Method for detecting phosphorylated Tau-231 protein in urine of patient with Alzheimer's disease

Abstract

The invention relates to the field of protein detection, in particular to a method for detecting phosphorylated Tau-231 protein in urine of a patient with Alzheimer's disease, which comprises the following steps: firstly, immobilizing a non-phosphorylated anti-Tau-231 antibody on a magnetic particle microsphere, immobilizing a phosphorylated anti-Tau-p-231 antibody on a fluorescent microsphere, and then, immobilizing a phosphorylated anti-Tau-p-231 antibody on the fluorescent microsphere. Forming a sandwich compound of magnetic particle-antigen-fluorescent probe by the detected object; the method specifically comprises the following steps: using fresh morning urine; the method comprises the following steps: taking out 100 [mu] l of urine by using a pipette, and adding the urine into a 5.0 ml centrifugal tube in which a phosphorylated anti-Tau-p-231 antibody which is prefabricated with a fluorescence label and a non-phosphorylated anti-Tau-p-231 antibody which is connected with nano magnetic particle microspheres are prefabricated; 4.9 ml of a urine diluent is added to dilute the urine; repeatedly reversing and uniformly mixing, reacting at room temperature for 8-12 hours, and placing a reaction tube on a separation test tube rack with a magnet for more than 20 minutes; irradiating the test tube by using a highlighter with the wavelength of about 340nm in a dark environment to judge a result; finally, the purposes of early screening, early intervention and early diagnosis of the Alzheimer's disease are achieved.

Description

Technical Field

[0001] This invention belongs to the field of phosphorylated Tau-231 protein detection technology, specifically a method for detecting phosphorylated Tau-231 protein in the urine of Alzheimer's patients. Background Technology

[0002] Alzheimer's disease is a degenerative disease of the central nervous system, primarily affecting middle-aged and elderly individuals. The main characteristics of the disease include progressive cognitive impairment and behavioral disturbances. Research indicates that some specific biological markers of Alzheimer's disease begin to be released into the bloodstream and excreted in urine 5-10 years before the onset of clinical symptoms. Among these, phosphorylated Tau-231 protein is closely related to the course of Alzheimer's disease; however, the concentration of phosphorylated Tau-231 protein is relatively low, making detection difficult.

[0003] In existing technologies, one of the core neuropathological features of Alzheimer's disease (AD) is the abnormal accumulation of paired helical filaments in neurofibrillary tangles within neurons, the main component of which is hyperphosphorylated microtubule-associated protein Tau. Tau protein contains an N-terminal projection domain, a proline-rich domain, a microtubule-binding repeat domain, and a C-terminal domain, each containing multiple potential phosphorylation sites. An immunoassay was used to quantitatively detect phosphorylated Tau at threonine 231 in the cerebrospinal fluid of AD patients. This indicator, used alone or in combination with Aβ-42, can accurately distinguish AD from normal controls and can predict cognitive decline in the preclinical and prodromal stages. Furthermore, Tau-p-231 can cross the blood-brain barrier and enter the bloodstream, and its peripheral blood concentration has been shown to be useful for the differential diagnosis of dementia. Tau-p-231 in the blood can also be excreted in urine, providing the possibility of detecting Tau-p-231 in urine.

[0004] In the aforementioned techniques, Tau-p-231 in the blood enters the kidneys through physiological metabolism and is eventually partially excreted in urine. However, its concentration in urine is only one-thousandth or less of that in cerebrospinal fluid, far below the detection limit of conventional immunological methods, thus making false negatives highly likely. Furthermore, Tau-p-231 in urine does not exist in a single form but coexists in two forms: a free form and a membrane-bound form encapsulated by neurogenic exosomes. The free form has a small molecular weight and is easily degraded, while the membrane-bound form encapsulated by neurogenic exosomes has higher stability but is difficult to be directly recognized by traditional antibodies, thus making accurate detection difficult.

[0005] Therefore, the present invention provides a method for detecting phosphorylated Tau-231 protein in the urine of Alzheimer's disease patients. Summary of the Invention

[0006] In order to overcome the shortcomings of the prior art, at least one technical problem raised in the background art is solved.

[0007] The technical solution adopted by this invention to solve its technical problem is: a method for detecting phosphorylated Tau-231 protein in the urine of Alzheimer's patients, comprising the following steps: S1: First, the non-phosphorylated anti-Tau-231 antibody is immobilized on magnetic particle microspheres, which then specifically capture the antigen in the reaction solution; S2: Then, phosphorylated anti-Tau-p-231 antibody is immobilized on fluorescent microspheres, so that the analyte forms a sandwich complex of magnetic particles-antigen-fluorescent probe; S3: Finally, the reaction complex is enriched by a magnetic field, and the fluorescence signal can be directly read to determine whether it is positive or negative.

[0008] Preferably, the step of linking the anti-Tau-p-231 antibody to the carboxyl magnetic particles using a chemical cross-linking method in S1 includes the following methods: S101: Activation of magnetic particles; S102: Conjugation of magnetic particles with non-phosphorylated anti-Tau-231 antibody; S103: Sealing and preservation of magnetic particles.

[0009] Preferably, the step of linking the anti-Tau antibody to the tracer fluorescent microspheres using a chemical cross-linking method in S2 includes the following methods: S201: Activation of microspheres; S202: Conjugation of microspheres with phosphorylated anti-Tau-p-231 antibody; S203: Sealing and preservation of microspheres.

[0010] Preferably, the anti-Tau antibody and anti-Tau-p-231 antibody are diluted with 25 mM phosphate buffer to a concentration of 1.0 mg / ml.

[0011] Preferably, the magnetic particles are used to enrich and separate microspheres, and the particle size of the enriched and separated magnetic particles is 100nm-3000nm.

[0012] Preferably, the fluorescent microspheres are used as tracers for fluorescent microspheres, and the particle size of the fluorescent microspheres is 100nm-3000nm.

[0013] Preferably, 500 μL of blocking solution is added to S101, the blocking solution comprising Hepes at pH 7.4, BSA glycine at a concentration of 5.0% and Proclin at a concentration of 0.1%.

[0014] Preferably, 2.0 ml of reconstitution solution is added to S102, the reconstitution solution comprising Hepes, 10% trehalose, 2% BSA, glycine and 0.1% Proclin.

[0015] The beneficial effects of this invention are as follows: This invention discloses a method for detecting phosphorylated Tau-231 protein in the urine of Alzheimer's patients. It employs a dual-probe strategy of "magnetic particle enrichment-fluorescent tracing." Anti-Tau-p-231 antibodies are immobilized on carboxyl magnetic particles, removing impurities and capturing the antigen in one step. Then, high-brightness fluorescent microspheres are used to label the anti-Tau antibody for secondary recognition, forming a sandwich complex of magnetic particles, antigen, and fluorescent probe. Finally, a magnetic field enriches the complex into the detection window, and the fluorescence signal is directly read to determine whether the result is positive or negative. Samples without fluorescent aggregation points are negative, and samples with fluorescent points are positive. The stronger the fluorescence intensity, the higher the content of phosphorylated Tau protein-231 in the sample. Attached Figure Description

[0016] The invention will now be further described with reference to the accompanying drawings.

[0017] Figure 1 This is a flowchart of the phosphorylated Tau-231 protein detection method in this invention; Figure 2 This is a flowchart illustrating the method for linking anti-Tau-p-231 antibody to carboxyl magnetic particles in this invention. Figure 3 This is a flowchart of the method for linking anti-Tau antibody to tracer fluorescent microspheres in this invention; Figure 4 This is a schematic diagram of the field test results in this invention; Figure 5 This is a schematic diagram of the measurement results of the fluorescence spectrophotometer in this invention. Detailed Implementation

[0018] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below in conjunction with specific embodiments.

[0019] 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.

[0020] 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.

[0021] Add 1.0 ml of 200 mM ethanesulfonic acid buffer to the centrifuge tube.

[0022] Add 200 μL of carbodiimide solution (10 mg / ml) and shake well.

[0023] Place the centrifuge tubes on a gyroscope and activate at room temperature for 20 minutes.

[0024] After activation, immediately add 100 μL of the diluted anti-Tau antibody solution from step 1 and mix quickly.

[0025] Place the mixture on a rotary mixer and couple it at room temperature in the dark for 2 hours.

[0026] 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 a "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 can achieve rapid and effective mixing and reduce operating costs. Using a gyroscope for mixing can achieve steps that require long mixing operations. 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.

[0027] 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-231 protein) and its formed immune complexes in the urine sample from a large amount of liquid 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.

[0028] 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.

[0029] 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).

[0030] Store in a refrigerator at 4°C for later use.

[0031] 1.2 Preparation of fluorescent microsphere-labeled anti-Tau-p-231 antibody The antiphosphorylated Tau-231 (Tau-p-231) specific monoclonal antibody was diluted with 25 mM phosphate buffer to a concentration of 1.0 mg / ml.

[0032] 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.

[0033] The subsequent activation, coupling, and blocking steps are the same as steps 3-8 in 1.1.

[0034] After the coupling and sealing are completed, centrifuge the centrifuge tubes at 4℃ and 15000r / min for 30 minutes, and carefully discard the supernatant.

[0035] The precipitated fluorescent microsphere-antibody complex was resuspended in 2.0 ml of reconstitution solution.

[0036] Store at 4℃ away from light for later use.

[0037] Example 2: Detection procedure for Tau-p-231 in urine samples 2.1 Sample Pretreatment The first midstream urine of the subject in the morning was collected in a clean container.

[0038] Gently invert the urine sample 10 times to mix.

[0039] Accurately pipette 100 μL of the mixed urine and add it to a 5.0 ml transparent centrifuge tube.

[0040] 2.2 Detection reaction These 5.0ml centrifuge tubes are pre-packaged reaction tubes, and the contents are lyophilized and contain: Example 1: Fluorescent microsphere-labeled anti-Tau-p-231 antibody (equivalent to 0.1 μg antibody); The magnetic particle-labeled anti-Tau antibody (equivalent to 0.1 μg antibody) prepared in Example 1.

[0041] 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.

[0042] Tighten the cap and manually invert the tube 20 times to ensure the lyophilized reagent is completely dissolved and mixed evenly.

[0043] Allow the reaction to stand at room temperature (approximately 25°C) for 8-12 hours.

[0044] Freeze-dried reagents, also known as lyophilized reagents, are solid, porous biological or chemical reagents prepared by freeze-drying technology. It involves removing liquid reagents by directly sublimating them from ice into water vapor under extremely low temperature and vacuum conditions, ultimately obtaining a dry solid powder or cake.

[0045] 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.

[0046] 2. Allow to stand for 30 minutes to enrich the magnetic particles. At this time, the magnetic particles and the immune complexes they capture are adsorbed onto the side of the tube wall near the magnet.

[0047] 3. Irradiate the reaction tube wall with a portable ultraviolet lamp with a wavelength of 340nm in a light-protected environment.

[0048] 4. Observation results: Negative: No visible red fluorescent clusters on the tube wall or only extremely weak uniform background fluorescence.

[0049] Positive: Obvious red fluorescent spots appear on the tube wall.

[0050] The brightness and intensity of the fluorescent spots can be used for preliminary semi-quantitative assessment of antigen concentration (e.g., weak positive, positive, strong positive).

[0051] 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.

[0052] The reaction results remain stable for 24 hours at room temperature and can be repeatedly interpreted or recorded.

[0053] Method B: Quantitative detection method 1. The enrichment steps are the same as in method A.

[0054] 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.

[0055] 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.

[0056] 4. Calculate the actual concentration of Tau-p-231 in the urine sample based on the standard curve pre-plotted using known concentrations of Tau-p-231 antigen.

[0057] 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.

[0058] 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-231 protein in urine, reducing the operation threshold and economic cost.

[0059] Example 3: We used this invention to test 200 random samples aged under 40 years old, and found no positive samples for Tau-p-231. We also tested 78 samples from elderly hospitalized patients aged 63-96 years; the results were: 6 negative, 30 suspicious, and 42 positive. See the table below: In summary, 100nm is suitable for fine detection and is often used in scientific research; 1550nm is suitable for routine detection; and 3000nm is suitable for rapid screening.

[0060] During the test, fresh morning urine is first collected in a suitable container. The mixture is then thoroughly mixed by inverting the tube. 100 μL of urine is transferred to a 5.0 mL centrifuge tube using a pipette with pre-filled tracer fluorescent microspheres and enrichment microspheres. Next, 4.9 mL of sample diluent is added to the 5.0 mL centrifuge tube using a pipette, and the tube is tightly capped. The centrifuge tube is then inverted 10-20 times to mix. The mixture is then incubated at room temperature for 10-12 hours, during which time the tube is inverted 3-5 times, 10-20 times each time. A magnetic block is then placed on the outside of the 5.0 mL reaction tube for enrichment. After 30 minutes of enrichment, the mixture is irradiated with a UV laser pen at approximately 340 nm wavelength to observe the reaction results. (Details follow...) Figure 4 As shown, Samples without fluorescent aggregation points are negative, while samples with fluorescent points are positive; the stronger the fluorescence intensity, the higher the content of phosphorylated Tau protein-231 in the sample, and the reaction results can be retained for 24 hours.

[0061] Working principle: First, non-phosphorylated anti-Tau-231 antibody is immobilized on magnetic microspheres, specifically capturing the antigen in the reaction solution. Then, phosphorylated anti-Tau-p-231 antibody is immobilized on fluorescent microspheres, forming a sandwich complex of magnetic particles, antigen, and fluorescent probe. Finally, the reaction complex is enriched by a magnetic field, and the fluorescence signal is directly read to determine the positivity or negativeness. Samples without fluorescent aggregation points are negative, and samples with fluorescent points are positive. The stronger the fluorescence intensity, the higher the content of phosphorylated Tau protein-231 in the sample. The steps of linking anti-Tau-p-231 antibody to carboxyl magnetic particles using chemical cross-linking include activation of the magnetic particles; coupling of the magnetic particles with anti-Tau-p-231; and blocking and preservation of the magnetic particles. The steps of linking anti-Tau antibody to tracer fluorescent microspheres using chemical cross-linking include activation of the microspheres; coupling of the microspheres with anti-Tau antibody; and blocking and preservation of the microspheres.

[0062] 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 present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting phosphorylated Tau-231 protein in the urine of Alzheimer's disease patients, characterized in that... Includes the following steps: S1: First, the non-phosphorylated anti-Tau-231 antibody is immobilized on magnetic particle microspheres, which then specifically capture the antigen in the reaction solution; S2: Then, phosphorylated anti-Tau-p-231 antibody is immobilized on fluorescent microspheres, so that the analyte forms a sandwich complex of magnetic particles-antigen-fluorescent probe; S3: Finally, the reaction complex is enriched by a magnetic field, and the fluorescence signal can be directly read to determine whether it is positive or negative.

2. The method for detecting phosphorylated Tau-231 protein in the urine of Alzheimer's patients according to claim 1, characterized in that... The step in S1 of linking the non-phosphorylated anti-Tau-231 antibody to the carboxyl magnetic particles using a chemical cross-linking method includes the following methods: S101: Activation of magnetic particles; S102: Conjugation of magnetic particles with non-phosphorylated anti-Tau-231 antibody; S103: Sealing and preservation of magnetic particles.

3. The method for detecting phosphorylated Tau-231 protein in the urine of Alzheimer's patients according to claim 1, characterized in that... The step in S2 of linking the phosphorylated anti-Tau-p-231 antibody to the tracer fluorescent microspheres using a chemical cross-linking method includes the following methods: S201: Activation of microspheres; S202: Conjugation of microspheres with phosphorylated anti-Tau-p-231 antibody; S203: Sealing and preservation of microspheres.

4. The method for detecting phosphorylated Tau-231 protein in the urine of Alzheimer's patients according to claim 1, characterized in that... The anti-Tau antibody and anti-Tau-p-231 antibody were diluted to 1.0 mg / ml with 25 mM phosphate buffer.

5. The method for detecting phosphorylated Tau-231 protein in the urine of Alzheimer's patients according to claim 2, characterized in that... The magnetic particles are used to enrich and separate microspheres, and the particle size of the enriched and separated magnetic microspheres is 100nm-3000nm.

6. The method for detecting phosphorylated Tau-231 protein in the urine of Alzheimer's patients according to claim 3, characterized in that... The fluorescent microspheres are used as tracers for fluorescent microspheres, and the particle size of the fluorescent microspheres is 100nm-3000nm.

7. The method for detecting phosphorylated Tau-231 protein in the urine of Alzheimer's patients according to claim 2, characterized in that... 500 μL of blocking solution is added to S101. The blocking solution includes Hepes at pH 7.4, BSA glycine at a concentration of 5.0%, and Proclin at a concentration of 0.1%.

8. The method for detecting phosphorylated Tau-231 protein in the urine of Alzheimer's patients according to claim 2, characterized in that... 2.0 ml of reconstitution solution is added to S102. The reconstitution solution includes Hepes, 10% trehalose, 2% BSA, glycine and 0.1% Proclin.

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