Method and system for detecting migration body

Abstract

The invention provides a method and a system for detecting a migration body. The invention provides a method for detecting a migration body. The method comprises the following steps: (a) providing a sample to be detected; (b) mixing the sample to be detected with a solid-phase carrier to form a'migration body-agglutinin-solid-phase carrier 'compound; (c) separating the'migration body-lectin-solid phase carrier 'compound; and (d) detecting the type and / or number of migrators in the "migrator-lectin-solid phase carrier" complex with a detection agent that binds to a migrator-specific marker in a targeted manner. The method disclosed by the invention can be used for conveniently, quickly, accurately and efficiently detecting the migrators in various body fluids such as cell culture fluid, urine and blood.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to methods and systems for detecting migrasomes, especially methods and systems for detecting migrasomes using a flow cytometer. Background Art

[0002] Migrasomes are single-membrane vesicle structures with a diameter of 0.5 - 2 μm generated by contractile filaments at the cell tail during cell directed migration. Migrasomes contain a large number of bioactive substances such as nucleic acids, proteins, fats, etc., and play an important role in cell-cell communication, participating in and regulating various physiological and pathological activities.

[0003] Recent studies have shown that migrasomes also exist in blood and various body fluids, such as serum, urine, etc. The content of migrasomes in some blood or body fluids is closely related to certain diseases (such as diabetic nephropathy, etc.), and is expected to become biomarkers for various diseases.

[0004] Currently, methods for specifically detecting migrasomes are limited to techniques such as electron microscopy, Western Blotting, etc. However, due to various reasons such as complex operation and inability to quantify, the above methods cannot be frequently applied to daily scientific research and clinical detection. This not only greatly limits the basic research on migrasomes, but also restricts the clinical application of migrasomes.

[0005] Therefore, there is an urgent need in the art to develop a convenient, fast, accurate, and efficient method for detecting migrasomes. Summary of the Invention

[0006] The object of the present invention is to provide a convenient, fast, accurate, and efficient method for detecting migrasomes.

[0007] In a first aspect of the present invention, there is provided a method for detecting migrasomes, comprising the steps of:

[0008] (a) providing a sample to be tested, the sample to be tested containing migrasomes;

[0009] (b) mixing the sample to be tested with a solid phase carrier to form a first mixture, wherein the solid phase carrier is conjugated or bound with lectin, and when the sample to be tested contains migrasomes, a "migrasome - lectin - solid phase carrier" complex is formed;

[0010] (c) optionally separating the "migrasome - lectin - solid phase carrier" complex; and

[0011] (d) using a detection agent that specifically binds to a migrasome specific marker to detect the type and / or quantity of migrasomes in the "migrasome - lectin - solid phase carrier" complex.

[0012] In another preferred example, the sample includes: urine sample, serum sample, cell culture medium, saliva, cerebrospinal fluid, tissue isolation fluid, or a combination thereof.

[0013] In another preferred example, the sample includes: blood, plasma, urine.

[0014] In another preferred example, the sample is in a liquid state.

[0015] In another preferred example, the solid-phase carrier includes: solid particles, microfluidic chips, cellulose acetate membranes, nylon membranes, agarose microbeads.

[0016] In another preferred example, the solid-phase carrier includes magnetic beads or non-magnetic microspheres.

[0017] In another preferred example, the solid-phase carrier includes fluorescence-coded microspheres (beads), and more preferably, the microspheres include polymer microspheres.

[0018] In another preferred example, the average particle size of the solid-phase carrier is 10 nm - 100 mm, preferably 0.1 - 1000 μm, and more preferably 1 - 100 μm.

[0019] In another preferred example, the surface of the solid-phase carrier is conjugated or bound with lectin.

[0020] In another preferred example, the lectin is conjugated to the surface of the solid-phase carrier through a chemical bond.

[0021] In another preferred example, the lectin is selected from the group consisting of: wheat germ agglutinin (WGA), concanavalin A (ConA), peanut agglutinin (PNA), or a combination thereof.

[0022] In another preferred example, the lectin carries a first detectable label.

[0023] In another preferred example, the first detectable label is selected from the group consisting of: fluorophore, chromophore, or a combination thereof. More preferably, the first detectable label is Alexa 594 or a similar fluorescent label.

[0024] In another preferred example, in step (b), the sample to be tested is contacted with the solid-phase carrier to form a first mixture, and in step (c), the "migrator - lectin - solid-phase carrier" complex is separated from the first mixture.

[0025] In another preferred example, in step (c), the separation includes separation by a magnetic field.

[0026] In another preferred embodiment, in step (d), the detection includes: flow cytometry, fluorescence imaging technology, and fluorescence immunoassay technology.

[0027] In another preferred embodiment, in step (d), the detection is performed using a flow cytometer.

[0028] In another preferred embodiment, the detection agent carries a second detectable label.

[0029] In another preferred embodiment, the detectable label is selected from the group consisting of: fluorophore, chromophore, or a combination thereof.

[0030] In another preferred embodiment, the detection agent that specifically binds to the migrasome-specific marker includes an antibody.

[0031] In another preferred embodiment, the antibody is selected from the group consisting of: an antibody against TSPAN4, an antibody against Integrinα5β1, a PIGK antibody, an NDST1 antibody, a CPQ antibody, an EOGT antibody, or a combination thereof.

[0032] In another preferred embodiment, in step (d), it is quantitatively detected whether the migrasome in the "migrasome-lectin-solid phase carrier" complex is a migrasome derived from tubular cells or podocytes.

[0033] In another preferred embodiment, the method is a non-diagnostic and non-therapeutic method.

[0034] In a second aspect of the present invention, a system for detecting migrasomes is provided, the system comprising:

[0035] (M1) A sample loading module, the sample loading module being configured to perform the following operations: mixing a sample to be tested with a solid phase carrier to form a first mixture, wherein the solid phase carrier is conjugated or bound with a lectin, and when the sample to be tested contains migrasomes, a "migrasome-lectin-solid phase carrier" complex is formed;

[0036] (M2) An optional separation module, the separation module being configured to perform the following operations: separating the "migrasome-lectin-solid phase carrier" complex from the first mixture; and

[0037] (M3) A detection module, the detection module being configured to perform the following operations: using a detection agent that specifically binds to a migrasome-specific marker to detect the type and / or quantity of migrasomes in the "migrasome-lectin-solid phase carrier" complex.

[0038] In another preferred embodiment, the separation module is further configured to: wash the separated "migrasome-lectin-solid phase carrier" complex.

[0039] In another preferred embodiment, the detection module includes:

[0040] (M3a) a detection agent adding submodule, wherein the detection agent adding submodule is configured to perform the following operations: mixing the detection agent that targets and binds to the migrating body specific marker with the "migrating body-lectin-solid phase carrier" complex to form a "detection agent-migrating body-lectin-solid phase carrier" quaternary complex; and

[0041] (M3b) A signal detection submodule, wherein the signal detection submodule is configured to perform the following operations: detecting a unique signal of the "detection agent-migrator-lectin-solid phase carrier" quaternary complex.

[0042] In another preferred embodiment, the specific signal comes from the detectable label carried by the detection agent.

[0043] In another preferred embodiment, the system further comprises:

[0044] (M4) a data processing module configured to process the detection data from the detection module; and

[0045] (M5) An output module, which is used to output the processing results of the data processing module.

[0046] In another preferred embodiment, the data processing module is configured to perform the following operations: comparing the detection data from the detection module with a predetermined value, thereby providing a disease risk result or a disease diagnosis result.

[0047] In another preferred embodiment, the diseases include: kidney disease, diabetic nephropathy, IgA nephropathy, and membranous nephropathy.

[0048] In a third aspect of the present invention, there is provided a lectin-solid phase carrier complex for capturing migrating bodies in a sample, comprising: a solid phase carrier and a lectin, wherein the solid phase carrier is coupled to or bound to the lectin;

[0049] Wherein, the lectin is selected from the following group: wheat germ agglutinin (WGA), concanavalin A agglutinin (ConA), peanut agglutinin (PNA), or a combination thereof.

[0050] In another preferred embodiment, the lectin carries a first detectable label.

[0051] In another preferred embodiment, the first detectable label is selected from the following group: a fluorophore, a chromophore, or a combination thereof.

[0052] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 Schematic diagram showing capture of migratsomes by lectin-coupled magnetic beads.

[0054] Figure 2 The following figure shows the construction of lectin-coupled magnetic beads, (A) fluorescence microscopy observation of WGA, conA, and PNA-coupled magnetic beads, and (B) flow cytometry observation of WGA, conA, and PNA-coupled magnetic beads.

[0055] Figure 3 Flow cytometric analysis of migrating bodies captured by lectin-conjugated magnetic beads. (A) Scanning electron microscopy of migrating bodies captured by WGA-conjugated magnetic beads. (B) Fluorescence microscopy of migrating bodies captured by WGA-conjugated magnetic beads; green indicates TSPAN4 antibody staining. (C) Flow cytometric analysis of migrating bodies captured by magnetic beads conjugated with WGA, ConA, and PNA.

[0056] Figure 4 Shown are migrating bodies captured by WGA-coupled magnetic beads labeled with TSPAN4 or Integrin α5β1 antibodies.

[0057] Figure 5 Figure 3 shows flow cytometric detection of migrasomes captured with WGA-coupled magnetic beads to distinguish the cell-of-origin of the migrasomes. (A) Western blotting analysis of nephrin and AQP1 protein expression in migrasomes derived from podocyte HPCs and tubular cells HK2. (B) Flow cytometric analysis of TSPAN4 expression in migrasomes derived from podocyte HPCs and tubular cells HK2. (C) Flow cytometric analysis of nephrin and AQP1 protein expression in migrasomes derived from podocyte HPCs. (D) Flow cytometric analysis of nephrin and AQP1 protein expression in migrasomes derived from tubular cells HK2.

[0058] Figure 6 Flow cytometry analysis shows a dose-dependent effect of migrasomes captured by WGA-coupled magnetic beads. (A) Western blotting analysis of migrasome protein expression in podocyte HPC-derived migrasomes. (B) Western blotting analysis of PIGK protein expression in podocyte HPC-derived migrasomes. (C) Flow cytometry analysis of TSPAN4 expression in podocyte HPC-derived migrasomes. C: Flow cytometric plot. D: Statistical graph.

[0059] Figure 7 It shows that the migrasomes captured by WGA-conjugated magnetic beads detected by flow cytometry exhibit a dose-dependent effect. (A) Western blotting was used to detect the protein content of urinary-derived migrasomes. (B) Western blotting was used to detect the protein content of PIGK in urinary-derived migrasomes. (C-D) Flow cytometry was used to detect TSPAN4 expressed by urinary-derived migrasomes. C: Flow cytometry plot. D: Statistical chart.

[0060] Figure 8 It shows that the migrasomes captured by WGA-conjugated magnetic beads detected by flow cytometry exhibit a dose-dependent effect. (A) Western blotting was used to detect the protein content of blood-derived migrasomes. (B-C) Flow cytometry was used to detect TSPAN4 expressed by blood-derived migrasomes. B: Flow cytometry plot. C: Statistical chart.

[0061] Figure 9 It shows the detection of urinary migrasome content in healthy volunteers (HV), diabetic nephropathy (DN), membranous nephropathy (MN), IgA nephropathy, and FSGS (focal segmental glomerulosclerosis) patients.

[0062] Figure 10 It shows the detection of PLA2R on the surface of migrasomes captured by WGA-conjugated magnetic beads. (A-B) Western blotting (A) and flow cytometry (B) were used to detect PLA2R expressed by migrasomes. (C) Flow cytometry was used to detect PLA2R expressed by urinary migrasomes in patients with membranous nephropathy.

[0063] Figure 11 It shows the observation of migrasomes captured by TSPAN4 antibody-conjugated magnetic beads by scanning electron microscopy. Detailed implementation methods

[0064] Through extensive and in-depth research and a large number of screenings, the present inventors unexpectedly developed, for the first time, an efficient, rapid, and accurate method and system for detecting migrasomes. The present inventors synthesized a class of migrasome capture carriers with unique structures, and the migrasome capture carriers are solid-phase carriers modified with lectins on the surface (such as microspheres (beads) or magnetic beads, etc.). Taking the migrasome capture magnetic beads as an example, the migrasome capture carriers of the present invention can efficiently and specifically capture migrasomes in a sample, and in combination with flow cytometry and other detections, can quickly, simply, and accurately detect the types and quantities of migrasomes in the sample. On this basis, the present invention was completed.

[0065] Migrasome

[0066] As described in this article, migrasomes are single-layer membrane vesicles with a diameter of 0.5 to 2 μm, produced by contractile filaments at the cell tail during directional cell migration. Migrasomes contain a large number of bioactive substances, such as nucleic acids, proteins, and fats, and play an important role in intercellular communication, participating in and regulating various physiological and pathological activities. Migrasomes are present in blood and various body fluids, such as serum and urine. The content of migrasomes in some blood or body fluids is closely associated with certain diseases, such as diabetic nephropathy.

[0067] Lectins

[0068] As used herein, the term "lectin" specifically recognizes glycosylated proteins. Lectins are a class of proteins that bind to polysaccharide groups and are primarily found in the extracellular matrix (ECM). The ECM is rich in glycoproteins and polysaccharide groups, and lectins mediate strong connections between cells by binding to these groups.

[0069] Preferred lectins include plant-derived lectins, such as concanavalin A (ConA), wheat germ agglutinin (WGA), peanut agglutinin (PNA) and soybean agglutinin (SBA), which can efficiently bind to polysaccharide groups.

[0070] In the present invention, a preferred class of lectins is one that carries a first detectable label (particularly a fluorescent label). Compared to antibodies, the lectins of the present invention that carry a detectable label (particularly a fluorescent label) not only have higher stability and anti-interference properties, but also unexpectedly possess highly specific capture capabilities for migratory bodies, enabling more specific capture of different types of migratory bodies in a variety of samples.

[0071] Solid phase carrier

[0072] Solid phase carriers refer to composite magnetic beads whose surface is embedded with a polymer material, mainly including: any one of composite magnetic beads such as dextran magnetic beads, agarose magnetic beads, resin or epoxy resin, polystyrene magnetic beads, or a mixture of two or more magnetic carriers. The particle size distribution range of the solid phase carrier is 1um-200um, preferably 10um-200um, and more preferably 30um-150um. In the production of solid phase carriers, the particle size of solid phase carriers produced in the same batch is not uniform. Therefore, the particle size description of the solid phase carrier can usually be described in terms of average particle size or particle size distribution range in the present invention.

[0073] Flow cytometry

[0074] Flow cytometry (FCM) is a high-throughput technique for simultaneously detecting and analyzing multiple labeled molecules in a single sample. It is a functional, multi-parameter, rapid, and accurate quantitative analysis and sorting of individual cells or other particles. It boasts rapid detection speed, multiple detection parameters, a large amount of collected data, comprehensive analysis, high sorting purity, and flexible methods. Currently, the highest sorting speed has reached 30,000 cells per second. The rapid development of this technology is primarily due to the powerful properties of fluorescent dyes, particularly the ability to distinguish between different fluorescent markers or fluorescence intensities in a flow cytometer. This unique property enables the simultaneous detection of multiple markers in the same sample, achieving rapid and convenient detection. Flow cytometry is primarily used for detecting cell surface markers and cell clustering. Detecting a specific molecule requires the use of other particles as carriers, and these carriers must be nanometer-sized and have excellent size uniformity. Magnetic microspheres are currently a well-established material in magnetic field-assisted separation technology, particularly biomagnetic separation. These materials possess characteristics such as uniform particle size, strong magnetic responsiveness, and good dispersibility in water. FCM technology, with its high accuracy and high-throughput detection capabilities, can be applied to protein concentration detection, including its rapid, high-throughput, multi-parameter, and accurate quantitative analysis. Nanosized, uniform magnetic microspheres are selected as carriers for flow cytometry, enabling the development of a fluorescent immunomagnetic microsphere-based flow cytometer system to detect the type and number of migratory bodies.

[0075] application

[0076] The present invention provides uses of the migsome-capturing vector of the present invention, for example, for preparing biomarkers for various diseases, especially for detecting or diagnosing diseases associated with migsome content.

[0077] Representative diseases associated with migratoria include, but are not limited to, renal diseases such as diabetes, membranous nephropathy, IgA nephropathy, and FSGS (focal segmental glomerulosclerosis).

[0078] In addition, the migratoria capturing carrier of the present invention can efficiently and specifically capture migratoria in a sample, and when used in conjunction with flow cytometry for detection, can quickly, simply and accurately detect the types and quantities of migratoria in a sample.

[0079] In a preferred example, the inventors constructed lectin-coupled magnetic beads that can specifically recognize surface proteins of migratory bodies. The magnetic beads were used to efficiently capture migratory bodies in various body fluids such as cell culture fluid, urine and blood. The migratory bodies were then labeled with migratory body-specific antibodies to identify the type and content of the migratory bodies.

[0080] The advantages of the present invention mainly include:

[0081] (a) The present invention is simple to operate and captures migratory bodies in various body fluids such as cell culture fluid, urine and blood by constructing lectin-coupled magnetic beads that can specifically recognize glycosylated proteins.

[0082] (b) Labeling migratoria with migratoria-specific antibodies enables identification and quantitative detection of migratoria properties, facilitating basic research and clinical applications related to migratoria.

[0083] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0084] Materials and general methods

[0085] Fluorescent lectin protein

[0086] WGA:WGA,Alexa 594 conjugate, Invitrogen, Cat. No. W11262

[0087] ConA:ConA,Alexa 594conjugate, Invitrogen, Cat. No. C11253

[0088] PNA:PNA,Alexa 594 conjugate, Invitrogen, Cat. No. L32459

[0089] Example 1

[0090] Preparation of fluorescently labeled lectins

[0091] 1. Weigh the lyophilized powder of lectin and resuspend it to 5 mg / mL in PBS buffer.

[0092] 2. Take 500 μL of the lectin solution resuspended in the first step and add 10 mg / mL Alexa 594NHSester. After mixing well, react at room temperature in the dark for 1 hour.

[0093] 3. Pass the reaction solution from the second step through a gel filtration column (BioSpin #732 - 6008, BioRad) to remove the Alexa 594 fluorescent probe that has not been conjugated to the lectin. 594 fluorescent probe

[0094] 4. Lyophilize the obtained lectin protein conjugated with Alexa 594 in the dark and store it at 4 °C in the refrigerator for later use. 594 in the refrigerator for later use.

[0095] Example 2

[0096] Construct lectin-conjugated magnetic beads

[0097] 1. Purchase NHS magnetic beads with a particle size of 2 μm (Solarbio, product number M2450). Take 500 μL of the magnetic bead suspension into a 1.5 mL EP tube. Place the EP tube in a magnetic separation rack to enrich the magnetic beads and remove the supernatant. Add 1 mL of pre-cooled Washing Buffer A at 4 °C to the 1.5 mL EP tube and vortex for 15 s to mix the magnetic beads evenly. Place the EP tube in the magnetic separation rack to enrich the magnetic beads and remove the supernatant.

[0098] 2. Dissolve three kinds of lectin proteins conjugated with Alexa Fluor 594 red fluorescence (WGA, Alexa 594 conjugate, Invitrogen, product number W11262; conA, Alexa 594 conjugate, Invitrogen, product number C11253; PNA, Alexa 594 conjugate, Invitrogen, product number L32459) with Coupling Buffer to prepare a protein solution with a concentration of 3.0 mg / mL. Add 500 μL of the lectin protein solution conjugated with Alexa Fluor 594 red fluorescence to the EP tube in the first step and vortex for 30 s to mix it evenly. Vortex the EP tube for 15 s and place it on a mixer to mix at room temperature for 2 h. If the mixing is uneven, then 30 min before the reaction, remove the EP tube every 5 min and vortex for 15 s. After that, remove the EP tube every 15 min and vortex for 15 s. TM 594 red fluorescence of the lectin protein (WGA, Alexa 594 conjugate, Invitrogen, product number W11262; conA, Alexa 594 conjugate, Invitrogen, product number C11253; PNA, Alexa 594 conjugate, Invitrogen, product number L32459) with Coupling Buffer to prepare a protein solution with a concentration of 3.0 mg / mL. Add 500 μL of the lectin protein solution conjugated with Alexa Fluor TM 594 red fluorescence to the EP tube in the first step and vortex for 30 s to mix it evenly. Vortex the EP tube for 15 s and place it on a mixer to mix at room temperature for 2 h. If the mixing is uneven, then 30 min before the reaction, remove the EP tube every 5 min and vortex for 15 s. After that, remove the EP tube every 15 min and vortex for 15 s.

[0099] 3. Place the EP tube in a magnetic separation rack to enrich the magnetic beads and remove the supernatant. Add 1 mL of Blocking Buffer to the EP tube and vortex for 30 s. Place the EP tube in the magnetic separation rack to enrich the magnetic beads and discard the supernatant.

[0100] 4. Repeat step 3 four times. Add 1 mL of Blocking Buffer to an EP tube, vortex for 30 s, place the EP tube in a mixer and react at room temperature for 2 h. Place the EP tube in a magnetic separation rack to enrich the magnetic beads, discard the supernatant. Add 1 mL of ultrapure water to the EP tube, mix well, use the magnetic rack to enrich the magnetic beads, and discard the supernatant.

[0101] 5. Add 1 mL of PBS solution (pH 7.2) to the EP tube, mix well, use the magnetic rack to enrich the magnetic beads, and discard the supernatant. Repeat this operation 2 times, then resuspend in 500 μL of PBS solution, mix well, and store at 4 °C for later use. The final concentration of the magnetic beads conjugated with protein is 10 mg / mL.

[0102] 6. Observe the magnetic beads conjugated with lectin labeled with red fluorescence using a fluorescence microscope and a flow cytometer.

[0103] Results

[0104] The results of the fluorescence microscope are as Figure 2 shown in A. The three kinds of WGA, ConA, and PNA labeled with red fluorescence can all be linked to the magnetic beads, and WGA has the best effect. It can be seen from the figure that the signal of the fluorescently labeled WGA is the strongest in the flow cytometer. The coupling efficiency of WGA with the fluorescent group and magnetic beads is higher than that of ConA and PNA.

[0105] The results observed by the flow cytometer are as Figure 2 shown in B. For the magnetic beads conjugated with WGA, ConA, and PNA, the results show that the above three kinds of lectins can all be successfully linked to the surface of the magnetic beads ( Figure 2 B).

[0106] Example 3

[0107] Detecting migrasomes captured by lectin-conjugated magnetic beads using a flow cytometer

[0108] 1. Collect the culture medium of human podocyte HPC cells, centrifuge at 4000 g for 20 minutes at 4 °C to remove cell debris. Take 5 mL of the cell culture medium and add 5 μL of lectin-conjugated magnetic beads (the WGA, conA, or PNA-conjugated magnetic beads constructed in 2.1), and incubate together on a mixer at 4 °C for 18 h.

[0109] 2. Place the EP tube on a magnetic separation rack to magnetically separate and remove the supernatant. After washing 3 times with 1000 μL of PBS solution (pH 7.2) containing 0.1% BSA, resuspend in 100 μL of PBS solution containing 1% BSA and incubate at room temperature for 30 min.

[0110] 3. Subsequently, place the EP tube in a magnetic separation rack to enrich the magnetic beads, and discard the supernatant. Resuspend in 100 μL of PBS solution containing 0.1% BSA.

[0111] 4. Add 0.5 μL of TSPAN4 antibody (abcam, catalog number ab181995, rabbit source) to the EP tube, incubate at room temperature for 1 h on a mixer, then place the EP tube in a magnetic separation rack to enrich the magnetic beads, and discard the supernatant. After washing 3 times with 1000 μL of PBS solution containing 0.1% BSA, resuspend in 250 μL of PBS solution containing 0.1% BSA.

[0112] 5. Add 0.5 μL of Alexa Fluor 647-donkey anti-rabbit fluorescent secondary antibody to the EP tube, incubate at room temperature for 1 h on a mixer, then place the EP tube in a magnetic separation rack to enrich the magnetic beads, and discard the supernatant. After washing 3 times with 1000 μL of PBS solution containing 0.1% BSA, resuspend in 300 μL of PBS solution and detect using a fluorescence microscope and a flow cytometer.

[0113] Results

[0114] The migrasomes captured by WGA-conjugated magnetic beads were observed using a scanning electron microscope. The results are shown in Figure 3 A, and the migrasomes captured by WGA-conjugated magnetic beads are indicated by the red arrows.

[0115] The migration captured by WGA-conjugated magnetic beads was observed using a fluorescence microscope. It can be seen from the fluorescence microscope that WGA-conjugated magnetic beads can capture migrasomes, where the magnetic beads appear red, and the migrasomes captured by WGA-conjugated magnetic beads can be labeled with the surface-specific protein TSPAN4 antibody (green) of the migrasomes (green fluorescence) ( Figure 3 B).

[0116] Migrasomes were captured using magnetic beads conjugated with WGA, ConA, and PNA respectively, and then the surface-specific protein TSPAN4 antibody (green) of the migrasomes was labeled. The results observed by flow cytometry are shown in Figure 3 C, indicating that magnetic beads conjugated with WGA, ConA, and PNA can all capture migrasomes.

[0117] Example 4

[0118] Detection of migrasomes captured by WGA-conjugated magnetic beads using a flow cytometer

[0119] 1. Collect the culture medium of human podocyte HPC cells, centrifuge at 4000 g for 20 minutes at 4 °C to remove cell debris. Take 5 mL of the cell culture medium and add 5 μL of WGA-conjugated magnetic beads, and incubate together at 4 °C on a mixer for 18 h.

[0120] 2. The following experimental procedures were repeated for steps 2-5 in Example 3, where 0.5 μL of TSPAN4 antibody (abcam, catalog number ab181995, rabbit-derived) or Integrin α5β1 antibody (CST, catalog number 4705T, rabbit-derived) was added to the EP tube.

[0121] Results

[0122] Migrasomes were captured using WGA-conjugated magnetic beads, and then the surface-specific proteins TSPAN4 or Integrin α5β1 antibody on the migrasomes were labeled. The results observed by flow cytometry were as Figure 4 shown. WGA-conjugated magnetic beads could capture migrasomes and could be detected by flow cytometry using the specific protein antibodies on the surface of migrasomes.

[0123] Example 5

[0124] Detecting migrasomes captured by WGA-conjugated magnetic beads using flow cytometry to distinguish the source cells of migrasomes

[0125] 1. The culture media of human podocytes HPC and tubular cells HK2 were collected, and the treatment process was the same as step 1 in Example 4.

[0126] 2. The following experimental operations were repeated for 2-5 in Example 3, where 0.5 μL of TSPAN4 antibody (abcam, catalog number ab181995, rabbit-derived), Podocin antibody (sigma, catalog number P0372, rabbit-derived), or AQP1 antibody (abcam, catalog number ab168387, rabbit-derived) was added to the EP tube.

[0127] Results

[0128] Migrasomes from podocytes HPC and tubular cells HK2 were captured using WGA-conjugated magnetic beads, and then the surface-specific protein TSPAN4 antibody was used to label the migrasomes. The migrasomes were also labeled with the podocyte-specific marker protein Nephrin antibody and the tubular cell-specific protein AQP1 antibody. The results detected by flow cytometry and western blotting were as Figure 5 shown in A. Nephrin was expressed in podocyte migrasomes, and AQP1 was expressed in tubular cell migrasomes. Only the podocyte-specific marker protein Nephrin could be detected in migrasomes derived from podocytes, while only the tubular cell-specific protein AQP1 could be detected in migrasomes derived from tubular cells ( Figure 5 B-C).

[0129] Example 6

[0130] Flow cytometry detection of WGA-coupled magnetic beads captured migrating bodies showed a dose-dependent effect

[0131] 1. Collect human podocyte HPC cell culture medium and centrifuge at 4000g for 20 minutes at 4°C to remove cell debris. Discard the pellet and recover the supernatant. Centrifuge at 20,000g for 30 minutes at 4°C to obtain the crude migratoria. Resuspend the pellet in PBS and assay the protein concentration using a BCA assay. Prepare migratoria resuspensions of varying concentrations by serial dilution according to protein concentration. Detect the number of migratoria particles using Nano-sight.

[0132] 2. After gradient dilution, the final volume of the migratoria sample was 100 μL. 5 μL of WGA-coupled magnetic beads were added and incubated at 4°C on a mixer for 18 h.

[0133] 3. The following experimental procedures were repeated as in 2-5 of Example 3.

[0134] result

[0135] The migratory bodies derived from podocyte HPCs were separated and graded diluted, and then the protein concentration was detected. It was found that the protein concentration changed with the amount of migratory bodies ( Figure 6 A), and then the content of the migration-specific protein makerPIGK was detected by western blotting. The results are shown in Figure 6 As shown in B, the PIGK content changes with the amount of migratory bodies. The TSPAN4 content of migratory bodies with different concentrations was detected by flow cytometry using WGA-coupled magnetic beads. The results showed that the TSPAN4 content increased with the increase in the amount of migratory bodies and was positively correlated with the protein content ( Figure 6 CD). However, when the amount of migration body added is saturated, it will affect the flow cytometer detection ( Figure 6 CD). The above results indicate that migrasomes can be captured by WGA-coupled magnetic beads and quantified in cell culture medium using flow cytometry.

[0136] Example 7

[0137] Flow cytometry detection of WGA-coupled magnetic beads capturing migratory bodies in urine and showing a dose-dependent effect

[0138] 1. Collect urine from a patient with membranous nephropathy and centrifuge at 4000g for 20 minutes at 4°C to remove cellular debris. Discard the precipitate and recover the supernatant. Centrifuge at 20,000g for 30 minutes at 4°C to obtain the crude migratoria. Resuspend the precipitate in PBS and assay the protein concentration using a BCA assay. Prepare a gradient of migratoria suspensions based on protein concentration.

[0139] 2. Repeat step 2 in Experimental Example 6.

[0140] 3. Repeat steps 2 - 5 in Example 3.

[0141] Results

[0142] The migrasomes in urine were separated, gradient - diluted, and then the protein concentration was detected. It was found that the protein concentration changed with the amount of migrasomes ( Figure 7 A). The content of TSPAN4 in migrasomes at different concentrations was detected by flow cytometry using WGA - conjugated magnetic beads. The results showed that the content of TSPAN4 increased with the amount of migrasomes and was positively correlated with the protein content ( Figure 7 B, 7C). The above results indicate that migrasomes captured by WGA - conjugated magnetic beads can be quantified in urine using flow cytometry.

[0143] Example 8

[0144] Flow cytometry detects migrasomes captured by WGA - conjugated magnetic beads in serum and shows a dose - dependent effect

[0145] Repeat Example 7, with the difference that: serum from patients with membranous nephropathy was collected as the test sample to replace the urine sample of patients with membranous nephropathy in Example 7.

[0146] Results

[0147] The migrasomes in serum were separated, gradient - diluted, and then the protein concentration was detected. It was found that the protein concentration changed with the amount of migrasomes ( Figure 8 A). Subsequently, the content of the migrasome - specific protein maker PIGK was detected by western blotting. As Figure 8 shown in B, the content of PIGK changed with the amount of migrasomes. The content of TSPAN4 in migrasomes at different concentrations was detected by flow cytometry using WGA - conjugated magnetic beads. The results showed that the content of TSPAN4 increased with the amount of migrasomes and was positively correlated with the protein content ( Figure 8 C - D). The above results indicate that migrasomes captured by WGA - conjugated magnetic beads can be quantified in blood using flow cytometry.

[0148] Example 9

[0149] Migrasomes captured by WGA - conjugated magnetic beads in urine as a marker of kidney injury

[0150] Urine samples were collected from healthy people, patients with diabetic nephropathy, membranous nephropathy, IgA nephropathy, and FSGS (focal segmental glomerulosclerosis), and the experiment in Example 3 was repeated.

[0151] Results

[0152] Isolate urinary migrasomes from healthy individuals (HV), patients with diabetic nephropathy (DN), membranous nephropathy (MN), IgA nephropathy, and focal segmental glomerulosclerosis (FSGS). The results of flow cytometry detection are as follows Figure 9 shown. There are basically no migrasomes in the urine of HV, while the urine of patients with diabetic nephropathy (DN), membranous nephropathy (MN), IgA nephropathy, and focal segmental glomerulosclerosis (FSGS) contains abundant migrasomes.

[0153] Example 10

[0154] Use WGA-conjugated magnetic beads to capture migrasomes in urine and detect PLA2R on the surface of migrasomes

[0155] 1. Collect the serum and corresponding urine of patients with membranous nephropathy, and repeat steps 1-3 in Example 3.

[0156] 2. Add 0.5 μL of TSPAN4 antibody (abcam, catalog number ab181995, rabbit-derived) and 20% of the corresponding patient's serum in PBS (containing 0.1% BSA) to the EP tube, incubate at room temperature for 1 h on a mixer, then place the EP tube in a magnetic separation rack to enrich the magnetic beads, and discard the supernatant. After washing 3 times with 1000 μL of PBS solution containing 0.1% BSA, resuspend in 250 μL of PBS solution containing 0.1% BSA.

[0157] 3. Add 0.5 μL of Alexa Fluor 647-donkey anti-rabbit fluorescent secondary antibody and FITC-Goat Anti-Human IgG antibody to the EP tube, incubate at room temperature for 1 h on a mixer, then place the EP tube in a magnetic separation rack to enrich the magnetic beads, and discard the supernatant. After washing 3 times with 1000 μL of PBS solution containing 0.1% BSA, resuspend in 300 μL of PBS solution and detect using a fluorescence microscope and flow cytometry.

[0158] Results

[0159] Collect the culture medium of podocyte HPC cells, isolate migrasomes, exosomes, and podocyte proteins. The results of western blotting detection show that abundant PLA2R is expressed on the surface of migrasomes ( Figure 10 A).

[0160] The results of flow cytometry detection are as follows Figure 10 shown in B, further confirming the expression of PLA2R on the surface of migrasomes.

[0161] Subsequently, three pairs of membranous nephropathy samples were obtained: 1) negative for PLA2R autoantibody detection in blood and negative for renal biopsy; 2) negative for PLA2R autoantibody detection in blood and positive for renal biopsy; 3) positive for PLA2R autoantibody detection in blood and positive for renal biopsy.

[0162] The migrasomes in urine were detected by flow cytometry, and the results are as Figure 10 shown in C. The migrasomes secreted by podocytes from patients with negative serum and tissue were still negative; the migrasomes secreted by podocytes from patients with negative serum and positive tissue were still positive; the migrasomes secreted by podocytes from patients with positive serum and positive tissue were still positive. Thus, it can be seen that the migrasomes in urine can be used as a substitute for renal tissue for the detection of PLA2R.

[0163] Comparative Example C1

[0164] The migrasomes captured by TSPAN4 antibody-conjugated magnetic beads were detected by flow cytometry

[0165] 1. Commercially available NHS magnetic beads with a particle size of 2 μm (Solarbio, product number M2450) were used. 100 μL of magnetic bead suspension was taken into a 1.5 mL EP tube, and the EP tube was placed in a magnetic separation rack to enrich the magnetic beads and remove the supernatant. 200 μL of pre-cooled Washing Buffer A at 4 °C was added to the 1.5 mL EP tube, and it was vortexed for 15 s to mix the magnetic beads evenly. The EP tube was placed in a magnetic separation rack to enrich the magnetic beads and remove the supernatant.

[0166] 2. 100 μL of TSPAN4 antibody (abcam, product number ab181995) was added to the EP tube containing the magnetic beads, and it was vortexed for 30 s to mix evenly. The EP tube was vortexed for 15 s and then placed on a mixer and mixed at room temperature for 2 h. If the mixture was not uniform, the EP tube was taken down and vortexed for 15 s every 5 min for the first 30 min before the reaction. Thereafter, the EP tube was taken down and vortexed for 15 s every 15 min.

[0167] 3. The EP tube was placed in a magnetic separation rack to enrich the magnetic beads and remove the supernatant. 200 μL of Blocking Buffer was added to the EP tube, and it was vortexed for 30 s. The EP tube was placed in a magnetic separation rack to enrich the magnetic beads, and the supernatant was discarded.

[0168] 4. Step 3 was repeated four times. 200 μL of Blocking Buffer was added to the EP tube, and it was vortexed for 30 s. The EP tube was placed in a mixer and reacted at room temperature for 2 h. The EP tube was placed in a magnetic separation rack to enrich the magnetic beads, and the supernatant was discarded. 200 μL of ultrapure water was added to the EP tube, mixed thoroughly, and the magnetic beads were enriched with a magnetic rack, and the supernatant was discarded.

[0169] 5. Add 200 μL of PBS (pH 7.2) to the EP tube, mix thoroughly, collect the beads using a magnetic rack, and discard the supernatant. Repeat this procedure twice, then resuspend the beads in 200 μL of PBS, mix thoroughly, and store at 4°C until needed. The final protein-coupled magnetic bead concentration is 10 mg / mL.

[0170] 6. The method for detecting whether the TSPAN4 antibody-coupled magnetic beads have captured migrating bodies is the same as that in Example 3, and the detection is performed using a scanning electron microscope and a flow cytometer.

[0171] result

[0172] Scanning electron microscopy was used to observe the migrating bodies captured by magnetic beads coupled to TSPAN4 antibodies. Figure 11 As shown, the red arrows indicate the migrating bodies captured by magnetic beads coupled to TSPAN4 antibodies.

[0173] Migrating bodies were captured using magnetic beads conjugated with TSPAN4 antibodies, and then labeled with antibodies against the surface-specific protein Integrin α5β1, and observed by flow cytometry. The results showed that although magnetic beads conjugated with TSPAN4 antibodies could capture migrating bodies, there were significant differences between different batches or sources of antibodies.

[0174] The above results show that compared with using magnetic beads coupled to lectins to capture migrating bodies, using magnetic beads coupled to TSPAN4 antibodies to capture migrating bodies has the following disadvantages:

[0175] 1) Lectins are isolated and purified from natural products such as wheat germ, while antibodies require the synthesis of specific antigens and isolation and purification from animals such as immunized mice. Therefore, compared to antibodies, lectins are not only much less expensive but also easier to quality control, ensuring consistent purity, properties, and efficacy across batches of lectins. Given the complexity of the antibody generation, isolation, and purification process, maintaining consistent consistency across batches of antibodies is difficult. This results in differences in the efficiency and specificity of capturing migratory bodies using magnetic beads conjugated to TSPAN4 antibodies.

[0176] 2) Compared to lectins, the capture of migrating bodies using magnetic beads conjugated to TSPAN4 antibodies is more dependent on the antibody's structure. Only when the light chain of the antibody conjugated to the magnetic beads is exposed can it bind to the specific protein on the surface of the migrating body, thereby adsorbing the migrating body. However, during the process of conjugating the antibody to the magnetic beads, it is difficult to ensure the direction of the link. This not only reduces the amount of antibody that can effectively capture the migrating body, resulting in waste, but also makes it difficult to ensure the consistency of the antibody content that can effectively capture the migrating body when conjugated to magnetic beads in each batch of antibodies. This also leads to differences in the efficiency and specificity of migrating body capture when using TSPAN4 antibody-conjugated magnetic beads from each batch of prepared magnetic beads. In comparison, the binding of lectins to migrating bodies is less dependent on the lectin's structure. Therefore, the efficiency and specificity of migrating body capture can be guaranteed to be consistent from batch to batch of prepared lectin-conjugated magnetic beads.

[0177] discuss

[0178] Migrasomes, a newly discovered organelle, are emerging as a new field in cell biology. Migrasomes contain proteins, RNA, and other proteins that can be taken up by recipient cells to exert their functions. Recent studies have shown that migrasomes are also present in body fluids such as blood and urine, suggesting that migrasomes may also serve as carriers of long-distance signaling, playing a role in the crosstalk between different tissues and organs.

[0179] Based on their mechanisms of production and their contents, migrasomes are believed to play a crucial role in development, immune response, tumor metastasis, and other processes. However, due to the limitations of current migrasome isolation, purification, and detection technologies, research on the mechanisms and applications of migrasomes is severely limited.

[0180] The detection method and system of migratory bodies developed by the present invention based on the biochemical properties of migratory bodies can not only be used for the separation and purification of migratory bodies, making it possible to comprehensively explore the types and quantities of contents such as proteins and RNA contained in migratory bodies, but can also be used for the determination of migratory body content, which can explore the content of migratory bodies in body fluids under different physiological and pathological conditions or the number of migratory bodies released by cells under different conditions.

[0181] The method of the present invention greatly simplifies the steps of separation, purification and detection of migratory bodies. It not only provides a convenient means for the study of the mechanism of migratory bodies and helps to fully reveal the generation, regulation and function of migratory bodies, but also enables migratory bodies to move from laboratory research to clinical research, making it possible to be used for disease diagnosis and treatment.

[0182] All documents mentioned in this invention are cited herein by reference as if each individual document was cited by reference. In addition, it should be understood that after reading the above teachings of this invention, those skilled in the art can make various changes or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A method for detecting migrasomes, characterized in that, Comprising the steps of: (a) Providing a sample to be tested, the sample to be tested containing migrasomes; (b) Mixing the sample to be tested with a solid-phase carrier to form a first mixture, wherein the solid-phase carrier is conjugated or bound with lectin, and when the sample to be tested contains migrasomes, a "migrasome-lectin-solid-phase carrier" complex is formed; (c) Optionally separating the "migrasome-lectin-solid-phase carrier" complex; and (d) Detecting the type and / or quantity of migrasomes in the "migrasome-lectin-solid-phase carrier" complex with a detection agent that specifically binds to a migrasome marker.

2. The method according to claim 1, wherein The sample includes: urine sample, serum sample, cell culture medium, saliva, cerebrospinal fluid, tissue dissociation solution, or a combination thereof.

3. The method according to claim 1, characterized in that, The solid-phase carrier includes: solid particles, microfluidic chips, cellulose acetate membranes, nylon membranes, agarose microbeads.

4. The method according to claim 1, characterized in that The surface of the solid-phase carrier is conjugated or bound with lectin.

5. The method according to claim 1, characterized in that In step (b), the sample to be tested is contacted with the solid-phase carrier to form a first mixture, and in step (c), the "migrasome-lectin-solid-phase carrier" complex is separated from the first mixture.

6. The method according to claim 1, characterized in that The detection includes: flow cytometry, fluorescence imaging technology, fluorescence immunoassay technology.

7. The method according to claim 1, wherein The detection agent is labeled with a detectable label.

8. A system for detecting migratory bodies, characterized in that: The system includes: (M1) A sample loading module configured to perform the following operations: Mix the sample to be tested with a solid-phase carrier to form a first mixture, wherein the solid-phase carrier is conjugated or bound with lectin, and when the sample to be tested contains migrasomes, a "migrasome-lectin-solid-phase carrier" complex is formed; (M2) An optional separation module configured to perform the following operations: Separate the "migrasome-lectin-solid-phase carrier" complex from the first mixture; and (M3) A detection module configured to perform the following operations: Detect the type and / or quantity of migrasomes in the "migrasome-lectin-solid-phase carrier" complex with a detection agent that specifically binds to a migrasome marker.

9. The system according to claim 8, wherein The detection module includes: (M3a) A detection agent adding sub-module configured to perform the following operations: Mix the detection agent that specifically binds to a migrasome marker with the "migrasome-lectin-solid-phase carrier" complex to form a "detection agent-migrasome-lectin-solid-phase carrier" quaternary complex; and (M3b) A signal detection sub-module configured to perform the following operations: Detect the specific signal of the "detection agent-migrasome-lectin-solid-phase carrier" quaternary complex.

10. The system according to claim 8, wherein The system further includes: (M4) A data processing module configured to process the detection data from the detection module; and (M5) An output module for outputting the processing result of the data processing module.

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