Method for detecting related proteins in urine of alzheimer's disease patient and application thereof

Abstract

The application provides a detection method of related proteins in urine of Alzheimer's disease patients and application thereof, and relates to the field of biological medicine, and the detection method comprises the following steps: taking a reaction tube in which fluorescent microsphere-labeled protein antibody 2 and magnetic particle-labeled protein antibody 1 are pre-prepared; adding a urine sample into the reaction tube; adding a urine diluent into the reaction tube, uniformly mixing, and placing at room temperature for 8-12 hours; using a magnet to enrich the immune complex, irradiating the complex with purple light, and judging the fluorescence intensity of the complex. By using a pair of matched antibodies (capture antibody and detection antibody) to specifically capture the ultra-low concentration target protein in the urine, the magnetic beads are used to pick up and concentrate the target protein from the complex matrix, the high-brightness fluorescent microspheres are used to convert the biological recognition event into a strong light signal, and finally the qualitative or quantitative detection is realized by naked eyes or instruments, so that the purpose of detecting the related proteins in the urine is achieved, and the operation threshold and economic cost are reduced.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a method for detecting related proteins in the urine of Alzheimer's patients and its application. Background Technology

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

[0003] Current technology indicates that some specific biological markers of Alzheimer's disease (AD) begin to be released into the bloodstream and excreted in urine 5-10 years before the onset of clinical symptoms. Research on the clinical symptoms of AD has a history of over a century, but only in recent years have relatively low-risk blood immunological testing methods (such as single-molecule immunoassay and chemiluminescent immunoassay) emerged to detect several key biological markers of AD.

[0004] Blood immunology testing for Alzheimer's disease (AD) utilizes ultra-high sensitivity detection technology to "capture" and "amplify" the core pathological signals of AD, which are normally only observable 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 application in large-scale population screening, repeated dynamic monitoring, and in resource-scarce areas. Summary of the Invention

[0005] The purpose of this invention is to provide a method for detecting related proteins in the urine of Alzheimer's patients and its application, thereby solving at least one of the technical problems mentioned in the background.

[0006] In a first aspect, the present invention provides a method for detecting related proteins in the urine of Alzheimer's disease patients, the detection method comprising the following steps: S1: Take the reaction tube containing pre-prepared protein antibody 2 labeled with fluorescent microspheres and protein antibody 1 labeled with magnetic particles; S2: Take a urine sample and add it to the above reaction tube; S3: Add urine diluent to the above reaction tube, mix well, and let it stand at room temperature for 8-12 hours. S4: Use a magnet to enrich the immune complex, irradiate the complex with ultraviolet light, and judge the fluorescence intensity of the complex. Samples with no fluorescent aggregation points are negative, and samples with fluorescent aggregation points are positive.

[0007] Preferably, the related proteins include Aβ-42, AD7c-NTP, Aβ-40, or NFL proteins.

[0008] Preferably, the method for preparing the fluorescent microsphere-labeled protein antibody 2 is as follows: Protein antibody 2 was diluted with phosphate buffer to obtain a protein antibody 2 solution; Take a suspension of carboxyl quantum dot fluorescent microspheres and place it in a centrifuge tube; Add morpholine ethanesulfonic acid buffer to the centrifuge tube; Add the carbodiimide solution and shake well; Place the centrifuge tubes on a gyroscope and activate them at room temperature; After activation, add protein antibody 2 solution to the centrifuge tube and mix well to obtain a mixture; Place the mixture on a rotary mixer, couple it at room temperature in the dark, and then add the blocking solution.

[0009] Preferably, the method for preparing the protein antibody 1 with magnetic particle labeling is as follows: Protein antibody 1 was diluted with phosphate buffer to obtain a protein antibody 1 solution.

[0010] Take a suspension of carboxyl magnetic particles and place it in a centrifuge tube; Add morpholine ethanesulfonic acid buffer to the centrifuge tube; Add the carbodiimide solution and shake well; Place the centrifuge tubes on a gyroscope and activate them at room temperature; After activation, add protein antibody 1 solution to the centrifuge tube and mix well to obtain a mixture; Place the mixture on a rotary mixer, couple it at room temperature in the dark, and then add the blocking solution. After the coupling and blocking process is completed, the supernatant is removed by centrifugation, and the precipitate is resuspended in a reconstitution solution.

[0011] Preferably, the fluorescent microspheres are 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.

[0012] Preferably, the magnetic particles have a particle size of 100nm-3000nm.

[0013] 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.

[0014] 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%.

[0015] Preferably, the complex solution comprises Hepes, 10% trehalose, 2% BSA, glycine, and 0.1% Proclin.

[0016] In the first aspect, a protein detection product related to Alzheimer's disease is also provided, including a protein antibody 2 containing fluorescent microspheres, a protein antibody 1 containing magnetic particles, and 25 mM phosphate containing 0.2% Tween-20.

[0017] Technical effects: 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 pick them up from 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 related proteins in urine, reducing the operational threshold and economic cost. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a schematic diagram of the detection device according to an embodiment of the present invention; Figure 2 This is a schematic diagram of the fluorescence results for Example 1; Figure 3 This is a schematic diagram of the fluorescence results in Example 2; Figure 4 This is a schematic diagram of the fluorescence results in Example 3; Figure 5 This is a schematic diagram of the fluorescence results in Example 4. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0021] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0022] This invention provides a method for detecting related proteins in the urine of Alzheimer's patients, the method comprising the following steps: This invention provides a method for detecting related proteins in the urine of Alzheimer's patients, the method comprising the following steps: S1: Take the reaction tube containing pre-prepared protein antibody 2 labeled with fluorescent microspheres and protein antibody 1 labeled with magnetic particles; S2: Take a urine sample and add it to the above reaction tube; S3: Add urine diluent to the above reaction tube, mix well, and let it stand at room temperature for 8-12 hours. S4: Use a magnet to enrich the immune complex, irradiate the complex with ultraviolet light, and judge the fluorescence intensity of the complex. Samples with no fluorescent aggregation points are negative, and samples with fluorescent aggregation points are positive.

[0023] When using a magnet to enrich immune complexes during the above-mentioned process, the magnet should enrich the magnetic particles for more than 20 minutes, and the results should be valid within 24 hours. After the enriched area is irradiated by an ultraviolet light source, positive samples show clearly visible fluorescent aggregation points on the reaction tube wall, while negative samples do not produce any signal, making the interpretation intuitive and clear. The above embodiments combine the highly selective enrichment capability of magnetic separation with the strong signal amplification effect of fluorescent microspheres, effectively overcoming the technical bottleneck of extremely low target protein concentrations in urine samples. By changing the pre-placed antibody combination in the reaction tube, it can flexibly adapt to the detection needs of various Alzheimer's disease-related proteins such as Aβ-42, AD7c-NTP, Aβ-40, or NFL, demonstrating good platform scalability. These embodiments do not require expensive instruments, provide stable and reliable detection results, lower the technical threshold and economic cost, and provide a non-invasive, rapid, and highly specific screening tool for primary healthcare scenarios and home self-testing, effectively promoting the widespread and accessible development of early Alzheimer's disease identification.

[0024] The enrichment process described above utilizes magnetic separation technology. The purpose of this step is to prepare magnetic particles with specific antibodies to form immune complexes with extremely low concentrations of target proteins (Aβ-42, AD7c-NTP, Aβ-40, or NFL) in urine samples. This is a key step in capturing, separating, and concentrating these proteins from a large volume 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 quantification by fluorescence instrument possible.

[0025] In one implementation, the method for preparing the fluorescent microsphere-labeled protein antibody 2 is as follows: Protein antibody 2 was diluted with phosphate buffer to obtain a protein antibody 2 solution; Take a suspension of carboxyl quantum dot fluorescent microspheres and place it in a centrifuge tube; Add morpholine ethanesulfonic acid buffer to the centrifuge tube; Add the carbodiimide solution and shake well; Place the centrifuge tubes on a gyroscope and activate them at room temperature; After activation, add protein antibody 2 solution to the centrifuge tube and mix well to obtain a mixture; Place the mixture on a rotary mixer, couple it at room temperature in the dark, and then add the blocking solution.

[0026] In one implementation, the method for preparing the protein antibody 1 with magnetic particle labeling is as follows: Protein antibody 1 was diluted with phosphate buffer to obtain a protein antibody 1 solution.

[0027] Take a suspension of carboxyl magnetic particles and place it in a centrifuge tube; Add morpholine ethanesulfonic acid buffer to the centrifuge tube; Add the carbodiimide solution and shake well; Place the centrifuge tubes on a gyroscope and activate them at room temperature; After activation, add protein antibody 1 solution to the centrifuge tube and mix well to obtain a mixture; Place the mixture on a rotary mixer, couple it at room temperature in the dark, and then add the blocking solution. After the coupling and blocking process is completed, the supernatant is removed by centrifugation, and the precipitate is resuspended in a reconstitution solution.

[0028] The preparation of the aforementioned magnetically labeled protein antibody 1 employs a carbodiimide chemical activation method. Magnetic particles rich in carboxyl groups on their surface are activated in a suitable buffer environment, converting the surface carboxyl groups of the magnetic particles into highly reactive intermediates. Specific capture antibodies targeting the target protein are then introduced, and under rotational conditions, the amino groups of the antibody are directionally covalently coupled to the activated carboxyl groups. After coupling, unreacted sites are blocked using a buffer system containing multiple blocking components, and non-specific adsorption sites are inhibited. Magnetic separation technology is used to wash away free antibodies and reaction byproducts. The purified immunomagnetic beads are resuspended in a reconstitution solution containing biological stabilizers and preservatives for cryogenic storage. The resulting product exhibits excellent magnetic response performance, retention of antibody biological activity, and long-term storage stability.

[0029] The preparation of the fluorescent microsphere-labeled protein antibody 2 follows the same covalent coupling chemical principle. The entire coupling process must be carried out in the dark, a buffer system that is friendly to fluorescent groups is selected, and gentle centrifugation is used in the blocking and purification steps to avoid fluorescence quenching or microsphere damage caused by mechanical stress.

[0030] It can form a highly specific pair with magnetically labeled antibodies, and the two work together to construct a dual antibody recognition system. After the immune complex is magnetically enriched, it can be visualized and interpreted through intuitive fluorescence signals. It has bioconjugation efficiency, signal fidelity and operational versatility, providing a reliable core reagent foundation for non-invasive detection platforms.

[0031] Furthermore, in the above embodiments, the fluorescent microspheres can be selected from time-resolved fluorescent microspheres, quantum dot fluorescent microspheres, aggregated luminescent microspheres, or fluorescein microspheres, with a particle size of 100nm-3000nm.

[0032] Furthermore, in the above embodiments, the magnetic particle size is 100nm-3000nm.

[0033] Furthermore, the urine diluent is a 25mM phosphate buffer containing 0.2% Tween 20, which dilutes the urine at a ratio of 1:40 to 1:200. The urine diluent and dilution ratio are key factors in ensuring reaction specificity and eliminating interference.

[0034] 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%.

[0035] After mixing, continue to rotate and seal 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 that require rapid shaking, manual shaking is used. This can achieve rapid and effective shaking and reduce operating costs. Using a gyroscope for shaking can achieve steps that require long shaking 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.

[0036] Furthermore, the complex solution comprises Hepes, 10% trehalose, 2% BSA, glycine, and 0.1% Proclin.

[0037] Secondly, a product for detecting Alzheimer's disease-related proteins is also provided, comprising a protein antibody 2 labeled with fluorescent microspheres, a protein antibody 1 labeled with magnetic particles, and 25 mM phosphate containing 0.2% Tween 20. The above product can be a kit, and the aforementioned protein antibody 2 labeled with fluorescent microspheres and protein antibody 1 labeled with magnetic particles are prepared according to the above embodiments.

[0038] Furthermore, in order to better implement the above embodiments, the present invention also provides a detection device. The main body of the device is a closed box structure, which is equipped with a light source, a magnet, a test tube rack and a viewing window to realize the whole process from sample enrichment to signal observation.

[0039] Specifically, a ring magnet (such as a neodymium iron boron magnet) is installed at the bottom of the device. Its surface magnetic field strength is sufficient to directionally adsorb the immune complex containing magnetically labeled antibodies onto the tube wall within a short time, thereby achieving the enrichment and separation of the target protein-antibody complex. Above the magnet is an adjustable-height and adjustable-angle tube rack to stably hold the test tubes, ensuring that the tubes remain tilted under magnetic force, causing the complex to accumulate along the tube wall near the observation window. The tube rack is made of corrosion-resistant, low-adsorption material to avoid contamination or interference with the samples.

[0040] A light source module is set on the side of the device. The light source uses a 340nm ultraviolet LED lamp. The light is focused by a reflector system and projected into the test tube, which excites the detection antibody labeled with fluorescent microspheres to emit a visible light signal.

[0041] The device has a transparent window on the front, which can be made of UV-resistant glass or polycarbonate material, ensuring good optical transparency while also having a certain mechanical strength and protective performance. Users can directly observe whether fluorescent aggregation points appear in the test tube through the window, thereby achieving visual qualitative interpretation.

[0042] The entire device is made of lightweight engineering plastic, providing excellent sealing and dustproof capabilities. The internal space is rationally laid out, with no physical interference between the various structures. After the user places the completed immunization tubes into the tube rack, they activate the magnetic power supply (or directly use a permanent magnet) for enrichment, then turn on the light source. The result is then visible through the viewing window to determine if the sample is positive.

[0043] The following detailed explanation is provided with reference to specific embodiments: Example 1 A method for detecting Aβ-42 in the urine of Alzheimer's patients includes the following steps: S1: Take a 5ml reaction tube pre-prepared with fluorescent microsphere-labeled Aβ-42-2 antibody and magnetic particle-labeled Aβ-42-1 antibody; S2: Add 100ul of urine sample to the above 5ml reaction tube; 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 judge the fluorescence intensity of the complex; samples without fluorescent aggregation points are negative, and samples with fluorescent aggregation points are positive.

[0044] (1) Preparation of detection reagents Preparation of magnetically labeled Aβ-42-1 antibody: Dilute the Aβ-42-1 antibody with 25 mM phosphate buffer (pH 7.4) to a concentration of 1.0 mg / mL.

[0045] 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.

[0046] Add 1.0 mL of 200 mM morpholine ethanesulfonic acid buffer to the centrifuge tube.

[0047] Add 200 μL of carbodiimide solution (10 mg / mL) and shake well.

[0048] Place the centrifuge tubes on a gyroscope and activate at room temperature for 20 minutes.

[0049] After activation, immediately add 100 μL of the diluted Aβ-42-1 antibody solution from step 1 and mix quickly.

[0050] Place the mixture on a rotary mixer and couple it at room temperature in the dark for 2 hours.

[0051] 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 and block for 6 hours. Place the centrifuge tubes on a magnetic rack and let them stand for 5 minutes until the magnetic particles are fully enriched. Then carefully discard the supernatant.

[0052] 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.

[0053] The precipitated magnetic particle-antibody complex was resuspended in 2.0 mL of a reconstitution solution (formulation: 50 mM Hepes, pH=7.4, containing 10% trehalose, 2.0% BSA, 500 mM glycine, 0.1% Proclin-300).

[0054] Store in a refrigerator at 4°C for later use.

[0055] Preparation of Aβ-42-2 antibody labeled with fluorescent microspheres: Dilute the Aβ-42-2 antibody with 25mM phosphate buffer to a concentration of 1.0 mg / mL.

[0056] 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.

[0057] The subsequent activation, coupling, and blocking steps are the same as those in the preparation of magnetic particle-labeled Aβ-42-1 antibody.

[0058] 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.

[0059] The precipitated fluorescent microsphere-antibody complex was resuspended in 2.0 mL of reconstitution solution.

[0060] Store at 4℃ away from light for later use.

[0061] The detection process of Aβ-42 in urine samples: Sample pretreatment: Collect the first midstream urine of the subject in the morning in a clean container.

[0062] Gently invert the urine sample 10 times to mix.

[0063] Accurately pipette 100 μL of the mixed urine and add it to a 5.0 mL transparent centrifuge tube.

[0064] Detection reaction: These 5.0 mL centrifuge tubes are pre-packaged reaction tubes, and the contents are lyophilized and contain: The fluorescent microsphere-labeled Aβ-42-2 antibody prepared above (equivalent to 0.1 μg antibody); The magnetic particle-labeled Aβ-42-1 antibody prepared above (equivalent to 0.1 μg antibody).

[0065] 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.

[0066] Tighten the cap and manually invert the tube 20 times to ensure the lyophilized reagent is completely dissolved and mixed evenly.

[0067] Allow the reaction to stand at room temperature (approximately 25°C) for 8-12 hours.

[0068] 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.

[0069] After the reaction is complete, the reaction tube is vertically inserted into a specially designed test tube rack, which has a built-in ring magnet at the bottom.

[0070] After standing for 30 minutes to enrich, the magnetic particles and the immune complexes they capture are adsorbed onto the side of the tube wall near the magnet.

[0071] Irradiate the reaction tube using a portable 340nm wavelength UV lamp close to the wall in a light-protected environment. The table below shows the measurement results from the fluorescence spectrophotometer: Observation results, such as Figure 2 As shown: Negative: No visible red fluorescent clusters on the tube wall or only extremely weak uniform background fluorescence.

[0072] Positive: Obvious red fluorescent spots appear on the tube wall.

[0073] 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).

[0074] 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 spatial concentration of the target substance by tens of thousands of times. This concentrates the scattered weak signals into a single point, making previously undetectable signals detectable.

[0075] The reaction results remain stable for 24 hours at room temperature and can be repeatedly interpreted or recorded.

[0076] In Example 1 above, 200 random samples aged under 40 years were tested, and no Aβ-42 positive samples were found. 78 random samples aged 63-96 years were tested, and the results were: 29 negative, 24 questionable, and 25 positive. See the table below: 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 the 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 purpose of detecting Aβ-42 in urine, reducing the operation threshold and economic cost.

[0077] Example 2 The fluorescent microsphere-labeled Aβ-42-2 antibody and the magnetic particle-labeled Aβ-42-1 antibody were replaced with fluorescent microsphere-labeled AD7c-NTP-2 antibody and magnetic particle-labeled AD7c-NTP-1 antibody, respectively. All preparation methods, reaction processes and result interpretation methods were the same. Irradiate the reaction tube using a portable 340nm wavelength UV lamp close to the wall in a light-protected environment. The table below shows the measurement results from the fluorescence spectrophotometer: Observation results, such as Figure 3 As shown: Negative: No visible red fluorescent clusters on the tube wall or only extremely weak uniform background fluorescence.

[0078] Positive: Obvious red fluorescent spots appear on the tube wall.

[0079] In Example 2 above, 200 random samples aged under 40 years were tested, and no AD7c-NTP positive samples were found. Samples from 78 elderly hospitalized patients aged 63-96 years were tested; the results were: 10 negative, 37 suspicious, and 31 positive. See the table below: Example 3 The fluorescent microsphere-labeled Aβ-42-2 antibody and the magnetic particle-labeled Aβ-42-1 antibody were replaced with fluorescent microsphere-labeled Aβ-40-2 antibody and magnetic particle-labeled Aβ-40-1 antibody, while all preparation methods, reaction processes, and result interpretation methods remained the same. Irradiate the reaction tube using a portable 340nm wavelength UV lamp close to the wall in a light-protected environment. The table below shows the measurement results from the fluorescence spectrophotometer: Observation results, such as Figure 4 As shown: Negative: No visible red fluorescent clusters on the tube wall or only extremely weak uniform background fluorescence.

[0080] Positive: Obvious red fluorescent spots appear on the tube wall.

[0081] In Example 3 above, 200 random samples aged under 40 years were tested, and no Aβ-40 positive samples were found. 78 random samples aged 63-96 years were tested, and the results were: 18 negative, 27 questionable, and 33 positive. See the table below: Example 4 The fluorescent microsphere-labeled Aβ-42-2 antibody and the magnetic particle-labeled Aβ-42-1 antibody were replaced with fluorescent microsphere-labeled NFL-2 antibody and magnetic particle-labeled NFL-1 antibody, and all preparation methods, reaction processes and result judgment methods were the same; Irradiate the reaction tube using a portable 340nm wavelength UV lamp close to the wall in a light-protected environment. The table below shows the measurement results from the fluorescence spectrophotometer: Observation results, such as Figure 5 As shown: Negative: No visible red fluorescent clusters on the tube wall or only extremely weak uniform background fluorescence.

[0082] Positive: Obvious red fluorescent spots appear on the tube wall.

[0083] In Example 4 above, 200 random samples aged under 40 years were tested, and no NFL-positive samples were found. Samples from 78 elderly hospitalized patients aged 63-96 years were tested, and the results were: 3 negative, 22 suspicious, and 53 positive. See the table below: This invention utilizes a pair of paired antibodies (capture antibody and detection antibody) to specifically capture ultra-low concentrations of target proteins in urine. These proteins are then "fished out" from a complex matrix and concentrated using magnetic beads. High-brightness fluorescent microspheres are then used to convert the biorecognition event into a strong light signal, ultimately enabling qualitative or quantitative detection by the naked eye or instruments. This achieves the goal of detecting related proteins in urine, reducing the operational threshold and economic cost.

[0084] The foregoing describes the basic principles, main features, and advantages of this invention. Those skilled in the art should understand that this 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 this invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the 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 related proteins in the urine of Alzheimer's disease patients, characterized in that, The detection method includes the following steps: S1: Take the reaction tube containing pre-prepared protein antibody 2 labeled with fluorescent microspheres and protein antibody 1 labeled with magnetic particles; S2: Take a urine sample and add it to the above reaction tube; S3: Add urine diluent to the above reaction tube, mix well, and let it stand at room temperature for 8-12 hours. S4: Use a magnet to enrich the immune complex, irradiate the complex with ultraviolet light, and judge the fluorescence intensity of the complex. Samples with no fluorescent aggregation points are negative, and samples with fluorescent aggregation points are positive.

2. The method for detecting Alzheimer's disease-related proteins according to claim 1, characterized in that, The related proteins include Aβ-42, AD7c-NTP, Aβ-40, or NFL proteins, etc.

3. The method for detecting Alzheimer's disease-related proteins according to claim 1, characterized in that, The preparation method of the fluorescent microsphere-labeled protein antibody 2 is as follows: Protein antibody 2 was diluted with phosphate buffer to obtain a protein antibody 2 solution; Take a suspension of carboxyl quantum dot fluorescent microspheres and place it in a centrifuge tube; Add morpholine ethanesulfonic acid buffer to the centrifuge tube; Add the carbodiimide solution and shake well; Place the centrifuge tubes on a gyroscope and activate them at room temperature; After activation, add protein antibody 2 solution to the centrifuge tube and mix well to obtain a mixture; Place the mixture on a rotary mixer, couple it at room temperature in the dark, and then add the blocking solution.

4. The method for detecting related proteins in the urine of Alzheimer's patients according to claim 1, characterized in that, The method for preparing the protein antibody 1 with magnetic particle labeling is as follows: Protein antibody 1 was diluted with phosphate buffer to obtain a protein antibody 1 solution; Take a suspension of carboxyl magnetic particles and place it in a centrifuge tube; Add morpholine ethanesulfonic acid buffer to the centrifuge tube; Add the carbodiimide solution and shake well; Place the centrifuge tubes on a gyroscope and activate them at room temperature; After activation, add protein antibody 1 solution to the centrifuge tube and mix well to obtain a mixture; Place the mixture on a rotary mixer, couple it at room temperature in the dark, and then add the blocking solution. After the coupling and blocking process is completed, the supernatant is removed by centrifugation, and the precipitate is resuspended in a reconstitution solution.

5. The method for detecting related proteins in the urine of Alzheimer's patients according to claim 1, characterized in that, The fluorescent microspheres are 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.

6. The method for detecting related proteins in the urine of Alzheimer's patients according to claim 1, characterized in that, The magnetic particles have a particle size of 100nm-3000nm.

7. The method for detecting related proteins 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.

8. A method for detecting related proteins in the urine of Alzheimer's patients according to claim 4 or 5, 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%.

9. The method for detecting related proteins in the urine of Alzheimer's patients according to claim 5, characterized in that: The reconstitution solution comprises Hepes, 10% trehalose, 2% BSA, glycine, and 0.1% Proclin.

10. A product for detecting related proteins in the urine of Alzheimer's patients, characterized in that, This includes protein antibody 2 labeled with fluorescent microspheres, protein antibody 1 labeled with magnetic particles, and 25 mM phosphate containing 0.2% Tween 20.

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