CN120385818A - Method and system for detecting migratory bodies - Google Patents
Method and system for detecting migratory bodiesInfo
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- CN120385818A CN120385818A CN202210195731.0A CN202210195731A CN120385818A CN 120385818 A CN120385818 A CN 120385818A CN 202210195731 A CN202210195731 A CN 202210195731A CN 120385818 A CN120385818 A CN 120385818A
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Abstract
The invention provides a method and a system for detecting a migration body. The invention provides a method for detecting a migration body, which comprises the steps of (a) providing a sample to be detected, (b) mixing the sample to be detected with a solid carrier to form a migration body-lectin-solid carrier complex, (c) separating the migration body-lectin-solid carrier complex, and (d) detecting the type and/or the quantity of the migration body in the migration body-lectin-solid carrier complex by using a detection agent bound to a migration body specific marker in a targeting manner. The method can conveniently, rapidly, accurately and efficiently detect the migration bodies in various body fluids such as cell culture fluid, urine, blood and the like.
Description
Technical Field
The invention belongs to the field of biological medicine, and particularly relates to a method and a system for detecting a migration body, in particular to a method and a system for detecting the migration body by using a flow cytometer.
Background
The migration body (migrasome) is a single-layer membrane vesicle structure with the diameter of 0.5-2 um, which is generated by a shrinkage wire at the tail of a cell in the cell directional migration process. The mobile body contains a large amount of bioactive substances such as nucleic acid, protein, fat and the like, plays an important role in the intercellular communication process, and participates in and regulates various physiological and pathological activities.
Recent studies have shown that mobile bodies are also present in blood and various body fluids, such as serum, urine, etc. The content of the migration body in some blood or body fluid is closely related to certain diseases (such as diabetic nephropathy and the like), and is expected to become a biomarker of various diseases.
The current methods for specifically detecting the migrates are limited to electron microscopy, western Blotting and other techniques. However, the above-described method cannot be frequently applied to daily scientific research and clinical detection due to various reasons such as complicated operation, inability to quantify, and the like. This not only greatly limits the basic research of the mobile body, but also restricts the clinical application of the mobile body.
Therefore, there is an urgent need in the art to develop a method for detecting a mobile body that is convenient, fast, accurate, and efficient.
Disclosure of Invention
The invention aims to provide a method for conveniently, rapidly, accurately and efficiently detecting a migration body.
In a first aspect of the present invention, there is provided a method of detecting a mobile body, comprising the steps of:
(a) Providing a sample to be tested, wherein the sample to be tested contains a migration body;
(b) Mixing the sample to be tested with a solid carrier to form a first mixture, wherein the solid carrier is coupled or combined with lectin, and when the sample to be tested contains a migration body, a migration body-lectin-solid carrier complex is formed;
(c) Optionally isolating said "mobile-lectin-solid support" complex, and
(D) Detecting the type and/or number of migrates in the "migrate-lectin-solid support" complex with a detection agent that targets binding to a specific marker of the migrates.
In another preferred embodiment, the sample comprises a urine sample, a serum sample, a cell culture fluid, saliva, cerebrospinal fluid, a tissue separation fluid, or a combination thereof.
In another preferred embodiment, the sample comprises blood, plasma, urine.
In another preferred embodiment, the sample is in a liquid state.
In another preferred example, the solid phase carrier comprises solid particles, microfluidic chips, glass cellulose membranes, nylon membranes and agarose microbeads.
In another preferred embodiment, the solid support comprises magnetic beads or non-magnetic microspheres.
In another preferred embodiment, the solid support comprises fluorescent-encoded microspheres (beads), more preferably the microspheres comprise polymeric microspheres.
In another preferred embodiment, the average particle size of the solid support is from 10nm to 100mm, preferably from 0.1 to 1000. Mu.m, more preferably from 1 to 100. Mu.m.
In another preferred embodiment, the surface of the solid support is coupled to or bound to lectin.
In another preferred embodiment, the lectin is coupled to the surface of the solid support through a chemical bond.
In another preferred embodiment, the lectin is selected from the group consisting of Wheat Germ Agglutinin (WGA), canavalia gladiata agglutinin (ConA), peanut agglutinin (PNA), or a combination thereof.
In another preferred embodiment, the lectin carries a first detectable label (detectable label).
In another preferred embodiment, the first detectable label is selected from the group consisting of a fluorophore, a chromophore, or a combination thereof. More preferably, the first detectable label is Alexa594 Or similar fluorescent markers.
In another preferred embodiment, in step (b), the sample to be tested is contacted with a solid support to form a first mixture, and in step (c) the "mobile-lectin-solid support" complex is separated from the first mixture.
In another preferred embodiment, in step (c), said separating comprises separating with a magnetic field.
In another preferred embodiment, in step (d), the detection comprises flow cytometry, fluorescence imaging techniques, fluorescence immunoassay reading techniques.
In another preferred embodiment, in step (d), the detection is performed using a flow cytometer.
In another preferred embodiment, the detection agent carries a second detectable label (detectable label).
In another preferred embodiment, the detectable label is selected from the group consisting of a fluorophore, a chromophore, or a combination thereof.
In another preferred embodiment, the detection agent that targets binding to a specific marker of a mobile body comprises an antibody.
In another preferred embodiment, the antibody is selected from the group consisting of an anti-TSPAN 4 antibody, an anti-Integrin β1 antibody, a PIGK antibody, a NDST1 antibody, a CPQ antibody, a EOGT antibody, or a combination thereof.
In another preferred embodiment, in step (d), it is quantitatively determined whether the mobile in the "mobile-lectin-solid support" complex is a mobile derived from a tubule cell or podocyte.
In another preferred embodiment, the method is a non-diagnostic and non-therapeutic method.
In a second aspect of the present invention, there is provided a system for detecting a mobile body, the system comprising:
(M1) a sample addition module configured to mix a sample to be tested with a solid support to form a first mixture, wherein the solid support is coupled or bound with a lectin and a "transporter-lectin-solid support" complex is formed when a transporter is contained in the sample to be tested;
(M2) an optional separation module configured to separate the "mobile lectin-solid support" complex from the first mixture, and
(M3) a detection module configured to detect the type and/or amount of the mobile agent in the "mobile-lectin-solid support" complex with a detection agent that targets binding to a mobile-specific marker.
In another preferred embodiment, the separation module is further configured to wash the separated "mobile-lectin-solid support" complex.
In another preferred embodiment, the detection module includes:
(M3 a) a detector reagent addition sub-module configured to mix the detector reagent targeted for binding to the transporter-specific marker with a "transporter-lectin-solid support" complex, thereby forming a "detector reagent-transporter-lectin-solid support" quaternary complex, and
(M3 b) a signal detection sub-module configured to detect a characteristic signal of the "detector-transporter-lectin-solid phase carrier" quaternary complex.
In another preferred embodiment, the characteristic signal is derived from a detectable label carried by the detection agent.
In another preferred embodiment, the system further comprises:
(M4) a data processing module configured to process the detection data from the detection module, and
And (M5) an output module for outputting the processing result of the data processing module.
In another preferred embodiment, the data processing module is configured to compare the detection data from the detection module with a predetermined value to give a disease risk result or a disease diagnosis result.
In another preferred embodiment, the disease comprises kidney disease, diabetic nephropathy, igA nephropathy, membranous nephropathy.
In a third aspect of the invention there is provided a lectin-solid support complex for capturing a mobile body in a sample comprising a solid support and a lectin, wherein the solid support is coupled or bound to the lectin;
wherein the lectin is selected from the group consisting of Wheat Germ Agglutinin (WGA), canavalia gladiata agglutinin (ConA), peanut agglutinin (PNA), and combinations thereof.
In another preferred embodiment, the lectin carries a first detectable label (detectable label).
In another preferred embodiment, the first detectable label is selected from the group consisting of a fluorophore, a chromophore, or a combination thereof.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 shows a schematic representation of lectin-coupled magnetic bead capture migrates.
FIG. 2 shows construction of lectin-coupled magnetic beads, (A) fluorescent microscopy of WGA, conA and PNA-coupled magnetic beads. (B) Flow cytometry observed magnetic beads coupled to WGA, conA and PNA.
FIG. 3 flow cytometer detects migration bodies captured by lectin-coupled magnetic beads. (A) Scanning electron microscopy observed the mobility captured by the WGA coupled magnetic beads. (B) The mobility captured by the WGA-coupled magnetic beads was observed by fluorescence microscopy, and the TSPAN4 antibody was stained green. (C) Flow cytometry observed the mobilities captured by the magnetic beads coupled to WGA, conA and PNA.
Figure 4 shows the mobilities captured by using TSPAN4 or Integrin β1 antibodies to label WGA coupled magnetic beads.
FIG. 5 shows detection of WGA-coupled magnetic bead captured mobilities using a flow cytometer to distinguish between migratory-derived cells. (A) western blotting detected the expression of Nephrin and AQP1 proteins by the podocyte HPC and the tubule cell HK 2-derived mobile. (B) Flow cytometry detected podocyte HPC and tubule cell HK 2-derived migratory expressed TSPAN4. (C) Flow cytometry detected podocyte HPC-derived migrates expressed Nephrin and AQP1 proteins. (D) Flow cytometry detected migration-expressed Nephrin and AQP1 proteins from tubular cell HK 2.
Figure 6 shows that flow cytometry detects WGA coupled bead captured migrates exhibiting a dose dependent effect. (A) western blotting detected the content of podocyte HPC-derived migratory protein. (B) western blotting detected the protein content of podocyte HPC-derived migrates PIGK. (C-D) flow cytometry detected podocyte HPC-derived migratory expressed TSPAN4. And C, a flow chart. And D, statistics.
Figure 7 shows that flow cytometry detects WGA coupled bead captured migrates exhibiting a dose dependent effect. (A) western blotting detected the content of urine-derived migratory protein. (B) western blotting detected the protein content of urine-derived migrates PIGK. (C-D) flow cytometry detected urine-derived migratory expressed TSPAN4. And C, a flow chart. And D, statistics.
Figure 8 shows that flow cytometry detects WGA coupled bead captured migrates exhibiting a dose dependent effect. (A) western blotting detects the amount of blood-derived migratory protein. (B-C) flow cytometry to detect blood-derived migratory expressed TSPAN4. And B, a flow chart. And C, counting.
Figure 9 shows urine mobility content assays for Healthy (HV), diabetic Nephropathy (DN), membranous Nephropathy (MN), igA nephropathy and FSGS (focal segmental glomerulosclerosis) patients.
FIG. 10 shows WGA-coupled magnetic beads capture PLA2R on the surface of a sufficient mobile detection mobile. (A-B) western blotting (A) and flow cytometry (B) were used to detect the PLA2R expressed by the migrates. (C) Flow cytometry detects PLA2R expressed by urine migrates from patients with membranous nephropathy.
Fig. 11 shows scanning electron microscopy of TSPAN4 antibody-conjugated magnetic bead captured migrates.
Detailed Description
The present inventors have made extensive and intensive studies, and have unexpectedly developed a method and system for detecting a mobile body with high efficiency, rapidity, and accuracy for the first time through mass screening. The inventor synthesizes a kind of migration body capturing carrier with unique structure, wherein the migration body capturing carrier is a solid phase carrier (such as a microsphere (bead) or magnetic bead) with the surface modified by lectin. Taking the migration body capturing magnetic beads as an example, the migration body capturing carrier can capture migration bodies in a sample efficiently and specifically, can detect migration bodies in the sample in cooperation with flow cytometry and the like, and can detect the types and the quantity of the migration bodies in the sample rapidly, simply, conveniently and accurately. The present invention has been completed on the basis of this finding.
Migration body
The migration body (migrasome) is a single-layer membrane vesicle structure with a diameter of 0.5-2 um generated by the contractile filaments at the tail of the cell during cell directional migration, as described herein. The mobile body contains a large amount of bioactive substances such as nucleic acid, protein, fat and the like, plays an important role in the intercellular communication process, and participates in and regulates various physiological and pathological activities. Mobile bodies are present in blood and various body fluids, such as serum, urine, etc. Some of the mobile body content in blood or body fluids is closely related to certain diseases (e.g., diabetic nephropathy, etc.).
Lectin
As used herein, the term "lectin" is capable of specifically recognizing glycosylation modified proteins. Lectins are a class of proteins capable of binding polysaccharide groups, predominantly distributed in the extracellular matrix. Numerous glycoprotein and polysaccharide groups are distributed in the extracellular matrix, and lectins are able to mediate firm links between cells by binding to polysaccharide groups.
Preferred lectins include plant-derived lectins such as canavalin a (Concocnvalina, conA), wheat germ (WHEAT GERM agglutinin, WGA), peanut lectin (Peanut agglutinin, PNA), and soybean lectin (Soybean agglutinin, SBA), etc., which are capable of binding polysaccharide groups with high efficiency.
In the present invention, a preferred class of lectins are those bearing a first detectable label, in particular a fluorescent label. Compared with antibodies, the lectin with the detectable label (especially fluorescent label) has higher stability and anti-interference performance, and unexpectedly has high specific capturing capability for the migration body, and can more favorably and specifically capture different types of migration bodies in various different samples.
Solid phase carrier
The solid carrier is a composite magnetic bead with the surface embedded by a polymer material, and mainly comprises any one of dextran magnetic beads, agarose magnetic beads, resin or epoxy resin, polystyrene magnetic beads and other composite magnetic beads, or a mixture of two or more magnetic carriers, wherein the particle size distribution range of the solid carrier is 1um-200um, preferably 10um-200um, and more preferably 30um-150um. In the production of the solid phase carriers, the particle size of the solid phase carriers produced in the same batch is not uniform, so that the particle size of the solid phase carriers can be generally described in terms of average particle size or particle size distribution range in the present invention.
Flow cytometry
In scientific research, flow Cytometry (FCM) has the characteristics of simultaneously detecting and analyzing and marking a plurality of molecules in a single sample, and is a high-throughput detection technology for carrying out one-by-one, multi-parameter, rapid, accurate and quantitative analysis and separation on single-row cells or other particles at a functional level. The highest sorting speed has reached 3 ten thousand cells per second. The rapid development of this technology is mainly dependent on the powerful nature of fluorescent dyes, in particular the fact that different fluorescent labels or different intensities of fluorescence can be distinguished in flow cytometry. The unique property suggests that the flow cytometer can be used to detect a plurality of differently labeled targets simultaneously in the same sample, thereby achieving the purpose of rapid and convenient detection. The flow cytometer is mainly used for cell surface labeling detection and cell grouping, other particles are needed to be used as carriers for detecting a certain molecule, and the carriers have to be nano-sized and have good particle size uniformity. At present, the magnetic microsphere is a magnetic microsphere which is mature when applied to a magnetic field auxiliary separation technology, particularly a biological magnetic separation technology, and the magnetic microsphere material has the characteristics of uniform particle size, strong magnetic response, good dispersibility in water and the like. The FCM technology has high accuracy and high flux detection characteristics, can apply the characteristics of FCM such as high speed, high flux, multiple parameters, accurate quantitative analysis and the like to the detection of protein concentration, and simultaneously selects magnetic microsphere materials with nanoscale and uniform particle size as carriers of a flow cytometer to establish the type and quantity of the fluorescent immunomagnetic microsphere combined flow cytometer detection migration body.
Application of
The present invention provides for the use of the inventive carrier for capturing a mobile body, for example for the preparation of biomarkers for a variety of diseases, in particular for the detection or diagnosis of diseases which are related to the content of mobile bodies.
Representative diseases associated with migratory body content include, but are not limited to, kidney disease such as diabetes, membranous kidney disease, igA kidney disease, FSGS (focal segmental glomerulosclerosis) and the like.
In addition, the carrier for capturing the migration body can capture the migration body in the sample efficiently and specifically, and can detect the type and the quantity of the migration body in the sample rapidly, simply, conveniently and accurately by combining with flow cytometry.
In a preferred embodiment, the present inventors constructed lectin-coupled magnetic beads capable of specifically recognizing a surface protein of a mobile body, efficiently captured mobile bodies in various body fluids such as cell culture fluid, urine, blood, etc., and then labeled the mobile bodies by a mobile body-specific antibody, thereby identifying the type and content of the mobile bodies.
The invention has the advantages that:
(a) The invention has simple operation, and can specifically identify the migration bodies in various body fluids such as cell culture fluid, urine, blood and the like by constructing the lectin-coupled magnetic beads capable of specifically identifying the glycosylation modification protein.
(B) The identification and quantitative detection of the properties of the migration body can be realized by labeling the migration body by the migration body specific antibody, and the identification and quantitative detection are beneficial to basic scientific research and clinical application related to the migration body.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedures, which do not address the specific conditions in the examples below, are generally followed by conventional conditions, such as those described in Sambrook et al, molecular cloning, a laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or by the manufacturer's recommendations. Percentages and parts are weight percentages and parts unless otherwise indicated.
Materials and general methods
Fluorescent lectin proteins
WGA:WGA,Alexa594Conjugate, invitrogen, cat# W11262
ConA:ConA,Alexa594Conjugate, invitrogen, cat# C11253
PNA:PNA,Alexa594Conjugate, invitrogen, cat# L32459
Example 1
Preparation of fluorescently labeled lectin
1. Lectin lyophilized powder was weighed and resuspended to 5mg/mL with PBS buffer.
2. Mu.L of the lectin solution resuspended in the first step was taken and 10mg/mL Alexa was added594NHS ester. After being mixed uniformly, the mixture reacts for 1 hour at room temperature in a dark place.
3. The reaction solution of the second step was passed through a gel filtration column (Biospin #732-6008, bioRad) to remove Alexa which was not coupled with lectin594 Fluorescent probes
4. Coupling Alexa to the obtained594 Is stored in a 4-degree refrigerator in a freeze-dried manner in the absence of light.
Example 2
Construction of lectin-coupled magnetic beads
1. NHS magnetic beads (Solarbio, cat. M2450) with particle size of 2 μm were purchased, 500. Mu.L of the magnetic bead suspension was taken in a 1.5mL EP tube, the EP tube was placed in a magnetic separation rack, the beads were enriched, and the supernatant was removed. 1mL of 4℃precooled Washing Buffer A was added to a 1.5mL EP tube, and the mixture was vortexed for 15s to mix the beads uniformly. The EP tube was placed in a magnetic separation rack, the beads were enriched, and the supernatant was removed.
2. Three lectin proteins (WGA, alexa) coupled to Alexa Fluor TM 594 94 red fluorescence594Conjugate, invitrogen, cat# W11262; conA, alexa594Conjugate, invitrogen, cat# C11253, PNA, alexa594Conjugate, invitrogen, cat# L32459) was dissolved in a Coupling Buffer to prepare a protein solution at a concentration of 3.0 mg/mL. To the EP tube of the first step, 500. Mu.L of a lectin protein solution coupled with Alexa Fluor TM 594 94 red fluorescence was added, and vortexed for 30s to mix well. The EP tube was vortexed for 15s and placed on a mixer and mixed for 2h at room temperature. If the mixing is not uniform, the EP tube is removed by vortexing for 15s at 5min intervals for 30min prior to the reaction. Thereafter, the EP tube was removed at 15min intervals by vortexing for 15s.
3. The EP tube was placed in a magnetic separation rack, the beads were enriched, and the supernatant was removed. Add 1mL Blocking Buffer to the EP tube, vortex for 30s, place the EP tube in a magnetic separation rack, enrich the beads, discard the supernatant.
4. The procedure 3 was repeated four times. Add 1mL Blocking Buffer to the EP tube, vortex for 30s and place the EP tube in a mixer for 2h at room temperature. The EP tube was placed in a magnetic separation rack, the beads were enriched, and the supernatant was discarded. Add 1mL of ultrapure water to the EP tube, mix well, enrich the beads with a magnetic rack, discard the supernatant.
5. 1ML of PBS solution (pH 7.2) was added to the EP tube, mixed well, the beads were concentrated with a magnetic rack, and the supernatant was discarded. After repeating this operation 2 times, resuspended in 500. Mu.L of PBS solution, thoroughly mixed and stored at 4℃for further use. The final conjugated protein had a magnetic bead concentration of 10mg/mL.
6. Magnetic beads coupled with red fluorescent-labeled lectin were observed using fluorescence microscopy and flow cytometry.
Results
The results of fluorescence microscopy are shown in FIG. 2A, where three WGA, conA and PNA, red fluorescence labelled, can be linked to the magnetic beads, with WGA being the most effective. From the figure it can be seen that the fluorescent labelled WGA signals most strongly in the flow cytometer. The coupling efficiency of WGA with fluorophores and magnetic beads is higher compared to ConA and PNA.
The results of flow cytometry observations are shown in FIG. 2B for magnetic beads coupled with WGA, conA and PNA, showing that all three lectins can be successfully linked to the bead surface (FIG. 2B).
Example 3
Detection of lectin-coupled magnetic bead-captured mobilities using flow cytometry
1. Human podocyte HPC cell culture broth was collected and centrifuged at 4000g for 20 min at 4℃to remove cell debris. 5mL of the cell culture broth was added to 5. Mu.L of lectin-coupled magnetic beads (2.1-constructed WGA, conA or PNA-coupled magnetic beads) and incubated on a mixer at 4℃for 18h.
2. The EP tube was placed on a magnetic separation rack, the supernatant was removed by magnetic separation, and after washing 3 times with 1000. Mu.L of PBS solution containing 0.1% BSA (pH 7.2), resuspended in 100. Mu.L of PBS solution containing 1% BSA and incubated at room temperature for 30min.
3. The EP tube was then placed in a magnetic separation rack, the beads were enriched, and the supernatant was discarded. Resuspended in 100. Mu.L of PBS containing 0.1% BSA.
4. To the EP tube, 0.5. Mu.L of TSPAN4 antibody (abcam, cat No. ab181995, rabbit source) was added, incubated on a mixer at room temperature for 1h, then the EP tube was placed in a magnetic separation rack, the beads were enriched, and the supernatant was discarded. After washing 3 times with 1000. Mu.L of PBS containing 0.1% BSA, resuspended in 250. Mu.L of PBS containing 0.1% BSA.
5. 0.5 Mu LAlexa Fluor 647-donkey anti-rabit fluorescent secondary antibody was added to the EP tube, placed on a mixer and incubated for 1h at room temperature, then the EP tube was placed in a magnetic separation rack, the magnetic beads were enriched, and the supernatant was discarded. After washing 3 times with 1000. Mu.L of PBS containing 0.1% BSA, the solution was resuspended in 300. Mu. LPBS and examined by fluorescence microscopy and flow cytometry.
Results
The mobility captured by the WGA-coupled magnetic beads was observed using a scanning electron microscope, and the results are shown in fig. 3A, with red arrows showing the mobility captured by the WGA-coupled magnetic beads.
Migration of the WGA-coupled magnetic bead capture was observed with a fluorescence microscope, from which it was seen that the WGA-coupled magnetic bead could capture the migrates, wherein the magnetic bead appeared red, whereas the migration captured by the WGA-coupled magnetic bead could be labeled with a migration surface-specific protein TSPAN4 antibody (green) (green fluorescence) (fig. 3B).
The migration was captured by using magnetic beads coupled to WGA, conA and PNA, respectively, and then the migration surface-specific protein TSPAN4 antibody was labeled (green), and the results of flow cytometry observation are shown in FIG. 3C, in which the magnetic beads coupled to WGA, conA and PNA all captured the migration.
Example 4
Detection of WGA-coupled magnetic bead-captured mobilities using flow cytometry
1. Human podocyte HPC cell culture broth was collected and centrifuged at 4000g for 20min at 4℃to remove cell debris. 5mL of the cell culture broth was added to 5. Mu.L of WGA-conjugated magnetic beads and incubated on a mixer at 4℃for 18h.
2. The next experimental procedure was repeated for steps 2-5 of example 3, wherein an EP tube was added with a solution containing 0.5. Mu.L of TSPAN4 antibody (abcam, cat# ab181995, rabbit) or Integrin. Alpha.5β1 antibody (CST, cat# 4705T, rabbit).
Results
The mobility body was captured by using WGA-coupled magnetic beads, and then the specific protein TSPAN4 or Integrin α5β1 antibody on the surface of the mobility body was labeled, and the results of flow cytometry observation are shown in fig. 4, and the mobility body was captured by using WGA-coupled magnetic beads and flow detection was performed by using specific protein antibodies on the surface of the mobility body.
Example 5
Detection of WGA-coupled magnetic bead captured mobilities using flow cytometry to differentiate migratory-derived cells
1. Human podocyte HPC and tubule cell HK2 cell culture broth were collected and the procedure was identical to step 1 of example 4.
2. The following experimental procedure was repeated for examples 2-5, in example 3, wherein an EP tube was charged with 0.5. Mu.L of TSPAN4 antibody (abcam, cat# ab181995, rabbit source), podocin antibody (sigma, cat# P0372, rabbit source) or AQP1 antibody (abcam, cat# ab168387, rabbit source).
Results
The mobilities from the HPC and HK2 cells were captured using WGA-conjugated magnetic beads, followed by labeling of the mobilities with the TSPAN4 antibody, and detection using flow cytometry using the Nephrin antibody and the AQP1 antibody, which are marker proteins specific to the podocytes. The result of western blotting is shown in FIG. 5A, where podocyte migrates to express Nephrin and tubule cell migrates to express AQP1. The podocyte-derived migrates only detected podocyte-specific marker protein Nephrin, while the tubule-cell-derived migrates only detected the tubule cell-specific protein AQP1 (fig. 5B-C).
Example 6
Flow cytometry detection of WGA-coupled magnetic bead-captured mobilities exhibited dose-dependent effects
1. Collecting human podocyte HPC cell culture solution, centrifuging for 20min at 4000g at 4 ℃ to remove cell fragments, discarding the precipitate to recover supernatant, centrifuging for 30min at 20000g at 4 ℃, and obtaining the precipitate as a crude extract. The pellet was then resuspended in PBS and the protein concentration was detected using BCA kit. And (3) carrying out gradient dilution according to the protein concentration, preparing migration body weight suspensions with different concentration gradients, and detecting the number of migration body particles by utilizing Nano-sight.
2. The final volume of the mobile sample after gradient dilution was 100. Mu.L, 5. Mu.L of WGA-conjugated magnetic beads were added and incubated on a mixer for 18h at 4 ℃.
3. The following experimental procedure was repeated for examples 2-5 in example 3.
Results
The podocyte HPC-derived migrates were isolated, subjected to gradient dilution, and then assayed for protein concentration, which was found to vary with the amount of the migrates (FIG. 6A), and then the content of the migration-specific protein MAKER PIGK was assayed by western blotting, as shown in FIG. 6B, and the PIGK content varied with the amount of the migrates. The levels of TSPAN4 at different concentrations were measured by WGA coupled magnetic beads using a flow cytometer and as a result it was found that the TSPAN4 levels increased with increasing amounts of the mobile and exhibited positive correlation with protein levels (fig. 6C-D). However, when the amount of mobile addition was saturated, it could affect flow cytometry detection (FIGS. 6C-D). The above results indicate that the migration bodies can be captured by WGA-coupled magnetic beads and quantified in the cell culture broth by flow cytometry.
Example 7
Flow cytometry detects migratory bodies in WGA-coupled magnetic beads-captured urine and exhibits a dose-dependent effect
1. Collecting urine of a patient with membranous nephropathy, centrifuging for 20 minutes at the temperature of 4000g at 4 ℃ to remove cell fragments, discarding the precipitate to recover supernatant, centrifuging for 30 minutes at the temperature of 20000g at 4 ℃, and obtaining the precipitate as a crude extracted mobile body. The pellet was then resuspended in PBS and the protein concentration was detected using BCA kit. And (3) carrying out gradient dilution according to the protein concentration to prepare migration weight suspensions with different concentration gradients.
2. Step 2 in experimental example 6 was repeated.
3. Steps 2-5 of example 3 were repeated.
Results
The mobile bodies in urine were separated, subjected to gradient dilution, and then the protein concentration was measured, and the protein concentration was found to vary with the amount of the mobile bodies (fig. 7A). The TSPAN4 content of the different concentrations was examined by WGA-coupled magnetic beads using a flow cytometer, and the results showed that the TSPAN4 content increased with increasing amounts of the mobile and exhibited positive correlation with the protein content (fig. 7B, 7C). The above results indicate that the mobility bodies can be captured by WGA-coupled magnetic beads and quantified in urine using a flow cytometer.
Example 8
Flow cytometry detects migratory bodies in WGA-coupled magnetic bead capture serum and exhibits a dose-dependent effect
Example 7 was repeated except that the urine sample of the patient with membranous nephropathy in example 7 was replaced with the serum of the patient with membranous nephropathy as the sample to be measured.
Results
The migrates in serum were separated, subjected to gradient dilution, and then the protein concentration was measured, and the protein concentration was found to vary with the amount of the migrates (FIG. 8A). The level of the migration specific protein MAKER PIGK was then measured using western blotting, as shown in FIG. 8B, and PIGK levels varied with the amount of the migration. The TSPAN4 content of the different concentrations was examined by WGA coupled magnetic beads using a flow cytometer and the results showed that the TSPAN4 content increased with increasing amounts of the mobile and exhibited a positive correlation with the protein content (fig. 8C-D). The above results indicate that the migration bodies were captured by WGA-coupled magnetic beads and quantified in blood using a flow cytometer.
Example 9
WGA-coupled magnetic beads capture migration bodies in urine as markers of kidney injury
Urine from healthy persons, diabetic nephropathy, membranous nephropathy, igA nephropathy and FSGS (focal segmental glomerulosclerosis) patients was collected, and the experiment in example 3 was repeated.
Results
Urine migrates from Healthy (HV), diabetic Nephropathy (DN), membranous Nephropathy (MN), igA nephropathy and FSGS (focal segmental glomerulosclerosis) patients were isolated, and as shown in fig. 9, HV urine was substantially free of migrates, while urine from Diabetic Nephropathy (DN), membranous Nephropathy (MN), igA nephropathy and FSGS (focal segmental glomerulosclerosis) patients was rich in migrates.
Example 10
WGA-coupled magnetic beads capture of mobile detection of PLA2R on the surface of mobile in urine
1. The serum and corresponding urine from patients with membranous nephropathy were collected and steps 1-3 of example 3 were repeated.
2. To the EP tube, 0.5. Mu.L of TSPAN4 antibody (abcam, cat No. ab181995, rabbit) and 20% PBS (containing 0.1% BSA) corresponding to the patient's serum were added, incubated on a mixer at room temperature for 1h, then the EP tube was placed in a magnetic separation rack, the magnetic beads were enriched, and the supernatant was discarded. After washing 3 times with 1000. Mu.L of PBS containing 0.1% BSA, resuspended in 250. Mu.L of PBS containing 0.1% BSA.
3. 0.5 Mu LAlexa Fluor 647-donkey anti-rabit fluorescent secondary antibody and FITC-Goat Anti-Human IgG antibody were added to the EP tube, incubated on a mixer at room temperature for 1h, then the EP tube was placed in a magnetic separation rack, the magnetic beads were enriched, and the supernatant was discarded. After washing 3 times with 1000. Mu.L of PBS containing 0.1% BSA, the mixture was resuspended in 300. Mu.L of PBS and examined by fluorescence microscopy and flow cytometry.
Results
Podocyte HPC cell culture broth was collected, and the migrates and exosomes were separated, and podocyte proteins were detected by western blotting, which showed that the surface of the migrates expressed rich PLA2R (FIG. 10A).
The results of the flow cytometer are shown in fig. 10B, again confirming that the surface of the mobile body expressed PLA2R.
Three pairs of membranous nephropathy samples were then obtained, 1) PLA2R autoantibodies detected blood negative, renal puncture test negative, 2) PLA2R autoantibodies detected blood negative, renal puncture test positive, 3) PLA2R autoantibodies detected blood positive, renal puncture test positive.
As shown in FIG. 10C, the result of detecting the migrates in urine by using a flow cytometer is that the migrates secreted by the foot cells of the patient with negative serum and tissue are still negative, the migrates secreted by the foot cells of the patient with negative serum and positive tissue are still positive, and the migrates secreted by the foot cells of the patient with positive tissue are still positive. From this, it is clear that the mobile body in urine can be used as a substitute for kidney tissue for PLA2R detection.
Comparative example C1
Detection of TSPAN4 antibody-conjugated magnetic bead captured mobilities using flow cytometry
1. Using commercially available NHS magnetic beads (Solarbio, cat. M2450) with a particle size of 2. Mu.m, 100. Mu.L of the magnetic bead suspension was placed in a 1.5mL EP tube, the EP tube was placed in a magnetic separation rack, the beads were enriched, and the supernatant was removed. 200. Mu.L of 4℃precooled Washing Buffer A was added to a 1.5mL EP tube, and the mixture was vortexed for 15s to mix the beads uniformly. The EP tube was placed in a magnetic separation rack, the beads were enriched, and the supernatant was removed.
2. 100 Mu LTSPAN antibody (abcam, cat. No. ab 181995) was added to the EP tube containing the magnetic beads and vortexed for 30s to mix well. The EP tube was vortexed for 15s and placed on a mixer and mixed for 2h at room temperature. If the mixing is not uniform, the EP tube is removed by vortexing for 15s at 5min intervals for 30min prior to the reaction. Thereafter, the EP tube was removed at 15min intervals by vortexing for 15s.
3. The EP tube was placed in a magnetic separation rack, the beads were enriched, and the supernatant was removed. 200 mu LBlocking Buffer was added to the EP tube, vortexed for 30s, the EP tube was placed in a magnetic separation rack, the beads were enriched, and the supernatant was discarded.
4. The procedure 3 was repeated four times. 200 mu L Blocking Buffer was added to the EP tube, vortexed for 30s, and the EP tube was placed in a mixer and reacted at room temperature for 2h. The EP tube was placed in a magnetic separation rack, the beads were enriched, and the supernatant was discarded. 200. Mu.L of ultrapure water was added to the EP tube, thoroughly mixed, the beads were concentrated with a magnetic rack, and the supernatant was discarded.
5. 200 Mu LPBS of solution (pH 7.2) was added to the EP tube, mixed well, the beads were enriched with a magnetic rack, and the supernatant was discarded. This procedure was repeated 2 times, resuspended in 200. Mu. LPBS solution, thoroughly mixed and stored at 4℃for further use. The final conjugated protein had a magnetic bead concentration of 10mg/mL.
6. The method for detecting whether or not the TSPAN4 antibody-conjugated magnetic beads captured the mobile bodies was consistent with example 3, and detection was performed using a scanning electron microscope and a flow cytometer.
Results
The migration bodies captured by the TSPAN4 antibody-conjugated magnetic beads were observed by a scanning electron microscope, and the results are shown in fig. 11, with red arrows showing the migration bodies captured by the TSPAN4 antibody-conjugated magnetic beads.
The migrates were captured using TSPAN4 antibody-conjugated magnetic beads, followed by labeling of the migrate surface specific protein Integrin β1 antibody and flow cytometry observations. The results indicate that although beads conjugated with TSPAN4 antibodies can capture the mobile, there is a large difference between different batches or different sources of antibodies.
The above results indicate that the use of a magnetic bead-captured transporter coupled to a TSPAN4 antibody has the following disadvantages compared to the capture of a transporter with a magnetic bead coupled to a lectin:
1) The lectin is obtained by separating and purifying natural products such as wheat germ, and the antibody is obtained by separating and purifying animals such as immunized mice by synthesizing specific antigens. Therefore, compared with antibodies, the lectin is not only far less in cost, but also easier to control quality, and the purity, the property and the effect of each batch of lectin can be ensured to be consistent. Given the complexity of the antibody generation, isolation and purification processes, the consistency of each batch of antibodies is relatively difficult to maintain consistent, which can result in differences in efficiency and specificity of capturing the migrates from batch to batch of different antibodies when capturing the migrates using TSPAN4 antibody-conjugated magnetic beads.
2) Compared with lectin, the magnetic beads coupled with TSPAN4 antibody have stronger dependence on the structure of the antibody when capturing the migration body, and the antibody coupled with the magnetic beads can be possibly combined with specific proteins on the surface of the migration body only when exposing the light chain, so that the migration body is adsorbed. However, in the process of coupling the antibody to the magnetic beads, it is difficult to ensure the direction of linking, which not only results in a reduction of the antibody that effectively captures the mobile body and causes waste, but also it is difficult to ensure uniformity of the antibody content of the effective mobile body capture that can be obtained when each batch of antibody is coupled to the magnetic beads, which also makes the magnetic beads prepared per batch have a difference in efficiency and specificity of mobile body capture when the magnetic beads coupled to the TSPAN4 antibody capture the mobile body. In contrast, lectin binding to the migrates is not strongly dependent on lectin structure, and therefore, lectin-coupled magnetic beads prepared per batch can be ensured to remain consistent in efficiency and specificity of capturing the migrates.
Discussion of the invention
The migration body is gradually developing as a new newly discovered new organelle into a new field of cell biology. Contents such as proteins and RNAs contained in the mobile body can be taken up by the recipient cells to function in the recipient cells. Recent researches show that the migration body also exists in body fluids such as blood, urine and the like, and the migration body possibly can also serve as a carrier for long-distance signal transmission and plays a role in the interactive regulation and control process between different tissues/organs.
Based on the mechanism of production and inclusion of the migratory body, the migratory body is thought to play a very important role in development, immune response, tumor metastasis, etc. However, due to limitations of the current techniques for separation, purification and detection of the mobile body, research on mechanisms and application related to the mobile body are greatly limited.
The detection method and the detection system of the migration body based on the biochemical property development of the migration body can be used for separating and purifying the migration body, so that the comprehensive exploration of the types and the quantity of the protein, RNA and other content species contained in the migration body is possible, the detection method and the detection system of the migration body can also be used for measuring the content of the migration body, and the content of the migration body in body fluid under different physiological and pathological states or the quantity of the migration body released by cells under different states can be explored.
The method greatly simplifies the steps of separation, purification and detection of the migration body, not only can provide a convenient means for mechanism research of the migration body and comprehensively reveal the generation, regulation and function exertion process of the migration body by assistance, but also enables the migration body to go to clinical research from laboratory research and become possible for disease diagnosis and treatment.
All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.
Claims (10)
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