Ultracentrifugation-free urine extracellular vesicle separation reagent and method

Abstract

The invention relates to the technical field of biology, in particular to an ultracentrifugation-free urine extracellular vesicle separation reagent and method, and the ultracentrifugation-free urine extracellular vesicle separation reagent is prepared from the following raw materials in percentage by mass: 0.2-0.4% of triethanolamine, 8-12% of cane sugar and the balance of distilled water. In addition, the invention also provides an ultracentrifugation-free urine extracellular vesicle separation method, which comprises the following steps: taking a urine sample, carrying out primary centrifugation, and reserving supernate; centrifuging for the second time at room temperature, and retaining supernate S1 and precipitate; mixing the obtained precipitate with the urine separation reagent, incubating, centrifuging for the third time, and retaining supernate S2 and precipitate P1; finally, uniformly mixing the supernate S1 and the supernate S2 to obtain urine supernate containing uEVs; the centrifugal forces of the first centrifugation, the second centrifugation and the third centrifugation are all less than 100000g, and the problems that the separation efficiency is low, the yield is low and the subsequent immunodetection is influenced when the urine extracellular vesicles are separated at present are solved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a reagent and method for separating urinary extracellular vesicles without ultracentrifugation. Background Art

[0002] Extracellular vesicles (EVs) are membranous vesicle structures secreted by almost all cells and play important roles in numerous pathophysiological processes. EVs are widely distributed and exist in biological fluids such as blood, urine, and saliva, becoming an important source of disease biomarkers. Among them, urinary extracellular vesicles (uEVs) show great potential in the field of disease biomarker research due to their non-invasive collection and rich content information.

[0003] The expression of uEVs-specific membrane proteins is rich in disease information, which not only helps to deeply understand the secretion and sorting mechanisms of uEVs but also helps to screen various disease biomarkers. Before detecting uEVs membrane proteins, uEVs need to be separated. Currently, the most commonly used method is ultracentrifugation. However, the protein abundance in urine spans five orders of magnitude, including the highest-content Tamm-Horsfall protein (THP), albumin, and 20 other serum filtration proteins. During ultracentrifugation, THP polymerizes into double-helix rope-like structures several micrometers long in urine, enclosing many uEVs, which precipitate together with uEVs during separation, affecting the purity of the obtained uEVs. Moreover, the encapsulation by the polymer reduces the yield of uEVs, limiting the clinical application prospects of uEVs, with low separation efficiency, affecting subsequent detection work, and affecting the development of rapid immunological detection techniques. Summary of the Invention

[0004] Aiming at the above defects, the purpose of the present invention is to provide a reagent and method for separating urinary extracellular vesicles without ultracentrifugation, so as to solve the problems of low separation efficiency and low yield during the separation of current urinary extracellular vesicles, which affect subsequent immunoassays.

[0005] To achieve this purpose, the present invention adopts the following technical solutions: A urine separation reagent, calculated by mass percentage, comprises the following raw materials: 0.2 - 0.4% triethanolamine, 8 - 12% sucrose, and the balance distilled water.

[0006] A method for separating urinary extracellular vesicles without ultracentrifugation, comprising the following steps: Step (1): Take a urine sample and perform the first centrifugation, and retain the supernatant; Step (2): Take the supernatant obtained in step (1), perform a second centrifugation at room temperature, and retain the supernatant S1 and the precipitate. Step (3): Mix the precipitate obtained in step (2) with the above urine separation reagent, incubate, and then perform a third centrifugation to retain the supernatant S2 and the precipitate P1. Step (4): Mix the supernatant S1 and the supernatant S2 evenly to obtain a urine supernatant containing uEVs. The centrifugal forces of the first centrifugation, the second centrifugation, and the third centrifugation are all less than 100,000 g.

[0007] Preferably, in step (3), the incubation conditions are shaking incubation at room temperature for 30 - 60 s.

[0008] Preferably, in step (1), the conditions for the first centrifugation are centrifugation at 2000 - 4000 g for 5 - 10 minutes at 4°C.

[0009] Preferably, in steps (2) and (3), the conditions for the second centrifugation and the third centrifugation are centrifugation at 16000 - 17000 g for 15 - 20 minutes at room temperature.

[0010] Preferably, the urine supernatant obtained in step (4) can be used for immunoassay after dilution.

[0011] Furthermore, the immunoassay method is immunochromatographic assay or chemiluminescent immunoassay.

[0012] Furthermore, the dilution factor is 10 - 20 times.

[0013] Preferably, in step (1), the urine sample is morning urine.

[0014] Preferably, the separated uEVs are used to distinguish diabetic kidney injury or prostate cancer.

[0015] The technical solution provided by the present invention may include the following beneficial effects: The urine separation reagent contains triethanolamine, sucrose, and distilled water, which dissociates THP polymers in urine and releases uEVs encapsulated therein, effectively increasing the yield of the separated uEVs. Moreover, the reaction conditions of this urine separation reagent are relatively mild, reducing the impact on the structure of the separated uEVs, improving the separation efficiency, and reducing the difficulty of separation operation. Description of the Drawings

[0016] Figure 1 It is the detection result diagram of Example Group 1 - 1 of the present invention.

[0017] Figure 2 It is the detection result diagram of Example Group 1 - 2 of the present invention.

[0018] Figure 3 It is the detection linear range result graph of Example Groups 1-3 of the present invention.

[0019] Figure 4 It is the detection linear range result graph of Example Groups 1-4 of the present invention.

[0020] Figure 5 It is the separation efficiency comparison graph of Example Group 2 and Comparative Example Groups 2-1 of the present invention.

[0021] Figure 6 It is the gel electrophoresis experiment graph and particle tracking analysis graph of Example Group 2, Comparative Example Groups 2-1 and 2-2 of the present invention.

[0022] Figure 7 It is the fluorescence intensity result graph of Example Group 3, Comparative Example Groups 3-1 and 3-2 of the present invention.

[0023] Figure 8 It is the luminescence value result graph of Example Group 4, Comparative Example Groups 4-1 and 4-2 of the present invention. Specific Embodiments

[0024] The technical solution of the present invention will be further described below through specific embodiments.

[0025] For the convenience of understanding the present invention, the present invention will be described more comprehensively below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0026] For those technologies or conditions not specified in the examples, they shall be carried out according to the technologies or conditions described in the literature in this field or according to the product specifications. For the reagents or instruments not indicated with the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0027] A urine separation reagent, calculated by mass percentage, includes the following raw materials: 0.2-0.4% triethanolamine, 8-12% sucrose and the balance distilled water.

[0028] To solve the problems existing in the prior art, the present invention provides a urine separation reagent, which includes triethanolamine, sucrose and distilled water. During the process of separating uEVs, in view of the uroregulatory protein rich in urine, namely Tamm-Horsfall protein (THP), the urine separation reagent contains triethanolamine, which can break the disulfide bonds between THP polymers in urine, dissociate them into monomeric forms, prevent the formation of double-helix rope-like structures and co-precipitate with uEVs, and can also release uEVs wrapped by THP polymers, improving the yield of uEVs, that is, increasing the recovery rate of uEVs. Moreover, triethanolamine can act as a pH buffer to maintain the stability of the pH value in the reduction reaction, ensuring the normal depolymerization of THP polymers.

[0029] Meanwhile, it also includes sucrose, which can increase the stability of extracellular vesicle membrane proteins. Because sucrose will preferentially bind to water molecules, reducing the unstable hydration of proteins and water molecules, and sucrose will form a protective hydration shell around proteins, maintaining the natural folded structure of proteins and playing a stabilizing role.

[0030] Compared with the currently used strong reducing agent dithiothreitol (DTT), the urine separation reagent proposed by the present invention has milder reaction conditions and can react at room temperature, reducing the temperature limitation and the difficulty of separation operation. Moreover, while having the advantage of high efficiency in dissociating THP polymers, it will not damage the structures of more extracellular vesicle membrane proteins like DTT, ensuring the normal progress of subsequent immunoassays and helping to solve the problem that the yield of urine extracellular vesicles is low during the current separation of urine extracellular vesicles, which in turn affects subsequent immunoassays.

[0031] In addition, calculated by mass percentage, it includes 0.2 - 0.4% triethanolamine, 8 - 12% sucrose and the balance of distilled water. At this content, the stability of the urine separation reagent is ensured, and the interference with the separation process is reduced, preventing the viscosity of the reagent from affecting the diffusion of particles and thus affecting the separation efficiency.

[0032] A method for separating urine extracellular vesicles without ultracentrifugation includes the following steps: Step (1): Take a urine sample and perform the first centrifugation, retaining the supernatant. Step (2): Take the supernatant obtained in step (1) and perform the second centrifugation at room temperature, retaining the supernatant S1 and the precipitate. Step (3): Mix the precipitate obtained in step (2) with the above-mentioned urine separation reagent, incubate it, and then perform the third centrifugation, retaining the supernatant S2 and the precipitate P1. Step (4): Mix the supernatant S1 and the supernatant S2 evenly to obtain a urine supernatant containing uEVs. The centrifugal force of the first centrifugation, the second centrifugation, and the third centrifugation is less than 100,000 g.

[0033] Specifically, in a urine sample, there are urinary residues, cells, THP, and the required uEVs. Currently, the most commonly used method for separation and enrichment internationally is the ultracentrifugation method. The specific steps are as follows: in the first centrifugation, large particulate matters such as cells, cell debris, and bacteria are removed by low-speed centrifugation. The second is ultracentrifugation (RCF > 100,000 g) to precipitate EVs for separation and enrichment. However, due to the large amount of THP in urine, the concentration of the uEVs sample obtained by the ultracentrifugation method is low, and it contains THP, and its purity is also greatly affected. In the separation method of the present invention, in step (1), the first centrifugation is carried out, and the precipitate formed by the first centrifugation is removed, and the supernatant is retained. Under the action of the first centrifugation, the retained supernatant does not include cells and urinary residues; in step (2), the second centrifugation is carried out. Under the action of the second centrifugation, the supernatant obtained in step (1) is further separated. The supernatant S1 obtained by the second centrifugation already contains uEVs. However, in the precipitate at this time, the THP with a high content polymerizes into a double-helical rope-like structure and wraps the uEVs therein, and they coexist in the precipitate and cannot be applied to subsequent immunoassays. Only the yield of uEVs in the supernatant S1 is low. Therefore, in step (3), the precipitate obtained by the second centrifugation is mixed with a urine separation reagent and incubated. During the incubation, triethanolamine in the urine separation reagent breaks the disulfide bonds between THP polymers, dissociating them into monomeric forms, so that the uEVs wrapped in the THP polymers are released. After the incubation is completed, the third centrifugation is carried out. After the third centrifugation, the uEVs and the dissociated THP monomers coexist in the supernatant S2. Finally, the supernatant S1 and the supernatant S2 are mixed evenly. At this time, the yield of uEVs is high. Compared with the separation and enrichment method of ultracentrifugation, the present invention releases the uEVs originally wrapped in the THP polymers. The higher concentration of uEVs facilitates subsequent immunoassays. At the same time, because the urine separation reagent used is relatively mild, it will not more affect the membrane protein structure of uEVs, further ensuring the concentration of uEVs for immunoassays and improving the separation efficiency. Moreover, the centrifugal force of the first centrifugation, the second centrifugation, and the third centrifugation is less than 100,000 g, that is, the first centrifugation, the second centrifugation, and the third centrifugation are all low-speed centrifugations, effectively avoiding the damage of protein structure caused by ultracentrifugation and thus affecting the yield of uEVs that can be detected subsequently, and can reduce costs, reduce the operation complexity, and be safer, solving the problems of low separation efficiency and low yield during the separation of urinary extracellular vesicles at present, which affect subsequent immunoassays.

[0034] Preferably, the placement time of the urine sample is not more than 2 hours.

[0035] Preferably, in step (3), the incubation conditions are shaking incubation at room temperature for 30 - 60 s.

[0036] Specifically, a urine separation reagent is added to the precipitate obtained in step (2), and shaken for 30 - 60 s at room temperature. Under the shaking condition, it promotes the urine separation reagent to contact more fully with the precipitate obtained in step (2), ensuring the full dissociation of THP aggregates and releasing the uEVs wrapped therein, which is beneficial for subsequent immunoassays. Under this condition, the incubation time is 30 - 60 s. This incubation time is relatively short, effectively shortening the time of the entire separation process and improving the separation efficiency.

[0037] Moreover, because the reaction conditions of the urine separation reagent used are relatively mild and do not require other operations such as heating, the separation steps are simplified, the operation is simple, and the safety is higher.

[0038] Preferably, in step (1), the conditions for the first centrifugation are centrifugation at 2000 - 4000 g for 5 - 10 minutes at 4°C.

[0039] Specifically, under the conditions of the first centrifugation, cells and large particle residues in the urine can be effectively removed, improving the purity of the sample. While facilitating the subsequent separation of uEVs, it does not affect the structure of uEVs, and has a relatively high centrifugation efficiency.

[0040] Meanwhile, under the low temperature condition of 4°C, the stability of biomolecules in the urine sample can be ensured, ensuring that uEVs can stably exist during the first centrifugation process.

[0041] Preferably, in steps (2) and (3), the conditions for the second centrifugation and the third centrifugation are centrifugation at 16000 - 17000 g for 15 - 20 minutes at room temperature.

[0042] In steps (2) and (3), the centrifugation conditions are both centrifugation at 16000 - 17000 g for 15 - 20 minutes at room temperature, that is, both separate uEVs from other components to ensure that uEVs are present in the supernatant.

[0043] Preferably, the urine supernatant obtained in step (4) can be used for immunoassay after dilution.

[0044] Specifically, the urine supernatant containing uEVs obtained after separation can be used for immunoassay after being diluted by an appropriate multiple, reducing the step of resuspending uEVs with buffer, and more quickly and conveniently performing subsequent immunoassays, which is beneficial for large-scale clinical use.

[0045] Furthermore, the method of immunoassay is immunochromatographic assay or chemiluminescent immunoassay.

[0046] Both immunochromatographic and chemiluminescent immunoassays can obtain test results in a relatively short period of time. Although Western blot (WB) is the most commonly used protein analysis technique in biology and can detect the presence of certain target proteins in EVs, the method has a cumbersome operation process and a long preparation and processing time (>10 hours). Immunochromatographic and chemiluminescent immunoassays are more suitable for actual clinical work. In addition, the above two immunoassay methods have high sensitivity and are very suitable for early diagnosis and disease monitoring, which is suitable for the purpose of isolating uEVs in urine for disease diagnosis.

[0047] Preferably, the dilution ratio is 10-20 times.

[0048] In the subsequent immunoassay process, since normal or healthy people also have uEVs with specific membrane proteins in their urine, this specific membrane protein refers to the corresponding membrane protein used for disease diagnosis. In order to widen the gap between the detection values of patients and healthy people as much as possible and improve the signal-to-noise ratio, it is necessary to dilute the sample 10-20 times to make the test results more accurate and improve the sensitivity of the immunoassay.

[0049] Preferably, in step (1), the urine sample is morning urine.

[0050] Specifically, after a night of accumulation and concentration, the concentrations of components such as cells, proteins and metabolites in morning urine are relatively high, and the concentration of uEVs is correspondingly high, which is conducive to the separation and acquisition of uEVs. In addition, because morning urine stays in the bladder for a long time, its composition is relatively stable and is less affected by factors such as daily activities, diet and medication. It can better reflect the true state of the body and help improve the accuracy of clinical diagnosis.

[0051] Preferably, the isolated uEVs are used to differentiate diabetic kidney damage or prostate cancer.

[0052] Specifically, uEVs isolated by the present invention can be used to distinguish diabetic kidney damage or prostate cancer. The membrane proteins of uEVs isolated by the present invention are not destroyed, which facilitates the differential diagnosis of the above diseases.

[0053] Among them, the CD63 / CD9+ EVs subpopulation can be used as an auxiliary indicator for disease staging diagnosis in patients with diabetic nephropathy, and urine CD63 / PSMA+ EVs can be used as a new diagnostic marker for prostate cancer patients.

[0054] The technical solution of the present invention is further illustrated below through specific implementation methods.

[0055] Example Group 1 Example Group 1-1 Immunochromatographic detection method for urine EVs subpopulations in diabetic nephropathy: 1. Preparation of labeled antibody and quality control antibody Preparation of labeled antibody: Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to 1 mL of europium fluorescent latex microspheres at a ratio of 1:5 to activate the surface of the microspheres. After sufficient reaction, centrifuge, discard the supernatant, resuspend the precipitate with 2 mL of buffer, and add 100 μg of anti-CD9 antibody for sufficient reaction. Subsequently, add 50% BSA blocking agent for blocking, resuspend the precipitate with 2 mL of labeled antibody complex solution (containing 1-10% BSA; 0.1-2% Tween-20) to obtain a labeled antibody suspension, and store it at 4 °C for later use.

[0056] Preparation of quality control antibody: Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to 1 mL of europium fluorescent latex microspheres at a ratio of 1:5 to activate the surface of the microspheres. After sufficient reaction, centrifuge, discard the supernatant, resuspend the precipitate with 2 mL of buffer, and add 100 μg of DNP antibody for sufficient reaction. Subsequently, add 20% BSA blocking agent for blocking, resuspend the precipitate with 2 mL of labeled antibody complex solution (containing 1-20% BSA; 0.1-2% Tween-20) to obtain a labeled antibody suspension, and store it at 4 °C for later use.

[0057] 2. Preparation of NC membrane In the direction from the conjugate pad to the absorbent pad, coat anti-CD63 antibody and DNP antigen onto the chromatographic membrane (nitrocellulose membrane) with a spray gold membrane coater at a rate of 2 μL / cm as the test line and the quality control line respectively. After it is completely dried, seal it with an aluminum foil bag and store it for later use.

[0058] 3. Assembly of test strip All parts are assembled on the PVC bottom plate in the following order: sample pad, conjugate pad, NC membrane and absorbent pad. Each part overlaps by 2 mm. Cut the assembled strip into a width of 4 mm with a strip cutter and put it into a plastic cartridge to form a complete rapid test strip for CD63 / CD9+ EVs in urine for kidney injury detection.

[0059] 4. Detection of clinical samples Step (1): Collect equal-volume early morning urine samples from people in good health, patients with stage A1, A2, and A3 diabetic nephropathy, separate them respectively, centrifuge at 3000 g for 10 minutes at 4 °C, and retain the supernatant; Step (2): Take the supernatant obtained in step (1), centrifuge at 17000 g for 20 minutes at room temperature, and retain the supernatant S1 and the precipitate; Step (3): Mix the precipitate obtained in step (2) with the urine separation reagent, incubate with shaking at room temperature for 60 s, then centrifuge at 17,000 g for 20 minutes at room temperature, and retain the supernatant S2 and the precipitate P1; The urine separation reagent includes 0.25 g of triethanolamine, 10.5 g of sucrose and 100 ml of distilled water; Step (4): Mix the supernatant S1 and the supernatant S2 evenly and dilute 15 times.

[0060] Detect the concentration of urine CD63 / CD9+ EVs subset with the kidney injury urine CD63 / CD9+ EVs rapid detection test strip prepared above, as Figure 1 shown in (A) of Figure 1 and draw an ROC curve according to the detection result, as

[0061] Example groups 1-2 Immunochromatographic detection method for urine EVs subset derived from prostate cancer tissue: 1. Preparation of labeled antibody and quality control antibody Preparation of labeled antibody: Add carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to 1 mL of europium fluorescent latex microspheres at a ratio of 1:5 for surface activation of the microspheres. After sufficient reaction, centrifuge, discard the supernatant, resuspend the precipitate with 2 mL of buffer, and add 100 μg of anti-PSMA antibody for sufficient reaction. Subsequently, add 50% BSA blocking agent for blocking, resuspend the precipitate with 2 mL of labeled antibody complex solution (containing 1-10% BSA; 0.1-2% Tween-20) to obtain a labeled antibody suspension, and store it at 4 °C for later use.

[0062] Preparation of quality control antibody: Add carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to 1 mL of europium fluorescent latex microspheres at a ratio of 1:5 for surface activation of the microspheres. After sufficient reaction, centrifuge, discard the supernatant, resuspend the precipitate with 2 mL of buffer, and add 100 μg of DNP antibody for sufficient reaction. Subsequently, add 20% BSA blocking agent for blocking, resuspend the precipitate with 2 mL of labeled antibody complex solution (containing 1-20% BSA; 0.1-2% Tween-20) to obtain a labeled antibody suspension, and store it at 4 °C for later use.

[0063] 2. Preparation of NC membrane In the direction from the conjugate pad to the absorbent pad, coat anti-CD63 antibody and DNP antigen onto the chromatography membrane (nitrocellulose membrane) with a gold spraying membrane instrument at a rate of 2 μL / cm respectively as the test line and the quality control line. After it is completely dried, seal it with an aluminum foil bag and store it for later use.

[0064] 3. Assembly of test strip All parts are assembled on the PVC bottom plate in the following order: sample pad, conjugate pad, NC membrane, and absorbent pad. Each part overlaps by 2 mm. The assembled strip is cut into a width of 4 mm by a strip cutter and loaded into a plastic cartridge to form a complete rapid detection test strip for CD63 / PSMA+ EVs in urine derived from prostate cancer tissue.

[0065] 4. Detection of clinical samples Step (1): Collect equal volumes of early morning urine samples from healthy individuals and prostate cancer patients for separation. At 4°C, centrifuge at 3000 g for 10 minutes and retain the supernatant. Step (2): Take the supernatant obtained in step (1) and centrifuge at 17000 g for 20 minutes at room temperature. Retain the supernatant S1 and the precipitate. Step (3): Mix the precipitate obtained in step (2) with the urine separation reagent, incubate with shaking at room temperature for 60 s, then centrifuge at 17000 g for 20 minutes at room temperature. Retain the supernatant S2 and the precipitate P1. The urine separation reagent includes 0.25 g of triethanolamine, 10.5 g of sucrose, and 100 ml of distilled water. Step (4): Mix the supernatant S1 and the supernatant S2 evenly and dilute 15 times.

[0066] Detect the concentration of CD63 / PSMA+ EVs subgroup in urine using the rapid detection test strip for CD63 / PSMA+ EVs in urine derived from prostate cancer tissue prepared above. The detection results are as shown in (A) of Figure 2 and draw an ROC curve based on the detection results, as shown in (B) of Figure 2

[0067] Example groups 1 - 3 Detect EVs subgroup in urine derived from prostate cancer tissue by direct chemiluminescence method: 1. Biotinylated CD63 antibody Weigh a certain amount of biotin, dissolve and dilute biotin with sterile water to a final concentration of 5.5 mg / mL. The mass ratio of biotin to antibody is 15:1. Mix the calculated corresponding masses of biotin and the antibody to be tested, and the total volume should be greater than 100 μL. React with rotation in the dark at room temperature for 1 h. After the reaction, dialyze overnight on a shaker at 4°C with PBS solution. The next day, change to a new PBS solution and continue dialysis for 5 h. After dialysis, collect and aliquot, and store in the dark at -80°C.

[0068] 2. Acridinium ester - modified PSMA antibody ​Take 0.5 mg of PSMA antibody and add it to 100 μL of PBS buffer. Then add 20 μL of acridinium ester solution and mix well. React at 37 °C, and purify the reaction mixture using a gel column. Add 10% BSA to the purified solution until the final concentration of bovine serum albumin in the purified solution is 0.5% - 2% to obtain acridinium ester-labeled anti-PSMA antibody.

[0069] 3. Streptavidin-modified solid phase Dissolve streptavidin in citrate buffer to prepare a streptavidin solution with a concentration of 520 μg / mL. Coat the solid phase with the streptavidin solution at 4 °C for 12 hours, and then block it at 37 °C for 2 h with a blocking solution to obtain a streptavidin-coated solid phase.

[0070] 4. Detection of clinical samples Step (1): Collect morning urine samples from healthy people and prostate cancer patients and separate them respectively. Centrifuge at 3000 g for 10 minutes at 4 °C and retain the supernatant. Step (2): Take the supernatant obtained in step (1), centrifuge at 17000 g for 20 minutes at room temperature, and retain the supernatant S1 and the precipitate. Step (3): Mix the precipitate obtained in step (2) with a urine separation reagent, incubate with shaking at room temperature for 60 s, then centrifuge at 17000 g for 20 minutes at room temperature, and retain the supernatant S2 and the precipitate P1. The urine separation reagent includes 0.25 g of triethanolamine, 10.5 g of sucrose and 100 ml of distilled water. Step (4): Mix the supernatant S1 and the supernatant S2 evenly and dilute them 15 times.

[0071] Dilute the mother solution of PSMA antibody modified with a chemiluminescent label to an antibody working solution with a concentration of 2 μg / mL using Tris buffer. Take 100 μL of the urine supernatant obtained in step (4), biotin-labeled CD63 antibody and 100 μL of acridinium ester-modified PSMA antibody and add them to the streptavidin-coated solid phase. Incubate at 37 °C for 30 min, then detect the chemiluminescent signal. The analysis results of the corresponding particle concentration are as Figure 3 shown.

[0072] Example groups 1 - 4 Detection of urinary EVs subsets in prostate cancer by enzyme-catalyzed chemiluminescence method: 1. Biotin-modified CD63 antibody Weigh a certain amount of biotin, dissolve it with sterile water and dilute the biotin to a final concentration of 5.5 mg / mL. The mass ratio of biotin to antibody is 15:1. Mix the calculated corresponding mass of biotin and the antibody to be tested. The total volume should be greater than 100 μL. React with rotation in the dark at room temperature for 1 h. After the reaction, dialyze overnight on a shaker at 4 °C with PBS solution. The next day, change to a new PBS solution and continue dialysis for 5 h. After dialysis, collect and aliquot, and store at -80 °C in the dark.

[0073] 2. PSMA antibody modified with alkaline phosphatase Mix 0.5 mg of PSMA antibody with 0.5 mg of alkaline phosphatase, then add 0.125 mL of 2% glutaraldehyde solution, and react in the dark at room temperature for 6 h; then add 20 μL of 2 mol / L monoethanolamine solution, and react with blocking in the dark at room temperature for 3 h; stir and dialyze the reacted mixture overnight at 4 °C with PBS buffer; add 10% BSA to the dialyzed solution, adjust the final concentration of BSA to 2%, and then add an equal volume of glycerol and mix well to obtain alkaline phosphatase-labeled anti-PSMA antibody.

[0074] 3. Streptavidin-modified solid phase Dissolve streptavidin in citrate buffer to prepare a streptavidin solution with a concentration of 520 μg / mL. Coat the solid phase with the streptavidin solution at 4 °C for 12 h, and block at 37 °C for 2 h with the blocking solution to obtain a streptavidin-coated solid phase.

[0075] 4. Detection of clinical samples Step (1): Separate the early morning urine samples of healthy people and prostate cancer patients respectively. Centrifuge at 3000 g for 10 minutes at 4 °C, and retain the supernatant. Step (2): Take the supernatant obtained in step (1), centrifuge at 17000 g for 20 minutes at room temperature, and retain the supernatant S1 and the precipitate. Step (3): Mix the precipitate obtained in step (2) with the urine separation reagent, incubate with shaking at room temperature for 60 s, then centrifuge at 17000 g for 20 minutes at room temperature, and retain the supernatant S2 and the precipitate P1. The urine separation reagent includes 0.25 g of triethanolamine, 10.5 g of sucrose and 100 mL of distilled water. Step (4): Mix the supernatant S1 and the supernatant S2 evenly and dilute 15 times.

[0076] The mother solution of the PSMA antibody modified with the chemiluminescent label was diluted with Tris buffer to an antibody working solution with a concentration of 2 μg / mL. 100 μL of the urine supernatant obtained in step (4), the biotinylated CD63 antibody, and 100 μL of the alkaline phosphatase-modified PSMA antibody were added to the streptavidin-coated solid phase. After incubation at 37 °C for 30 min, it was washed four times with 200 μL of TBST. 200 μL of AMPPD was added, the parameters of the microplate reader were set, and after incubation at 37 °C for 30 min, the chemiluminescent signal was detected. The analysis results of the corresponding particle concentration are as Figure 4 shown.

[0077] Example Group 2 Denoted as the IS group. Step (1): Equal volumes of early morning urine samples from three normal individuals were collected and separated respectively. At 4 °C, centrifuged at 3000 g for 10 minutes, and the supernatant was retained; Step (2): Take the supernatant obtained in step (1), and at room temperature, centrifuge at 17000 g for 20 minutes, and retain the supernatant S1 and the precipitate; Step (3): Mix the precipitate obtained in step (2) with the urine separation reagent, incubate with shaking at room temperature for 60 s, and then at room temperature, centrifuge at 17000 g for 20 minutes, and retain the supernatant S2 and the precipitate P1; The urine separation reagent includes 0.25 g of triethanolamine, 10.5 g of sucrose, and 100 ml of distilled water; Step (4): Mix the supernatant S1 and the supernatant S2 evenly and dilute 15 times.

[0078] To detect the particle concentration of uEVs therein, the urine supernatant obtained in step (4) was centrifuged at 200000 g for 1 h at 4 °C to obtain a precipitate P2. The precipitate P2 was resuspended with 1000 μL of sterile PBS solution and stored at -80 °C. The particle concentration of uEVs therein was detected, and the detection results are as Figure 5 shown. And gel electrophoresis experiments and nanoparticle tracking analysis were carried out. The results of the gel electrophoresis experiment are as Figure 6 shown in (A) of Figure 6 shown in (B) of

[0079] Control Group 2-1 Compared with Example Group 2, the same urine samples were used. The difference was that in step (3), DTT was used to replace the urine separation reagent, and it needed to be incubated at 37 °C for 10 min, and in step (4), no dilution was required. It was denoted as the DTT group. The particle concentration of uEVs therein was detected, and the detection results are as Figure 5 shown. And gel electrophoresis experiments and nanoparticle tracking analysis were carried out. The results of the gel electrophoresis experiment are as Figure 6As shown in (A) of Figure 6 shown in (B) of

[0080] Control group 2-2 Denoted as the control group, take the same urine samples as those in Example group 2, centrifuge at 4 °C and 3000 g for 10 minutes, remove the cell precipitate, retain the supernatant, centrifuge the supernatant at 17000 g for 20 minutes at room temperature, retain the supernatant and precipitate P1, centrifuge the supernatant at 200000 g for 1 h at 4 °C to obtain precipitate P2, resuspend precipitate P2 with 1000 μL of sterile PBS solution, and perform gel electrophoresis experiments and nanoparticle tracking analysis. The gel electrophoresis results are as Figure 6 shown in (A) of Figure 6 shown in (B) of

[0081] Example group 3 Denoted as the IS group, prepare the labeled antibody and quality control antibody, prepare the NC membrane, and assemble the test strip according to Example group 1-1; Collect equal-volume early morning urine samples from five people in good health, denoted as sample 1, sample 2, sample 3, sample 4, and sample 5 respectively, and separate them respectively: Step (1): Centrifuge at 4 °C and 3000 g for 10 minutes, and retain the supernatant; Step (2): Take the supernatant obtained in step (1), centrifuge at 17000 g for 20 minutes at room temperature, and retain the supernatant S1 and the precipitate; Step (3): Mix the precipitate obtained in step (2) with the urine separation reagent, incubate at room temperature with shaking for 60 s, then centrifuge at 17000 g for 20 minutes at room temperature, and retain the supernatant S2 and precipitate P1; The urine separation reagent includes 0.25 g of triethanolamine, 10.5 g of sucrose, and 100 ml of distilled water; Step (4): Mix the supernatant S1 and supernatant S2 evenly and dilute 15 times. Detect through the prepared rapid detection test strip for urine CD63 / CD9+ EVs, and the results of each fluorescence intensity are as Figure 7 shown.

[0082] Control group 3-1 Denoted as the 37℃ DTT group, compared with Example Group 3, the same urine sample was used. The difference lies in that in step (3), DTT was used to replace the urine separation reagent, and the incubation condition was incubation at 37℃ for 10 min. Dilution was not required and step (5) was added: the obtained urine supernatant was centrifuged at 200,000g for 1 h at 4℃ to obtain precipitate P2, and the precipitate P2 was resuspended with 1000 μL of sterile PBS solution and detected by the prepared rapid detection test strip for urine CD63 / CD9+ EVs. The results of each fluorescence intensity are as Figure 7 shown.

[0083] Comparative Example Group 3-2 Denoted as the control group, compared with Example Group 3, the same urine sample was used. It was centrifuged at 3000g for 10 minutes at 4℃ to remove the cell precipitate, and the supernatant was retained. The supernatant was centrifuged at 17000g for 20 minutes at room temperature, and the supernatant and precipitate P1 were retained. The supernatant was centrifuged at 200,000g for 1 h at 4℃ to obtain precipitate P2, and the precipitate P2 was resuspended with 1000 μL of sterile PBS solution and detected by the prepared rapid detection test strip for urine CD63 / CD9+ EVs. The fluorescence intensity results are as Figure 7 shown.

[0084] Example Group 4 Denoted as the IS group, in accordance with Examples 1-3, biotinylated anti-CD63 antibody, acridinium ester-modified anti-PSMA antibody, and streptavidin-modified solid phase were carried out; Five equal-volume early morning urine samples from prostate cancer patients were collected and denoted as Sample 6, Sample 7, Sample 8, Sample 9, and Sample 10 respectively, and were separated respectively: Step (1): Centrifuge at 3000g for 10 minutes at 4℃ and retain the supernatant; Step (2): Take the supernatant obtained in step (1) and centrifuge at 17000g for 20 minutes at room temperature, and retain the supernatant S1 and the precipitate; Step (3): Mix the precipitate obtained in step (2) with the urine separation reagent, incubate with shaking at room temperature for 60 s, and then centrifuge at 17000g for 20 minutes at room temperature to retain the supernatant S2 and the precipitate P1; The urine separation reagent includes 0.25 g of triethanolamine, 10.5 g of sucrose, and 100 ml of distilled water; Step (4): Mix the supernatant S1 and supernatant S2 evenly and dilute by 15 times. Dilute the mother liquor of PSMA antibody modified with chemiluminescent label with Tris buffer to an antibody working solution with a concentration of 2 μg / mL. Take 100 μL of the urine supernatant obtained in step (4), biotin-labeled CD63 antibody, and 100 μL of acridinium ester-modified PSMA antibody and add them to the streptavidin-coated solid phase. After incubating at 37 °C for 30 min, detect the chemiluminescent signal, and detect its luminescence value through a direct chemiluminescence immunoassay platform. The luminescence intensity results of each group are as Figure 8 shown.

[0085] Control group 4-1 Recorded as the 37 °C DTT group. Compared with Example group 4, the urine samples used are the same. The difference is that in step (3), DTT is used to replace the urine separation reagent, and the incubation condition is to incubate at 37 °C for 10 min. No dilution is required and step (5) is added: Centrifuge the obtained urine supernatant at 200,000 g at 4 °C for 1 h to obtain precipitate P2, and resuspend precipitate P2 with 1000 μL of sterile PBS solution. Detect its luminescence value through a direct chemiluminescence immunoassay platform. The luminescence value results are as Figure 8 shown.

[0086] Control group 4-2 Recorded as the control group. Compared with Example group 4, the urine samples used are the same. Centrifuge at 4 °C and 3000 g for 10 minutes to remove the cell precipitate and retain the supernatant. Centrifuge the supernatant at 17,000 g at room temperature for 20 minutes, retain the supernatant and precipitate P1. Centrifuge the supernatant at 200,000 g at 4 °C for 1 h to obtain precipitate P2, and resuspend precipitate P2 with 1000 μL of sterile PBS solution. Detect its luminescence value through a direct chemiluminescence immunoassay platform. The luminescence value results are as Figure 8 shown.

[0087] As shown by Figures 1 to 4 the uEVs separated by the present invention can be used to distinguish diabetic nephropathy and prostate cancer and can be used in clinical applications.

[0088] Among them, as Figure 1 shown in (A) therein, the vertical axis is the ratio of the signal values of the test line and the quality control line (T / C value). The concentration of the CD63 / CD9+ EVs subset in the group with good physical condition (HD) is significantly higher than that in patients with stage A1, A2, and A3 diabetic nephropathy. And from Figure 1As shown in (B) therein, for differentiating patients in stage A1 and stage A3, the area under the receiver operating characteristic curve (ROC curve) of the chromatographic test strip for detecting extracellular vesicles can reach 0.869. Therefore, under the separation method of the present invention, among the isolated uEVs, the CD63 / CD9+ EVs subset can be used as an auxiliary index for diagnosing the disease stage of diabetic nephropathy patients, that is, the uEVs isolated by the present invention can be used to distinguish diabetic nephropathy patients from healthy people, and help to distinguish patients in stage A1 and stage A3; similarly, as Figure 2 shown in (A) therein, the fluorescence intensity of prostate cancer patients (PCa) is significantly higher than that of people in good health (Healthy), and as Figure 2 shown in (B) therein, the area under the receiver operating characteristic curve (ROC curve) of the chromatographic test strip for detecting extracellular vesicles can reach 0.9428. Therefore, under the separation method of the present invention, among the isolated uEVs, urinary CD63 / PSMA+ EVs can be used as a novel diagnostic marker for prostate cancer patients and is expected to be used in large-scale population screening work. And Figure 3 and Figure 4 are the linear equations obtained by two chemiluminescence methods in Example Group 1-3 and Example Group 1-4, Figure 3 the linear equation in is 8 , R2 = 0.9916 (>0.9). It can be seen from the figure that there is a good linear relationship between the detection signal and the concentration of urinary CD63 / PSMA+ EVs within the concentration range of 0.2 - 8.2×10 Figure 4 the linear equation in is 8 , R2 = 0.9891 (>0.9). There is a good linear relationship between the detection signal and the concentration of urinary CD63 / PSMA+ EVs within the concentration range of 0.5 - 3.3×10 Figure 3 and Figure 4 show that the uEVs isolated by the present invention can be effectively applied to the above two chemiluminescence immunoassays.

[0089] According to the results obtained in Example Group 1, it can be seen that the isolated uEVs can be effectively applied to immunochromatographic detection and chemiluminescence immunoassay for differentiating diabetic nephropathy and prostate cancer. It can be seen that under the separation method of the present invention, the obtained urine supernatant can be effectively applied to immunoassay for clinical application.

[0090] From the detection results obtained in Example Group 2 and Comparative Example Group 2-1, that is, as Figure 5As shown, for the same urine sample, the particle concentration of uEVs in Example Group 2 (IS) was significantly higher than that of uEVs in Comparative Example Group 2-1. It can be seen that under the condition of consistent uEVs separation operation, the particle concentration of uEVs obtained by selecting the urine separation reagent of the present invention is higher than that treated with DTT, that is, the separation efficiency of the present invention is higher.

[0091] The results of gel electrophoresis experiments and nanoparticle tracking analysis obtained from Example Group 2, Comparative Example Group 2-1, and Comparative Example Group 2-2 are respectively as Figure 6 shown in (A) of Figure 6 and (B) of Figure 6 shown. The molecular weight of THP is 92 kDa. For the same urine sample, compared with Example Group (IS) and Comparative Example 2-1 (37°C DTT), in the case of untreated THP, almost all THP will appear in P1, that is, the low-speed centrifugation precipitate, and there is almost no THP in the ultracentrifugation precipitate P2. However, at this time, P1 removed includes uEVs wrapped by THP, and the yield of uEVs in P2 is low. After digestion treatment with DTT or the urine separation reagent, THP monomers will not appear in P1, and subsequent ultracentrifugation will appear in P2 together with extracellular vesicles, without affecting subsequent immunoassays. Therefore, adding a step of treating THP polymers with a reducing agent can effectively degrade THP polymers into monomer form and release the encapsulated urine EVs. As Figure 6 shown in (B) of

[0092] shown, the detection results of Example Group 3 (IS), Comparative Example Group 3-1 (37°C DTT), and Comparative Example Group 3-2 (control) are as Figure 7 shown, and the detection results of Example Group 4 (IS), Comparative Example Group 4-1 (37°C DTT), and Comparative Example Group 4-2 (control) are as Figure 8 shown. Among them, the reason for adding step (5) to the DTT treatment compared with Example Group 3 is that the separation efficiency is low, and it is necessary to enrich uEVs by ultracentrifugation separation before it is convenient for detection. From Figure 7It can be seen that for the same urine sample, after different separation operations of Example Group 3, Comparative Example Group 3-1 and Comparative Example Group 3-2, it can be seen that the fluorescence signal value of uEVs obtained in Example Group 3 is higher. Therefore, compared with DTT treatment or ultracentrifugation treatment, uEVs treated with the urine separation reagent of the present invention are more suitable for use in the immunochromatographic detection platform. Figure 8 It can be seen that for the same urine sample, after different separation operation treatments of Example Group 4, Comparative Example Group 4-1 and Comparative Example Group 4-2, the uEVs obtained after treatment with the urine separation reagent of the present invention have a higher luminescence value and are more suitable for use in the chemiluminescence immunoassay platform.

[0093] In summary, the separation method using the urine separation reagent of the present invention according to this scheme has a higher particle concentration of uEVs than that obtained using DTT reducing agent or ultracentrifugation separation method, is more suitable for immunochromatographic detection platform and chemiluminescent immunoassay platform, and is clinically used to distinguish diabetic nephropathy from prostate cancer.

[0094] Similarly, the technical principles of the present invention have been described above in conjunction with specific embodiments. These descriptions are intended solely to illustrate the principles of the present invention and are not to be construed in any way as limiting the scope of protection of the present invention. Based on the explanations herein, those skilled in the art will be able to devise other specific embodiments of the present invention without inventive effort, and such embodiments will fall within the scope of protection of the present invention.

Claims

1. A urine separation reagent, characterized in that, Calculated by mass percentage, it includes the following raw materials: 0.2-0.4% of triethanolamine, 8-12% of sucrose and the balance of distilled water.

2. A method for separating urine extracellular vesicles without ultracentrifugation, characterized in that, It includes the following steps: Step (1): Take a urine sample and centrifuge it for the first time, retaining the supernatant. Step (2): Take the supernatant obtained in step (1) and centrifuge it for the second time at room temperature, retaining the supernatant S1 and the precipitate. Step (3): Mix the precipitate obtained in step (2) with the urine separation reagent described in claim 1, incubate it, and then centrifuge it for the third time, retaining the supernatant S2 and the precipitate P1. Step (4): Mix the supernatant S1 and the supernatant S2 evenly to obtain a urine supernatant containing uEVs. The centrifugal forces of the first centrifugation, the second centrifugation and the third centrifugation are all less than 100,000 g.

3. The method for separating urine extracellular vesicles without ultracentrifugation according to claim 2, wherein: In step (3), the incubation conditions are oscillating incubation at room temperature for 30-60 s.

4. The method for separating urinary extracellular vesicles without ultracentrifugation according to claim 2, characterized in that: In step (1), the conditions for the first centrifugation are centrifugation at 2,000-4,000 g for 5-10 minutes at 4 °C.

5. The separation method of urine extracellular vesicles without ultracentrifugation according to claim 2, wherein: In steps (2) and (3), the conditions for the second centrifugation and the third centrifugation are centrifugation at 16,000-17,000 g for 15-20 minutes at room temperature.

6. The isolation method of urine extracellular vesicles without ultracentrifugation according to claim 2, characterized in that: The urine supernatant obtained in step (4) can be used for immunoassay after dilution.

7. The method for separating urinary extracellular vesicles without ultracentrifugation according to claim 6, wherein: The immunoassay method is immunochromatographic assay or chemiluminescent immunoassay.

8. The separation method of urine extracellular vesicles without ultracentrifugation according to claim 6, characterized in that: The dilution factor is 10-20 times.

9. The separation method of urine extracellular vesicles without ultracentrifugation according to claim 2, wherein: In step (1), the urine sample is morning urine.

10. The method for separating urinary extracellular vesicles without ultracentrifugation according to claim 2, wherein: The separated uEVs are used to distinguish diabetic kidney injury or prostate cancer.

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