Test strips, kits and methods for detecting exosomes

Abstract

The application provides an exosome detection and classification test strip, which comprises a sample pad, a chromatography membrane, a water absorption pad and a base plate; the chromatography membrane is coated with an optional quality control line and a plurality of detection lines, each detection line is coated with at least two different functional proteins, one of the at least two different functional proteins is an exosome capture protein, and the other is an enzyme; and the exosome capture proteins on each detection line are different. The application also provides a kit comprising the test strip and a method for detecting and classifying exosomes by using the test strip or the kit. The method of the application is simple to operate and does not require special instruments, thereby meeting the demand for instant detection; meanwhile, the method of the application can not only detect the presence or absence of exosomes, but also distinguish a plurality of subtypes of exosomes.

Description

Technical Field

[0001] This invention relates to the field of detection. Specifically, it relates to test strips, kits, and detection methods for detecting exosomes. Background Technology

[0002] Exosomes are vesicles secreted by cells, ranging in size from 30 nm to 150 nm. Their surface contains abundant tetraspan membrane proteins such as CD9, CD63, and CD81, which are involved in exosome formation. Exosomes contain a wealth of information about their parent cells, including proteins, nucleic acids, transcription factors, and cell surface receptors, thus making them a novel biomarker. However, the number of exosomes is dynamic and highly variable. The proportions of different exosome subtypes vary between normal individuals and patients, between different normal individuals, and even within the same individual at different times. Therefore, suitable methods for detecting and classifying exosomes are needed.

[0003] Currently, mainstream methods for quantifying exosomes include Nanosight nanoparticle tracking analysis (NTA), tunable resistance pulse sensing (TRPS), high-resolution flow cytometry, and electron microscopy. However, these methods either require expensive equipment, produce results that deviate from actual conditions, or cannot distinguish between exosome subtypes.

[0004] System Biosciences has developed exosome detection kits based on enzyme-linked immunosorbent assay (ELISA). However, these kits require prior isolation and purification of exosomes using other methods; if multiple indicators of exosomes need to be detected, different kits need to be used for multiple experiments; and the above kits also require the use of an ELISA reader for detection, which cannot meet the needs of point-of-care testing (POCT).

[0005] Patent application CN113567668A discloses the preparation and application of a fluorescent immunochromatographic test strip for exosome quantification. However, this method still requires a fluorescent immunoassay analyzer; it also requires the use of fluorescent microspheres for detection; furthermore, because there are many antigens on the surface of exosomes, this method of exosome quantification may result in missed detections; finally, this method can only detect one subtype of exosomes and cannot reflect the overall results that include multiple exosome subtypes.

[0006] Therefore, there is an urgent need in this field for an exosome detection method that is simple to operate, low in cost, and capable of classifying exosome subtypes while detecting the presence or absence of exosomes. Summary of the Invention

[0007] The purpose of this invention is to provide a test strip for exosome detection, which can be easily operated to detect exosomes and can also classify exosome subtypes.

[0008] Another object of the present invention is to provide a kit for exosome detection.

[0009] Another object of the present invention is to provide a method for detecting exosomes in a sample using the test strip or kit.

[0010] In a first aspect, the present invention provides an exosome detection and classification test strip, the test strip comprising a sample pad, a chromatography membrane, an absorbent pad, and a base plate;

[0011] The chromatography membrane is coated with an optional control line and multiple detection lines. Each detection line is coated with at least two proteins with different functions. One of the proteins with different functions is an exosome capture protein, preferably a specific antibody against an exosome antigen; the other is an enzyme, preferably an enzyme that catalyzes the color development of a substrate. The exosome capture protein coated on each detection line is different.

[0012] In a specific embodiment, the enzyme is horseradish peroxidase, alkaline phosphatase, catalase, galactosidase, or glucose oxidase; catalase is preferred.

[0013] In a specific embodiment, the specific antibody against the exosome antigen is an antibody specifically targeting CD9 on exosomes, a specific antibody targeting CD63, a specific antibody targeting CD81, an HSPA8 / Hsc70 antibody, an HSP90AA1 / Hsp90α antibody, an HSP90AB1 / Hsp90β antibody, an ALIX antibody, an FLOT2 antibody, an FLOT1 antibody, an HSPA4 / Hsp70 antibody, an ACTB antibody, a RAP1B antibody, a Tim4 antibody, a Moesin antibody, an A2M antibody, a Gelsolin antibody, a B2M antibody, a Filamin A antibody, etc.; preferably, it is an antibody specifically targeting CD9 on exosomes, a specific antibody targeting CD63, or a specific antibody targeting CD81.

[0014] In a preferred embodiment, the detection lines may be three, fewer than three, or more than three.

[0015] In a specific embodiment, the enzyme may also be present on the chromatographic membrane at other locations outside the detection line.

[0016] In a specific embodiment, the sample pad partially overlaps with the chromatography membrane, the chromatography membrane partially overlaps with the absorbent pad, and the sample pad and absorbent pad do not overlap. The sample pad, chromatography membrane, and absorbent pad are placed on the base plate.

[0017] In a preferred embodiment, the spacing between the plurality of detection lines is 4-8 mm, preferably 6 mm.

[0018] In a preferred embodiment, the exosome detection and classification test strip is used to detect the presence or absence of exosomes and to classify exosome subtypes.

[0019] In a second aspect, the present invention provides an exosome detection and classification kit, the kit comprising the exosome detection and classification test strips described in the first aspect, and instructions for use of the test strips to detect and classify exosomes in a sample.

[0020] In a preferred embodiment, the exosome detection and classification test strip is placed in a card box, which consists of a lower cover and an upper cover. The upper cover is provided with a sample application window and a card reading window. The exosome detection and classification test strip is placed in the lower cover. The sample application window and the card reading window correspond to the sample pad, control line, and detection line on the exosome detection and classification test strip, respectively.

[0021] In a specific embodiment, the kit further includes a chromogenic substrate corresponding to the enzyme coated on the test strip and an exosome separation column.

[0022] In a preferred embodiment, the chromogenic substrate is a solution containing a substance that is not chromogenic or has a light color on its own, but will produce color and / or darken and / or change color after being catalyzed by an enzyme.

[0023] In a preferred embodiment, the chromogenic substrate is lipid-soluble.

[0024] In a specific embodiment, the chromogenic substrate is a solution of 3,3',5,5'-tetramethylbenzidine (TMB) and / or diaminobenzidine (DAB) and / or 3-amino-9-ethylcarbazole (AEC) and / or 2,2'-hydrazine-bis-[3-ethylbenzothiazoline-6-sulfonic acid]-diamine salt (ABST) and / or o-phenylenediamine dihydrochloride (OPD) and / or p-nitrophenyl phosphate (PNPP) and / or o-nitrophenyl-β-D-galactopyranoside (ONPG).

[0025] In a specific implementation, the kit also includes auxiliary colorimetric reagents and / or eluents.

[0026] In a specific embodiment, the auxiliary colorimetric reagent is a reagent that assists in the color development of the colorimetric substrate; preferably, the auxiliary colorimetric reagent is a reagent that assists in the color development of the colorimetric substrate bound to exosomes; more preferably, the auxiliary colorimetric reagent is a solution containing peroxides, such as a solution of hydrogen peroxide (H2O2).

[0027] In a specific embodiment, the eluent is an isotonic solution; preferably physiological saline (0.9% NaCl solution), 5% glucose solution, phosphate buffer (PBS buffer), or tris(hydroxymethyl)aminomethane buffer (Tris buffer).

[0028] In a third aspect, the present invention provides a method for preparing the exosome detection and classification test strip described in the first aspect, comprising the following steps:

[0029] (1) Coating of the chromatographic membrane: The chromatographic membrane undergoes at least two rounds of coating; in the first round, a membrane scrubbing apparatus is used to coat multiple exosome capture proteins onto multiple detection lines of the chromatographic membrane; in the second round, an enzyme is used to coat the enzyme onto the aforementioned multiple detection lines of the chromatographic membrane, so that each detection line contains both exosome capture proteins and enzymes; optionally, the enzyme may also be coated on other areas of the chromatographic membrane that are not detection lines while being coated on the detection lines.

[0030] (2) Assembly of test strips: The sample pad, the chromatography membrane prepared in step (1) and the absorbent pad are attached to the base plate one after another to obtain the assembled test strips;

[0031] (3) Cutting the test strip: Cut the assembled test strip after step (2) into test strips with a width of 6-8mm, preferably 6mm, to obtain the exosome detection and classification test strip.

[0032] In a preferred embodiment, in step (1), the concentration of the exosome capture protein coated on the chromatography membrane is 0.6-1.5 mg / mL, preferably 1 mg / mL; and the concentration of the enzyme coated on the chromatography membrane is 0.1-1.5 mg / mL, preferably 0.6 mg / mL.

[0033] In a preferred embodiment, in step (1), the spacing between the plurality of detection lines is 4-8 mm, preferably 6 mm.

[0034] In a fourth aspect, the present invention provides the use of the test strip described in the first aspect or the reagent kit described in the second aspect in the detection and classification of exosomes.

[0035] In a preferred embodiment, the use is for non-diagnostic purposes.

[0036] In a fifth aspect, the present invention provides a method for detecting and classifying exosomes using the kit described in the second aspect, comprising one or more of the following steps:

[0037] S1. Separate exosomes from the sample using the exosome separation column in the kit, and elute the exosomes using the eluent in the kit.

[0038] S2. Co-incubate the isolated exosomes with the chromogenic substrate in the kit.

[0039] S3. Purify the exosomes after incubation with the chromogenic substrate using the exosome separation column in the kit to remove the free chromogenic substrate, and elute the exosomes after incubation with the chromogenic substrate using the eluent in the kit.

[0040] S4. Mix the exosomes incubated with the chromogenic substrate with the auxiliary chromogenic reagent in the kit.

[0041] S5. Add the mixed solution to the exosome detection, classification test strip or test card in the kit for detection.

[0042] In a preferred embodiment, the method is used for non-diagnostic purposes.

[0043] In a specific implementation, the method further includes judging the detection results according to the following criteria:

[0044] 1) If both the control line and the test line show color, and the test line shows clear color, then the sample contains exosomes;

[0045] 2) If the control line shows color but the test line does not, the sample does not contain exosomes;

[0046] 3) If the control line or one or more of the test lines show color, it indicates that the sample contains the exosome subtype corresponding to the antibody;

[0047] 4) If the control line does not develop color, the detection and classification kit is invalid.

[0048] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described 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 described in detail here. Attached Figure Description

[0049] Figure 1 The operating procedure of the exosome detection and classification kit of the present invention is shown;

[0050] Figure 2 This is a structural description of the exosome test strip of the present invention;

[0051] Figures 3A-3H The diagrams showing the test results for samples 1 through 8 are displayed respectively. Detailed Implementation

[0052] Through research, the inventors have developed a novel exosome detection and classification kit. This kit enables the complete process of exosome isolation, purification, and detection. It not only determines the presence of exosomes in a sample but also allows for further classification of exosome subtypes. The kit utilizes enzyme catalysis to develop colorimetric properties on the substrate of the exosome, eliminating the need for fluorescence detection, fluorescent microspheres, or instruments. Detection results can be visually interpreted, making it more intuitive and convenient. This kit meets the needs of ordinary consumers, laboratories in remote areas, and those requiring point-of-care testing (POCT) in emergency situations. This invention is based on these findings.

[0053] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed invention pertains. For ease of understanding, the relevant terms involved in this invention are defined as follows, but the scope of this invention is not limited to these specific definitions.

[0054] Exosomes and the detection and classification test strips of the present invention

[0055] The term "exosome" as used in this article has the meaning conventionally understood by those skilled in the art: it refers to nanoscale (30-150 nm) lipid membrane vesicles originating from multivesicular bodies (MVBs), containing various bioactive molecules such as proteins, lipids, and nucleic acids. All cultured cell types can secrete exosomes, and exosomes are naturally present in body fluids, including blood, saliva, urine, cerebrospinal fluid, and breast milk. Exosomes are secreted by most cells in the body and participate in intercellular communication. Currently, research on exosomes is increasing, and they have significant implications for liquid biopsy.

[0056] To detect and classify exosomes, this invention provides an exosome detection and classification test strip, which includes a sample pad, a chromatography membrane, an absorbent pad, and a base plate. The chromatography membrane is coated with a control line and a detection line. The detection line is coated with at least two proteins with different functions, one of which is an exosome capture protein; the other is used to detect the captured exosomes.

[0057] Based on the teachings of this invention, those skilled in the art will understand that the exosome capturing proteins described herein are proteins used to capture exosomes. Those skilled in the art will also be aware of various forms of proteins used to capture exosomes. For example, the exosome capturing protein can be various specific antibodies against exosome antigens, including but not limited to specific antibodies against CD9, CD63, or CD81 on exosomes. Based on the teachings of this invention, those skilled in the art will also understand that the protein used to detect the captured exosomes can be various enzymes, such as enzymes that catalyze substrate color development, including but not limited to horseradish peroxidase, alkaline phosphatase, catalase, galactosidase, and glucose oxidase; preferably catalase. Those skilled in the art will understand that, in addition to being coated on the detection line, the enzyme can also be coated at other locations on the chromatographic membrane outside the detection line, but it must be present on the detection line.

[0058] To classify exosomes in a sample, the chromatography membrane can be coated with multiple detection lines. Each detection line is coated with a different exosome-capturing protein, thereby capturing exosomes with different antigens. The captured exosomes can then be classified by detecting the proteins in the captured exosomes.

[0059] Therefore, in a preferred embodiment, the exosome detection and classification test strip of the present invention has three detection lines, each detection line being coated with a specific antibody against CD9, CD63, or CD81 on the exosome. The enzymes coated on each detection line may be the same or different, but preferably the same.

[0060] Based on the teachings of this invention and common knowledge in the field, those skilled in the art will know how to assemble the exosome detection and classification test strip of this invention. In a specific embodiment, in the exosome detection and classification test strip of this invention, the sample pad partially overlaps with the chromatography membrane, the chromatography membrane partially overlaps with the absorbent pad, and the sample pad and absorbent pad do not overlap. The sample pad, chromatography membrane, and absorbent pad are placed on a base plate. The spacing between the detection lines can be 4-8 mm, preferably 6 mm.

[0061] Based on common knowledge in the art, those skilled in the art will know that the exosome detection and classification test strip of the present invention may also include a control line. In a specific embodiment, the control line may be set on the chromatographic membrane, near the absorbent pad, to ensure that the test strip or the colloidal gold reagent itself has no quality problems.

[0062] Based on common knowledge in the art, those skilled in the art know how to set up a control line and its specific components. In a specific embodiment, similar to the test line, the control line is coated with an enzyme and a protein highly expressed in vivo. The term "protein highly expressed in vivo" includes, but is not limited to, proteins expressed by housekeeping genes. In a specific embodiment, the term "protein highly expressed in vivo" refers to GAPDH protein, β-actin protein, and ACTB protein.

[0063] The detection and classification kit of the present invention

[0064] Based on the test strip of the present invention, the present invention further provides an exosome detection and classification kit, the kit comprising the exosome detection and classification test strip of the present invention and instructions for use of the test strip to detect and classify exosomes in a sample.

[0065] For ease of use, the exosome detection and classification test strip can be placed in a card box, which consists of a lower cover and an upper cover. The upper cover has a sample application window and a card reading window. The exosome detection and classification test strip is placed in the lower cover. The sample application window and the card reading window correspond to the sample pad, control line, and test line on the exosome detection and classification test strip, respectively.

[0066] To isolate exosomes from samples, the kit of the present invention may further include an exosome separation column. Simultaneously, to enable the detection of exosomes, the kit of the present invention also includes a chromogenic substrate corresponding to the enzyme coated on the test strip. The chromogenic substrate may be a solution containing substances that are not colorless or have a light color on their own, but produce color and / or darken and / or change color upon enzyme catalysis. The chromogenic substrate is lipid-soluble. For example, the chromogenic substrate may be a solution of 3,3',5,5'-tetramethylbenzidine (TMB) and / or diaminobenzidine (DAB) and / or 3-amino-9-ethylcarbazole (AEC) and / or 2,2'-hydrazine-bis-[3-ethylbenzothiazoline-6-sulfonic acid]-diamine (ABST) and / or o-phenylenediamine dihydrochloride (OPD) and / or p-nitrophenyl phosphate (PNPP) and / or o-nitrophenyl-β-D-galactopyranoside (ONPG).

[0067] In a preferred embodiment, the test strip in the kit of the present invention is coated with catalase, and the chromogenic substrate may be 3,3',5,5'-tetramethylbenzidine (TMB).

[0068] Furthermore, the kit of the present invention may also include auxiliary chromogenic reagents and / or eluents to enhance the chromogenic effect of the substrate and elute exosomes from the exosome separation column. In a specific embodiment, the auxiliary chromogenic reagent is a solution containing hydrogen peroxide (H2O2); the eluent is an isotonic solution, preferably physiological saline (0.9% NaCl solution), 5% glucose solution, phosphate buffer (PBS buffer), or tris(hydroxymethyl)aminomethane buffer (Tris buffer).

[0069] Based on the above description of the test strips and kits of the present invention, those skilled in the art will understand that, unlike other test strips or kits in the prior art, the exosome test strips of the present invention do not require a binding pad; to achieve the detection and classification of exosomes, the present invention does not require the use of fluorescent microspheres for color development; the exosome test strips of the present invention utilize enzyme catalysis of the chromogenic substrate on the exosomes for color development, without the use of colloidal gold for color development.

[0070] Preparation of the test strips and reagent kits of the present invention

[0071] Based on the teachings of this invention, those skilled in the art will know how to prepare the test strips and reagent kits of this invention. In a specific embodiment, the test strips of this invention are prepared through the following steps:

[0072] (1) In at least two rounds of coating, a membrane scrubbing apparatus is used to coat multiple exosome capture proteins and enzymes onto multiple detection lines of the chromatography membrane, so that each detection line contains both exosome capture proteins and enzymes; optionally, the enzymes can be coated on other areas of the chromatography membrane that are not detection lines while being coated on the detection lines.

[0073] (2) The sample pad, the chromatography membrane prepared in step (1) and the absorbent pad are attached to the base plate one after another to obtain the assembled test strip;

[0074] (3) Cut the assembled test strips processed in step (2) into test strips.

[0075] In a specific embodiment, the width of the test strip is 6-8 mm, preferably 6 mm; the concentration of the exosome capture protein coated on the chromatography membrane is 0.6-1.5 mg / mL, preferably 1 mg / mL; and the concentration of the enzyme coated on the chromatography membrane is 0.1-1.5 mg / mL, preferably 0.6 mg / mL.

[0076] Those skilled in the art can determine the spacing between the detection lines as needed, but 4-8 mm is preferred, and 6 mm is more preferred.

[0077] Detection and classification of exosomes

[0078] Using the detection and classification test strips and kits of the present invention, those skilled in the art will know how to detect and classify exosomes. In a specific embodiment, exosomes are first isolated from the sample, then co-incubated with a chromogenic substrate, followed by purification of the incubated exosomes, and then dropped onto the exosome detection test strip along with an auxiliary chromogenic reagent. After chromatography on the test strip's chromogenic membrane, the exosome capture antibody on the detection line captures the exosomes, and finally, the enzyme on the detection line catalyzes color development to display the detected exosomes.

[0079] After performing the above-described detection on exosomes in the sample, a further judgment can be made based on whether or not color development occurs. In a specific implementation, the detection results are judged according to the following criteria:

[0080] 1) If both the control line and the test line show color, and the test line shows clear color, then the sample contains exosomes;

[0081] 2) If the control line and test line do not show color, the sample does not contain exosomes;

[0082] 3) If the control line or one or more of the test lines show color, it indicates that the sample contains the exosome subtype corresponding to the antibody.

[0083] 4) If the control line does not develop color, the detection and classification kit is invalid.

[0084] Based on the teachings of this invention and common knowledge in the field, those skilled in the art will understand that the method for detecting and classifying exosomes of this invention can be used for diagnostic purposes, but it can also be used for non-diagnostic purposes, such as scientific research and drug screening.

[0085] The advantages of this invention include:

[0086] 1. This invention develops an exosome detection technology based on test strips, which eliminates the need for instruments in exosome detection, making it more intuitive and convenient, and can meet the needs of point-of-care testing (POCT);

[0087] 2. This invention utilizes enzyme-catalyzed colorimetric methods for the detection of exosomes, overcoming the limitations of exosome size and the requirement of maintaining a high concentration of colloidal gold on the detection line for colloidal gold colorimetric methods. It also eliminates the need for fluorescent microspheres, allowing for direct visual interpretation of the results.

[0088] 3. The exosome detection and classification kit of the present invention can not only detect the presence or absence of exosomes, but also distinguish multiple subtypes of exosomes simultaneously using multiple indicators.

[0089] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2001), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

[0090] Example

[0091] The materials used in the following examples are all commercially available. Specifically,

[0092] CD9 antibody, CD63 antibody and CD81 antibody were all purchased from Abogen (Shanghai) Trading Co., Ltd.

[0093] The chromatography membrane was purchased from Whatman. TM The product number is 3001-861;

[0094] Sample pads, absorbent pads, and base plates were purchased from Shanghai Jinbiao Biotechnology Co., Ltd.

[0095] The exosome separation column is IZON's qEV original columns (#SP1, Izon Science).

[0096] Example 1

[0097] This embodiment describes the preparation method of the exosome detection test strip of the present invention.

[0098] The preparation of the exosome test strip of the present invention is as follows: Figure 2 As shown, the exosome test strip of this embodiment includes a sample pad, a chromatography membrane, and an absorbent pad sequentially attached to a base plate; the sample pad is a glass cellulose membrane; the chromatography membrane is chromatographic chromatography paper, preferably Whatman paper. TM Grade 1 Chr Cellulose Chromatography Paper; the chromatography membrane is sequentially coated with one control line and three detection lines, the control line and the three detection lines being GAPDH antibody, CD9 antibody, CD63 antibody and CD81 antibody, respectively, and the control line and the three detection lines are also coated with HRP enzyme; the absorbent pad is absorbent paper.

[0099] The interval between each detection line is 6 mm, and the width of the exosome test strip is 6 mm. The sample pad overlaps with the chromatography membrane by 2 mm, and the chromatography membrane overlaps with the absorbent pad by 2 mm.

[0100] The preparation method of the above-mentioned exosome test strip is as follows:

[0101] (1) Coating of the chromatographic membrane: The chromatographic membrane undergoes at least two rounds of coating. The first round is antibody coating, which involves using a membrane scrubbing apparatus to coat multiple exosome-specific antibodies and internal control antibodies for quality control onto multiple detection lines of the chromatographic membrane. Preferably, CD9 antibody, CD63 antibody, and CD81 antibody are coated onto three detection lines of the chromatographic membrane (the concentration of these antibodies is 1 mg / mL), and GAPDH antibody is coated onto the quality control line of the chromatographic membrane (the concentration of the antibody is 1 mg / mL). The second round is enzyme coating, which involves using a membrane scrubbing apparatus to coat the enzyme onto the aforementioned multiple detection lines of the chromatographic membrane, so that each detection line contains both exosome-specific antibodies and enzymes (the concentration of the enzymes is 0.6 mg / mL). The parameters of the membrane scrubbing apparatus are set as follows: scrubbing interval of 6 mm and scrubbing concentration of 0.5 μL / cm.

[0102] (2) Assembly of test strips: The sample pad, the chromatography membrane prepared in step (1) and the absorbent pad are attached to the base plate one after another, with the adjacent parts overlapping by 2 mm, to obtain the assembled test strips.

[0103] (3) Cutting the test strips: Cut the assembled test strips after step (2) into strips with a width of 6mm using a strip cutter to obtain the exosome test strips.

[0104] Example 2

[0105] This embodiment illustrates the operation procedure of the exosome detection and classification kit of the present invention (e.g., Figure 1 (As shown). The specific steps are as follows:

[0106] S1 uses an exosome separation column to separate exosomes from the sample (taking IZON's qEV column as an example; other columns or self-made columns can also be used).

[0107] 1) Place the chromatographic column on the support and level it;

[0108] 2) Remove the cap of the column. When the liquid level of the storage solution provided with the column reaches the top of the filter, slowly add a total of 10 mL of PBS.

[0109] 3) When the PBS level reaches the top of the filter, add 1 mL of plasma sample (using plasma as an example, but other samples can be used);

[0110] 4) When the sample level reaches the top of the filter, slowly add a total of 10 mL of PBS;

[0111] 5) The outflowing liquids of 0-0.5mL, 0.5-1.0mL, 1.0-1.5mL, 1.5-2.0mL, 2.0-2.5mL, 2.5-3.0mL, 3.0-3.5mL, 3.5-4.0mL, 4.0-4.5mL, 4.5-5.0mL, 5.0-5.5mL, 5.5-6.0mL, 6.0-6.5mL, and 6.5-7.0mL are respectively labeled as components F1, F2, F3, F4, F5, F6, F7, F8, F9, F10, F11, F12, F13, and F14;

[0112] 6) Collect one or more components. Among the above components, the exosomes are collected as F8, F9, and F10 in some embodiments, with F9 (4.0–4.5 mL) being preferred.

[0113] S2. The isolated exosomes are co-incubated at room temperature for 5 minutes with a chromogenic substrate (such as TMB, DAB, AEC, etc., TMB is used as an example in this embodiment).

[0114] S3 uses an exosome separation column to purify the exosomes after incubation with the chromogenic substrate to remove the free chromogenic substrate, and elutes the exosomes after incubation with the chromogenic substrate using an eluent.

[0115] 1) Purify the exosomes after incubation with the chromogenic substrate according to steps S1 1) to 6).

[0116] S4 involves mixing exosomes that have been incubated with the chromogenic substrate with an auxiliary chromogenic reagent.

[0117] S5 involves adding the mixed solution to the sample pad of the exosome test strip for detection.

[0118] Example 3

[0119] This embodiment illustrates the detection results of the exosome test strip of the present invention, and explains the detection results.

[0120] Samples of plasma, serum, urine, breast milk, pleural and peritoneal fluid, bile, and cerebrospinal fluid were prepared after removing endogenous exosomes. The sample preparation methods are described below:

[0121] Centrifuge the above sample at 3000g for 10 minutes and collect the supernatant;

[0122] Centrifuge at 16000g for 10 minutes and collect the supernatant;

[0123] Pass the above sample through a 0.22 μm membrane and collect the supernatant;

[0124] Centrifuge at 120,000g for 2 hours, and collect the supernatant;

[0125] Centrifuge at 160,000g for 2 hours and collect the supernatant;

[0126] The supernatant is a sample from which endogenous exosomes have been removed.

[0127] Note: Removing endogenous exosomes is not a necessary step in using the exosome detection and classification kit of this invention. Removing endogenous exosomes here is to avoid the influence of endogenous exosomes already present in the sample and to verify the ability of this invention to detect various exosome subtypes. This step is not required in actual use.

[0128] Prepare the samples for the experiment:

[0129] Sample 1: Untreated plasma sample;

[0130] Sample 2: Plasma depleted of endogenous exosomes supplemented with exosomal subtypes containing CD9 surface antigen;

[0131] Sample 3: Serum depleted of endogenous exosomes supplemented with exosomal subtypes containing CD63 surface antigen;

[0132] Sample 4: Urine containing an exosomal subtype of CD81 surface antigen that has been depleted of endogenous exosomes;

[0133] Sample 5: Milk containing exosome subtypes of CD9 and CD63 surface antigens that had been depleted of endogenous exosomes;

[0134] Sample 6: Pleural and peritoneal fluid depleted of endogenous exosomes containing exosomal subtypes of CD9 and CD81 surface antigens;

[0135] Sample 7: Bile containing exosome subtypes of CD63 and CD81 surface antigens that had been depleted of endogenous exosomes;

[0136] Sample 8 depleted the cerebrospinal fluid of endogenous exosomes.

[0137] The above samples were subjected to the following steps as described in Example 2: separation of exosomes, incubation with chromogenic substrate, purification of exosomes after incubation with chromogenic substrate, and addition of samples to exosome test strips.

[0138] The structure of the exosome test strip is as follows: Figure 2 As shown, the sample pad and absorbent pad partially overlap with the chromatography membrane. The sample pad is where the sample is added, and it acts as a filter and buffer for the sample to be tested. The absorbent pad mainly absorbs water, causing the sample to move along the direction of the absorbent pad. The chromatography membrane has two main functions: first, it allows exosomes in the sample to undergo chromatographic reaction, thus enabling the exosomes to move along the membrane; second, it coats the detection line, which contains exosome-capturing antibodies and catalase (HRP).

[0139] The exosomes dropped onto the sample pad of the exosome test strip will move along the chromatography membrane and be captured by the exosome capture antibody on the detection line. The captured exosomes will develop color under the catalysis of catalase. The presence of exosomes and the subtype of exosomes enriched in the sample can be distinguished by the color band.

[0140] In this embodiment, the antibodies coated on the exosome test strip (prepared in Example 1) are, in order of increasing distance from the sample pad, CD9 antibody, CD63 antibody, and CD81 antibody. Schematic diagrams of the detection results for samples 1, 2, 3, 4, 5, 6, 7, and 8 are shown below. Figure 3A , Figure 3B , Figure 3C , Figure 3D , Figure 3E , Figure 3F , Figure 3G , Figure 3H As shown. Untreated plasma samples contain various exosome subtypes, therefore, Figure 3A CD9, CD63, and CD81 antibodies were all detected; for samples containing various exosome subtypes, they were captured by the corresponding antibodies and bands appeared at the corresponding positions. Figures 3B-3G For samples depleted of exosomes, the detection results are as follows: Figure 3H As shown, since there are no exosomes (and therefore no chromogenic substrate), no chromogenic substrate is captured by the exosome-capturing antibody, so the detection lines do not develop color. However, the samples depleted of exosomes still contain phospholipid bimolecular structures that bind to the backbone protein GAPDH. These phospholipid bimolecular structures bind to the lipid-soluble chromogenic substrate, so the control lines still develop color. The detection results of the exosome detection and classification kit of this invention for the exosome subtypes in various samples are shown in Table 1. The detection results are the same as the exosome subtypes added to the samples, verifying the feasibility of the exosome detection and classification kit of this invention.

[0141] Table 1. Capture results of different exosome subtypes in different samples by this invention.

[0142] CD9 CD63 CD81 Sample 1 + + + Sample 2 + - - Sample 3 - + - Sample 4 - - + Sample 5 + + - Sample 6 + - + Sample 7 - + + Sample 8 - - -

[0143] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An exosome detection and classification kit, the kit comprising an exosome detection and classification test strip and instructions for use of the test strip to detect and classify exosomes in a sample; The test strip includes a sample pad, a chromatography membrane, an absorbent pad, and a base plate; in, The chromatography membrane is coated with a control line and multiple detection lines. Each detection line is coated with at least two proteins with different functions. One of the at least two proteins with different functions is an exosome capture protein, and the other is an enzyme. The exosome capture protein coated on each detection line is different. The enzyme is an enzyme that catalyzes the color development of the substrate. The kit also includes a chromogenic substrate corresponding to the enzyme coated on the test strip and an exosome separation column.

2. The exosome detection and classification kit as described in claim 1, characterized in that, The exosome capture protein is a specific antibody against exosome antigens.

3. The exosome detection and classification kit as described in claim 1, characterized in that, The enzymes mentioned are horseradish peroxidase, alkaline phosphatase, catalase, galactosidase, and glucose oxidase.

4. The exosome detection and classification kit as described in claim 3, characterized in that, The enzyme in question is catalase.

5. The exosome detection and classification kit as described in claim 2, characterized in that, The specific antibodies against exosome antigens are specific antibodies against CD9 on exosomes, specific antibodies against CD63, specific antibodies against CD81, HSPA8 / Hsc70 antibodies, HSP90AA1 / Hsp90α antibodies, HSP90AB1 / Hsp90β antibodies, ALIX antibodies, FLOT2 antibodies, FLOT1 antibodies, HSPA4 / Hsp70 antibodies, ACTB antibodies, RAP1B antibodies, Tim4 antibodies, Moesin antibodies, A2M antibodies, Gelsolin antibodies, B2M antibodies, or Filamin A antibodies.

6. The exosome detection and classification kit as described in claim 5, characterized in that, The specific antibody against the exosome antigen is a specific antibody against CD9 on the exosome, a specific antibody against CD63, or a specific antibody against CD81.

7. The exosome detection and classification kit as described in claim 1, characterized in that, The enzyme is also present on the chromatographic membrane at other locations outside the detection line.

8. The exosome detection and classification kit as described in claim 1, characterized in that, The sample pad partially overlaps with the chromatography membrane, the chromatography membrane partially overlaps with the absorbent pad, and the sample pad and absorbent pad do not overlap. The sample pad, chromatography membrane, and absorbent pad are placed on the base plate.

9. The exosome detection and classification kit as described in claim 1, characterized in that, The chromogenic substrate is a solution of 3,3',5,5'-tetramethylbenzidine (TMB) and / or diaminobenzidine (DAB) and / or 3-amino-9-ethylcarbazole (AEC) and / or 2,2'-hydrazine-bis-[3-ethylbenzothiazoline-6-sulfonic acid]-diamine (ABTS) and / or o-phenylenediamine dihydrochloride (OPD) and / or p-nitrophenyl phosphate (PNPP) and / or o-nitrophenyl-β-D-galactopyranoside (ONPG).

10. The exosome detection and classification kit as described in claim 1, characterized in that, The kit also contains auxiliary colorimetric reagents and / or eluents.

11. The exosome detection and classification kit as described in claim 10, characterized in that, The auxiliary colorimetric reagent is a reagent that assists in the colorimetric development of the substrate.

12. The exosome detection and classification kit as described in claim 11, characterized in that, The auxiliary chromogenic reagent is a reagent that assists in the chromogenic development of a chromogenic substrate that binds to exosomes.

13. The exosome detection and classification kit as described in claim 12, characterized in that, The auxiliary colorimetric reagent is a solution containing peroxide.

14. The exosome detection and classification kit as described in claim 13, characterized in that, The auxiliary colorimetric reagent is a solution of hydrogen peroxide (H2O2).

15. The exosome detection and classification kit as described in claim 10, characterized in that, The eluent is an isotonic solution.

16. The exosome detection and classification kit as described in claim 15, characterized in that, The eluent is physiological saline (0.9% NaCl solution), 5% glucose solution, phosphate buffer (PBS buffer), or tris(hydroxymethyl)aminomethane buffer (Tris buffer).

17. A method for preparing an exosome detection and classification test strip, the test strip comprising a sample pad, a chromatography membrane, an absorbent pad, and a base plate; wherein, The chromatography membrane is coated with a control line and multiple detection lines. Each detection line is coated with at least two proteins with different functions. One of the at least two proteins with different functions is an exosome capture protein, and the other is an enzyme. The exosome capture protein coated on each detection line is different. The enzyme is an enzyme that catalyzes the color development of the substrate. Includes the following steps: (1) Coating of the chromatographic membrane: The chromatographic membrane undergoes at least two rounds of coating; in the first round, a membrane-spreading apparatus is used to coat multiple exosome capture proteins onto multiple detection lines of the chromatographic membrane; in the second round, a membrane-spreading apparatus is used to coat enzymes onto the aforementioned multiple detection lines of the chromatographic membrane, so that each detection line contains both exosome capture proteins and enzymes. (2) Assembly of test strips: The sample pad, the chromatography membrane prepared in step (1) and the absorbent pad are attached to the base plate one after another to obtain the assembled test strips; (3) Cutting the test strips: Cut the assembled test strips after step (2) into test strips with a width of 6-8 mm to obtain the exosome detection and classification test strips.

18. The method as described in claim 17, characterized in that, In step (1), the enzyme is coated not only on the detection line but also in other areas of the chromatography membrane outside the detection line.

19. The method as described in claim 17, characterized in that, In step (3), the assembled test strips processed in step (2) are cut into test strips with a width of 6 mm.

20. Use of the kit according to any one of claims 1-16 in the detection and classification of exosomes, wherein the use is for non-diagnostic purposes.

21. A method for detecting and classifying exosomes using an exosome detection and classification kit, the kit comprising an exosome detection and classification test strip and instructions for use of the test strip to detect and classify exosomes in a sample, the test strip comprising a sample pad, a chromatography membrane, an absorbent pad, and a base plate; in, The chromatography membrane is coated with a control line and multiple detection lines. Each detection line is coated with at least two proteins with different functions. One of the at least two proteins with different functions is an exosome capture protein, and the other is an enzyme. The exosome capture protein coated on each detection line is different. The enzyme is an enzyme that catalyzes the color development of the substrate. The kit also includes a chromogenic substrate corresponding to the enzyme coated on the test strip, an exosome separation column, auxiliary chromogenic reagents, and an eluent, and includes the following steps: S1. Separate exosomes from the sample using the exosome separation column in the kit, and elute the exosomes using the eluent in the kit; S2. Co-incubate the isolated exosomes with the chromogenic substrate in the kit. S3. Purify the exosomes after incubation with the chromogenic substrate using the exosome separation column in the kit to remove the free chromogenic substrate, and elute the exosomes after incubation with the chromogenic substrate using the eluent in the kit. S4. Mix the exosomes incubated with the chromogenic substrate with the auxiliary chromogenic reagent in the kit. S5. Add the mixed solution to the exosome detection and classification test strip in the kit for detection; The method is used for non-diagnostic purposes.

22. The method as described in claim 21, characterized in that, This also includes judging the test results according to the following criteria: 1) If both the control line and the test line show color, and the test line shows clear color, then the sample contains exosomes; 2) If the control line shows color but the test line does not, the sample does not contain exosomes; 3) If the control line or one or more of the test lines show color and the exosome capture protein is a specific antibody against the exosome antigen, then the sample contains the exosome subtype corresponding to that antibody. 4) If the control line does not develop color, the detection and classification kit is invalid.

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