η, and δ-secretases. This process produces multiple β-amyloid precursor fragments, including Aβ1-40 and Aβ1-42. The generation of neurotoxic Aβ1-42 through the continuous proteolysis of β-amyloid precursor is one of the key steps in the pathogenesis of Alzheimer's disease (AD).

[0004] Cerebrospinal fluid (CSF) contains many biomarkers that offer advantages in the diagnosis of Alzheimer's disease (AD) due to their high efficiency and specificity. However, CSF testing is invasive, and the inconvenience of sample collection makes it difficult for patients to accept, hindering its clinical implementation. Plasma, due to its simple and convenient collection, is one of the best options when routine examinations cannot definitively diagnose AD. However, plasma collection is invasive and increases the risk of infection, local hematoma, and puncture site pain. In recent years, increasing research has found that the β-amyloid precursor fragment Aβ can also be detected in urine, making urine a promising source of non-invasive, convenient, and repeatable early AD screening samples. However, the concentration of Aβ in urine is extremely low, and the urine matrix is ​​complex, containing various interfering substances (such as urea, uric acid, creatinine, and salt ions). Furthermore, Aβ concentration fluctuates significantly and is easily affected by factors such as diet, medication, and individual differences, placing high demands on the sensitivity, specificity, and resistance to matrix interference of the detection methods. Summary of the Invention

[0005] The present invention aims to overcome the problems of invasive, expensive, and complex operation of AD marker detection in the prior art, as well as the serious interference of urine matrix and the difficulty in accurately quantifying low concentrations of Aβ. It provides a reagent combination for detecting the precursor fragment Aβ of β-amyloid protein in urine based on chemiluminescence method.

[0006] To achieve the above objectives, the technical solution provided by this invention is as follows: The reagent combination for detecting the precursor fragment Aβ of β-amyloid protein in urine using chemiluminescence immunoassay consists of reagents R1, R2, and R3. Reagent R1 is prepared by diluting carboxyl magnetic beads conjugated with β-amyloid monoclonal antibody at a volume ratio of 1:30 to 1:60 using magnetic bead diluent. Reagent R2 is prepared by diluting acridine ester-labeled humanized IgG1 monoclonal antibody with acridine ester diluent to a luminescence value controlled above 60 million. Reagent R3 is prepared by diluting an inhibitor with an inhibitor diluent to a concentration of 8 μg / mL or higher. The inhibitor is mouse IgG+HAMA and RF-specific antibody.

[0007] Preferably, the magnetic bead diluent contains the following components and concentrations: 50-200 mmol / L PBS, 0.5-2% BSA, 0.05-0.5% Triton, 1-5% trehalose, 0.05-0.2% PC-300, 0.5-5% PEG-6000, and 0.1-2% histidine, with a pH of 7.2-7.4; the acridinium ester diluent contains the following components and concentrations: 10-100 mmol / L PBS, 0.5-2% BSA, 1-10% trehalose, 0.05-0.5% Triton, 0.05-0.2% PC-300, 0.5-2% PEG-20000, 0.5-2% histidine, and 0.5-2% mannitol, with a pH of 6.5-6.8; and the blocking agent diluent contains the following components and concentrations: 10-100 mmol / L. PBS, 0.5-2% BSA, 1-10% trehalose, 0.05-0.5% Triton, 0.05-0.2% PC-300, 0.5-2% PEG-20000, 0.5-2% histidine, 0.5-2% mannitol, pH 6.5-6.8; all percentages refer to w / v, where w is in g and v is in mL.

[0008] More preferably, the magnetic bead diluent contains the following components and concentrations: 100 mmol / L PBS, 1% BSA, 0.1% Triton, 3% trehalose, 0.1% PC-300, 2% PEG-6000, and 0.5% histidine, with a pH of 7.2-7.4; the acridinium ester diluent contains the following components and concentrations: 20 mmol / L PBS, 2% BSA, 5% trehalose, 0.1% Triton, 0.1% PC-300, 1% PEG-20000, 1% histidine, and 1% mannitol, with a pH of 6.8; and the blocking agent diluent contains the following components and concentrations: 20 mmol / L PBS, 2% BSA, 5% trehalose, 0.1% Triton, 0.1% PC-300, 1% PEG-20000, 1% histidine, and 1% mannitol, with a pH of 6.8.

[0009] Preferably, the volume ratio of the reagent combination when used is 1:1:1.

[0010] Preferably, when using the sample, the sample is first incubated with reagents R2 and R3, and then incubated with reagent R1.

[0011] More preferably, the volume ratio of reagents R1, R2, and R3 to the sample to be tested is 4:9 to 6:7.

[0012] More preferably, the volume ratio of reagent R1, reagent R2 and reagent R3 to the sample to be tested is 5:8.

[0013] Preferably, the time for incubating the sample to be tested with reagents R2 and R3 is 10 min, and the time for incubating the sample to be tested with reagent R1 is 20 min.

[0014] The reagent combination for detecting the precursor fragment Aβ of β-amyloid protein in urine based on chemiluminescence method described in this invention can be used to prepare reagents for Alzheimer's disease screening or auxiliary diagnosis.

[0015] The present invention will be further described below: The detection principle of this invention is a double-antibody sandwich chemiluminescent immunoassay. When the urine sample to be tested is mixed and incubated with reagents R2 and R3, the β-amyloid precursor fragment Aβ antigen in the sample binds to the humanized IgG1 monoclonal antibody, forming an Aβ antigen-humanized IgG1 monoclonal antibody complex. When this complex is mixed and incubated with reagent R1, it binds to the β-amyloid monoclonal antibody coupled to carboxyl magnetic beads, forming a sandwich structure of "magnetic microparticles-β-amyloid monoclonal antibody-Aβ antigen-acrididine ester-humanized IgG1 monoclonal antibody". After magnetic separation and washing, pre-excitation solution and excitation solution are added to generate a chemiluminescent signal. The signal intensity is positively correlated with the Aβ concentration in the urine sample. The Aβ content in the urine sample is quantitatively calculated using a calibrator standard curve.

[0016] The reagent combination for detecting β-amyloid precursor fragment Aβ in urine based on chemiluminescence immunoassay described in this invention can be used to detect β-amyloid precursor fragment Aβ in urine samples, which can significantly improve the sensitivity and accuracy of the detection results and has broad application prospects.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: 1) The sample source is non-invasive and easy to accept: it uses urine samples, which does not require venous blood collection or lumbar puncture, and has high patient acceptance, making it suitable for large-scale population screening and long-term dynamic monitoring; 2) High sensitivity and accurate quantification: Chemiluminescence detection can reach the pg / mL level, accurately detecting trace amounts of Aβ in urine, and can be used for early screening, disease tracking and efficacy evaluation; 3) Easy to operate and high detection efficiency: It can achieve automated, high-throughput detection on a fully automated chemiluminescence analyzer, with high detection efficiency and small human error; 4) High specificity and good anti-interference ability: The β-amyloid monoclonal antibody and the humanized IgG1 monoclonal antibody bind specifically, and the introduction of blocking agents effectively improves the accuracy of detection and anti-interference ability. Attached Figure Description

[0018] Figure 1 The figure shows the experimental results in Example 2, section 1.2.

[0019] Figure 2 The figure shows the experimental results in section 2.2 of Example 2.

[0020] Figure 3 and Figure 4 The figure shows the experimental results in section 3.2 of Example 2. Detailed Implementation

[0021] Example 1: Preparation of reagents R1, R2, and R3

[0022] 1. Preparation of reagent R1 Reagent R1 contains carboxyl magnetic beads and magnetic bead diluent conjugated with antibodies. The reagent containing carboxyl magnetic beads and magnetic bead diluent conjugated with β-amyloid monoclonal antibody is designated as Reagent R1-1, and the reagent containing carboxyl magnetic beads and magnetic bead diluent conjugated with humanized IgG1 monoclonal antibody is designated as Reagent R1-2.

[0023] 1) The coupling process between carboxyl magnetic beads and antibodies is as follows: Take 10 mg of carboxyl magnetic beads, and after magnetic separation and washing, resuspend them in 0.1 M MES (2-(N-morpholine) ethanesulfonic acid, pH 5.5). At the same time, add 60 μL of EDC solution (1-ethyl-(3-dimethylaminopropyl)carbodiimide, 10 mg / mL) and 60 μL of NHS solution (N-hydroxysuccinimide) and vortex mix. React at room temperature for 15 min to activate the magnetic beads. After magnetic separation and washing, add MES solution containing antibody with a final concentration of 200 µg / mL and react at room temperature for 3-4 h. Perform multiple blocking treatments on the magnetic bead complex. After magnetic separation, wash with magnetic bead washing buffer and store in magnetic bead preservation solution. During the entire process of magnetic bead-antibody conjugation, the magnetic beads are subjected to multiple ice bath sonication treatments. The first sonication treatment is before the addition of antibody, the second sonication treatment is before the first blocking treatment, the third sonication treatment is before the second blocking treatment, and the fourth sonication treatment is after the addition of magnetic bead preservation solution.

[0024] Multiple ultrasonic disruption processes ensure that the magnetic beads form a uniform monodisperse system. Effectively dispersed magnetic beads guarantee the uniformity of each reaction during coupling, thereby significantly improving coupling efficiency and consequently significantly improving detection sensitivity and specificity. At the same time, the standardized ultrasonic disruption process helps reduce batch-to-batch variations between experiments, resulting in more consistent and reliable results.

[0025] The preferred formulation of the magnetic bead diluent is 100 mmol / L PBS, 1% BSA, 0.1% Triton, 3% trehalose, 0.1% PC300, 2% PEG6000, 0.5% histidine, with a pH of 7.2-7.4. 3) Reagent R1 is prepared by diluting the carboxyl magnetic beads of the conjugated antibody at a ratio of 1:40 using magnetic bead diluent.

[0026] 2. Preparation of reagent R2 Reagent R2 contains a labeled acridinium ester antibody and acridinium ester diluent. The reagent containing a labeled acridinium ester humanized IgG1 monoclonal antibody and acridinium ester diluent is reagent R2-1, and the reagent containing a labeled acridinium ester β-amyloid monoclonal antibody and acridinium ester diluent is reagent R2-2.

[0027] 2) The acridinium ester and antibody labeling process is as follows: Label 0.1 mg of antibody and add a certain volume of 50 mM CB solution (a mixture of sodium carbonate and sodium bicarbonate, pH 9.0) to make the working solution concentration 0.25 mg / mL. Add a certain amount of acridine ester to the antibody at a molar mass ratio of 1:30, vortex to mix, and react at room temperature in the dark for 0.5-1 h. Add 1% glycine to the final concentration and react for 0.5 h. Centrifuge the solution at 12000 rpm for 10 min, and pass it through a G25 dextran column to remove free acridine ester. Detect the protein concentration and luminescence value of the eluent. Take the two eluent tubes with the highest protein concentration and luminescence value, and perform ultrafiltration centrifugation using a 50 kDa ultrafiltration tube at 3500 rpm for 1 h. After centrifugation, add 10 mM CB solution. PBS (pH 7.4), repeat 3-5 times until the amount of acridine ester in the waste liquid after ultrafiltration and centrifugation is small. Add an equal volume of glycerol to the target acridine ester-antibody and store at -20°C.

[0028] The acridine ester and antibody labeling process involved two separation and purification steps. In the acridine ester labeling reaction system, the reaction product (acidine ester-labeled antibody) coexisted with impurities (free acridine ester, by-products, salt ions, etc.). Using either method alone has certain limitations; however, combining the two methods can achieve a purification effect of "1+1>2".

[0029] First purification: After the labeling reaction is completed, a large amount of unreacted free acridine esters remaining in the system are the main source of impurities. If these free small molecules are not removed in time, they will bring serious non-specific luminescent signals, directly leading to increased detection background and false positive problems. The G25 dextran column can quickly remove free acridine esters.

[0030] After purification with G25, most of the free acridine esters have been removed from the labeled antibody solution, but the following problems may still exist: (1) The solution volume is large and the concentration is low, which is not conducive to aliquoting and storage; (2) There may be a small amount of residual low molecular weight impurities; (3) There may be aggregates generated during the labeling process, which can lead to unstable detection signals and large batch-to-batch differences. Therefore, a second purification is performed using an ultrafiltration tube.

[0031] Second purification: Based on G25 column purification, residual impurities are further removed, while precise control of concentration and storage buffer is achieved. More importantly, the high-concentration labeled antibody after ultrafiltration concentration can be stably stored at -20°C for a long time without activity decay or background increase after the addition of a protective agent (such as glycerol).

[0032] The G25 column and ultrafiltration tube purification methods, used in tandem, yield a high-purity, high-concentration, and highly uniform acrid ester-labeled antibody complex. For the application scenario of Aβ detection in urine samples, the synergistic effect of the two purification processes is particularly prominent, serving as a key guarantee for ultra-high sensitivity detection.

[0033] 2) The preferred formulation of acridinium ester diluent is 20 mmol / L PBS, 2% BSA, 5% trehalose, 0.1% Triton, 0.1% PC300, 1% PEG20000, 1% histidine, 1% mannitol, pH 6.8; 3) Reagent R2 is prepared by diluting acridinium ester-labeled antibody in a certain proportion using acridinium ester diluent, with the luminescence value controlled above 60 million; 8.1.3 Preparation of reagent R3 3. Reagent R3 contains the blocking agent and the blocking agent diluent. 1) The blocking agent is mouse IgG+HAMA and RF specific antibody, with a specificity of <50% (purchased from Hangzhou Boyue Biotechnology Co., Ltd.); 2) The preferred formulation of the blocking agent diluent is 20 mmol / L PBS, 2% BSA, 5% trehalose, 0.1% Triton, 0.1% PC300, 1% PEG20000, 1% histidine, 1% mannitol, pH 6.8; 3) Reagent R3 is prepared by diluting the blocking agent with a certain ratio using a blocking agent diluent to control the concentration of the blocking agent to above 8 μg / mL.

[0034] Example 2: Reagents R1, R2, and R3 are used to detect calibrator Aβ. 1. Testing Aβ1-42 calibrators 1.1 Experimental Procedure: 1) The combination of reagent R1-1 + reagent R2-1 + reagent R3 is designated as reagent combination 1 by default, and the combination of reagent R1-2 + reagent R2-2 + reagent R3 is designated as reagent combination 2 by default.

[0035] 2) Dilute the Aβ1-42 calibrator with sample diluent to a series of concentration gradients of 0, 1, 5, 10, 50, 100, 500, and 1000 pg / mL. Use reagent combination 1 and reagent combination 2 to detect the calibrator Aβ1-42 on a chemiluminescence analyzer.

[0036] 1.2 Experimental Results: The experimental results are shown in Table 1 and Figure 1 As shown: 1) From the perspective of background, the background of reagent combination 1 (luminescence value 415) is significantly lower than that of reagent combination 2 (luminescence value 1209). 2) Within the concentration range of 0-1000 pg / mL, both reagent combination 1 and reagent combination 2 showed a significant linear relationship, but the linear correlation coefficient r(R) of reagent combination 1 was lower. 2 =1) is better than the linear correlation coefficient r(R) of reagent combination 2. 2 =0.9999); 3) From the perspective of sensitivity, when the concentration of Aβ1-42 calibrator is 1 pg / mL, the reaction signal intensity of reagent combination 1 is significantly enhanced, while the reaction signal intensity of reagent combination 2 is not enhanced, indicating that the detection sensitivity of reagent combination 1 when detecting Aβ1-42 calibrator reaches 1 pg / mL. In summary, reagent combination 1 has significant advantages when testing Aβ1-42 calibrators.

[0037] Table 1. Testing Aβ1-42 calibrators

[0038] 2. Testing Aβ1-40 calibrators 2.1 Experimental Procedure: 1) The combination of reagent R1-1 + reagent R2-1 + reagent R3 is designated as reagent combination 1 by default, and the combination of reagent R1-2 + reagent R2-2 + reagent R3 is designated as reagent combination 2 by default.

[0039] 2) Dilute the Aβ1-40 calibrator with sample diluent to a series of concentration gradients of 0, 1, 5, 10, 50, 100, 500, and 1000 pg / mL. Use reagent combination 1 and reagent combination 2 to detect the calibrator Aβ1-40 on a chemiluminescence analyzer.

[0040] 2.2 Experimental Results: The experimental results are shown in Table 2 and Figure 2 As shown: 1) From the perspective of background, the background of reagent combination 1 (luminescence value 461) is significantly lower than that of reagent combination 2 (luminescence value 965). 2) Within the concentration range of 0-1000 pg / mL, both reagent combination 1 and reagent combination 2 showed a significant linear relationship, but the linear correlation coefficient r(R) of reagent combination 1 was lower. 2 =0.9996) is better than the linear correlation coefficient r(R) of reagent combination 2. 2 =0.9970); 3) From the perspective of sensitivity, when the concentration of Aβ1-40 calibrator is 10 pg / mL, the reaction signal intensity of both reagent combination 1 and reagent combination 2 is significantly enhanced, indicating that the detection sensitivity of Aβ1-40 calibrator can reach 10 pg / mL. In summary, reagent combination 1 has significant advantages when testing Aβ1-40 calibrators.

[0041] Table 2. Testing Aβ1-40 calibrators

[0042] 3. Detect the mixture of calibrators Aβ1-42 and Aβ1-40 3.1 Experimental Procedure 1) Use reagent combination 1 for testing.

[0043] 2) Mix Aβ1-42 and Aβ1-40 calibrators at a concentration ratio of 1:10, and dilute with sample diluent to create a series of concentration gradients of 0, 0.25, 0.5, 1, 5, 10, 50, 100, 500, and 1000 pg / mL for Aβ1-42 calibrator, and a series of concentration gradients of 0, 2.5, 5, 10, 50, 100, 500, 1000, 5000, and 10000 pg / mL for Aβ1-40 calibrator. Use reagents R1, R2, and R3 to detect the mixture of calibrators Aβ1-42 and Aβ1-40 on a chemiluminescence analyzer.

[0044] 3.2 Experimental Results The experimental results are shown in Table 3. Figure 3 and Figure 4 As shown, when Aβ1-42 and Aβ1-40 calibrators were mixed at a concentration ratio of 1:10, the reaction signal intensity was significantly enhanced when the concentration of Aβ1-42 calibrator was 1 pg / mL and the concentration of Aβ1-40 calibrator was 10 pg / mL. The detection sensitivity was the same before and after mixing.

[0045] For the Aβ1-42 calibrator, there is a clear linear relationship in the concentration range of 0-100 pg / mL, with a linear correlation coefficient r > 0.9999 and a detection sensitivity of 1 pg / mL.

[0046] For the Aβ1-40 calibrator, there is a clear linear relationship in the concentration range of 0-1000 pg / mL, with a linear correlation coefficient r > 0.9999 and a detection sensitivity of 10 pg / mL.

[0047] Table 3. Detection of mixtures of Aβ1-42 and Aβ1-40 calibrators

[0048] Example 3: Effect of reagent R3 1. Experimental Procedure 1) Test using reagent combination 1 and reagent combination 3. Reagent combination 3 is reagent R3 in reagent combination 1 replaced with water.

[0049] 2) Mix Aβ1-42 and Aβ1-40 calibrators at a concentration ratio of 1:10, and dilute with sample diluent to create a series of concentration gradients of 0, 1, 5, 10, 50, and 100 pg / mL for Aβ1-42 calibrator, and a series of concentration gradients of 0, 10, 50, 100, 500, and 1000 pg / mL for Aβ1-40 calibrator. Use reagent combination 1 and reagent combination 3 to detect the mixture of calibrators Aβ1-42 and Aβ1-40 on a chemiluminescence analyzer.

[0050] 2 Experimental Results The experimental results are shown in Table 4. After adding reagent R3 to the reagent combination, the background signal was significantly reduced, and the emission value dropped from 455 to 713, a decrease of about 36%. The sensitivity is generally evaluated by the C2 / C1 ratio. After adding reagent R3 to the reagent combination, the sensitivity was improved by 12.3%.

[0051] Table 4 Effects of Reagent R3

[0052] Example 4: Anti-interference ability of reagents R1, R2, and R3 1. Experimental Procedure Aβ1-42 and Aβ1-40 calibrators were mixed at a concentration ratio of 1:10 and diluted with sample diluent to create two concentration gradients of 5 and 50 pg / mL for Aβ1-42 calibrator and two concentration gradients of 50 and 500 pg / mL for Aβ1-40 calibrator. These were then divided into three groups. Group 1 was treated with 0 mmol / L urea and 600 mmol / L urea, respectively. Group 2 was treated with 0 mmol / L creatinine and 20 mmol / L creatinine, respectively. Group 3 was treated with 0 mmol / L uric acid and 500 μmol / L uric acid, respectively. The effect of reagent combination 1 on the reagents when the samples contained urea, creatinine, and uric acid was detected on the reagents using a chemiluminescence analyzer.

[0053] 2 Experimental Results The experimental results are shown in Table 5. When simulating samples containing different concentrations of interfering substances such as urea, creatinine, and uric acid, it was found that when 600 mmol / L of urea, 20 mmol / L of creatinine, and 500 μmol / L of uric acid were added to samples containing low to medium concentrations of Aβ1-42 and Aβ1-40 calibrators, the luminescence values ​​detected were within ±10% of the luminescence values ​​without the addition of interfering substances, which is within an acceptable range. Therefore, it is considered that no obvious interference reaction was detected when the urea concentration was ≤600 mmol / L, the creatinine concentration was ≤20 mmol / L, and the uric acid concentration was ≤500 μmol / L, indicating that at these concentrations of interfering substances, urea, creatinine, and uric acid in the sample had no effect on the detection.

[0054] Table 5. Anti-interference capability of reagent combination 1

[0055] Example 5: Detection of urine samples using reagents R1, R2, and R3. 1. Experimental Procedure Three positive urine samples and three negative urine samples were diluted with sample diluent in equal volumes. The resulting concentrations were: original urine, 1 / 2 original urine, 1 / 4 original urine, 1 / 8 original urine, and sample diluent. The samples were then tested using reagents R1, R2, and R3 on a chemiluminescence analyzer.

[0056] 2 Experimental Results The experimental results are shown in Table 6. Three positive urine samples and three negative urine samples were tested respectively. The positive urine samples showed a certain concentration gradient reaction, while the negative urine samples did not show an obvious concentration gradient reaction.

[0057] Table 6. Testing of urine samples

Claims

1. A reagent combination for detecting the precursor fragment Aβ of β-amyloid protein in urine using chemiluminescence immunoassay, characterized in that, The reagent combination consists of reagent R1, reagent R2, and reagent R3; reagent R1 is prepared by diluting carboxyl magnetic beads conjugated with β-amyloid monoclonal antibody at a volume ratio of 1:30-1:60 using magnetic bead diluent; reagent R2 is prepared by diluting acridine ester-labeled humanized IgG1 monoclonal antibody with acridine ester diluent until the luminescence value is controlled above 60 million; reagent R3 is prepared by diluting an inhibitor with an inhibitor diluent until the inhibitor concentration is above 8 μg / mL; the inhibitor is mouse IgG+HAMA and RF-specific antibody.

2. The reagent combination for detecting β-amyloid precursor fragment Aβ in urine using chemiluminescence immunoassay as described in claim 1, characterized in that, The magnetic bead diluent contains the following components and concentrations: 50-200 mmol / L PBS, 0.5-2% BSA, 0.05-0.5% Triton, 1-5% trehalose, 0.05-0.2% PC-300, 0.5-5% PEG-6000, and 0.1-2% histidine, with a pH of 7.2-7.4; the acridinium ester diluent contains the following components and concentrations: 10-100 mmol / L PBS, 0.5-2% BSA, 1-10% trehalose, 0.05-0.5% Triton, 0.05-0.2% PC-300, 0.5-2% PEG-20000, 0.5-2% histidine, and 0.5-2% mannitol, with a pH of 6.5-6.8; the blocking agent diluent contains the following components and concentrations: 10-100 mmol / L PBS. PBS, 0.5-2% BSA, 1-10% trehalose, 0.05-0.5% Triton, 0.05-0.2% PC-300, 0.5-2% PEG-20000, 0.5-2% histidine, 0.5-2% mannitol, pH 6.5-6.8; all percentages refer to w / v, where w is in g and v is in mL.

3. The reagent combination for detecting β-amyloid precursor fragment Aβ in urine using chemiluminescence immunoassay as described in claim 2, characterized in that, The magnetic bead diluent contains 100 mmol / L PBS, 1% BSA, 0.1% Triton, 3% trehalose, 0.1% PC-300, 2% PEG-6000, and 0.5% histidine, with a pH of 7.2-7.4; the acridinium ester diluent contains 20 mmol / L PBS, 2% BSA, 5% trehalose, 0.1% Triton, 0.1% PC-300, 1% PEG-20000, 1% histidine, and 1% mannitol, with a pH of 6.8; the blocking agent diluent contains 20 mmol / L PBS, 2% BSA, 5% trehalose, 0.1% Triton, 0.1% PC-300, 1% PEG-20000, 1% histidine, and 1% mannitol, with a pH of 6.

8.

4. The reagent combination for detecting β-amyloid precursor fragment Aβ in urine based on chemiluminescence immunoassay as described in any one of claims 1 to 3, characterized in that, The reagent combination is used in a volume ratio of 1:1:

1.

5. The reagent combination for detecting β-amyloid precursor fragment Aβ in urine using chemiluminescence immunoassay as described in claim 4, characterized in that, When using this product, first incubate the sample to be tested with a mixture of reagents R2 and R3, and then incubate the sample to be tested with reagent R1.

6. The reagent combination for detecting β-amyloid precursor fragment Aβ in urine using chemiluminescence immunoassay as described in claim 5, characterized in that, The volume ratio of reagents R1, R2, and R3 to the sample to be tested is 4:9 to 6:

7.

7. The reagent combination for detecting β-amyloid precursor fragment Aβ in urine using chemiluminescence immunoassay as described in claim 6, characterized in that, The volume ratio of reagents R1, R2, and R3 to the sample to be tested is 5:

8.

8. The reagent combination for detecting β-amyloid precursor fragment Aβ in urine using chemiluminescence immunoassay as described in claim 5, characterized in that, The test sample was incubated with reagents R2 and R3 for 10 minutes, and the test sample was incubated with reagent R1 for 20 minutes.

9. The use of the reagent combination for detecting β-amyloid precursor fragment Aβ in urine based on chemiluminescence as described in any one of claims 1 to 3 in the preparation of reagents for screening or auxiliary diagnosis of Alzheimer's disease.

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