A single-molecule detection method for the Alzheimer's disease biomarker P-tau181

Abstract

This invention provides a single-molecule detection method for the Alzheimer's disease biomarker P-tau181, belonging to the field of biotechnology. The method includes: preparing an enzyme-linked immunosorbent assay (ELISA) plate coated with a capture antibody; preparing fluorescent particles coated with a detection antibody; capturing the target protein P-tau181 using the ELISA plate; amplifying the fluorescence signal using the fluorescent particles; and performing digital imaging and analysis using a microscopic fluorescence imaging system. This invention utilizes 50-300 nm fluorescent particles to achieve efficient signal amplification, combined with a liquid-free microscopic imaging system for single-molecule counting, achieving a detection limit as low as 150 fg / mL. 2 =0.9996. This invention has the advantages of high sensitivity, low instrument cost, medium to high throughput, simple operation, and good visibility, and is suitable for large-scale early screening, early warning, clinical auxiliary diagnosis, and long-term monitoring of Alzheimer's disease.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a single-molecule detection method for the Alzheimer's disease biomarker P-tau181. Background Technology

[0002] Alzheimer's disease is a common neurodegenerative disease, and early diagnosis is crucial for slowing disease progression and improving patients' quality of life. As a key blood biomarker for Alzheimer's disease, P-tau181 has attracted significant attention due to the research on highly sensitive detection methods. Currently, traditional methods for detecting P-tau181, a bodily fluid biomarker for Alzheimer's disease, mainly rely on cerebrospinal fluid samples, which suffers from complex sampling procedures, high levels of specialization, and expensive equipment, making them unsuitable for large-scale screening. Furthermore, the concentration of P-tau181 in blood is low (approximately 1.65 pg / mL) and easily affected by blood-brain barrier permeability and changes in systemic physiological state, placing higher demands on the sensitivity of detection methods.

[0003] Existing immunoassay methods for detecting P-tau181 have the following main shortcomings: First, insufficient detection sensitivity. Traditional immunosorbent assays (such as ELISA) use enzyme-catalyzed substrate color development for signal amplification. The signal generated by a single enzyme molecule is limited, making it difficult to detect low concentrations of P-tau181 (approximately 1.65 pg / mL) in blood. This easily leads to false negative results, limiting its application in early screening and accurate diagnosis. Second, high instrument costs. While some high-sensitivity detection methods (such as Simoa single-molecule array technology) can achieve single-molecule level detection, they rely on complex microfluidic systems to isolate individual molecules and react them in independent microchambers. The equipment is expensive and has high maintenance costs, making it difficult to promote and popularize in ordinary laboratories and primary hospitals. Third, difficulty in balancing throughput and cost. Existing high-sensitivity detection methods mostly use dedicated microwell chips or microfluidic chips, resulting in high consumable costs and limited sample throughput per test. This makes it difficult to meet the urgent need for medium-to-high throughput and low-cost detection technologies for large-scale population screening. Fourth, cumbersome operation procedures. Traditional cerebrospinal fluid testing methods require lumbar puncture, which is complex and has low patient acceptance. Some high-sensitivity testing methods involve multiple complex steps or special reagent preparation processes, requiring a high degree of professional expertise from operators, which is not conducive to routine clinical testing. Fifth, the visibility of test results is poor. Most existing testing methods are "black box" tests, only outputting the final reading without visually presenting the reaction process and results. It is difficult for laboratory personnel to make real-time judgments on test quality and trace the source of problems, which is not conducive to the visualization and quality control of results.

[0004] In summary, existing technologies lack a P-tau181 detection method that can simultaneously address the five issues mentioned above. Therefore, developing a P-tau181 single-molecule detection method that is highly sensitive, low-cost, has medium-to-high throughput, is easy to operate, and offers good visibility is of great significance for large-scale early screening, early warning, clinical auxiliary diagnosis, and long-term monitoring of Alzheimer's disease. Summary of the Invention

[0005] The purpose of this invention is to provide a single-molecule detection method for the Alzheimer's disease biomarker P-tau181. This method has advantages such as ultra-high sensitivity, strong specificity, low cost, medium to high throughput, and ease of operation.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A single-molecule detection method for the Alzheimer's disease biomarker P-tau181 includes the following steps: S1: Prepare an ELISA plate coated with an antibody corresponding to the capture of P-tau181 protein; S2: Prepare fluorescent particles coated with the detection antibody corresponding to P-tau181 protein; S3: Capture the target protein P-tau181 using the coated ELISA plate; S4: Amplify the fluorescence signal of the target protein P-tau181 using the coated fluorescent particles; S5: Digital imaging and analysis of the target protein P-tau181 using a micro-fluorescence imaging system.

[0007] As a preferred technical solution, step S1 specifically includes: S1.1 Prepare the washing buffer for the microplate: Weigh 1.59 g sodium carbonate, 2.94 g sodium bicarbonate and 5 mL Tween-20, add ultrapure water to a final volume of 1 L, and stir thoroughly to dissolve; S1.2 Prepare the coating buffer for the ELISA plate: Weigh 1.59 g of sodium carbonate and 2.94 g of sodium bicarbonate, add ultrapure water to a final volume of 1 L, and stir thoroughly to dissolve; S1.3 Prepare the blocking buffer for ELISA plates: Weigh 3.40 g sodium hydrogen phosphate, 6.32 g sodium dihydrogen phosphate, 20.0 g bovine serum albumin and 1.05 g Proclin 300, add ultrapure water to a final volume of 1 L, and stir thoroughly to dissolve. S1.4 Preparation of capture antibody-coated ELISA plates: Dilute the capture antibody corresponding to P-tau181 protein to 1-5 μg / mL with coating buffer, add 100 μL of the diluted capture antibody solution to each well of the ELISA plate, react at 4 ℃ for 16 hours, then wash the plate 5 times with washing buffer and pat dry for later use.

[0008] As a preferred technical solution, step S2 specifically includes: S2.1 Preparation of fluorescent particle coupling buffer: Dissolve 195 mg 2-(N-morpholino)ethanesulfonic acid and 52.5 mg Proclin 300 in 80 mL of ultrapure water, adjust the pH to 6.3 with 280 mg / mL KOH aqueous solution, and bring the volume to 100 mL. S2.2 Preparation of EDC solution: Dissolve 100 mg EDC in 10 mL of fluorescent particle coupling buffer; S2.3 Preparation of NHS solution: Dissolve 100 mg NHS in 10 mL of fluorescent particle coupling buffer; S2.4 Preparation of fluorescent particle blocking solution: Dissolve 31 mg boric acid, 427 mg sodium tetraborate decahydrate, 500 μL Tween-20, 1.00 g bovine serum albumin and 240 mg ethanolamine in 100 mL of ultrapure water; S2.5 Preparation of fluorescent particle coupling washing solution: Dissolve 606 mg of tris(hydroxymethyl)aminomethane, 500 mg of bovine serum albumin, 250 μL of Tween-20, and 30 mg of Proclin 300 in 100 mL of ultrapure water.

[0009] S2.6 Preparation of fluorescent particle preservation solution: Dissolve 303 mg tris(hydroxymethyl)aminomethane, 877 mg sodium chloride, 250 μL Tween-20, 1.00 g bovine serum albumin, 5.00 g trehalose and 100 mg Proclin 300 in 100 mL of ultrapure water.

[0010] S2.7 Activation of fluorescent particles: Add 0.05 mL of fluorescent particle dispersion (10 mg / mL) to 1 mL of coupling buffer, sonicate for 3 minutes, centrifuge at 2000 g for 10 minutes, and remove the supernatant; repeat the washing once, add 1 mL of coupling buffer, sonicate for 3 minutes, add 3.5 μL of EDC solution and 3.5 μL of NHS solution in sequence, sonicate for 3 minutes, and react on a shaker at 37 ℃ in the dark for 30 minutes; centrifuge to remove the supernatant, and wash twice with coupling buffer; S2.8 Conjugation of fluorescent particles to antibodies: Add 0.75 mL of conjugation buffer, sonicate for 3 minutes, add 0.25 mL of detection antibody with a concentration of 0.2 mg / mL, vortex for 1 minute to mix, and react on a shaker at 37 ℃ for 2 hours in the dark. S2.9 Blocking and preservation of fluorescent particles: Add 0.5 mL of blocking solution, vortex for 1 minute, and react on a shaker at 37 ℃ for 1 hour in the dark; remove the supernatant by centrifugation, wash once with coupling buffer, and finally add 500 μL of preservation solution, sonicate to mix, and store at 4 ℃ for later use.

[0011] As a preferred technical solution, step S3 specifically includes: S3.1 Preparation of phosphate buffer washing solution: Dissolve 3.40 g sodium hydrogen phosphate, 6.32 g sodium dihydrogen phosphate and 5 mL Tween-20 in 1 L of ultrapure water and stir thoroughly to dissolve; S3.2 Capture the target protein: Take 45 μL of calibrator, quality control or body fluid sample containing P-tau181 protein, add it to the well of the ELISA plate coated with capture antibody, react on a shaker at 37 ℃ for 0.5 hours, then wash 5 times with 300 μL of phosphate buffer and pat dry.

[0012] As a preferred technical solution, step S4 specifically involves: The fluorescent particles coated with the detection antibody were diluted 20 times with fluorescent particle preservation solution, and 100 μL was added to the wells of the ELISA plate. The plate was incubated on a shaker at 37 °C for 0.5 hours. The plate was then washed 5 times with 300 μL of phosphate buffer and patted dry.

[0013] As a preferred technical solution, step S5 specifically includes: Place the ELISA plate on the sample stage of the fluorescence microscopy system, and use 10x, 20x, 50x, or 100x objectives to acquire digital images via transmission or reflection. Use counting and data processing software to plot a standard curve, substitute the signal values ​​of the quality control sample and the test body fluid sample into the standard curve, and calculate the concentration of P-tau181 protein in the sample. Perform statistical analysis on the number of fluorescent particles in the acquired images to obtain the detection results.

[0014] Furthermore, in the method, the concentration of P-tau181 protein in the range of 0.15 pg / mL to 1000 pg / mL shows a positive correlation with the number of fluorescent particles in the imaging, with a detection limit of 150 fg / mL and an R-value of [missing value] for the fitted curve. 2 The value is 0.9996.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. Detection sensitivity reaches the single-molecule level, with a detection limit as low as 150 fg / mL. This invention enables single-molecule quantitative detection of P-tau181 protein with a detection limit as low as 150 fg / mL, far below its concentration in blood (approximately 1.65 pg / mL), effectively meeting the detection requirements for low-abundance biomarkers. Detection antibodies are coupled with 50–300 nm fluorescent particles. Each fluorescent particle generates an extremely strong fluorescence signal under excitation light. Compared to the limited signal generated by a single enzyme molecule catalyzing the substrate in traditional ELISA, this achieves efficient signal amplification for single antigen-antibody binding events, allowing even extremely low-abundance target proteins to be reliably captured by subsequent imaging systems. Direct counting of fluorescent particles, i.e., digital analysis, is performed using a yes / no signal reading method instead of the grayscale reading of traditional methods, effectively avoiding interference from background noise and signal intensity fluctuations. Experimental results show that within the P-tau181 protein concentration range of 0.15 pg / mL to 1000 pg / mL, the number of fluorescent particles imaged is positively correlated with the protein concentration, and the R-value of the fitted curve is [value missing]. 2 The value reached 0.9996, proving that the digital analysis method has extremely high linear correlation and detection accuracy.

[0016] 2. Instrument costs are significantly reduced, eliminating the need for complex liquid circuit systems. This invention significantly reduces the cost of core detection equipment by employing digital imaging and analysis using reflection or transmission imaging without the need for a liquid path detection module. Unlike mainstream single-molecule detection platforms (such as Simoa) that rely on complex microfluidic liquid path systems, this invention requires only a standard research-grade fluorescence microscope and a conventional stage to complete all imaging and detection. The entire reaction is performed within a standard 96-well or 384-well ELISA plate, eliminating the need for any microfluidic chips or special liquid path rinsing modules. This design greatly reduces the procurement, operation, and maintenance costs of the instrument, making the widespread application of this technology in ordinary laboratories and primary hospitals possible.

[0017] 3. It balances medium-to-high throughput with low cost, making it suitable for large-scale screening. This invention balances high sensitivity with detection efficiency and cost control, employing 96-well or 384-well transparent substrate ELISA plates (made of glass, polystyrene, or polymethyl methacrylate), the most standard and mature consumable form in the field of bioassay. Using standard plates means that dozens to hundreds of samples can be processed simultaneously, and the entire capture, washing, and reaction process can be completed using conventional equipment such as multi-channel pipettes and plate washers, making operation simple and easily automated. Compared to expensive dedicated single-molecule detection chips, the consumable cost of standard ELISA plates is extremely low. This combination of a high-sensitivity method and a low-consumable platform gives this invention the dual advantages of high throughput and low cost, making it particularly suitable for large-scale early screening of Alzheimer's disease.

[0018] 4. Simple to operate, serum samples can be used directly. Compared to traditional cerebrospinal fluid (CSF) testing or complex single-molecule detection procedures, this invention significantly simplifies the operational steps and allows for the direct use of blood samples. Using the body fluid sample to be tested (including serum), it eliminates the need for invasive lumbar puncture to obtain CSF, simplifying sampling, increasing patient acceptance, and requiring only 45 μL of sample, resulting in a small volume. In the entire immune reaction, the capture and fluorescence amplification steps are each reacted for 0.5 hours at 37 °C, totaling a core reaction time of 1 hour. Combined with overnight coating (which can be pre-prepared in batches), the overall process is highly efficient and rapid. The washing step involves five washes with standard phosphate buffer, a mature and reliable procedure. Furthermore, the EDC / NHS chemical method is used to covalently couple the antibody to the surface of the fluorescent particles. This is a classic and mature technology in the field of bioconjugation. The entire preparation process can be completed within one day, and the prepared fluorescent particles can be stored long-term at 4 °C, enabling batch and large-scale preparation of the reagents for immediate use and reducing batch-to-batch variability.

[0019] 5. Good visibility, facilitating result quality control and problem troubleshooting. The visualization feature of this invention is a significant advantage over traditional black-box detection methods. The digital imaging process directly transforms the series of microscopic immune reaction events—antibody capture of target proteins and binding of fluorescent particles—into fluorescent dot images that can be observed and analyzed by the human eye or software. Researchers can directly observe the morphology, distribution, and quantity of fluorescent dots in each well, intuitively judging experimental quality, such as background cleanliness and particle aggregation, which helps to eliminate false positives or false negatives. If the results are abnormal, the problematic step can be quickly located by reviewing the original images, facilitating process optimization. The standard curve not only provides the calculation results, but the error bars at each point are derived from statistical analysis of multiple images. This WYSIWYG characteristic greatly enhances the persuasiveness of the data and the traceability of the results.

[0020] In summary, this invention systematically solves the pain points of existing technologies, such as low sensitivity, high instrument cost, and cumbersome operation, by combining specific capture based on standard well plates, efficient signal amplification mediated by fluorescent particles, and digital imaging of a liquid-free microscopy system. It provides a single-molecule detection method for P-tau181 that is highly sensitive, low-cost, easy to operate, has medium to high throughput, and good visibility. It is suitable for large-scale early screening, early warning, clinical auxiliary diagnosis, and long-term monitoring of Alzheimer's disease. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the specific embodiments of the present invention, the accompanying drawings used in the specific embodiments will be briefly introduced below. Obviously, the drawings described below are only some specific embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 A schematic diagram illustrating the basic principle of the single-molecule detection method for the Alzheimer's disease biomarker P-tau181 provided by the present invention; Figure 2 The standard curve for detecting P-tau181, a biomarker for Alzheimer's disease, using a digital protein detection method based on fluorescence immunoadsorption. Detailed Implementation

[0023] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The same or similar reference numerals denote the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0024] like Figure 1 As shown, a single-molecule detection method for the Alzheimer's disease biomarker P-tau181 includes the following steps: S1: Preparation of ELISA plates coated with capture antibodies corresponding to the P-tau181 protein. S1.1 Prepare the washing buffer for the ELISA plate: Weigh 1.59 g sodium carbonate, 2.94 g sodium bicarbonate and 5 mL Tween-20 into a beaker, add ultrapure water to make up to 1 L, and stir thoroughly to dissolve.

[0025] S1.2 Prepare the coating buffer for the ELISA plate: Weigh 1.59 g of sodium carbonate and 2.94 g of sodium bicarbonate into a beaker, add ultrapure water to a final volume of 1 L, and stir thoroughly to dissolve.

[0026] S1.3 Prepare the blocking buffer for ELISA plates: Weigh 3.40 g sodium hydrogen phosphate, 6.32 g sodium dihydrogen phosphate, 20.0 g bovine serum albumin and 1.05 g Proclin 300 into a beaker, add ultrapure water to a final volume of 1 L, and stir thoroughly to dissolve.

[0027] S1.4 Preparation of enzyme-labeled plates coated with capture antibodies: Dilute the capture antibody corresponding to P-tau181 protein to 1–5 μg / mL using coating buffer, add 100 μL of the diluted capture antibody solution to each well of the ELISA plate, and incubate at 4 °C for 16 hours. Then wash the plate five times with washing buffer, blot dry, and set aside.

[0028] S2: Preparation of fluorescent particles coated with the detection antibody corresponding to P-tau181 protein. S2.1 Preparation of fluorescent particle coupling buffer: Dissolve 195 mg 2-(N-morpholino)ethanesulfonic acid and 52.5 mg Proclin 300 in 80 mL of ultrapure water, adjust the pH to 6.3 with 280 mg / mL KOH aqueous solution, and bring the volume to 100 mL. S2.2 Preparation of EDC solution: Dissolve 100 mg EDC in 10 mL of fluorescent particle coupling buffer; S2.3 Preparation of NHS solution: Dissolve 100 mg NHS in 10 mL of fluorescent particle coupling buffer; S2.4 Preparation of fluorescent particle blocking solution: Dissolve 31 mg boric acid, 427 mg sodium tetraborate decahydrate, 500 μL Tween-20, 1.00 g bovine serum albumin, and 240 mg ethanolamine in 100 mL of ultrapure water. S2.5 Preparation of fluorescent particle coupling washing solution: Dissolve 606 mg of tris(hydroxymethyl)aminomethane, 500 mg of bovine serum albumin, 250 μL of Tween-20, and 30 mg of Proclin 300 in 100 mL of ultrapure water.

[0029] S2.6 Preparation of fluorescent particle preservation solution: Dissolve 303 mg tris(hydroxymethyl)aminomethane, 877 mg sodium chloride, 250 μL Tween-20, 1.00 g bovine serum albumin, 5.00 g trehalose and 100 mg Proclin 300 in 100 mL of ultrapure water.

[0030] S2.7 Activation of fluorescent particles: Add 0.05 mL of fluorescent particle dispersion (10 mg / mL) to 1 mL of fluorescent particle conjugation buffer, sonicate for 3 minutes, centrifuge at 2000 g for 10 minutes, and remove the supernatant. Add another 1 mL of conjugation buffer, sonicate for 3 minutes, centrifuge under the same conditions, and remove the supernatant. Repeat the above operation once, add 1 mL of conjugation buffer, sonicate for 3 minutes, then add 3.5 μL of EDC solution and 3.5 μL of NHS solution, sonicate for 3 minutes. Incubate in the dark on a shaker at 37 °C for 30 minutes. Then centrifuge at 2000 g for 10 minutes and remove the supernatant. Add 1.5 mL of conjugation buffer, sonicate for 3 minutes, centrifuge, remove the supernatant, and repeat the washing once.

[0031] S2.8 Conjugation of fluorescent particles to antibodies: Add 0.75 mL of fluorescent particle conjugation buffer and sonicate for 3 minutes. Add 0.25 mL of 0.2 mg / mL detection antibody (conjugation buffer as solvent), vortex for 1 minute, and incubate in the dark on a shaker at 37 °C for 2 hours.

[0032] S2.9 Blocking and preservation of fluorescent particles: Add 0.5 mL of fluorescent particle blocking solution, vortex for 1 minute to mix, and incubate on a shaker at 37 °C for 1 hour in the dark. Then centrifuge at 2000 g for 10 minutes and remove the supernatant. Add 1 mL of coupling buffer, sonicate for 3 minutes, centrifuge, and remove the supernatant. Finally, add 500 μL of fluorescent particle preservation solution, sonicate to mix, and store at 4 °C for later use.

[0033] S3: Capture the target protein P-tau181 using the coated ELISA plate. S3.1 Prepare phosphate buffer washing solution: Dissolve 3.40 g sodium hydrogen phosphate, 6.32 g sodium dihydrogen phosphate and 5 mL Tween-20 in 1 L of ultrapure water and stir thoroughly to dissolve.

[0034] S3.2 Capture target protein: Take 45 μL of calibrator, quality control, or test body fluid sample containing P-tau181 protein and add it to the well of an ELISA plate coated with capture antibody. Place the plate on a shaker and incubate at 37 °C for 0.5 hours. Then wash five times with 300 μL of phosphate buffer and pat dry.

[0035] S4: Utilize fluorescent particles to amplify the fluorescence signal of the target protein P-tau181. The fluorescent particles coated with the detection antibody were diluted 20-fold with fluorescent particle preservation solution, and 100 μL was added sequentially to the wells of the above-mentioned ELISA plate. The plate was placed on a shaker and incubated at 37 °C for 0.5 hours. Then, the plate was washed 5 times with 300 μL of phosphate buffer and patted dry.

[0036] S5: Digital Imaging and Analysis Using Microfluorescence Imaging Systems Place the ELISA plate on the sample stage of the fluorescence microscopy system, and use 10x, 20x, 50x, or 100x objectives to acquire digital images via transmission or reflection. Use counting and data processing software to plot a standard curve, and substitute the signal values ​​of the quality control sample and the test fluid sample into the standard curve to calculate the concentration of P-tau181 protein in the sample.

[0037] Statistical analysis of the number of fluorescent particles in the S5 acquired images showed that, within the P-tau181 protein concentration range of 0.15 pg / mL to 1000 pg / mL, the number of fluorescent particles was positively correlated with the protein concentration. The detection limit for P-tau181 protein using this method was 150 fg / mL, and the R-value of the fitted curve was [value missing]. 2 The value is 0.9996, such as Figure 2 As shown.

[0038] In the description of this specification, references to the terms "an embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0039] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. A single-molecule detection method for the Alzheimer's disease biomarker P-tau181, characterized in that, Includes the following steps: S1: Prepare an ELISA plate coated with an antibody corresponding to the capture of P-tau181 protein; S2: Prepare fluorescent particles coated with the detection antibody corresponding to P-tau181 protein; S3: Capture the target protein P-tau181 using the coated ELISA plate; S4: Amplify the fluorescence signal of the target protein P-tau181 using the coated fluorescent particles; S5: Digital imaging and analysis of the target protein P-tau181 using a micro-fluorescence imaging system.

2. The method according to claim 1, characterized in that, The enzyme-labeled plate mentioned in step S1 is a transparent substrate enzyme-labeled plate with 96 wells or 384 wells, made of glass, polystyrene or polymethyl methacrylate.

3. The method according to claim 1, characterized in that, The fluorescent particles described in step S2 have a size of 50 to 300 nanometers and can generate fluorescent signals when excited by a light source of a suitable wavelength.

4. The method according to claim 1, characterized in that, The micro-fluorescence imaging system described in step S5 performs digital imaging and analysis using reflective or transmissive imaging without being equipped with a liquid path detection module.

5. The method according to claim 1, characterized in that, Step S1 specifically includes: diluting the capture antibody to 1-5 μg / mL with coating buffer, adding it to the wells of an ELISA plate, reacting at 4 °C for 16 hours, washing the plate, and then using it for later use.

6. The method according to claim 1, characterized in that, Step S2 specifically includes: using EDC / NHS chemical methods to covalently couple the detection antibody to the surface of fluorescent particles, and then storing it for later use after blocking.

7. The method according to claim 1, characterized in that, Step S3 specifically includes: taking 45 μL of calibrator, quality control or body fluid sample containing P-tau181 protein, adding it to the well of the ELISA plate, reacting at 37 ℃ for 0.5 hours, washing and patting dry.

8. The method according to claim 1, characterized in that, Step S4 specifically includes: diluting the fluorescent particles coated with the detection antibody by 20 times, adding 100 μL to the wells of the ELISA plate, reacting at 37 ℃ for 0.5 hours, washing and patting dry.

9. The method according to claim 1, characterized in that, Step S5 specifically includes: placing the ELISA plate on the sample stage of the microluminescence system, selecting a 10x to 100x objective lens, acquiring digital images by transmission or reflection, plotting a standard curve using counting software, and calculating the concentration of P-tau181 protein in the sample.

10. The method according to any one of claims 1 to 9, characterized in that, The method has a detection limit of 150 fg / mL for P-tau181 protein. Within a concentration range of 0.15 pg / mL to 1000 pg / mL, the number of fluorescent particles is positively correlated with protein concentration. The R-squared value of the fitted curve is... 2 The value is 0.9996.

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