Single molecule joint detection method for Alzheimer's disease markers A beta 1-42, A beta 1-40 and P-Tau181

Abstract

The invention discloses a single-molecule joint detection method for Alzheimer's disease markers A beta 1-42, A beta 1-40 and P-Tau181, which comprises the following steps: 1, activating microspheres, and coupling the activated microspheres with antibodies to prepare fluorescent microsphere labeled antibodies, the fluorescent microsphere labeled antibodies comprise microspheres labeled with A beta 1-42 detection antibodies, microspheres labeled with A beta 1-40 detection antibodies, and microspheres labeled with P-Tau181 detection antibodies; 2, preparing a coated antibody immobilization plate, wherein the coated antibody immobilization plate is coated with an A beta 1-42 capture antibody, an A beta 1-40 capture antibody and a P-Tau181 capture antibody; and 3, adding a sample containing A [beta] 1-42, A [beta] 1-40 and P-Tau, a quality control product or a calibration product into the plate coated with the antibody for immobilization, adding the fluorescent microsphere labeled antibody into the plate, detecting by using a digital fluorescence immunoassay instrument, and quantifying the content of A [beta] 1-42, A [beta] 1-40 and P-Tau by analyzing the number of fluorescent particles and matching with a standard curve. According to the kit, the digital fluorescence immunoadsorption technology is adopted, and the three markers A [beta] 1-42, A [beta] 1-40 and P-Tau181 are combined for detection, so that the kit has important clinical values on early diagnosis of AD, risk stratification of diseases and timely symptomatic treatment and prognosis judgment of patients.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a single-molecule joint detection method for Alzheimer's disease markers Aβ1-42, Aβ1-40, and P-Tau181. Background Art

[0002] Alzheimer's disease (AD) is a common dementia, also known as neurodegenerative disease or neurodegenerative disease. The current diagnostic methods used in clinical practice include clinical evaluation, neuropsychological testing, and imaging examinations (such as magnetic resonance imaging and positron emission tomography). However, the above methods have certain subjectivity and limitations and cannot provide sufficient accuracy and sensitivity for early diagnosis.

[0003] For patients with Alzheimer's disease, commonly used biological markers in clinical practice include amyloid-β (Aβ40, amyloid-β, Aβ), phosphorylated tau (p-tau), etc. Aβ is a polypeptide containing 39 to 43 amino acids produced by the proteolysis of APP by β- and γ-secretases, mainly Aβ1-40 and Aβ1-42. It can be produced by a variety of cells and circulates in the blood, cerebrospinal fluid, and brain interstitium. Most of them are bound to chaperone protein molecules, and a few exist in a free state. The Aβ amyloid cascade hypothesis believes that the imbalance between the production and clearance of Aβ in the brain is the main cause of Alzheimer's disease. Aβ oligomers will directly inhibit long-term potentiation in the hippocampus and damage synapses, ultimately leading to neurotransmitter disorders and neurological dysfunction.

[0004] Phosphorylated tau protein (P-Tau) is formed by abnormal overphosphorylation of tau protein, also known as the pathological protein of AD. Studies have found that AD patients have 21 abnormal phospho-sites. Phosphorylation of multiple phospho-sites is more likely to lead to the loss of microtubule binding function of tau protein. Among them, Tau181, Tau199, Tau231, Tau396 and Tau404 are more studied. High concentrations of p-tau protein at the above sites have been detected in the cerebrospinal fluid of AD patients with high specificity and sensitivity. Studies have shown that in the early pathological changes of AD, the level of p-tau (181P) in plasma and cerebrospinal fluid is low, and it also increases significantly as the disease progresses.

[0005] The above markers will be released or aggregated when the brain changes abnormally. Currently, their concentration or presence can be detected through blood, cerebrospinal fluid and other channels. The presence and increase of these markers can be used as important indicators for early diagnosis, disease progression monitoring and prognosis evaluation. However, for the clinical diagnosis of AD patients, although the accuracy of invasive measurement of cerebrospinal fluid can reach 80% to 90%, the sampling of cerebrospinal fluid will cause trauma to the patients, especially for the elderly with weak constitution, which is quite restrictive and not suitable for clinical promotion.

[0006] At present, the commonly used detection methods are as follows: enzyme-linked immunosorbent assay, colloidal gold method, immunoturbidimetry, chemiluminescence method, fluorescent immunochromatography and SiMoA single molecule detection technology. Among them, the colloidal gold method can quickly obtain diagnostic results, but the sensitivity is low, and the detection results of different manufacturers vary greatly. At present, this method is mostly used for qualitative analysis and cannot be further quantitatively analyzed, and there are certain limitations in the results; the traditional enzyme-linked immunosorbent assay has problems such as low sensitivity and large measurement differences, and it is difficult to achieve high sensitivity and stable detection; the amount of antigen or antibody in the immunoturbidimetry method must be greatly excessive, and soluble complexes may appear to cause errors, and the test results are also easily affected by lipemia; although the chemiluminescence method has a high degree of automation, a significant limitation of chemiluminescence products is its high cost, which has led to some hospitals being unable to purchase corresponding equipment and reagents due to budget constraints, which has greatly limited its promotion in some hospitals, and the detection sensitivity cannot reach the single molecule level. SiMoA single-molecule detection technology is a relatively new detection method. It is relatively expensive and the detection operation process is too cumbersome and complicated. Therefore, considering a series of factors such as detection cost, detection efficiency, detection reagent stability, and subsequent maintenance costs, the SiMoA system will inevitably face obvious obstacles when it is promoted to the clinical diagnosis market.

[0007] Therefore, designing a method for detecting biomarkers (Aβ1-42, Aβ1-40, P-Tau181) in serum or plasma, which can improve the detection sensitivity, detect multiple biomarkers, and improve the detection specificity, is a technical problem that needs to be solved urgently. Summary of the invention

[0008] To solve the above problems, the present invention provides a single-molecule joint detection method for Alzheimer's disease markers Aβ1-42, Aβ1-40, and P-Tau181, which has important clinical value for the early diagnosis of Alzheimer's disease, disease typing, and evaluation of the therapeutic effect after medication.

[0009] The present invention is achieved through the following technical solutions:

[0010] A single molecule joint detection method for Alzheimer's disease markers Aβ1-42, Aβ1-40, and P-Tau181, comprising the following steps:

[0011] Step 1, after activating the microspheres, coupling with antibodies to prepare fluorescent microsphere-labeled antibodies, the fluorescent microsphere-labeled antibodies include microspheres labeled with Aβ1-42 detection antibodies, microspheres labeled with Aβ1-40 detection antibodies, and microspheres labeled with P-Tau181 detection antibodies, which are set aside;

[0012] Step 2: prepare an antibody-coated solid-phase plate coated with Aβ1-42, Aβ1-40, and P-Tau181 capture antibodies;

[0013] Step 3. After adding samples, quality control products or calibrators containing Aβ1-42, Aβ1-40, and P-Tau into the corresponding antibody-coated plate wells, add fluorescent microsphere-labeled antibodies into the corresponding plate wells, and use a digital fluorescence immunoassay to quantify the content of Aβ1-42, Aβ1-40, and P-Tau by analyzing the number of fluorescent particles and matching the standard curve.

[0014] Furthermore, the specific method of step one is:

[0015] S1. Take the microsphere suspension and add it to the coupling buffer for ultrasonic mixing, centrifuge and remove the supernatant, then repeat the ultrasonic and centrifugal washing in the coupling buffer for several times; add the EDC solution, vortex mix, then add the NHS solution, ultrasonically mix and place it on a turntable for rotation, activate it in the dark for 15-30 minutes, centrifuge and remove the supernatant, then repeat the ultrasonic and centrifugal washing in the coupling buffer for several times to obtain the activated microspheres;

[0016] S2, adding the activated microspheres to the coupling buffer and mixing by ultrasonication; adding the coupling buffer with the detection antibody to the mixed microspheres, vortex mixing; placing them on a turntable for rotation, coupling in the dark for 2 hours, and obtaining the coupled microspheres and antibodies;

[0017] S3. Add microsphere blocking solution to the centrifuge tube, vortex mix and place the centrifuge tube on a turntable for rotation. After blocking for 1 hour in the dark, centrifuge to remove the supernatant; add microsphere washing solution, ultrasonically mix and centrifuge to remove the supernatant; add microsphere washing solution again and repeat the washing steps; finally, add microsphere preservation solution to the centrifuge tube, ultrasonically mix and store in the dark at 2-8℃ for later use.

[0018] Further, the coupling buffer included 10 mM MES at pH 6.2 ± 0.05 and 0.05% ProClin300;

[0019] The preparation method of EDC solution and NHS solution is as follows: dissolve EDC and NHS in coupling buffer respectively, and the concentrations thereof are both 10 mg / mL;

[0020] The microsphere blocking solution includes: borate buffer, 1% BSA, 0.24% ethanolamine; wherein the borate buffer includes 5mM boric acid, 11.2mM sodium tetraborate decahydrate, and 0.05% Tween-20;

[0021] The microsphere washing solution included: 50 mM Tris with a pH of 8.0 ± 0.05, 0.5% BSA, 0.05% Tween-20, and 0.03% ProClin 300;

[0022] The microsphere storage solution includes: 25mM Tris with a pH of 7.2±0.05, 150mM NaCI, 0.05% Tween-20, 1% BSA, 5% trehalose, and 0.1% ProClin 300.

[0023] Furthermore, the plate described in step 2 is a detection plate, and the steps of antibody coating the detection plate are: coating according to the Aβ1-42, Aβ1-40, and P-Tau181 capture antibody concentration of 0.1-0.5ug / T, respectively, static coating of 100ul / T coating solution at 2-8°C for 16±1h, washing with washing solution for several times, adding 150ul / well blocking solution after patting dry, blocking at 37°C for 2h, washing with washing solution for several times, putting desiccant into the tinfoil bag after patting dry, and setting aside for use.

[0024] Further, the coating solution includes 0.1M sodium carbonate and sodium bicarbonate;

[0025] The blocking solution includes: 0.05-0.1M PBS with a pH of 7.4, 1-2% BSA, 0.1% ProClin 300, and 0.1% protein protectant;

[0026] The washing solution includes PBS and TWEEN.

[0027] Furthermore, the plate in step 2 is a carboxyl-based plate or a polymethyl methacrylate plate, and the steps of antibody coating the carboxyl-based plate or the polymethyl methacrylate plate are: firstly activate the carboxyl-based plate or the polymethyl methacrylate plate, wash the plate with PBS and pat dry, add 100ul / T MES solution to dilute the antibody and place it in a 37°C incubator for coating for 2h, wash the plate with PBS and pat dry, add 150ul / T blocking solution and place it in a 37°C incubator for coating for 1-2h, wash the plate with PBS and pat dry for standby use.

[0028] Furthermore, the method for activating the carboxyl-based substrate is as follows: adding 20 mg / ml EDC and NHS solutions to the carboxyl-based substrate respectively and placing the substrate in an incubator for activation.

[0029] Furthermore, the method for activating the polymethyl methacrylate plate is: placing the polymethyl methacrylate plate in a plasma instrument for plasma surface activation.

[0030] Furthermore, the preparation method of the quality control / calibrator in step three is: Aβ1-42, Aβ1-40, P-Tau181 protein, PBS buffer, bovine serum albumin, protein protective agent and 0.2% ProClin 300 are mixed.

[0031] The beneficial effects of the present invention are:

[0032] (1) The kit prepared by the present invention contains fluorescent microsphere-labeled antibodies, namely, microspheres labeled with Aβ1-42 detection antibodies, microspheres labeled with Aβ1-40 detection antibodies, and microspheres labeled with P-Tau181 detection antibodies; when Aβ1-42, Aβ1-40, and P-Tau in the sample to be tested bind to the solid-phase antibody coated on the detection plate, the fluorescent microsphere-labeled antibody will bind to another antigenic determinant on the antigen, thereby forming a "sandwich" complex between the solid-phase antibody and the fluorescent microsphere-labeled antibody. The higher the antigen concentration, the more fluorescent particles there are;

[0033] (2) By analyzing the number of fluorescent particles and matching the standard curve to quantify the content of (Aβ1-42, Aβ1-40, P-Tau), the antigen concentration can be judged by the number of fluorescent particles, thus avoiding the influence of lipemia / hemolysis on the test results.

[0034] (3) The present invention integrates sample addition, incubation, washing and detection into one, which can reduce human operation errors, accurately detect antigen concentration through digital fluorescence technology, and can detect Aβ1-42, Aβ1-40, P-Tau181 markers at the femtogram (fg / mL) level in plasma, and greatly reduce the detection cost, achieving a much lower cost than chemiluminescence and SiMoA single molecule detection technology, while saving patients' expenses and laboratory resources;

[0035] In summary, the present invention adopts digital fluorescent immunoadsorption technology, and the combined detection of three markers, Aβ1-42, Aβ1-40, and P-Tau181, has important clinical value for the early diagnosis of AD, risk stratification of the disease, timely symptomatic treatment of patients, and prognosis judgment. The technology has high detection sensitivity that can reach the single molecule detection level (fg / mL), high specificity, and convenient and fast automated detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1Schematic diagram of the plate of the present invention, the plate is coated with three items of antibodies, different items of calibration area, quality control area, sample detection area, wherein S0-S5: calibration area; Q1-Q2: quality control area; P1-P16: sample area;

[0037] Figure 2 is the standard curve of Aβ1-42;

[0038] Figure 3 is the standard curve of Aβ1-40;

[0039] Figure 4 is the P-Tau181 standard curve;

[0040] Figure 5 This is a comparison chart of P-tau181 detection results and existing technologies. DETAILED DESCRIPTION

[0041] The present invention is further described below in conjunction with the accompanying drawings and specific preferred embodiments, but the protection scope of the present invention is not limited thereby.

[0042] Example 1

[0043] A single molecule joint detection method for Alzheimer's disease markers Aβ1-42, Aβ1-40, and P-Tau181, comprising the following steps:

[0044] Step 1, after activating the microspheres, coupling with antibodies to prepare fluorescent microsphere-labeled antibodies, the fluorescent microsphere-labeled antibodies include microspheres labeled with Aβ1-42 detection antibodies, microspheres labeled with Aβ1-40 detection antibodies, and microspheres labeled with P-Tau181 detection antibodies, which are set aside;

[0045] The specific method of step one is:

[0046] S1. Take 0.05 mL of microspheres (1% solid content) into a 2 mL centrifuge tube with 1 mL of coupling buffer, mix by ultrasonication, centrifuge at 15°C and 20000 g for 10 min, and remove the supernatant; repeat washing several times, add 3.5 μL of LEDC solution, vortex mix, add 33 μL of NHS solution, mix by ultrasonication, place it on a turntable, activate it at 37°C, 40 r / min, protect from light for 15-30 min, centrifuge at 15°C and 20000 g for 10 min, remove the supernatant, add 1.5 mL of coupling buffer, mix by ultrasonication, centrifuge at 15°C and 20000 g for 10 min, and remove the supernatant; add 1.5 mL of coupling buffer and repeat washing several times to obtain activated microspheres;

[0047] S2. Add 0.75 mL of coupling buffer to the activated microspheres and mix by ultrasonication; add 50 μg of the antibody to be labeled to 0.25 mL of coupling buffer, add the coupling buffer with the antibody to the mixed microspheres, and vortex mix; place the centrifuge tube on a turntable, at 37°C, 40 r / min, and couple for 2 h in the dark to obtain the coupled microspheres and antibodies;

[0048] S3. Add 0.5 mL of microsphere blocking solution to the centrifuge tube, vortex mix, place the centrifuge tube on a turntable, and seal it for 1 hour at 37°C, 40 r / min in the dark. Centrifuge it at 15°C, 20,000 g for 10 min, remove the supernatant; add 1.5 mL of microsphere washing solution, mix it by ultrasonic, centrifuge it at 15°C, 20,000 g for 10 min, remove the supernatant; add 1.5 mL of microsphere washing solution again, and repeat the washing steps; finally, add 0.5 mL of microsphere preservation solution (microsphere final concentration 1 mg / mL) to the centrifuge tube, mix it by ultrasonic, and store it at 2-8°C in the dark for later use.

[0049] The coupling buffer included 10 mM MES with a pH of 6.2 ± 0.05 and 0.05% ProClin 300;

[0050] The preparation method of EDC solution and NHS solution is as follows: dissolve EDC and NHS in coupling buffer respectively, and the concentrations thereof are both 10 mg / mL;

[0051] The microsphere blocking solution includes: borate buffer, 1% BSA, 0.24% ethanolamine; wherein the borate buffer includes 5mM boric acid, 11.2mM sodium tetraborate decahydrate, and 0.05% Tween-20;

[0052] The microsphere washing solution included: 50 mM Tris with a pH of 8.0 ± 0.05, 0.5% BSA, 0.05% Tween-20, and 0.03% ProClin 300;

[0053] The microsphere storage solution includes: 25mM Tris with a pH of 7.2±0.05, 150mM NaCI, 0.05% Tween-20, 1% BSA, 5% trehalose, and 0.1% ProClin 300.

[0054] Step 2: prepare an antibody-coated solid-phase plate (coated with Aβ1-42, Aβ1-40, P-Tau181 capture antibodies; different project calibration areas, quality control areas, and sample detection areas);

[0055] The plate is a detection plate, and the steps of antibody coating the detection plate are as follows: coating according to the concentration of Aβ1-42, Aβ1-40, and P-Tau181 capture antibodies of 0.1-0.5ug / T, static coating of 100ul / T coating solution at 2-8°C for 16±1h, washing with washing solution (PBS, 0.05%wt TWEEN) for several times, adding 150ul / well blocking solution after patting dry, blocking at 37°C for 2h, washing with washing solution for several times, putting desiccant into the tinfoil bag after patting dry, and setting aside for use.

[0056] The coating solution includes 0.1M sodium carbonate and sodium bicarbonate, with a pH of 9.6; the blocking solution includes: 0.05-0.1M PBS with a pH of 7.4, 1-2% BSA, 0.1% ProClin 300, and 0.1% protein protective agent;

[0057] The plate can also be replaced with a carboxyl-based plate or a polymethyl methacrylate plate. The steps of antibody coating on the carboxyl-based plate or the polymethyl methacrylate plate are as follows: first, the carboxyl-based plate or the polymethyl methacrylate plate is activated, the plate is washed with PBS and patted dry, 100ul / TMES solution is added to dilute the antibody and placed in a 37°C incubator for coating for 2h, the plate is washed with PBS and patted dry, 150ul / T blocking solution is added to place in a 37°C incubator for coating for 1-2h, and the plate is washed with PBS and patted dry for standby use.

[0058] Furthermore, the method for activating the carboxyl-based substrate is as follows: adding 20 mg / ml EDC and NHS solutions to the carboxyl-based substrate respectively and placing the substrate in an incubator for activation.

[0059] Furthermore, the method for activating the polymethyl methacrylate plate is: placing the polymethyl methacrylate plate in a plasma instrument for plasma surface activation.

[0060] Step 3. After adding samples, quality control products or calibrators containing Aβ1-42, Aβ1-40, and P-Tau into the corresponding antibody-coated plate wells, add fluorescent microsphere-labeled antibodies into the corresponding plate wells and place them into the self-developed instrument of the Institute of Physics, Henan Academy of Sciences, the digital fluorescence immunoassay analyzer: FX100 for detection. The content of Aβ1-42, Aβ1-40, and P-Tau is quantified by analyzing the number of fluorescent particles and matching the standard curve. The instrument is calibrated according to the information on the reagent calibration card. After successful calibration, the subsequent clinical sample testing can be carried out only when the quality control test is within the range.

[0061] The preparation method of the quality control / calibrator is as follows: Aβ1-42, Aβ1-40, P-Tau181 protein, PBS buffer, bovine serum albumin, protein protective agent and 0.2% ProClin 300 are mixed. The quality control (freeze-dried state, two levels), the calibrator (freeze-dried state, six-point calibration).

[0062] The results of the standard curves of Aβ1-42, Aβ1-40, and P-Tau181 are shown in Figures 2 to 4 As shown, from Figures 2 to 4 It can be seen that the Aβ1-40 standard curve: within the measurement range of (0-300) pg / mL, the correlation coefficient (r) of the kit should be ≥0.9900; the Aβ1-42 standard curve: within the measurement range of (0-300) pg / mL, the correlation coefficient (r) of the kit should be ≥0.9900; the P-tau181 standard curve: within the measurement range of (0-300) pg / mL, the correlation coefficient (r) of the kit should be ≥0.9900;

[0063] A specific test was performed on it, and Aβ and Tau cross-reactants (Aβ1-38, Aβ1-43, Tau217, Tau231) with high antigen concentration (1000 pg / mL) were prepared. The test results are shown in Table 1;

[0064] Table 1 Specificity test results

[0065] Cross-reactants Interference concentration Test results Crossover rate Aβ1-38 1000pg / mL 0.00 0.00％ Aβ1-43 1000pg / mL 0.00 0.00％ tau217 1000pg / mL 0.00 0.00％ tau231 1000pg / mL 0.00 0.00％ GFAP 1000pg / mL 0.00 0.00％ Nf 1000pg / mL 0.00 0.00％

[0066] The kit was used to detect the contents of Aβ1-38, Aβ1-43, Tau-217, Tau-231, GFAP and NfL, and the crossover rates were 0.00% with high specificity when compared with the theoretical concentrations.

[0067] The present invention has good sensitivity, and the sensitivity assessment method is as follows: blank limit (LOB): 5 samples with a value of 0 (samples without the object to be tested), repeated 3 times, and assessed for 4 days; minimum detection limit (LOD): 5 samples with a value of 1 to 4 times the LoB, repeated 3 times, and assessed for 4 days;

[0068] 1) First establish the sensitivity of Aβ1-42:

[0069] Table 2 Aβ1-42 blank limit data table

[0070]

[0071] According to the data in Table 2, the normal distribution analysis of 60 groups of data showed that sig<0.05, which belongs to non-normal distribution. Referring to the EP17-A2 file, LOB was calculated by non-parametric method, and the test results of all B blank samples were arranged from small to large, LOB=NB(p / 100)+0.5 (i.e., arrangement position=0.5+number of measurements×0.95), then the Aβ1-42 established LOB=(p / 100)+0.5=57+0.5=57.5, and the 95th percentile would be the average of the 57th and 58th observations=(0.06+0.06)=0.12 / 2=0.06pg / mL.

[0072] 2) Aβ1-40 sensitivity establishment:

[0073] Table 3 Aβ1-40 blank limit data table

[0074]

[0075] According to the data in Table 3, the normal distribution analysis of 60 groups of data showed that sig<0.05, which belongs to non-normal distribution. Referring to the EP17-A2 file, LOB was calculated by non-parametric method, and the test results of all B blank samples were arranged from small to large, LOB=NB(p / 100)+0.5 (i.e., arrangement position=0.5+number of measurements×0.95), then the Aβ1-40 established LOB=(p / 100)+0.5=57+0.5=57.5, and the 95th percentile would be the average of the 57th and 58th observations=(0.09+0.10)=0.19 / 2=0.095pg / mL, which is significantly lower than the minimum detection limit of 50.00pg / ml of a listed company.

[0076] 3) P-tau181 sensitivity establishment:

[0077] Table 4P-tau181 blank limit data table

[0078]

[0079] According to the data in Table 4, the normal distribution analysis of 60 groups of data showed that sig<0.05, which belongs to non-normal distribution. Referring to the EP17-A2 file, LOB was calculated by non-parametric method, and the test results of all B blank samples were arranged from small to large, LOB=NB(p / 100)+0.5 (i.e., arrangement position=0.5+number of measurements×0.95), then the P-tau181 established LOB=(p / 100)+0.5=57+0.5=57.5, and the 95th percentile would be the average of the 57th and 58th observations=(0.04+0.04) / 2=0.04pg / mL, which is significantly lower than the minimum detection limit of 4.00pg / mL of a listed company.

[0080] The present invention has high accuracy. The assessment method is: collect 40 clinical samples with a concentration range of 8.0-120.0pg / mL, perform the test according to the instructions, and perform self-calibration according to the reagent calibration card information. After the calibration is successful, the quality control test can be carried out within the range before the subsequent clinical sample test. The Tau181 test results are compared with those of a well-known listed company as follows:

[0081] Table 5 Clinical comparison results

[0082]

[0083] Correlation R between P-tau181 test results and test results of well-known listed companies 2 All of them are greater than 0.95, and the results are accurate and reliable.

[0084] It should be noted that although the present invention is described by the above embodiments, the present invention may also have other various embodiments. Without departing from the spirit and scope of the present invention, it is obvious that those skilled in the art may make various corresponding changes and deformations to the present invention, but these changes and deformations should all fall within the scope of protection of the appended claims of the present invention and their equivalents.

Claims

1. A single molecule joint detection method for Alzheimer's disease markers Aβ1-42, Aβ1-40, and P-Tau181, characterized by: The following steps are involved: Step 1, after activating the microspheres, coupling with antibodies to prepare fluorescent microsphere-labeled antibodies, the fluorescent microsphere-labeled antibodies include microspheres labeled with Aβ1-42 detection antibodies, microspheres labeled with Aβ1-40 detection antibodies, and microspheres labeled with P-Tau181 detection antibodies, which are set aside; Step 2: prepare an antibody-coated solid-phase plate coated with Aβ1-42, Aβ1-40, and P-Tau181 capture antibodies; Step 3. After adding samples, quality control products or calibrators containing Aβ1-42, Aβ1-40, and P-Tau into the corresponding antibody-coated plate wells, add fluorescent microsphere-labeled antibodies into the corresponding plate wells, and use a digital fluorescence immunoassay to quantify the content of Aβ1-42, Aβ1-40, and P-Tau by analyzing the number of fluorescent particles and matching the standard curve.

2. The single molecule joint detection method of Alzheimer's disease markers Aβ1-42, Aβ1-40, and P-Tau181 according to claim 1, characterized in that: The specific method of step one is: S1. Take the microsphere suspension and add it to the coupling buffer for ultrasonic mixing, centrifuge and remove the supernatant, then repeat the ultrasonic and centrifugal washing in the coupling buffer for several times; add the EDC solution, vortex mix, then add the NHS solution, ultrasonically mix and place it on a turntable for rotation, activate it in the dark for 15-30 minutes, centrifuge and remove the supernatant, then repeat the ultrasonic and centrifugal washing in the coupling buffer for several times to obtain the activated microspheres; S2, adding the activated microspheres into the coupling buffer and mixing by ultrasound; Add the coupling buffer containing the detection antibody to the mixed microspheres and vortex to mix; Place it on a rotating disk and rotate it, protect it from light and couple it for 2 hours to obtain the coupled microspheres and antibodies; S3. Add microsphere blocking solution, vortex mix, place on a turntable for rotation, block for 1 hour in the dark, and remove the supernatant by centrifugation; add microsphere washing solution, ultrasonically mix, and remove the supernatant by centrifugation; add microsphere washing solution again and repeat the washing steps; finally, add microsphere preservation solution to the centrifuge tube, ultrasonically mix, and store in the dark at 2-8℃ for later use.

3. A single molecule joint detection method for Alzheimer's disease markers Aβ1-42, Aβ1-40, and P-Tau181 according to claim 2, characterized in that: The coupling buffer included 10 mM MES, pH 6.2 ± 0.05, and 0.05% ProClin300; The preparation method of EDC solution and NHS solution is as follows: dissolve EDC and NHS in coupling buffer respectively, and the concentrations thereof are both 10 mg / mL; The microsphere blocking solution includes: borate buffer, 1% BSA, 0.24% ethanolamine; wherein the borate buffer includes 5mM boric acid, 11.2mM sodium tetraborate decahydrate, and 0.05% Tween-20; The microsphere washing solution included: 50 mM Tris with a pH of 8.0 ± 0.05, 0.5% BSA, 0.05% Tween-20, and 0.03% ProClin 300; The microsphere storage solution includes: 25mM Tris with a pH of 7.2±0.05, 150mM NaCI, 0.05% Tween-20, 1% BSA, 5% trehalose, and 0.1% ProClin 300.

4. The single molecule joint detection method of Alzheimer's disease markers Aβ1-42, Aβ1-40, and P-Tau181 according to claim 2, characterized in that: The plate described in step 2 is a detection plate, and the steps for antibody coating of the detection plate are as follows: coating according to the Aβ1-42, Aβ1-40, and P-Tau181 capture antibody concentration of 0.1-0.5ug / T, static coating at 2-8°C for 16±1h, washing with washing solution for several times, adding 150ul / well blocking solution after patting dry, blocking at 37°C for 2h, washing with washing solution for several times, putting desiccant into the tin foil bag after patting dry, and setting aside for use.

5. The single molecule joint detection method of Alzheimer's disease markers Aβ1-42, Aβ1-40, and P-Tau181 according to claim 4, characterized in that: The coating solution includes 0.1M sodium carbonate and sodium bicarbonate; The blocking solution includes: 0.05-0.1M PBS with a pH of 7.4, 1-2% BSA, 0.1% ProClin 300, and 0.1% protein protectant; The washing solution includes PBS and TWEEN.

6. A single molecule joint detection method for Alzheimer's disease markers Aβ1-42, Aβ1-40, and P-Tau181 according to claim 2, characterized in that: The plate described in step 2 is a carboxyl-based plate or a polymethyl methacrylate plate. The steps of coating the carboxyl-based plate or the polymethyl methacrylate plate with antibodies are as follows: first, the carboxyl-based plate or the polymethyl methacrylate plate is activated, the plate is washed with PBS and patted dry, 100ul / T of MES solution is added to dilute the antibody and placed in a 37°C incubator for coating for 2h, the plate is washed with PBS and patted dry, 150ul / T of blocking solution is added to place in a 37°C incubator for coating for 1-2h, and the plate is washed with PBS and patted dry for standby use.

7. A single molecule joint detection method for Alzheimer's disease markers Aβ1-42, Aβ1-40, and P-Tau181 according to claim 6, characterized in that: The method for activating the carboxyl-based substrate is as follows: adding 20 mg / ml EDC and NHS solutions to the carboxyl-based substrate respectively and placing the substrate in an incubator for activation.

8. The method for single-molecule joint detection of Alzheimer's disease markers Aβ1-42, Aβ1-40, and P-Tau181 according to claim 6, characterized in that: The method for activating the polymethyl methacrylate plate is as follows: placing the polymethyl methacrylate plate in a plasma instrument for plasma surface activation.

9. The single molecule joint detection method for Alzheimer's disease markers Aβ1-42, Aβ1-40, and P-Tau181 according to claim 1, characterized in that: The preparation method of the quality control / calibrator in step 3 is as follows: Aβ1-42, Aβ1-40, P-Tau181 protein, PBS buffer, bovine serum albumin, protein protective agent and 0.2% ProClin 300 are mixed.

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