Method for detecting phosphorylated Tau-181 protein in urine of patient with Alzheimer's disease

Abstract

The invention discloses a method for detecting phosphorylated Tau-181 protein in urine of a patient with Alzheimer's disease by utilizing a liquid-phase fluorescence immunoreaction principle, which is mainly characterized by comprising the following steps: by utilizing the amplification effect of fluorescent microspheres and magnetic particle microspheres, the fluorescent microspheres are tracers, the magnetic particle microspheres have enrichment and concentration effects, and the phosphorylated Tau-181 protein in urine of the patient with Alzheimer's disease can be detected; the method comprises the following steps: combining a fluorescence labeled Tau-181 antibody and a magnetic particle labeled Tau antibody with a Tau-p-181 antigen in urine to form an immune complex by utilizing a principle of specific combination of an immunology antigen and an immunology antibody; a pair of paired antibodies is used for specifically grabbing ultralow-concentration target protein in urine and concentrating the ultralow-concentration target protein, then a biological recognition event is converted into a strong optical signal by means of high-brightness fluorescent microspheres, finally qualitative or quantitative detection is achieved through naked eyes or an instrument, and finally detection of phosphorylated Tau-181 protein in the urine is achieved. Therefore, the purposes of early screening, early intervention and early diagnosis of the Alzheimer's disease are achieved, and the operation threshold and the economic cost are reduced.

Description

Technical Field

[0001] This invention relates to the field of Alzheimer's disease detection technology, specifically a method for detecting phosphorylated Tau-181 protein in the urine of Alzheimer's patients. Background Technology

[0002] The occurrence and development of Alzheimer's disease (AD) is a biological process. Various specific indicators slowly accumulate in asymptomatic individuals until neuropathological changes occur in the brain. These gradual changes eventually lead to the appearance and aggravation of severe clinical symptoms. The occurrence of Alzheimer's disease places a heavy burden on society and families. It is essential to establish an early, simple, effective and inexpensive immunological detection method for AD.

[0003] Current technology shows that some specific biological markers of Alzheimer's disease (AD) begin to be released into the blood and excreted in urine 5-10 years before the onset of clinical symptoms. Although research on the clinical symptoms of AD has a history of more than 100 years, only in recent years have low-risk blood immunological detection methods emerged to detect several key biological markers of AD, such as single-molecule immunoassay and chemiluminescence immunoassay.

[0004] Blood immunology testing for Alzheimer's disease (AD) uses ultra-high sensitivity detection technology to "capture" and "amplify" the core pathological signals of AD that can only be observed in cerebrospinal fluid or through imaging in a blood sample. However, blood immunology testing has a high operational threshold and high overall cost, which together limit its widespread application in large-scale population screening, repeated dynamic monitoring, and resource-scarce areas.

[0005] Therefore, in response to the above problems, we have invented a method for detecting phosphorylated Tau-181 protein in the urine of Alzheimer's patients. This method is non-invasive, simple, and inexpensive, suitable for the needs of the general public, and has broad application prospects. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies and address the limitations of invasive blood immunoassay tests, which are often cumbersome, have high operational barriers, and are costly, thus restricting large-scale population screening, this invention proposes a method for detecting phosphorylated Tau-181 protein in the urine of Alzheimer's patients. This method is suitable for use in primary healthcare institutions and at home.

[0007] A method for detecting phosphorylated Tau-181 protein in the urine of Alzheimer's patients includes the following steps: S1: Take a 5ml reaction tube containing pre-prepared fluorescent microsphere-labeled Tau-p-181 antibody and magnetic particle-labeled Tau antibody. S2: Add 100 μL of urine sample to the 5 mL reaction tube mentioned above. S3: Add 4.9 ml of urine diluent to this 5 ml reaction tube, mix by hand by inverting 10 times, and let it react at room temperature for 8-12 hours. S4: Use a magnet to enrich the immune complex, irradiate the complex with ultraviolet light at around 340nm, and determine the fluorescence intensity of the complex. S5: Samples without fluorescent aggregation points are negative, and samples with fluorescent aggregation points are positive.

[0008] Preferably, the fluorescent microspheres can be time-resolved fluorescent microspheres, quantum dot fluorescent microspheres, aggregated luminescent microspheres, or fluorescein microspheres, and the particle size of the fluorescent microspheres is 100nm-3000nm.

[0009] Preferably, the magnetic particles have a particle size of 100nm-3000nm.

[0010] Preferably, the urine diluent is a 25mM phosphate buffer containing 0.2% Tween-20, used to dilute the urine at a ratio of 1:40 to 1:200.

[0011] Preferably, the magnetically labeled Tau antibody mixture comprises anti-Tau antibody, phosphate buffer, carboxyl magnetic particles, ethanesulfonic acid solution, and carbodiimide solution.

[0012] Preferably, the blocking solution comprises Hepes at pH 7.4, BSA glycine at a concentration of 5.0%, and Proclin at a concentration of 0.1%.

[0013] Preferably, the complex solution comprises Hepes, 10% trehalose, 2% BSA, glycine, and 0.1% Proclin.

[0014] Preferably, the shaking includes manually inverting and mixing, and shaking on a rotator.

[0015] Preferably, the stronger the fluorescence intensity, the higher the content of phosphorylated Tau protein-181 in the urine sample, and the reaction result can be effectively retained for 24 hours at room temperature.

[0016] The advantages of this invention are: By utilizing a pair of paired antibodies (capture antibody and detection antibody) to specifically capture ultra-low concentrations of target proteins in urine, magnetic beads are used to "fish" them out of a complex matrix and concentrate them, and then high-brightness fluorescent microspheres are used to convert biorecognition events into strong light signals. Finally, qualitative or quantitative detection can be achieved by visual inspection or instruments, thereby realizing the detection of phosphorylated Tau-181 protein in urine, reducing the operational threshold and economic cost. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a flowchart of one embodiment of the present invention; Figure 2 This is a schematic diagram of an embodiment of the present invention before ultraviolet irradiation; Figure 3 This is a schematic diagram of ultraviolet irradiation according to an embodiment of the present invention; Figure 4 This is a schematic diagram of the measurement results of a fluorescence spectrophotometer according to an embodiment of the present invention. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] A method for detecting phosphorylated Tau-181 protein in the urine of Alzheimer's patients includes the following steps: S1: Take a 5ml reaction tube containing pre-prepared fluorescent microsphere-labeled Tau-p-181 antibody and magnetic particle-labeled Tau antibody. S2: Add 100 μL of urine sample to the 5 mL reaction tube mentioned above. S3: Add 4.9 ml of urine diluent to this 5 ml reaction tube, mix by hand by inverting 10 times, and let it react at room temperature for 8-12 hours. S4: Use a magnet to enrich the immune complex, irradiate the complex with ultraviolet light at around 340nm, and determine the fluorescence intensity of the complex. S5: Samples without fluorescent aggregation points are negative, and samples with fluorescent aggregation points are positive.

[0021] Furthermore, the fluorescent microspheres may be time-resolved fluorescent microspheres, quantum dot fluorescent microspheres, aggregated luminescent microspheres, or fluorescein microspheres, with a particle size of 100 nm to 3000 nm.

[0022] Furthermore, the magnetic particles have a particle size of 100nm-3000nm.

[0023] Furthermore, the urine diluent is a 25mM phosphate buffer solution containing 0.2% Tween-20, used to dilute the urine at a ratio of 1:40 to 1:200.

[0024] Furthermore, the magnetically labeled Tau antibody mixture comprises anti-Tau antibody, phosphate buffer, carboxyl magnetic particles, melamine ethanesulfonic acid solution, and carbodiimide solution.

[0025] Furthermore, the blocking solution comprises Hepes at pH 7.4, BSA glycine at a concentration of 5.0%, and Proclin at a concentration of 0.1%.

[0026] Furthermore, the complex solution comprises Hepes, 10% trehalose, 2% BSA, glycine, and 0.1% Proclin.

[0027] Furthermore, the mixing includes manually inverting and mixing, and shaking on a gyroscope.

[0028] Furthermore, the stronger the fluorescence intensity, the higher the content of phosphorylated Tau protein-181 in the urine sample, and the reaction results can be retained at room temperature for 12-24 hours.

[0029] Existing blood immune tests are highly invasive, have high operational barriers, and are costly in general. These factors together limit their widespread application in large-scale population screening, repeated dynamic monitoring, and resource-scarce areas.

[0030] Example 1: Preparation of detection reagents 1.1 Preparation of Tau antibody labeled with magnetic microspheres The anti-Tau antibody was diluted with 25 mM phosphate buffer (pH 7.4) to a concentration of 1.0 mg / ml.

[0031] Take 100 μL of a suspension of carboxylated magnetic particles with a particle size of 500 nm and place it in a 2.0 ml centrifuge tube.

[0032] Add 1.0 ml of 200 mM ethanesulfonic acid buffer to the centrifuge tube.

[0033] Add 200 μL of carbodiimide solution (10 mg / mL) and shake well.

[0034] Place the centrifuge tubes on a gyroscope and activate at room temperature for 20 minutes.

[0035] After activation, immediately add 100 μL of the anti-Tau antibody solution diluted in step 1 and mix quickly.

[0036] Place the mixture on a rotary mixer and couple it at room temperature in the dark for 2 hours.

[0037] Add 500 μL of blocking buffer (formulation: 50 mM Hepes, pH 7.4, containing 5.0% BSA, 200 mM glycine, 0.1% Proclin-300), mix well, and continue to rotate for 6 hours. Hepes is the "gold standard" buffer designed to maintain and simulate physiological pH environments. It plays an irreplaceable role in cell culture and most biochemical experiments that need to be conducted at near-neutral pH. BSA is one of the most widely used and almost ubiquitous proteins in biomedical laboratories. Proclin is a very important class of preservatives in biological and diagnostic laboratories, used to prevent liquid reagents (especially ready-to-use reagents and buffers) from being contaminated and deteriorated by microorganisms (bacteria, fungi). For steps requiring rapid mixing, manual mixing is used. This allows for quick and effective mixing and reduces operating costs. Using a gyroscope for mixing can achieve steps that require long mixing times. Coupling refers to the establishment of a stable, usually covalent bond, between two molecules (such as proteins, nucleic acids, small molecules, and polymers) through chemical methods.

[0038] Place the centrifuge tube on a magnetic rack and let it stand for 5 minutes. After the magnetic particles are fully enriched, carefully discard the supernatant. Enrichment specifically refers to the use of magnetic separation technology. The purpose of this step is to prepare magnetic particles with specific antibodies to capture, separate, and concentrate the extremely low concentration of the target protein (phosphorylated Tau-181 protein) and its formed immune complexes from a large amount of liquid in the urine sample to a specific location on the tube wall. It is the core technology guarantee for achieving highly sensitive, non-invasive urine detection, making subsequent visual observation or fluorescence quantification possible.

[0039] Add 1 ml of PBST washing buffer (containing 0.2% Tween-20 phosphate buffer), gently tap the tube wall to resuspend the particles, place them on a magnetic rack again to separate, discard the supernatant, and repeat the washing once. Tween is the most commonly used nonionic surfactant (detergent) in biochemistry and molecular biology laboratories.

[0040] The precipitated magnetic particle-antibody complex was resuspended in 2.0 ml of reconstitution solution (formulation: 50 mM Hpeps, pH 7.4, containing 10% trehalose, 2.0% BSA, 500 mM glycine, 0.1% Proclin-300).

[0041] Store in a refrigerator at 4°C for later use.

[0042] 1.2 Preparation of fluorescent microsphere-labeled anti-Tau-p-181 antibody The antiphosphorylated Tau-181 (Tau-p-181) specific monoclonal antibody was diluted with 25 mM phosphate buffer to a concentration of 1.0 mg / ml.

[0043] Take 100 μL of a suspension of carboxylated quantum dot fluorescent microspheres with a particle size of 200 nm (excitation / emission wavelength: 340 nm / 610 nm) and place it in a 2.0 mL centrifuge tube.

[0044] The subsequent activation, coupling, and blocking steps are the same as steps 3-8 in 1.1.

[0045] After the coupling and sealing are completed, centrifuge the centrifuge tubes at 4°C and 15000r / min for 30 minutes, and carefully discard the supernatant.

[0046] The precipitated fluorescent microsphere-antibody complex was resuspended in 2.0 ml of reconstitution solution.

[0047] Store at 4℃ away from light for later use.

[0048] Example 2: Detection procedure for Tau-p-181 in urine samples 2.1 Sample Pretreatment The first midstream urine of the subject in the morning was collected in a clean container.

[0049] Gently invert the urine sample 10 times to mix.

[0050] Accurately pipette 100 μL of the mixed urine and add it to a 5.0 mL clear centrifuge tube.

[0051] 2.2 Detection reaction These 5.0ml centrifuge tubes are pre-packaged reaction tubes, and the contents are lyophilized and contain: The fluorescent microsphere-labeled anti-Tau-p-181 antibody (equivalent to 0.1 μg antibody) prepared in Example 1. The magnetic particle-labeled anti-Tau antibody (equivalent to 0.1 μg antibody) prepared in Example 1.

[0052] Add 4.9 ml of sample diluent (25 mM phosphate buffer, pH 7.4, containing 0.2% Tween-20 and 0.1% Proclin-300) to the reaction tube. At this point, the urine dilution ratio is 1:50. The urine diluent and dilution ratio in this step are one of the key factors to ensure the specificity of the reaction and eliminate interference.

[0053] Tighten the cap and manually invert the tube 20 times to ensure the lyophilized reagent is completely dissolved and mixed evenly.

[0054] Allow the reaction to stand at room temperature (approximately 25°C) for 8-12 hours.

[0055] Freeze-dried reagents, also known as lyophilized reagents, are solid, porous biological or chemical reagents prepared by freeze-drying technology. This process involves removing liquid reagents by directly sublimating them from ice into water vapor under extremely low temperatures and vacuum conditions, ultimately yielding a dry solid powder or cake.

[0056] 2.3 Result Interpretation Method A: Qualitative / Semi-quantitative visual inspection 1. After the reaction is complete, insert the reaction tube vertically into a specially designed test tube rack, which has a built-in ring magnet at the bottom.

[0057] 2. Allow to stand for 30 minutes to enrich the magnetic particles, at which point the magnetic particles and the immune complexes they capture are adsorbed onto the side of the tube wall near the magnet.

[0058] 3. Irradiate the reaction tube wall with a portable ultraviolet lamp with a wavelength of 340nm in a light-protected environment.

[0059] 4. Observation results: Negative: No visible red fluorescent clusters on the tube wall or only extremely weak uniform background fluorescence.

[0060] Positive: Obvious red fluorescent spots appear on the tube wall.

[0061] The brightness and intensity of fluorescent spots can be used for preliminary semi-quantitative assessment of antigen concentration (e.g., weak positive, positive, strong positive).

[0062] The ring magnet built into the bottom of the test tube rack enables the antibody-labeled magnetic particles to act like a "smart magnetic fishing net," specifically binding to the target protein. The external force provided by the magnet can instantly drag all the "fishing nets" from the entire "swimming pool" of reaction liquid and gather them onto a single point on the tube wall, achieving tens of thousands of times spatial concentration of the target substance. This concentrates the scattered weak signals into a single point, making previously undetectable signals detectable.

[0063] The reaction results remain stable for 24 hours at room temperature and can be repeatedly interpreted or recorded.

[0064] Method B: Quantitative detection method 1. The enrichment steps are the same as in method A.

[0065] 2. Discard the supernatant, resuspend the magnetic particle-labeled Tau antibody mixture at the bottom of the tube in 100 μL PBS buffer (laboratory phosphate-buffered saline), and transfer it to the detection plate.

[0066] 3. Using a fluorescence microplate reader, set the excitation wavelength to 340nm and the emission wavelength to 610nm, and detect the fluorescence intensity (RFU) of each well.

[0067] 4. Calculate the actual concentration of Tau-p-181 in the urine sample based on the standard curve pre-plotted using known concentrations of Tau-p-181 antigen.

[0068] Resuspension refers to the process of re-dispersing the precipitate (usually particles or cells) formed after centrifugation or magnetic enrichment into a certain volume of liquid (such as buffer, culture medium or water). Its core purpose is not simply "dissolving", but to make the aggregated and compacted solid particles suspend uniformly in the liquid again to form a stable suspension.

[0069] Working principle: By using a pair of paired antibodies (capture antibody and detection antibody) to specifically capture ultra-low concentrations of target proteins in urine, magnetic beads are used to "fish" them out of the complex matrix and concentrate them. Then, high-brightness fluorescent microspheres are used to convert the biorecognition event into a strong light signal, which can be qualitatively or quantitatively detected by the naked eye or instruments. This achieves the purpose of detecting phosphorylated Tau-181 protein in urine, reducing the operation threshold and economic cost.

[0070] Example 3: We used this invention to test 200 random samples under the age of 40 years, and found no positive samples for Tau-p-181. We also tested 78 samples from elderly hospitalized patients aged 63-96 years; the results were: 3 negative, 1 suspicious, and 74 positive. See the table below: In the description of this specification, references to terms such as "an embodiment," "example," and "specific example" 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.

[0071] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. A method for detecting phosphorylated Tau-181 protein in the urine of Alzheimer's disease patients, characterized in that: Includes the following steps: S1: Take a 5ml reaction tube containing pre-prepared fluorescent microsphere-labeled Tau-p-181 antibody and magnetic particle-labeled Tau antibody. S2: Add 100 μL of urine sample to the 5 mL reaction tube mentioned above. S3: Add 4.9 ml of urine diluent to this 5 ml reaction tube, mix by hand by inverting 10 times, and let it react at room temperature for 8-12 hours. S4: Use a magnet to enrich the immune complex, irradiate the complex with ultraviolet light at around 340nm, and determine the fluorescence intensity of the complex. S5: Samples without fluorescent aggregation points are negative, and samples with fluorescent aggregation points are positive.

2. The method for detecting phosphorylated Tau-181 protein in the urine of Alzheimer's patients according to claim 1, characterized in that: The fluorescent microspheres may be time-resolved fluorescent microspheres, quantum dot fluorescent microspheres, aggregated luminescent microspheres, or fluorescein microspheres, with a particle size of 100 nm to 3000 nm.

3. The method for detecting phosphorylated Tau-181 protein in the urine of Alzheimer's patients according to claim 1, characterized in that: The magnetic particles have a particle size of 100nm-3000nm.

4. The method for detecting phosphorylated Tau-181 protein in the urine of Alzheimer's patients according to claim 1, characterized in that: The urine diluent is a 25mM phosphate buffer solution containing 0.2% Tween-20, used to dilute the urine at a ratio of 1:40 to 1:

200.

5. The method for detecting phosphorylated Tau-181 protein in the urine of Alzheimer's patients according to claim 1, characterized in that: The reaction time of the magnetic particle-labeled Tau antibody, the fluorescent microsphere-labeled Tau-p-181 antibody, and urine is 8-12 hours.

6. The method for detecting phosphorylated Tau-181 protein in the urine of Alzheimer's patients according to claim 1, characterized in that: The blocking solution comprises Hepes at pH 7.4, BSA glycine at a concentration of 5.0%, and Proclin at a concentration of 0.1%.

7. The method for detecting phosphorylated Tau-181 protein in the urine of Alzheimer's patients according to claim 1, characterized in that: The reconstitution solution comprises Hepes, 10% trehalose, 2% BSA, glycine, and 0.1% Proclin.

8. The method for detecting phosphorylated Tau-181 protein in the urine of Alzheimer's patients according to claim 1, characterized in that: The magnet should enrich the magnetic particles for more than 20 minutes, and the results should be valid for 24 hours.

9. The method for detecting phosphorylated Tau-181 protein in the urine of Alzheimer's patients according to claim 1, characterized in that: The reaction proceeds at room temperature for an extended period of time, eliminating the need to separate the reaction solution and preventing environmental pollution from waste liquid.

10. The method for detecting phosphorylated Tau-181 protein in the urine of Alzheimer's patients according to claim 1, characterized in that: The stronger the fluorescence intensity, the higher the content of phosphorylated Tau protein-181 in the urine sample, and the reaction results can be effectively retained for 24 hours at room temperature.

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