Compositions and methods for detecting analytes in exosomes

Abstract

This specification provides compositions and methods for detecting analytes in exosomes. In particular, the present invention relates to the capture and lysis of exosomes, as well as the detection of analytes in exosomes using immunoassays.

Description

Technical Field

[0001] Reference to Related Applications This application claims priority to U.S. Provisional Application No. 63 / 524,517, filed Jun. 30, 2023, and U.S. Provisional Application No. 63 / 571,733, filed Mar. 29, 2024. These applications are hereby incorporated by reference in their entirety.

[0002] This specification provides compositions and methods for detecting an analyte in exosomes. In particular, the present invention relates to the capture and lysis of exosomes and the detection of analytes in exosomes using immunoassays.

Background Art

[0003] Since the COVID-19 pandemic, testing at home or in a clinical setting has become widespread. In particular, lateral flow testing is a simple platform for detecting analytes in liquid samples and enables home testing in vulnerable populations. Generally, lateral flow testing is less sensitive compared to more complex and expensive clinical tests. However, by concentrating the analyte on the lateral flow test, the trade-off between sensitivity and simplicity can be reduced.

[0004] Exosomes are vesicles secreted by cells and are a type of extracellular vesicle (EV) with a size range of 40 - 200 nm. These EVs are a new source of biomarkers indicating disease, from specific surface proteins to cell origin-specific contents. EVs are not only widely present in the blood, but are tissue-derived, circulate throughout the body, and are detected in various body fluids. EVs contain specific membrane proteins such as integrins and tetraspanins, which are key factors in membrane transport and fusion. These membrane proteins have been used as EV "markers" to concentrate EVs, including exosomes, from samples. EVs derived from body fluids such as blood and urine are a non-invasive alternative to many current diagnostic tests.

[0005] In some infectious diseases, such as HIV, pathogen-derived proteins are contained within extracellular vesicles (EVs). In the case of HIV, the virus shares many characteristics with EVs, including the presence of tetraspanin in its outer membrane. See, for example, Jing et al., (2021) Exosomes in HIV infection, Current Opinion in HIV and AIDS 16(5):p 262-270. In the case of prostate cancer, prostate-specific antigen (PSA) is detected both in the blood and in extracellular vesicles. In this case, PSA levels in the blood are easily measured, but they do not correlate well with the true incidence of the disease. However, EV-related PSA levels can distinguish between prostate cancer and healthy individuals, as well as between prostate cancer and benign prostatic hyperplasia. See, for example, Salciccia et al., (2023) Exosome Analysis in Prostate Cancer: How They Can Improve Biomarkers' Performance, Curr Issues Mol Biol. 2023 Jul; 45(7): 6085-6096.

[0006] Previous studies have demonstrated the ability to capture extracellular organisms (EVs) from body fluids using antibodies against tetraspanins in lateral flow assays. See, for example, Oliveira-Rodriguez et al., (2016) Development of a rapid lateral flow immunoassay test for detection of exosomes previously enriched from cell culture medium and body fluids, J. Extracellular Vesicles 5(1):1-10. However, this study only measured the total amount of captured EVs and did not further investigate the surface proteins or contents proteins of the captured EVs. The invention described herein not only captures EVs from a liquid sample but also dissolves the captured EVs to release their contents, which are then detected on a lateral flow assay test line. [Overview of the project]

[0007] This specification describes compositions and methods for detecting analytes in extracellular vesicles such as exosomes. In particular, the present invention relates to the capture and lysis of exosomes, as well as the detection of analytes in extracellular vesicles using immunoassays.

[0008] The experiments described herein illustrate highly sensitive testing for analytes (e.g., human immunodeficiency virus (HIV) or PSA) by probing the EV load for HIV-specific antigens or PSA using EV enrichment and capture. Such methods result in the detection of various analytes at the required level.

[0009] For example, in some embodiments, a method is provided for detecting the presence of an analyte in a sample from a subject. This method includes obtaining a patient sample containing exosomes, capturing the exosomes from the patient sample, lysing the exosomes to release the proteins contained therein, contacting the proteins with one or more analyte-specific binders and / or one or more detection reagents suitable for detecting the analyte, and determining whether the exosomes contain the analyte.

[0010] A method for diagnosing human immunodeficiency virus (HIV) infection in a subject is also provided, which includes: obtaining a patient sample containing exosomes; capturing exosomes from the patient sample; lysing the exosomes to release the proteins contained therein; contacting the proteins with one or more HIV protein binders and / or one or more detection reagents suitable for detecting one or more HIV proteins; and determining whether the exosomes contain HIV protein p24 and at least one additional HIV protein selected from, for example, Nef, Tat, Vpu, and / or gp120; where the determination of the presence of p24 and at least one additional HIV protein indicates HIV infection in the subject. In some embodiments, the at least one additional HIV protein is Nef. In some embodiments, the at least one additional HIV protein is Tat. In some embodiments, the at least one additional HIV protein is Vpu. In some embodiments, the at least one additional HIV protein is gp120.

[0011] Furthermore, a method for diagnosing prostate cancer and / or differentiating prostate cancer from benign prostatic hyperplasia (BPH) in the subject is provided, which includes: collecting a patient sample containing exosomes; capturing exosomes from the patient sample; lysing the exosomes to release the proteins contained therein; contacting the proteins with one or more PSA binders and / or one or more detection reagents suitable for detecting PSA; and determining the PSA level in the exosomes compared to a reference level and / or control, where an elevated PSA level compared to a reference level and / or control suggests prostate cancer or BPA, or allows for differentiation between the presence of prostate cancer and BPA.

[0012] Furthermore, devices, systems, and kits used for detecting analytes in exosomes are provided. The analytes may be, for example, proteins or their fragments, or nucleic acids.

[0013] This disclosure is not limited to a specific analyte. In some embodiments, the analyte is a protein. In some embodiments, the protein is derived from an infectious pathogen, and the presence of the analyte in the sample indicates infection of the analyte in the subject. In some embodiments, the analyte is the HIV protein p24, and at least one additional HIV protein is one or more of Nef, Tat, Vpu, and / or gp120. In some embodiments, the analyte is a cancer antigen or cancer-specific protein (e.g., PSA). In other embodiments, the analyte is a nucleic acid, such as DNA, RNA, mRNA, rRNA, cell-free DNA, and other nucleic acids.

[0014] In some embodiments, subjects have previously received HIV vaccination. In some embodiments, subjects have previously received antiretroviral therapy.

[0015] In certain embodiments, exosomes are captured on a chromatography test strip (e.g., within a lateral flow assay device). In some embodiments, exosomes are captured using tetraspanins (e.g., CD9, CD63, or CD81). In some embodiments, exosomes are lysed on a chromatography test strip (e.g., within a lateral flow device).

[0016] This disclosure is not limited to any particular sample type. Examples include, but are not limited to, blood or serum samples.

[0017] This disclosure is not limited to specific detection reagents or analyte-binding molecules. Examples include, but are not limited to, antibodies, antibody fragments, nucleic acids, etc.

[0018] Accordingly, in some embodiments, the present invention provides a method for detecting the presence of an analyte in a sample from a subject, the method comprising: obtaining a patient sample containing extracellular vesicles; capturing extracellular vesicles from the patient sample; lysing the extracellular vesicles to release the proteins contained therein; contacting the proteins with one or more analyte-specific binders and / or one or more detection reagents suitable for detecting the analyte; and determining whether the extracellular vesicles contain the analyte.

[0019] In some embodiments, the analyte is a protein. In some embodiments, the analyte is a cancer antigen or cancer-specific protein. In some embodiments, the analyte is HIV protein p24, optionally comprising at least one additional HIV protein selected from the group consisting of Nef, Tat, Vpu, and gp120. In some embodiments, the analyte is prostate-specific antigen (PSA). In some embodiments, the protein is derived from an infectious pathogen, and the presence of the analyte in the sample indicates infection of the analyte in the subject. In some embodiments, the infectious pathogen is a virus or bacteria. In some embodiments, the analyte is nucleic acid.

[0020] In another embodiment, the present invention provides a method for diagnosing human immunodeficiency virus (HIV) infection in a subject, the method comprising: collecting a patient sample containing extracellular vesicles; capturing extracellular vesicles from the patient sample; lysing the extracellular vesicles to release the proteins contained therein; contacting the proteins with one or more HIV protein binders and / or one or more detection reagents suitable for detecting one or more HIV proteins; and determining whether the extracellular vesicles contain HIV protein p24 and optionally at least one additional HIV protein selected from the group consisting of Nef, Tat, Vpu, and gp120; where the determination of the presence of p24 and at least one additional HIV protein indicates HIV infection in the subject. In some embodiments, the at least one additional HIV protein is Nef. In some embodiments, the at least one additional HIV protein is Tat. In some embodiments, the at least one additional HIV protein is Vpu. In some embodiments, the at least one additional HIV protein is gp120. In some embodiments, the subject has been vaccinated against HIV infection. In some embodiments, the subjects have either previously received or are currently receiving antiretroviral therapy.

[0021] In some embodiments of the above-described model, extracellular vesicles are captured on a chromatography test strip. In some embodiments, the chromatography strip is located within a lateral flow assay device. In some embodiments, extracellular vesicles are captured using tetraspanin. In some embodiments, the tetraspanin is selected from the group consisting of CD9, CD63, and CD81. In some embodiments, extracellular vesicles are lysed on the chromatography test strip. In some embodiments, the captured extracellular vesicles are lysed by adding a lysis buffer or chase buffer to the chromatography test strip. In some embodiments, the lysis buffer or chase buffer includes a solvent selected from saponins, surfactants, detergents, and combinations thereof. In some embodiments, the patient sample is a blood or serum sample. In some embodiments, the detection reagent includes one or more antigen-binding molecules. In some embodiments, the antigen-binding molecule is an antibody or antibody fragment. In some embodiments, one or more analyte-specific binders and / or one or more detection reagents suitable for detecting the analyte are labeled antibodies or functional fragments thereof. In some embodiments, the labeled antibody or its functional fragment is labeled with colloidal gold or a colored microsphere. In some embodiments, the detection reagent comprises one or more nucleic acid molecules. In some embodiments, one or more analyte-specific binders and / or one or more detection reagents suitable for detecting the analyte are labeled nucleic acids. In some embodiments, the labeled nucleic acids are labeled with colloidal gold or a colored microsphere. In some embodiments, the extracellular vesicles are exosomes.

[0022] In another embodiment, the present invention provides an assay device comprising: a chromatographic test strip comprising a plurality of regions, wherein the chromatographic test strip allows a fluid test sample to flow from a first end to a second end of the chromatographic strip; wherein the chromatographic strip comprises a first region comprising an extracellular vesicle binder, a second region comprising a first binding partner that binds to a first analyte, and optionally, at least a second binding partner that binds to at least a second analyte; and further comprising a reagent for visualizing the binding of the analyte to the labeled binding partner.

[0023] In some embodiments, the extracellular vesicle binding agent in the first region is a tetraspanin. In some embodiments, the tetraspanin is selected from the group consisting of CD9, CD63, CD81 and combinations thereof. In some embodiments, the first analyte is HIV p24. In some embodiments, at least the second analyte is selected from the group consisting of HIV proteins selected from Nef, Tat, Vpu, gp120 and combinations thereof. In some embodiments, the first analyte is PSA. In some embodiments, the analyte is a protein or a fragment thereof. In some embodiments, the first binding partner that binds to the first analyte is an antibody or a functional fragment thereof. In some embodiments, at least the second binding partner that binds to at least the second analyte is an antibody or a functional fragment thereof. In some embodiments, the analyte is a nucleic acid. In some embodiments, the first binding partner that binds to the first analyte is a nucleic acid. In some embodiments, at least the second binding partner that binds to at least the second analyte is a nucleic acid. In some embodiments, the chromatography strip is a nitrocellulose strip. In some embodiments, the chromatography strip is in fluid communication with the sample pad. In some embodiments, the chromatography strip is in fluid communication with the conjugate pad. In some embodiments, the conjugate pad includes a first labeling-binding partner that binds to a first analyte. In some embodiments, the first labeling-binding partner is an antibody or a functional fragment thereof. In some embodiments, the first labeling-binding partner is a nucleic acid. In some embodiments, the first labeling-binding partner is labeled with colloidal gold or a colored microsphere. In some embodiments, the conjugate pad includes at least a second labeling-binding partner that binds to at least a second analyte. In some embodiments, the at least second labeling-binding partner is an antibody or a functional fragment thereof. In some embodiments, the first labeling-binding partner is labeled with colloidal gold or a colored microsphere.In some embodiments, the assay device further includes an absorption pad in fluid communication with the chromatography test strip. In some embodiments, the chromatography test strip further includes a control region. In some embodiments, the extracellular vesicle binder is an exosome binder.

[0024] In another aspect, the present invention provides a kit for detecting an analyte in extracellular vesicles, the kit comprising: a) an assay device as described in any of the various embodiments above; and b) a container of lysis buffer or chase buffer.

[0025] In another aspect, the kit of the present invention is used for detecting an analyte in extracellular vesicles. In some embodiments, the extracellular vesicles are exosomes.

[0026] Additional aspects of the present invention are described herein.

Brief Description of the Drawings

[0027] [Figure 1] Figures 1A-B show an overview of the EV-HIV test. (A) Whole blood or plasma contains antibodies, virus particles, viral proteins, and extracellular vesicles (EVs). (B) A blood / plasma sample is added to the sample pad and drawn across a nitrocellulose strip, enabling immunocapture of EVs via binding to tetraspanin proteins (CD9, CD63, CD81). [Figure 2] Figure 2 shows a Western blot analysis of the presence of p24 in H9 cells and H9MN full-length intact (H9MN FI) cells. [Figure 3] Figure 3 shows a quantitative ELISA for HIV p24 protein. Exosomes purified by the ExoQuick method or the MagCapture method from normal cells (H9, black bars) or cells chronically infected with the HIV MN strain (H9MN FI, gray bars). Error bars indicate the standard deviation of the mean of three measurements. [Figure 4A]Figures 4A-B show the NS300 analysis of exosomes purified using the ExoQuick method. A. Size distribution of extracellular vesicles derived from chronically infected H9MN FI cells. Average size: 191 nm. B. Size distribution of extracellular vesicles derived from chronically infected H9MN FI cells. Average size: 157 nm. [Figure 4B] Figures 4A-B show the NS300 analysis of exosomes purified using the ExoQuick method. A. Size distribution of extracellular vesicles derived from chronically infected H9MN FI cells. Average size: 191 nm. B. Size distribution of extracellular vesicles derived from chronically infected H9MN FI cells. Average size: 157 nm. [Figure 5] Figure 5 shows automated Western blotting analysis of exosomes purified from human plasma collected from HIV- and HIV-+ subjects. A. Detection of HIV p24. Measured by area under the curve (AUC) of the approximately 24 kDa peak. B. Detection of HIV Tat. Measured by AUC of the approximately 14 kDa peak. C. Detection of HIV Mef. Measured by AUC of the approximately 27 kDa peak. Exosomes of approximately 0.4 mg / mL purified from the plasma of HIV-negative or HIV-positive subjects were analyzed. HIV- n=10, HIV- n=40. [Figure 6] Figures 6A-D show immunocapture of exosomes on a lateral flow strip. A. Schematic diagram of the tetraspanin-capturing lateral flow strip. Purified exosomes are added to the sample well. B. Schematic diagram of the lateral flow capture device and tetraspanin-capturing region. C. Only the running buffer was tested on the exosome capture strip "buffer". The total amount of purified exosomes captured on the test line was 10 μg / mL. B. Scorecard quantifying the strength of the test strip on a scale of 1 to 10. [Figure 7] Figure 7 shows the detection limits of purified exosomes using anti-CD63 + anti-CD81 striped lateral flow strips. The vertical axis represents the cube reader score as a percentage of the test line's coloration, and the horizontal axis represents the exosome concentration (μg / mL). [Figure 8]Figures 8A-C show lateral flow detection of p24 from purified exosomes. A. Schematic diagram of lateral flow detection of p24 using lysed extracellular vesicles (EVs). B. Schematic diagram of nitrocellulose strips for p24 capture and detection from purified EVs. C. p24 measurement in EV lysis buffer. Buffer alone (score 3 out of 10). EVs derived from H9 cells lysed in EV lysis buffer, p24- (3 out of 10). EVs derived from H9MN FI cells lysed in EV lysis buffer, p24+ (6 out of 10). [Figure 9A] Figures 9A-B show lateral flow detection of exosome-derived p24. A. Schematic diagram of the lateral flow device. B. Photographs of negative control (first two panels) and p24 detection on the lateral flow device (last panel). EVs concentrated from each sample type were incubated in EV lysis buffer and flowed using the p24 lateral flow test. p24 was detected in the test line, and anti-species specificity was detected in the control line. The numbers below the panels indicate visual scores. [Figure 9B] Figures 9A-B show lateral flow detection of exosome-derived p24. A. Schematic diagram of the lateral flow device. B. Photographs of negative control (first two panels) and p24 detection on the lateral flow device (last panel). EVs concentrated from each sample type were incubated in EV lysis buffer and flowed using the p24 lateral flow test. p24 was detected in the test line, and anti-species specificity was detected in the control line. The numbers below the panels indicate visual scores. [Figure 10] Figure 10 shows the effect of various lysis buffer components on the release of p24 cargo from 20 μg of EV input. PBS represents the background of a system without EV input, H9 represents a nonspecific background derived from a p24-cell line, and H9 MNFI represents a specific p24 signal derived from a p24-expressing cell line. [Figure 11]Figure 11A-B shows an overview of the EV-PSA test. (A) Whole blood or plasma contains antibodies, immune cells, cellular proteins, and extracellular vesicles (EVs). (B) The blood / plasma sample is added to the sample pad and adsorbed onto a nitrocellulose strip. This allows for immunocapture of EVs via binding to tetraspanin proteins (CD9, CD63, CD81). After the addition of lysis buffer or chase buffer, the EV load is released, captured by the anti-PSA test line, and detected by an anti-PSA detection antibody. [Figure 12] Figure 12 shows an overview of the EV-PSA test. A. Whole blood, urine, or plasma contains extravasation biomarkers (EVs) including disease-specific biomarkers. B. Sequential EV capture and PSA detection. [Figure 13] Figures 13A-E show the detection of PSA biomarkers in EV-PSA testing. A. Detection of PSA less than 0.5 ng / mL in EV-PSA testing. B. Design of EV-PSA testing with multiple anti-tetraspanin capture lines placed in the EV capture region. C. Additional testing of low-concentration PSA down to the pg level. D. Score reading by concentration measurement using a cube reader to improve sensitivity. E. Score reading by visual inspection using a 1-10 scale. [Figure 14] Figure 14 shows that different types of exosomes contain tetraspanin in different proportions. The capture line is a 1:1:1 mixture of anti-CD9, anti-CD63, and anti-CD81 antibodies. The detected antibodies are labeled on the graph and show results corresponding to exosomes purified from human plasma, PC3 cell (PSA-) derived exosomes, and LNCaP cell (PSA+) derived exosomes. [Figure 15] Figure 15 shows the results of PSA ELISA from intact and lysed extracellular viable cells (EVs). PSA was detected in EVs derived from prostate cancer, but not in healthy controls. Purified EVs from normal (no prostate cancer) and LNCaP (prostate cancer) were tested at various total protein concentrations in PBS or lysis buffer. [Figure 16]Figure 16 shows the PSA detection results when purified exosomes derived from LNCaP cells (PSA+) were lysed. The visual scores (1-10) of three strips are graphed with standard error bars. [Modes for carrying out the invention]

[0028] definition To facilitate understanding of the present invention, the following terms and expressions are defined: In this specification, "exosome" refers to membrane-bound extracellular vesicles (EVs) produced in the endosomal compartment of most eukaryotic cells. In multicellular organisms, exosomes are found in bodily fluids such as saliva, blood, urine, and cerebrospinal fluid.

[0029] In this specification, “analyte” means any molecule detected by the devices and methods described herein. In a non-limiting example, the analyte may be a protein or other biological molecule.

[0030] The terms “detect,” “to detect,” or “to determine the level” refer to quantitatively or non-quantitatively determining the presence of the analyte being investigated (e.g., HIV). “Detecting the formation of a complex” refers to detecting a complex containing the detection reagent by any method suitable for observing a specific label associated with the detection reagent. Examples include visual observation of a colored (or other visible) label, or measurement or visual detection of fluorescence, chemiluminescence, or radioactive labeling.

[0031] In this specification, the term “sample” is used in its broadest sense. In a sense, it is intended to include specimens or cultures obtained from any source, as well as biological and environmental samples. Biological samples are obtained from animals (including humans) and include liquids, solids, tissues, and gases. Biological samples include blood products such as urine, saliva, cerebrospinal fluid, whole blood, plasma, and serum. However, these examples are not intended to limit the types of samples to which the present invention can be applied.

[0032] The term "specific binding partner (or binding partner)" refers to one of a pair of molecules that interact through a specific, non-covalent interaction that depends on the three-dimensional structure of the molecules involved. Typical specific binding partner pairs include antigen / antibody, hapten / antibody, hormone / receptor, nucleic acid chain / complementary nucleic acid chain, substrate / enzyme, inhibitor / enzyme, carbohydrate / lectin, biotin / (strept)avidin, and virus / cell receptor.

[0033] In this specification, the terms “immunoglobulin” or “antibody” refer to proteins that bind to a specific antigen. Immunoglobulins include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, Fab fragments, F(ab')2 fragments, and further include the following classes of immunoglobulins: IgG, IgA, IgM, IgD, IgB, IbE, and secretory immunoglobulin (sIg). Immunoglobulins generally consist of two identical heavy chains and two light chains. However, the terms “antibody” and “immunoglobulin” also encompass single-chain and double-chain antibodies.

[0034] "Label" refers to a molecule or composition bound to an analyte, an analogue of an analyte, a detection reagent, an antibody, or a binding partner, which is detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Examples of labels including enzymes, colloidal gold particles, and colored latex particles (e.g., microspheres) are disclosed (U.S. Patents 4,275,149, 4,313,734, 4,373,932, and 4,954,452, all incorporated herein by reference). Additional examples of useful labels include, but are not limited to, radioisotopes, cofactors, ligands, chemiluminescent or fluorescent agents, protein-adsorbed silver particles, protein-adsorbed iron particles, protein-adsorbed copper particles, protein-adsorbed selenium particles, protein-adsorbed sulfur particles, protein-adsorbed tellurium particles, protein-adsorbed carbon particles, and protein-binding pigment sacs. Methods for attaching compounds (e.g., detection reagents) to labels include covalent bonding, adsorption processes, hydrophobic and / or electrostatic bonding such as chelation, or combinations of these bonding and interactions, and may involve bonding groups.

[0035] Regarding antibodies, the expressions "specifically binds to the analyte" or "specifically elicits an immune response to the analyte" refer to a binding reaction that definitively indicates the presence of the analyte in the presence of a heterogeneous population of molecules such as proteins and other biological molecules. Therefore, under specified immunoassay conditions, a designated antibody will bind to a specific analyte and will not bind to other analytes present in the sample in significant amounts. Various immunoassay formats are used to select antibodies that specifically elicit an immune response to a particular analyte. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies that specifically elicit an immune response to proteins. For a list of immunoassay formats and conditions that can be used to determine specific immunoreactivity, see Harlow and Lane, Antibodies, A Laboratory Manual, CSHP, New York (1988).

[0036] The term “capture agent” refers to an unlabeled specific binding partner specific to any of the following: (i) extracellular vesicles (e.g., exosomes), (ii) the analyte in a sandwich assay, (iii) the detection reagent or analyte in a competitive assay, or (iv) a secondary specific binding partner that is itself specific to the analyte in an indirect assay. In this specification, “secondary specific binding partner” refers to a specific binding partner that binds to the specific binding partner of the analyte. For example, a secondary specific binding partner may include an antibody specific to another antibody (e.g., a goat anti-human antibody). “Capture area” refers to a region of a lateral flow device on which the capture reagent is immobilized. A lateral flow device may have multiple capture areas, for example, “primary capture area,” “secondary capture area,” etc. Often, different capture reagents are immobilized in the primary, secondary, and other capture areas. Multiple capture areas may have any orientation relative to each other on the lateral flow substrate. For example, the primary capture area may be distal or proximal to a secondary (or other) capture area, and vice versa. Alternatively, the primary and secondary (or other) capture areas may be positioned perpendicular to each other, and the two (or more) capture areas may form a cross, a plus sign, or other symbol.

[0037] A "detection reagent" refers to a specific binding partner that is bound to a label. Detection reagents include, for example, labeled analyte-specific binding members or labeled secondary specific binding members (such as enzyme conjugates or goat anti-human antibodies).

[0038] A "lateral flow device" refers to an assay device in the form of a test strip used in lateral flow chromatography. In this device, a suspected test sample solution containing the analyte flows (for example, by capillary action) through a strip (often made of absorbent material such as paper, nitrocellulose, or cellulose). The test solution and suspended analyte flow along the strip to the detection area, where the analyte (if present) interacts with the detection agent to indicate the presence, absence, and / or quantity of the analyte.

[0039] The "sample application area" refers to the area on an immunochromatography test strip (for example, an immunochromatography test strip present in a lateral flow device) where a liquid sample is introduced. For example, the sample may be introduced into the sample application area by external application using a dropper or other applicator. Another example is immersing the sample application area directly in the sample, such as dipping the test strip into a sample holding container. Yet another example is pouring or squeezing the sample into the sample application area.

[0040] A "solid support" or "substrate" refers to a material that is insoluble or can be made insoluble by a subsequent reaction. Solid supports include a wide variety of materials well known in the art, such as nitrocellulose, reaction tray well walls, multiwell plates, test tubes, polystyrene beads, magnetic beads, membranes, microparticles (such as latex particles), and sheep (or other animal) red blood cells. This term assumes any suitable porous material that has sufficient porosity to allow access to detection reagents and appropriate surface affinity to immobilize capture reagents. For example, the porous structure of nitrocellulose exhibits excellent absorption and adsorption properties for a variety of reagents, including capture reagents. Nylon has similar properties and is suitable. Microporous structures are also useful, as are materials that have a gel structure in a hydrated state.

[0041] Further examples of useful solid carriers include: natural polymeric carbohydrates and their synthetic modifications, crosslinking, and substitution derivatives, such as agar, agarose, crosslinked alginic acid, substituted crosslinked guar gum, especially cellulose esters with nitrates and carboxylic acids, mixed cellulose esters, and cellulose ethers; nitrogen-containing natural polymers, such as proteins and their derivatives, including crosslinked or modified gelatin; natural hydrocarbon polymers, such as latex and rubber; vinyl polymers, such as polyethylene, polypropylene, polystyrene, polyvinyl chloride, polyvinyl acetate and its partially hydrolyzed derivatives; copolymers and ternary copolymers of the above polycondensates, such as polyacrylamide, polymethacrylate, and polyester; and polyamides. Synthetic polymers that can be prepared with a suitable porous structure, such as polyurethane or other polymers such as polyepoxy; porous inorganic materials such as alkaline earth metal and magnesium sulfates or carbonates, including barium sulfate, calcium sulfate, calcium carbonate, alkali metal and alkaline earth metal silicates, and aluminum and magnesium silicates; oxides or hydrates of aluminum or silicon, such as clay, alumina, talc, kaolin, zeolite, silica gel, and glass (these materials can be used as filters in combination with the above polymer materials); and mixtures or copolymers of the above classifications, such as graft copolymers obtained by initiating the polymerization of synthetic polymers on existing natural polymers.

[0042] The porous solid carriers, such as nitrocellulose, described herein are preferably in the form of sheets or strips. The thickness of such sheets or strips can vary over a wide range, for example, from about 0.01 to 0.5 mm, about 0.02 to 0.45 mm, about 0.05 to 0.3 mm, about 0.075 to 0.25 mm, about 0.1 to 0.2 mm, or about 0.11 to 0.15 mm. The pore size of such sheets or strips can also vary over a wide range, for example, from about 0.025 to 15 microns, more specifically from about 0.1 to 3 microns. However, pore size is not intended to be a limiting factor in the selection of the solid carrier. The flow rate of the solid carrier can also vary over a wide range, where applicable. For example, the flow rate can vary in the range of approximately 12.5 to 90 seconds / cm (i.e., 50 to 300 seconds / 4cm), approximately 22.5 to 62.5 seconds / cm (i.e., 90 to 250 seconds / 4cm), approximately 25 to 62.5 seconds / cm (i.e., 100 to 250 seconds / 4cm), approximately 37.5 to 62.5 seconds / cm (i.e., 150 to 250 seconds / 4cm), or approximately 50 to 62.5 seconds / cm (i.e., 200 to 250 seconds / 4cm). In certain embodiments of the device described herein, the flow rate is approximately 62.5 seconds / cm (i.e., 250 seconds / 4cm). In other specific embodiments of the device described herein, the flow rate is approximately 37.5 seconds / cm (i.e., 150 seconds / 4cm).

[0043] The surface of a solid support may be activated by a chemical process that covalently bonds a drug (e.g., a capture reagent) to the support. However, other suitable methods, including ionic interactions, hydrophobic interactions, and covalent interactions, may also be used to immobilize the drug (e.g., a capture reagent) on the solid support. The specific forces resulting in the immobilization of the drug on the solid phase are not important for the methods and devices described herein.

[0044] Unless physically constrained, the solid support can be used in any suitable shape, such as a film, sheet, strip, or plate, or it can be coated, bonded, or laminated onto a suitable inert carrier such as paper, glass, plastic film, or textile.

[0045] "Lateral flow substrate" refers to any solid support or substrate useful in lateral flow devices.

[0046] Description of the Invention This specification describes compositions and methods for detecting analytes in extracellular vesicles (EVs). In particular, the present invention relates to the capture and lysis of EVs such as exosomes, and to the detection of analytes in EVs using immunoassays.

[0047] The experiments described herein developed a method for screening extracellular vesicles containing exosomes to detect disease-specific biomarkers. Example 1 describes only the HIV-1 / 2 (HIV) protein. While the results described herein are exemplified in the detection of the HIV protein, the devices, compositions, kits, and methods described herein are available for the detection of any number of analytes. Non-limiting examples of suitable analytes are described below.

[0048] Current "fourth-generation" HIV-1 / 2 combined tests screen for both the presence of the antigen (p24) and antibodies against envelope proteins (gp160, gp41, gp120). The assay described herein tests for the antigen only, includes the detection of p24, and adds at least one additional biomarker (e.g., Nef, Tat, Vpu, and / or gp120). These additional viral proteins, Nef, Tat, Vpu, and gp120, were traditionally thought to be expressed only during the short period of acute viral infection. However, recent studies have shown that these proteins can also be expressed in the chronic phase of infection, and even in patients receiving antiretroviral therapy (ART) where the viral genome is at undetectable levels. Imamichi et al., Defective HIV-1 proviruses produce viral proteins. PNAS. 2020 Feb; 117(7):3704-3710; Ferdin et al., Viral protein Nef is detected in plasma of half of HIV-infected adults with undetectable plasma HIV RNA. PLoS One. 2018 Jan 24;13(1):e0191613.

[0049] To develop diagnostic methods that detect only HIV-1 / 2 antigens, additional enrichment of these antigens was used to enable detection of low levels of expression in the majority of HIV-infected individuals. See, for example, Lee et al. HIV-Nef and ADAM17-Containing-Plasma Extracellular Vesicles Induce and Correlate with Immune Pathogenesis in Chronic HIV Infection. EBioMedicine 2016, (6) 103-113. shows that HIV proteins Nef and Vpu are persistently upexpressed and detectable in plasma extracellular vesicles (EVs). Other studies also support the presence of Nef within EVs and exosomes, and Nef is a known regulator of endosomal transport (see, for example, McNamara et al., Nef secretion into Extracellular Vesicles or Exosomes Is Conserved across Human and Simian Immunodeficiency Viruses. mBio. 2018 (9)e02344-17, and Roeth et al., Human Immunodeficiency virus type 1 Nef: Adapting to Intracellular Trafficking Pathways. Microbiol Mol Biol Rev 70:548-563, Figure 5).

[0050] Extracellular vesicles are mainly classified into three subgroups: apoptotic bodies, microvesicles, and exosomes. All three are rich in surface membrane proteins and can be distinguished by both size and membrane protein markers. EVs can be purified using tetraspanin cell surface markers such as CD9, CD63, and CD81. See, for example, Kowal et al., Proteomic Comparison Defines Novel Markers to Characterize Heterogeneous Populations of Extracellular Vesicle Subtypes. PNAS. 2016, 113, E968-977.20: 7. In HIV, EVs containing CD81 have been shown to contain Nef 4.

[0051] In one embodiment of this disclosure, immunocapture of extracellular proteins (EVs) via tetraspanins CD9, CD63, and CD81 was utilized in a lateral flow immunoassay test, enriching the EVs before lysis and release of the target proteins. This enabled capture on multiple assay lines for p24, Nef, Tat, and gp120.

[0052] Therefore, the present invention provides devices, kits, and methods for detecting the presence of analytes in EVs or exosomes. Representative devices and methods are described below.

[0053] I. Isolation of exosomes Extracellular vesicles (EVs) are small, cell-derived particles surrounded by a plasma membrane, containing exosomes, and ranging in diameter from 30 to 150 nm (Doyle et al, Cells. 2019 Jul; 8(7): 727). After taking up proteins, nucleic acids, and lipids, they are released from the cell's plasma membrane by forming projections directly outward. Exosomes are thought to be a means of intercellular communication that transports important transport molecules. Membrane proteins and intraluminal proteins contained in extracellular proteins (EVs) can be used as useful biomarkers for diseases including cancer and infectious diseases (Dai, J., Su, Y., Zhong, S. et al. Exosomes: key players in cancer and potential therapeutic strategy. Sig Transduct Target Ther 5, 145 (2020); Hosseini et al., Inflammation & Allergy-Drug Targets (Formerly Current Drug Targets - Inflammation & Allergy) (Discontinued), Volume 12, Number 1, 2013, pp. 29-37(9); Fleming et al., Pathogens and Disease, Volume 71, Issue 2, 1 July 2014, Pages 109-120). Exosomes are present in high concentrations in most body fluids (plasma, serum, urine, saliva, breast milk, bronchial lavage fluid, amniotic fluid, cerebrospinal fluid, and malignant ascites) (Raimondo et al., Proteomics. 2011 Feb;11(4):709-20. doi: 10.1002 / pmic.201000422. Epub 2011 Jan 17).

[0054] The HIV proteins Nef and Vpu are persistently upexpressed and detected in extracellular vesicles (EVs) in the plasma of HIV-infected individuals. Other studies also support the presence of Nef within EVs or exosomes, and Nef is a known regulator of endosomal transport. Extracellular vesicles are thought to be classified into three main subgroups: apoptotic bodies, microvesicles, and exosomes. All three are rich in surface membrane proteins and can be distinguished by both size and membrane protein markers. In some embodiments, EVs are purified using exosome-specific cell surface markers (e.g., tetraspanin cell surface markers including CD9, CD63, and CD81). In HIV, EVs containing CD81 have been shown to include Nef, gp120 (Env), Tat, Vpr, and Gag and Pol polypeptides (Anush Arakelyan, Wendy Fitzgerald, Sonia Zicari, Christophe Vanpouille & Leonid Margolis Scientific Reports volume 7, Article number: 1695 (2017); Benjamin J. Patters & Santosh Kumar Retrovirology volume 15, Article number: 79 (2018)).

[0055] This disclosure is not limited to any specific method for separating exosomes (EVs). Multiple methods for separating EVs have been developed. The three main categories of exosome separation are: ultracentrifugation and ultrafiltration for size-based separation, and immunoaffinity for separation using exosome-specific membrane proteins (e.g., tetraspanins) (Chen et al., Front. Bioeng. Biotechnol., 05 January 2022). In some embodiments, this disclosure uses immunocapture methods that utilize tetraspanin expression on exosomes. The tetraspanin superfamily is defined by four transmembrane domains and post-translational modifications (Stipp et al., Volume 28, ISSUE 2, P106-112, February 2003). Tetraspanins CD9, CD37, CD63, CD81, and CD82 are abundant in extravasation vessels (EVs) and have therefore been used as biomarkers for exosomes (Andreuet al., Front. Immunol., 16 September 2014 Sec. Vaccines and Molecular Therapeutics Volume 5 - 2014).

[0056] In certain embodiments, immunocapsulation of tetraspanin on a lateral flow strip (e.g., within a lateral flow assay device) is used for immunocapsulation and concentration of EVs derived from biological fluids. The EVs concentrated on the lateral flow strip are then lysed on the strip, releasing the internal cargo, which is then detected on a downstream test strip on the device. Typical devices are described below.

[0057] II. Assay Devices and Methods This invention provides devices, kits, and methods for detecting analytes in extravasation (EV). These devices, kits, and methods can be used in a variety of research, screening, and diagnostic applications.

[0058] In one embodiment, the assay is suitable for point-of-care applications in clinics or homes. In another embodiment, the assay is suitable for use in a clinical setting. In either embodiment, the assay is used to determine the presence and / or level of one or more analytes in a test sample taken from a subject. In some embodiments, the level of the analytes in the test sample is compared to a standard, such as a control sample or a standard curve.

[0059] This disclosure is not limited to a specific analyte. In some embodiments, the analyte is a protein or other biological molecule. In some embodiments, the analyte is a protein derived from an infectious pathogen (e.g., HIV or other viral, bacterial, or fungal pathogens), or a cancer-specific or cancer-associated protein. In some embodiments, the analyte is a nucleic acid. Detectable nucleic acids include, but are not limited to, DNA, RNA, mRNA, rRNA, and cell-free DNA. Lateral flow assays for nucleic acids include assays for amplified nucleic acids by PCR amplification, isothermal amplification methods such as LAMP, and other amplification techniques, as well as detection methods incorporating endonucleases such as Cas9 endonucleases. For example, Deng et al., Nature Communications volume 15, Article number: 1818 (2024); Zahra et al., Mol. Biotechnol. 65:699-714 (2023); Fang et al., Applied Microbiol. Biotech. 107:3983-3996 (2023); Sanchez et al., Plant Biotechnol J. 20(12):2418-2429 (2022); Islam et al., Trends Biotech. 41(2):144-146 (2023); Jiang et al., Front. Bioeng. Biotechnol., 11:1-10 (2023); Tang et al., BMC Infect. Dis. 24(1):81 (2024); Akalin et al., MethodsX, 11:102372 (2023); Law See et al., Biomedicines 11(9):2344 (2023); Saxena et al., IJMS 23(21):13105 (2022); all of these are incorporated herein by reference in their entirety. In embodiments, when the analyte is nucleic acid, the process of the present invention may further include an amplification step of amplifying the nucleic acid sequence to be detected, for example, by thermal amplification or isothermal amplification.

[0060] In some embodiments, the analyte is detected by binding to an analyte-specific capture molecule (e.g., an aptamer, or an antibody in an immunoassay). The present invention is not limited to a specific capture molecule or antibody. Any capture molecule or antibody, or functional fragment thereof, that can detect an analyte (e.g., monoclonal or polyclonal antibodies, Fab fragments, scFv, etc.) may be used. Exemplary methods of antibody production are described below. The antibody-analyte complex formed by antibody binding is detected by techniques well known to those skilled in the art. In some embodiments, a detection reagent is used. In some embodiments, the detection reagent is a labeled antibody or a functional fragment thereof that binds to the analyte. The present invention is not limited to a specific detection format. A variety of detection formats are conceivable. These include radioimmunoassays, ELISA (enzyme immunoassay), "sandwich" immunoassays, immunoradioassays, gel diffusion precipitation, immunodiffusion assays, precipitation assays, agglutination assays (e.g., gel agglutination assays, hemagglutination assays, etc.), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays.

[0061] In some embodiments, the immunoassay device of the present invention enables a relatively inexpensive, disposable membrane-based assay for visually identifying the presence (or absence) of an analyte in a liquid sample. Such devices are typically configured as a self-standing dipstick (e.g., a test strip) or as a device with some kind of housing. Typically, the immunoassay device of the present invention is usable with approximately 200 μl of liquid sample, and detection of the analyte in the sample can be completed within 2–5 minutes (though not required). In preferred embodiments, no auxiliary equipment is required to perform such tests, and they can be easily used by inexperienced individuals in clinics, laboratories, field, or homes.

[0062] Immunoassay devices have been developed for the routine identification and monitoring of physiological and pathological conditions (infections, pregnancy, cancer, endocrine disorders, etc.) using various biological samples (e.g., urine, serum, plasma, blood, saliva), or for contamination analysis of environmental samples (e.g., natural liquids and industrial plant wastewater). Many of these tests are based on highly specific interactions between particular binding pairs. Examples of such binding pairs include antigen / antibody, hapten / antibody, lectin / carbohydrate, apolipoprotein / cofactor, and biotin / (strept)avidin. Furthermore, many of these tests involve devices in which one or both of the binding pairs are immobilized on movable or immobile solid-phase materials such as latex beads, glass fibers, glass beads, cellulose strips, and nitrocellulose membranes (e.g., solid phase, lateral flow test strip, flow-through test) (U.S. Patents 4,703,017, 4,743,560, and 5,073,484).

[0063] In some embodiments, the assay device is a lateral flow assay device. Numerous commercially available lateral flow assays and patents disclose methods for detecting analytes. See, for example, U.S. Patents 5,229,073; 5,591,645; 4,168,146; 4,366,241; 4,855,240; 4,861,711; 4,703,017; 5,451,504; 5,451,507; 5,798,273; 6,001,658; and 5,120,643; European Patent No. 0296724; WO97 / 06439; and WO98 / 36278, all of which are incorporated herein by reference.

[0064] The lateral flow assay device of the present invention comprises strips of absorbent or porous material (e.g., microporous membrane (e.g., nitrocellulose)), which may be composed of different materials, each joined to one another in regions, and which may be adjacent and / or overlapping. In some examples, the absorbent strips may be fixed to a non-reactive supporting material (e.g., nonwoven polyester) to increase the rigidity of the strips, for example. Regions within each strip may contain different specific binding partners and / or other reagents necessary for the detection and / or quantification of the particular analyte to be detected. Thus, these regions can be considered functional sectors or functional regions within the test device.

[0065] In some embodiments, the assay strip of the present disclosure includes an EV capture region and one or more analyte capture regions.

[0066] In some embodiments, a liquid sample (or a sample suspended in liquid) is introduced to the proximal end of the strip, for example, by immersion or spotting. The sample is collected or obtained using methods well known to those skilled in the art. The sample containing the analyte to be detected can be obtained from any biological source. Examples of biological sources include human or animal serum, plasma, urine, cerebrospinal fluid, saliva, fermentation fluid, lymph, tissue culture fluid, and ascites. The sample may be diluted, purified, concentrated, filtered, dissolved, suspended, or otherwise manipulated before the immunoassay to optimize the immunoassay results. The liquid moves distally through the entire functional area of ​​the strip. The final distribution of the liquid in each functional area depends on the adsorption capacity and dimensions of the material used.

[0067] In some embodiments, the porous solid support, such as nitrocellulose described above, is preferably in the form of a sheet or strip. The thickness of such a sheet or strip can vary over a wide range, for example, from about 0.01 to 0.5 mm, about 0.02 to 0.45 mm, about 0.05 to 0.3 mm, about 0.075 to 0.25 mm, about 0.1 to 0.2 mm, or about 0.11 to 0.15 mm. The pore size of such a sheet or strip can also vary over a wide range, for example, from about 0.025 to 15 microns, more specifically about 0.1 to 3 microns. However, pore size is not intended to be a limiting factor in the selection of the solid support. The flow rate of the solid support can also vary over a wide range, where applicable. For example, the flow rate can vary in the range of approximately 12.5 to 90 seconds / cm (i.e., 50 to 300 seconds / 4cm), approximately 22.5 to 62.5 seconds / cm (i.e., 90 to 250 seconds / 4cm), approximately 25 to 62.5 seconds / cm (i.e., 100 to 250 seconds / 4cm), approximately 37.5 to 62.5 seconds / cm (i.e., 150 to 250 seconds / 4cm), or approximately 50 to 62.5 seconds / cm (i.e., 200 to 250 seconds / 4cm). In certain embodiments of the device described herein, the flow rate is approximately 62.5 seconds / cm (i.e., 250 seconds / 4cm). In other specific embodiments of the device described herein, the flow rate is approximately 37.5 seconds / cm (i.e., 150 seconds / 4cm).

[0068] In some embodiments, the assay device includes a detection reagent. The detection reagent provides a means for detecting the formation of a complex between the analyte and a capture reagent (e.g., a primary antibody specific to the analyte). The detector is either integrated into the immunoassay device (e.g., contained in a conjugate pad as described below) or applied to the device from an external source.

[0069] The detection agent may be a single reagent or a series of reagents that work together to achieve the detection objective. In some cases, the detection agent is a labeled binding partner specific to the analyte. In other cases, the detection agent may include an unlabeled first binding partner specific to the analyte and a labeled second binding partner specific to the first binding partner, etc. In either case, since the detection reagent specifically detects the bound analyte in the capture reagent complex, it is desirable that it does not substantially bind or react with the capture reagent or other components localized in the analyte capture area. Such nonspecific binding or reaction by the detection reagent can lead to false positive results. Optionally, the detection reagent can specifically recognize positive control molecules present in the secondary capture area (e.g., nonspecific human IgG for labeled protein A detection, labeled protein G detection molecule, labeled anti-human antibody (Fc), etc.). In preferred embodiments, the detection molecule may be bound to or associated with a label. Examples of labels including enzymes, colloidal gold particles, and colored latex particles (e.g., microspheres) are disclosed (U.S. Patents 4,275,149, 4,313,734, 4,373,932, and 4,954,452, each incorporated herein by reference). Additional examples of useful labels include, but are not limited to, radioisotopes, cofactors, ligands, chemiluminescent or fluorescent agents, protein-adsorbing silver particles, protein-adsorbing iron particles, protein-adsorbing copper particles, protein-adsorbing selenium particles, protein-adsorbing sulfur particles, protein-adsorbing tellurium particles, protein-adsorbing carbon particles, and protein-binding pigment sacs. Methods for attaching compounds (e.g., detection reagents) to labels include covalent bonding, adsorption processes, hydrophobic and / or electrostatic bonding such as chelation, or combinations thereof, and may also involve binding groups.

[0070] A lateral flow device is an assay device equipped with a test strip. A suspected test sample solution containing the analyte flows through this strip. The analyte, suspended in the test solution, flows along the strip and reaches the detection area. In this area, the analyte (if present) interacts with a capture agent and a detection agent, indicating the presence, absence, and / or quantity of the analyte. Many lateral flow devices are one-step lateral flow assays, where a biological fluid is dropped onto the sample area on the strip and allowed to flow along the strip until it comes into contact with a specific binding partner. This binding partner interacts with the analyte in the fluid. When the analyte interacts with the binding partner, a signal, such as a fluorescent or visible dye, appears to indicate the occurrence of the interaction. Multiple independent binding partners can also be placed on the strip (e.g., in parallel lines) to detect multiple analytes in the fluid. A control indicator can also be incorporated into the test strip, providing a signal that the test was performed correctly even if a positive signal indicating the presence (or absence) of the analyte is not observed on the strip.

[0071] The structure and design of lateral flow devices are described, for example, in the following literature: Millipore Corporation, A Short Guide Developing Immunochromatographic Test Strips, 2nd Edition, pp. 1-40, 1999, available by request at (800) 645-5476; and Schleicher & Schuell, Easy to Work with BioScience, Products and Protocols 2003, pp. 73-98, 2003, available by request at Schleicher & Schuell BioScience, Inc., 10 Optical Avenue, Keene, NH 03431, (603) 352-3810; both of these documents are incorporated herein by reference. Lateral flow devices exist in a variety of physical forms. This disclosure assumes any physical form that supports / houses the basic components of a lateral flow device in appropriate functional relationships.

[0072] In some embodiments, the lateral flow device of the present invention comprises an elongated housing containing a lateral flow strip extending substantially along the entire length of the housing. In some embodiments, the lateral flow strip is divided into a proximal sample application pad located below the sample introduction port, an intermediate test result membrane, and a distal absorption pad. This flow strip is interrupted by a conjugate pad containing a labeled conjugate. The flow path along the strip proceeds from the proximal pad through the conjugate pad to the test result membrane and finally to the absorption pad where it is collected. A selective binder (e.g., an exosome or an analyte-specific antibody) is positioned on the proximal test line within the test result membrane. A control line is provided within the test result membrane slightly distal to the test line. A liquid sample containing the target analyte is applied to the sample pad through the sample introduction port. In some embodiments, the sample is applied by dropping it into the sample introduction port or by immersing the end of the device having the sample introduction port into the sample. From the sample pad, the sample moves to the conjugate pad, for example, by capillary action. Within the conjugate pad, exosomes in the sample bind to the first capture region. After binding, a buffer to lyse or perforate the exosomes is added to the assay device. Next, the analyte binds (or is bound to) (or is bound to) a mobile or mobile detection reagent in the second capture region (and optionally a third (or more) capture region). For example, the analyte may bind to a labeled (e.g., gold-bound) antibody detection reagent contained in the conjugate pad. The analyte, having formed a complex with the detection reagent, then flows into the test result membrane. There, the complex may further interact with the capture reagent, such as an analyte-specific antibody immobilized on the proximal test line. The formation of an immunocomplex between the analyte, the labeled (e.g., gold-bound) detection reagent, and the immobilized antibody can be detected by the appearance of a visible line on the proximal test line. This is due to the accumulation of the label (e.g., gold) in a localized area of ​​the proximal test line. A detection reagent-specific binding partner may be immobilized on the control line. This can bind to the detection reagent with or without the analyte.Such coupling in the control line indicates that the test is functioning correctly, even when the target analyte is not present.

[0073] The materials used in a particular lateral flow device depend on several variables, including the analyte to be detected, the sample volume, and the desired flow rate. In some embodiments, a sample pad receives the sample and removes particles from it. In some embodiments, the sample pad is cellulose. The sample pad may be treated with release agents such as buffers, salts, proteins, detergents, and surfactants. These release agents are useful, for example, to promote the redissolution of the conjugate and pad components, and to block nonspecific binding sites in other components of the lateral flow device, such as nitrocellulose membranes. Typical release agents include, for example, trehalose or glucose (1-5%), PVP or PVA (0.5-2%), Tween20 or Triton X-100 (0.1-1%), casein (1-2%), SDS (0.02-5%), and PEG (0.02-5%).

[0074] The conjugate pad holds the detection reagent. In some embodiments, the detection reagent may be applied externally, for example, from a developer bottle, in which case the lateral flow device may not include a conjugate pad (see, for example, U.S. Patent No. 4,740,468). The detection reagent contained in the conjugate pad is typically released into the solution when the test sample is applied. The conjugate pad may be treated with various substances to influence the release of the detection reagent into the solution. For example, the conjugate pad may be treated with PVA or PVP (0.5% to 2%) and / or Triton X-100 (0.5%). Other release agents include hydroxypropyl methylcellulose, SDS, Brij, and β-lactose.

[0075] The absorbent pad plays a role in increasing the total volume of sample that enters the device. This increased volume is useful, for example, for washing away unbound analytes from a membrane. Various materials are useful for preparing the absorbent pad. Depending on the embodiment of the device, the absorbent pad may be paper (i.e., cellulose fiber). Those skilled in the art can select a paper absorbent pad based, for example, on thickness, compressibility, manufacturability, and uniformity of bed volume. The volume absorbed by the absorbent can be adjusted by changing the dimensions (usually length) of the absorbent pad.

[0076] Figure 1 shows a preferred embodiment of the lateral flow device of the present invention. As shown in Figure 1, the assay device comprises: a chromatographic test strip having multiple regions; the test strip allows a fluid test sample to flow from a first end to a second end of the chromatographic strip; the chromatographic strip comprises a first region containing an exosome conjugate, a second region containing a first binding partner that binds to a first analyte, and optionally, at least a second binding partner that binds to at least a second analyte; and further comprises a reagent for visualizing the binding of the analyte to the labeled binding partner, where the reagent for visualizing the binding of the analyte is preferably located within a conjugate pad.

[0077] In some preferred embodiments, the exosome binding agent in the first region is a tetraspanin. In particularly preferred embodiments, the tetraspanin is selected from the group consisting of CD9, CD63, CD81 and combinations thereof.

[0078] In some preferred embodiments, the second region includes one or more binding partners for at least one analyte, which bind to the exosome capture region and play a role in capturing the analyte released from exosomes lysed by contact with a chase buffer or lysis buffer. In some preferred embodiments, one or more binding partners are antibodies or functional fragments thereof that specifically bind to the desired analyte. In some embodiments, the first analyte is HIV p24. In preferred embodiments, the binding partner is an antibody or functional fragment thereof that binds to p24. In embodiments, at least the second analyte is selected from the group consisting of HIV proteins selected from Nef, Tat, Vpu, gp120 and combinations thereof. In preferred embodiments, at least the second binding partner is an antibody or functional fragment thereof that binds to Nef, Tat, Vpu, or gp120.

[0079] In another embodiment, the analyte is prostate-specific antigen (PSA). In some preferred embodiments, the assay of the present invention provides detection of PSA in EVs. In some preferred embodiments, an increase in PSA levels in EVs compared to a control indicates prostate cancer, and the measured PSA levels can be used to distinguish between patients with healthy prostates, patients with benign prostatic hyperplasia (BPH), and patients with prostate cancer.

[0080] In some preferred embodiments, the chromatography test strip is a nitrocellulose strip, but other materials can also be used as described above.

[0081] In some preferred embodiments, the chromatography strip is in fluid communication with the sample pad. In some embodiments, a sample, such as a biological sample containing exosomes, is applied to the sample pad when the device is used. In some embodiments, the biological sample may be diluted with running buffer.

[0082] In some preferred embodiments, the chromatography strip is in fluid communication with the conjugate pad. As described above, the conjugate pad preferably includes at least a first labeling-binding partner that binds to a first analyte (e.g., p24 or PSA). In some preferred embodiments, the first labeling-binding partner is an antibody or a functional fragment thereof. Suitable labeling has been described in detail above. In preferred embodiments, the first labeling-binding partner is labeled with colloidal gold. In preferred embodiments, the conjugate pad further includes at least a second labeling-binding partner that binds to at least a second analyte (e.g., Nef, Tat, Vpu, or gp120). In preferred embodiments, the at least second labeled-binding partner is an antibody or a functional fragment thereof. In preferred embodiments, the at least second labeled-binding partner is labeled with colloidal gold.

[0083] In some preferred embodiments, the device further includes an absorbent pad that communicates with a chromatography test strip and fluid.

[0084] Absorbent pads, conjugate pads, and sample pads may be formed from any suitable material. In a preferred embodiment, these pads are formed from an absorbent material (e.g., glass fiber, cellulose, etc.).

[0085] In some preferred embodiments, the chromatography test strip further includes a control region.

[0086] In some preferred embodiments, the user applies a biological sample (e.g., whole blood or plasma) containing extracellular vesicles with exosomes to a sample pad. The biological sample may be pre-diluted with running buffer, or the running buffer may be applied to the sample pad before, preferably after, applying the sample to the sample pad. The biological sample in the buffer flows from the sample pad to the chromatography strip and then to the absorption pad. The extracellular vesicles containing exosomes in the biological sample are captured as the buffer passes across the exosome capture region.

[0087] Next, the user applies a chase buffer or lysis buffer to the conjugate pad. The chase buffer or lysis buffer flows from the conjugate pad to the chromatography test strip. The chase buffer carries the labeled binding partner in the conjugate pad to the chromatography test strip while simultaneously containing components that dissolve or permeate the EVs captured in the EV capture region. In a preferred embodiment, the lysis buffer or chase buffer contains one or more reagents that dissolve or permeate the EVs. In a preferred embodiment, the reagents are selected from the group consisting of saponins, surfactants, and detergents, such as Triton, Triton X-100, Tween 20, sodium dodecyl sulfate, and / or deoxycholates.

[0088] Upon contact with the lysis buffer or chase buffer, the analyte is released from the EV, binds to its labeled binding partner (i.e., the detection reagent), and is captured in the second region (also called the detection region). This allows the user to confirm the detection of the analyte by visually analyzing the second region.

[0089] One embodiment of the present invention provides a kit used for detecting an analyte in a sample (e.g., a biological sample). Such a kit can be used, for example, to determine the presence of an infectious pathogen in a sample. Certain embodiments of the disclosed kit are generally portable and provide a simple, rapid, and / or cost-effective method for determining the presence or absence of an analyte, for example, in a clinical setting, without requiring laboratory equipment.

[0090] In some embodiments, the kit of the present invention includes one or more immunoassay devices disclosed herein, one or more antibodies and / or detection reagents, and means of transport such as boxes, bags, satchels, plastic cartons (such as molded plastic or other transparent packaging), packaging materials (such as sealed or sealable plastic, paper, or metal packaging materials), and other containers. In some examples, the components of the kit are housed in a single packaging unit such as a box or other container. This packaging unit may be provided with compartments capable of housing one or more components of the kit. In other examples, the kit may hold, for example, one or more biological samples to be tested, positive control and / or negative control samples or solutions (e.g., positive control serum containing the analyte), diluents (e.g., phosphate buffer or saline buffer), detection reagents (e.g., for external application to the kit device), substrate reagents for visualization of the detection reagent enzyme (e.g., 5-bromo-4-chloro-3-indolyl phosphate, nitroblue tetrazolium in dimethylformamide), and / or washing solutions (e.g., Tris buffer, saline buffer, or distilled water).

[0091] Other kit embodiments may include syringes, fingertip puncture devices, alcohol swabs, gauze, cotton balls, bandages, latex gloves, culture trays with a variable number of grooves, adhesive plate sealers, and data reporting sheets, which are useful for handling, collecting, and / or processing biological samples. The kit may optionally include instruments useful for introducing samples into the sample chamber of the immunoassay device. Examples include droppers, disposable pipettes, capillary tubes, and rubber bulbs (e.g., for capillary tubes). Further other kit embodiments may include disposal means for disposing of used immunoassay devices and / or other items used with the device (e.g., patient samples). Such disposal means may include, but are not limited to, containers capable of containing leakage from the waste, such as plastic, metal, or other impermeable bags, boxes, containers, etc.

[0092] In some embodiments, the kit of the present invention includes instructions for the use of an immunoassay device or antigen-coated plate. The instructions provide details on how to apply the sample to the test device or plate, the time required or recommended for waiting for the development of results, and how to read and interpret the test results. Such instructions may also include standards such as standard tables, graphs, and figures for comparing test results. These standards may optionally include information necessary for quantifying the analyte using the test device, such as standard curves showing the relationship between signal intensity or the number of signal lines and the amount of analyte in the sample.

[0093] III. Antibodies In some embodiments, the devices, kits, and methods of the present invention, as described in detail above, utilize antibodies that bind to an analyte (e.g., HIV protein) or other reagents in a sample to enable the measurement of the level of an analyte in the sample.

[0094] The antibody of the present invention may be either a monoclonal antibody or a polyclonal antibody produced against an analyte, as long as it can recognize the analyte. The antibody can be produced using a specific analyte or an analyte as an antigen, following a conventional antibody or antiserum production process.

[0095] The present invention envisions the use of both monoclonal antibodies and polyclonal antibodies, as well as their functional fragments. Any suitable method can be used to produce the antibodies used in the methods and compositions of the present invention, including but not limited to the methods disclosed herein. For example, in the preparation of monoclonal antibodies, the protein itself, or together with a suitable carrier or diluent, is administered to an animal (e.g., a mammal) under conditions that enable antibody production. A complete or incomplete Freund's adjuvant may be administered to enhance antibody production capacity. Typically, the protein is administered once every two to six weeks, for a total of about two to about ten times. Suitable animals for use in such methods include, but are not limited to, primates, rabbits, dogs, guinea pigs, mice, rats, sheep, and goats.

[0096] To prepare monoclonal antibody-producing cells, individuals with confirmed antibody titers (e.g., mice) are selected, their spleens or lymph nodes are collected 2 to 5 days after final immunization, and the antibody-producing cells contained therein are fused with myeloma cells to prepare the desired monoclonal antibody-producing hybridoma. The antibody titer in antiserum can be measured, for example, by reacting antiserum with a labeled protein (described later) and measuring the activity of the labeling agent bound to the antibody. Cell fusion can be performed according to known methods, such as the method described by Koehler and Milstein (Nature 256:495

[1975] ). As fusion promoters, polyethylene glycol (PEG) or Sendai virus (HVJ) can be used, with PEG being preferred.

[0097] Examples of myeloma cells include NS 1, P3U1, SP2 / 0, and AP 1. The ratio of antibody-producing cells (spleen cells) to the number of myeloma cells used is preferably about 1:1 to about 20:1. PEG (preferably PEG 1000 or PEG 6000) is preferably added at a concentration of about 10% to about 80%. Cell fusion can be efficiently performed by incubating the mixture of both cells at about 20°C to about 40°C, preferably about 30°C to about 37°C, for about 1 to 10 minutes.

[0098] Various methods are used to screen hybridomas that produce antibodies (e.g., antibodies against a specific analyte). For example, one method involves adding the supernatant of a hybridoma to a solid phase (e.g., a microplate) to which antibodies are adsorbed, either directly or together with a support. Subsequently, an anti-immunoglobulin antibody (anti-mouse immunoglobulin antibody is used if mouse cells are used for cell fusion) or protein A labeled with a radioactive substance or enzyme is added to detect monoclonal antibodies against the protein bound to the solid phase. Alternatively, the supernatant of a hybridoma is added to a solid phase to which anti-immunoglobulin antibodies or protein A are adsorbed, and then a protein labeled with a radioactive substance or enzyme is added to detect monoclonal antibodies bound to the solid phase.

[0099] The selection of monoclonal antibodies can be carried out by any known method or an improved method. Typically, animal cell culture media supplemented with HAT (hypoxanthine, aminopterin, thymidine) are used. Any selection and growth medium is acceptable as long as hybridomas can grow. For example, RPMI 1640 medium containing 1% to 20%, preferably 10% to 20%, fetal bovine serum, GIT medium containing 1% to 10%, fetal bovine serum, and serum-free hybridoma culture medium (SFM 101, Nissui Pharmaceutical) can be used. Typically, culture is carried out under approximately 5% CO2 gas at 20°C to 40°C, preferably 37°C, for about 5 days to 3 weeks, preferably 1 to 2 weeks. The antibody titer in the hybridoma culture supernatant can be measured in the same manner as described above for the antibody titer against antiproteins in antiserum.

[0100] The isolation and purification of monoclonal antibodies can be carried out in the same way as for conventional polyclonal antibodies, for example, in the same way as for immunoglobulins. Specifically, these methods include salting out, alcohol precipitation, isoelectric focusing, electrophoresis, adsorption and desorption using ion exchange resins (e.g., DEAE), ultracentrifugation, gel filtration, or specific purification methods that involve recovering only the antibody using an antigen-binding solid phase, active adsorbents such as protein A or protein G, and then dissociating the binding to obtain the antibody.

[0101] Polyclonal antibodies can be prepared by any known method or an improvement on such method, including methods for obtaining antibodies from patients. For example, a complex of an immunogen (antigen against a protein) and a carrier protein is prepared, and animals are immunized using this complex in a manner similar to that described for the preparation of monoclonal antibodies above. The antibody-containing material is recovered from the immunized animals, and the antibodies are separated and purified.

[0102] For immunogen-carrier protein complexes used in animal immunization, any carrier protein and any mixing ratio of carrier to hapten can be used, as long as antibodies against the hapten, which is cross-linked on the carrier and used for immunization, are efficiently produced. For example, bovine serum albumin, bovine cycloglobulin, keyhole limpet hemocyanin, etc., can be bound to hapten in a weight ratio of about 0.1 to about 20 parts, preferably about 1 to about 5 parts, per part of hapten.

[0103] Furthermore, various condensation agents can be used to bind the hapten to the carrier. For example, glutaraldehyde, carbodiimide, maleimide activated esters, and activated ester reagents containing thiol or dithiopyridyl groups are useful in the present invention. The condensation product is administered alone or with a suitable carrier and diluent to an animal site capable of antibody production. To enhance antibody production capacity, a complete or incomplete Freund's adjuvant may be administered. Typically, this protein is administered once every 2 to 6 weeks for a total of about 3 to 10 doses.

[0104] Polyclonal antibodies are recovered from the blood, ascites, etc., of animals immunized using the method described above. The antibody titer in the antiserum can be measured in the same way as described above for hybridoma culture supernatant. Antibody isolation and purification can be carried out in the same way as the immunoglobulin isolation and purification methods described above for monoclonal antibodies.

[0105] In addition to polyclonal and monoclonal antibodies, other antigen-binding proteins that bind to the target of analysis can be used. Examples of other antigen-binding proteins include chimeric antibodies, humanized antibodies, Fab fragments, F(ab')2 fragments, and single-chain antibodies (scFv). [Examples]

[0106] Example 1 - HIV detection This example describes a highly sensitive test for human immunodeficiency virus (HIV) that uses EV concentration and capture to search for EV cargo as an HIV-specific antigen. HIV viral proteins are present in EV (i.e., "EV cargo") isolated from blood 20,24-27 and urine 28 of viremia and non-viremia patients (<20 viral copies / mL). For example, Aqil et al., Transcriptomic Analysis of mRNAs in Human Monocytic Cells Expressing the HIV-1 Nef Protein and Their Exosomes. Biomed Res. Int. 2015, 1-10 (2015); Aqil et al., The HIV Nef protein modulates cellular and exosomal miRNA profiles in human monocytic cells. J. Extracell. Vesicles 3, 23129 (2014). Lenassi et al., HIV Nef is Secreted in Exosomes and Triggers Apoptosis in Bystander CD4+ T Cells. Traffic 11, 110-122 (2010). Khan et al., Nef exosomes isolated from the plasma of individuals with HIV associated dementia (HAD) can induce Aβ1-42 secretion in SH-SY5Y neural cells. J. Neurovirol. 22, 179-190 (2016). See Anyanwu et al., Detection of HIV-1 and Human Proteins in Urinary Extracellular Vesicles from HIV+ Patients. Adv. Virol. 2018, 1-16 (2018).The presence and persistence of HIV proteins in extracellular vesicles (EVs) have facilitated basic research into the mechanisms by which EVs contribute to HIV pathogenicity and disease progression. See, for example, Arakelyan et al., Extracellular Vesicles Carry HIV Env and Facilitate HIV Infection of Human Lymphoid Tissue. Sci. Rep. 7, 1695 (2017); Dias et al., The Ambiguous Roles of Extracellular Vesicles in HIV Replication and Pathogenesis. Front. Microbiol. 9, (2018); Perez et al., Extracellular vesicles and chronic inflammation during HIV infection. J. Extracell. Vesicles 8, 1687275 (2019).

[0107] This example illustrates the use of the presence or absence of HIV protein in extravasation cells (EVs) for diagnostic purposes.

[0108] Materials and methods: Figure 1 shows an overview of the EV-HIV assay.

[0109] Western blot material: 4-12% Bis-Tris Gradient Gel, Fisher Scientific NP0321BOX 20X MOPS SDS Running Buffer 4X NuPage LDS Sample Buffer DTT Recombinant p24 Novex Sharp Pre-Stained Ladder NuPage Transfer Buffer Methanol, Fisher Scientific Mouse anti-p24 monoclonal antibody Goat anti-mouse HRP conjugate Goat anti-mouse alkaline phosphatase conjugate 10X Washing Buffer dried milk Novex PVDF film SuperSignal West PicoPlus HRP Substrate BCIP / NBT alkaline phosphatase substrate

[0110] method: In short, samples were electrophoresed on a 4-12% gradient gel at 180V for 1 hour, then transferred to a PVDF membrane at 30V for 1 hour. The blots were blocked with 5% milk blotting buffer for at least 1 hour. After washing the blots three times, they were probed overnight with the target antibody diluted in blotting buffer. After washing the blots three times, they were probed for 1 hour with the detection antibody diluted in blotting buffer. After washing the blots, they were incubated with the detection substrate according to the substrate manufacturer's instructions.

[0111] ELISA material: HIV-1 p24 ELISA kit, Sinobiological

[0112] method: The p24 ELISA was performed according to the manufacturer's instructions. Briefly, a standard curve was created. Samples were appropriately diluted according to the sample (typically 1:100). Standards and samples were added to a 96-well plate and incubated for 2 hours. The wells were washed three times, and 100 μL of diluted detection antibody was added to each well. After incubating the plate for 1 hour, each well was washed three times. 100 μL of substrate was added to each well, and the plate was incubated for 30 minutes. The reaction was stopped, and each well was measured at 450 nm using a Tecan GenIOS SpectraFluor plus plate reader (Mannedorf, Switzerland).

[0113] Exosome purification method material: ExoQuick-TC, System Biosciences EXOTC10A-1 ExoQuick, System Biosciences EXOQ5A-1 Thrombin Plasma Prep Kit, System Biosciences TMEXO-1 MagCapture Exosome Isolation Kit V2, Fujifilm 294-84101 1X PBS, Intuitive Bioscience B111 Heparin sodium, Fisher Scientific AC411210010

[0114] method: Two different methods were used to purify exosomes: affinity separation using magnetic beads and polymer precipitation. Both methods were carried out according to the manufacturer's instructions, and a brief description is provided below. Both methods used different materials and processes for tissue culture and human plasma. A complete explanation is provided below.

[0115] Affinity isolation, MagCapture Exosome Isolation Kit V2 (for tissue culture): Magnetic beads are prepared by equilibrating them with washing buffer. Cells and cell residue are precipitated by continuous centrifugation with gradually increasing centrifugal force. The supernatant is mixed with an exosome binding enhancer, 5 U / mL heparin sodium, and the pre-equilibrium beads, and incubated for 1 hour. After washing the beads, bound exosomes are collected using exosome elution buffer.

[0116] Affinity separation, MagCapture Exosome Isolation Kit V2 (for human plasma): Prepare the magnetic beads by equilibrating them with washing buffer. Precipitate the cells and cell residue by continuous centrifugation with gradually increasing centrifugal force. Mix the supernatant with the exosome binding enhancer and the pre-equilibrium beads and incubate for 1 hour. After washing the beads, collect the bound exosomes using exosome elution buffer.

[0117] Precipitation separation method, ExoQuick-TC (for tissue culture): Collect the cell culture supernatant and centrifuge at 3,000xg for 15 minutes. Transfer the supernatant to a new tube and add 200 μL of ExoQuick-TC per 1 mL of supernatant. Mix the tubes and incubate overnight at 2–8°C. Centrifuge at 1,500xg for 30 minutes. Remove the supernatant by aspirate and resuspend the precipitate in 500 μL of 1X PBS.

[0118] Precipitation separation method, ExoQuick (for human plasma): To increase the exosome yield from human plasma, add 2 μL of thrombin plasma preparation to 250 μL of human plasma. Incubate for 5 minutes. Centrifuge the samples at 10,000 x g for 5 minutes and transfer the supernatant to a new tube. Add 63 μL of ExoQuick to each sample and incubate at 2-8°C for 30 minutes. After incubation, centrifuge the samples at 1,500 x g for 30 minutes. Remove the supernatant by aspirate and resuspend the precipitate in 200 μL of 1X PBS.

[0119] Isolation of exosomes from plasma Exosomes were separated using the Norgen resin purification method (Norgen Biotek 57400) according to the manufacturer's instructions. A standard volume of 250 μL of each plasma sample was centrifuged at 2,500 x g to remove cellular residue, and the precipitate was discarded. The sample was adjusted to 1 mL in 750 μL of ultrapure water. Next, 3 mL of nuclease-free water, 100 μL of ExoC buffer, and 200 μL of slurry E were added to each sample and allowed to stand at room temperature for 5 minutes. Then, the sample was vortexed for 10 seconds and centrifuged at 2,000 RPM for 2 minutes. After discarding the supernatant, 200 μL of ExoR buffer was added to the sample pellet and allowed to stand at room temperature for 5 minutes. The sample was vortexed for 10 seconds and centrifuged at 500 RPM for 2 minutes. The supernatant of each sample was transferred to a minifilter spin column assembled in a 2 mL tube and centrifuged at 6,000 rpm for 1 minute. The exosomes were present in the sample collected in a 2 mL tube.

[0120] Cells and serum material: H9MN full-length intact (FI) cells: Generously provided by Dr. Cliff Lane, Department of Research, National Institute of Allergy and Infectious Diseases, NIH. H9 cells, ATCC HTB-176 HIV-positive human plasma, manufactured by Precision for Medicine (multiple lots) HIV+ human plasma, manufactured by Precision for Medicine (multiple lots) RPMI 1640, Gibco 1187-085 Fetal bovine serum, Fisher Scientific 10437028 Glutamine, Fisher Scientific 25030081 Gentamicin, Gibco 15710-064 HEPES, Gibco 15630-080 Exosome-free fetal bovine serum, Fisher Scientific A27208-03

[0121] method: Cell lines were maintained at 37°C and 5% CO2 conditions using RPMI modified with fetal bovine serum, glutamine, HEPES, and gentamicin, as needed. Before exosome purification, cells were divided into medium prepared with exosome-depleted FBS and cultured for a minimum of 36 hours.

[0122] Preparation of cell lysate material: BCA Kit, Fisher Scientific 23225 RIPA buffer, Fisher Scientific 89900 HALT protease inhibitor, Fisher Scientific 87785

[0123] method: Tissue culture cells were lysed according to the manufacturer's instructions for RIPA buffer. Briefly, the lysis buffer was prepared by adding a HALT protease inhibitor to RIPA buffer. The cells were precipitated by centrifugation, resuspended in the prepared lysis buffer, and lysed at 2-8°C for 15 minutes. The lysis reaction mixture was centrifuged at 14,000xg for 15 minutes to precipitate insoluble debris. The supernatant was transferred to a new tube. The protein concentration of the lysed cell pellet was measured using a BCA kit according to the manufacturer's instructions.

[0124] Nanoparticle tracking analysis material: Purified exosomes PBS

[0125] method: The purified exosomes were analyzed using a NanoSight NS300 (Malvern Panalytical, Malvern, UK). Specifically, the particles were resuspended using a vortex pump, diluted with PBS, and then injected into the NS300 for size analysis and particle counting.

[0126] Measurement of exosome protein concentration. Concentration was measured using a micro-BCA assay. Standards were prepared according to the manufacturer's instructions. 2 μL of sample or standard was added to a 96-well plate, and 40 μL of freshly prepared BCA working reagent was added. After incubation at 37°C for 30 minutes, the samples were cooled to room temperature and measured using a Tecan Infinite M Nano (using a NanoQuant plate) or an Eppendorf Biophotometer (using a μCuvette).

[0127] Abby® Automated Chemiluminescence Western Protein Analyzer. The Abby Automated Western Protein Assay Device Platform (ProteinSimple, Bio-Techne) separates proteins by molecular weight using microvolume capillaries, followed by immunodetection. The separated proteins are immobilized on the capillaries by UV light, probed with antibodies and HRP-labeled secondary antibodies, and detected by chemiluminescence. A molecular weight ladder is performed for each full cartridge consisting of 25 capillaries, and the loading dye in each cartridge contains an additional molecular weight marker to compensate for molecular weight variations calculated between capillaries. A typical process involves preparing samples at the appropriate protein concentration, mixing them with loading dyes to denaturate them, and then loading them into the appropriate rows on a dedicated plate. Next, the target antibody is diluted and placed in the appropriate row on the same plate. Buffers, diluents, wash buffers, and detection reagents are added to the plate. The plate is then inserted into the Abby system along with the capillaries to begin the operation. Since the samples and antibodies are loaded into individual wells,

[0128] Exosome immunocapsulation lateral flow material: Anti-human CD9 antibody, 40nm gold colloid, CN140 nitrocellulose membrane Polyester conjugate pad Sample pad, Human anti-CD63 antibody Human CD81 antibody HEPES Running Buffer (10mM HEPES, pH 7.4, 150mM NaCl, 0.05% Tween20, 1% BSA) PBS-T running buffer (using PBS and 0.1% Tween20 buffer)

[0129] method: The CD9 antibody used for detection was adsorbed onto gold colloid. Anti-human CD63 antibody and anti-human CD81 antibody were stripped at a concentration of 0.1 μL / mm onto CN140 layered nitrocellulose membranes using a benchtop Isoflow device (Arista Biologicals). The stripped membranes were dried overnight at 37°C and then stored in a dry state. Purified exosomes were thawed and diluted to 10 μg / mL with HEPES running buffer. 100 μL of diluted exosomes or HEPES buffer alone was incubated in a microtiter plate with 30 GU of anti-CD9 gold conjugate. Nitrocellulose strips equipped with tetraspanin test lines and sample adsorption pads were added to the microtiter wells and allowed to flow for 15 minutes.

[0130] HIV p24 Lateral Flow and Test EV Lysis Buffer material: 40nm gold colloid CN140 Nitrocellulose Membrane Polyester conjugate pad Sample pad Anti-p24 antibody, used as a capture reagent. Anti-p24 antibody, used as a detection reagent. HEPES Running Buffer (10mM HEPES, pH 7.4, 150mM NaCl, 0.05% Tween20, 1% BSA) PBS-T running buffer (using PBS and 0.1% Tween20 buffer) Glass fiber conjugate pad Freeze-dried exosomes, various origins BBS Sucrose Recombinant p24 Triton X-100 saponin RIPA cell lysis buffer (25 mM Tris HCl (pH 7.6), 150 mM NaCl, 1% Tergitol NP 40, 1% sodium deoxycholic acid, 0.1% SDS) M-PER (trademark) Mammalian protein extraction reagent, 78501 HEPES buffer (10mM HEPES (pH 7.4), 150mM NaCl, 0.05% Tween20, 1% BSA) Running buffer (2mM Tris-HCl pH 7.6, 0.1% NP-40, 0.1% sodium deoxycholic acid, 0.01% SDS, 9mM HEPES pH 7.4, 150mM NaCl, 0.045% Tween20, 0.9% BSA) HALT 0.1X (AEBSF 0.1mM, aprotinin 80nM, bestatin 5μM, E64 1.5μM, leupeptin 2μM, pepstatin A 1μM, DMSO)

[0131] method: Both anti-p24 antibodies were diluted in sucrose borate buffer. The detection anti-p24 antibody was adsorbed onto gold colloid. 30 GU of 53M gold conjugate at pH 7.0 was spotted onto the conjugate pad and dried at 37°C for 30 minutes. The capture anti-p24 antibody was stripped at a concentration of 0.1 μL / mm onto a CN140 layered nitrocellulose membrane using a benchtop Isoflow device (Arista Biologicals, Pennsylvania). The stripped membrane was dried overnight at 37°C and then stored in a dry state. The prototype lateral flow test was assembled into a square cassette and sealed with a cassette press. Exosomes purified from H9(p24-) or H9MN FI(p24+) cells were diluted in HEPES running buffer with or without detergent or surfactant. For each condition, 100 μL of sample was added to each test and allowed to flow for 15 minutes. Evaluation was then performed using a visual scorecard or image quantification by concentration measurement analysis using ImageJ. Schneider, CA, Rasband, WS, & Eliceiri, KW (2012). NIH Image to ImageJ: 25 years of image analysis. Nature Methods, 9(7), 671-675.

[0132] Automated Western blotting was performed using an Abby (Simple Western) device with a 12-200 kDa separation module. Depending on the primary antibody, either an anti-mouse or anti-rabbit detection module was used. Peak values ​​corresponding to the target protein were quantified using Compass software, and the area under the curve (AUC) was measured for each band within each lane.

[0133] result Chronically infected H9MN FI cells express p24 in whole cell lysates and purified exosomes. H9 (parental strain, HIV-cell line) and H9MN FI (stable provirus, HIV-+ cell line) cells were cultured in culture medium supplemented with 10% FBS. At least 24 hours before harvesting, the cells were washed and the culture medium was replaced with exosome-free medium. After 24 hours, the cells were collected and centrifuged to precipitate the cell aggregate. The precipitate was treated with RIPA buffer to lyse the cells. Exosomes (EVs) were purified using the MagCapture method with the supernatant from centrifugation. 2.5 μg of lysate was flowed per lane, and 5 μg of purified exosomes per lane (Figure 2). Detection of p24 by mouse anti-p24 Gag monoclonal antibody was confirmed by flowing 0.5 μg of recombinant p24 protein in a separate lane.

[0134] Exosomes derived from H9MN FI cell lines, enriched using two different methods, contain high concentrations of p24 protein. To confirm that the detected p24 originated from exosomes, exosomes purified from H9 cells and H9MN FI cells were characterized, and their quantification and size distribution were confirmed. Exosome concentration and size distribution were measured using NanoSight NS300 (Malvern Panalytical, UK). EV particles derived from H9MN FI cells measured 3.5 × 10⁶. 9 Particles / mL, derived from H9 cells: 1.0 × 10⁶ 9 The particle count per mL was confirmed to be within the exosome size range (30-150 nm), and the particle count was also similar (Figures 3-4).

[0135] Exosomes purified from HIV-positive plasma contain HIV p24. Exosomes were purified from human plasma samples using the ExoQuick method and the MagCapture method for both HIV- and HIV-+ subjects. The size and quantity of the purified exosomes were analyzed using NanoSight 300. The results are summarized in Table 1. The number of particles per mL was similar for both exosome purification methods. When tested with p24 ELISA, exosomes derived from H9 cells showed a signal below the detection limit. Exosomes derived from H9MN FI cells had similar HIV p24 protein content in ng / mL units, regardless of the purification method.

[0136] [Table 1]

[0137] In addition to exosome characterization using NanoSight, CD63 ELISA was performed to confirm that the exosomes contained tetraspanin markers used in lateral flow. p24 ELISA was performed on the same samples to measure the amount of detected HIV-specific protein. In HIV-plasma samples, exosomes were detected by both NanoSight and CD63 ELISA, but no measurable amount of p24 was detected. In HIV-+ plasma, NanoSight and CD63 ELISA confirmed the presence of exosomes, while p24 ELISA confirmed the presence of HIV protein in the exosome preparation.

[0138] [Table 2]

[0139] Confirmation of HIV protein detection in purified extracellular viable (EV). To further demonstrate the presence of HIV proteins in extracellular vesicles (including exosomes) derived from human plasma, extracellular vesicles (EVs) were concentrated from plasma samples using the silicon carbide resin method. Extracellular vesicles were concentrated from 0.2 mL of plasma from HIV- and HIV-+ subjects and analyzed using an automated Western blotting system. A total of 0.4 mg of concentrated EV per sample was analyzed. The levels of HIV proteins p24, Nef, and Tat in the samples were measured using specific antibodies. As shown in Figure 5, EVs concentrated from HIV-+ samples contained significantly higher levels of p24, Nef, and Tat compared to HIV- samples. This confirms the presence of HIV proteins within EVs and demonstrates their usefulness as diagnostic biomarkers for HIV infection.

[0140] Detection of extracellular viable cells (EVs) by lateral flow precipitation. To purify and concentrate exosomes using the point-of-care method, a lateral flow assay for exosome immunocapture was performed according to the method of Oliveria-Rodriguez et al. (J Extracell Vesicles. 2016; 5: 10.3402 / jev.v5.31803.). Briefly, CD63 and CD81 capture antibodies were coated onto nitrocellulose lateral flow strips. Exosome capture was determined using an anti-CD9 gold conjugate as the detection antibody. As shown in Figure 6, the tetraspanin lateral flow assay was capable of capturing 10 μg / mL of exosome protein, and the calculated LOD was 2.5–5 μg / mL, or approximately 5 × 10⁶. 7 This corresponds to one exosome particle (Figure 7). The LOD was calculated from the signal intensity measured using the portable lateral flow reader, the DPP® MicroReader (ChemBio Diagnostics, Medford, New York). This demonstrated successful capture of exosomes by CD63 / CD81 immunocapture on the lateral flow device, with low nonspecific binding and a visible specific signal.

[0141] We demonstrate lateral flow detection of HIV p24 from purified exosomes released using a lysis buffer. When p24 is present within exosomes, it is encapsulated as a cargo protein within the exosome. To more effectively detect HIV cargo proteins within exosomes, we conducted tests to identify buffer formulations that can release exosomal cargo without interfering with the function of capture and detection antibodies. To test these formulations, we developed standard p24 lateral flow strips using anti-p24 capture antibodies and gold-labeled detection antibodies.

[0142] We will demonstrate that EVs can be dissolved and their cargo can be captured by lateral flow. We investigated whether the amount of p24 contained in exosomes isolated from H9 (HIV-) cells and H9MN FI (HIV-+) cells could be detected by a lateral flow assay. Lateral flow technology is not considered a highly sensitive method for biomarker detection. However, the isolation of extracellular vesicles should concentrate p24, increasing the detectable amount. To verify this, we screened several buffers with properties that disrupt lipid rafts present in high concentrations in extracellular vesicles and exosomes, and identified formulations that release EV cargo while being compatible with lateral flow technology.

[0143] Purified exosomes were incubated with various buffers containing surfactants and chemicals known to disrupt membranes. Saponins, in particular, have been used in exosome loading methods due to their ability to create pores in exosomes (Chen et al., Front. Cell Dev. Biol., 08 October 2021). The main property of saponins is their interaction with membrane-bound cholesterol, forming pores in the exosome membrane. Furthermore, they can even lyse red blood cells (Jamur and Oliver, 2010) or HIV virus particles (Bonisch et al., J Virol. 2020 Apr; 94(7): e01024-19). For HIV virus particles, saponin treatment causes the viral membrane to become permeable, releasing the viral contents. Triton also interacts with the membrane, creating permeability, thus releasing detectable cargo (contents). Other surfactants such as Triton X-100, Tween20, sodium dodecyl sulfate, and sodium deoxycholate can also be used (Xabier Osteikoetxea et al., Org Biomol Chem. 2015 Oct 14;13(38):9775-82. doi: 10.1039 / c5ob01451d.).

[0144] The effectiveness of exosome lysis was tested by incubating enriched exosomes from H9(p24-) and H9MN FI(p24+) cells in various buffers. An example of a standard p24 lateral flow test is shown in Figure 8. Briefly, exosomes purified from H9 and H9MN FI cells were incubated in lysis buffer (2.5 mM bicine, 9 mM HEPES, 135 mM NaCl, 0.045% Tween20, 0.9% BSA, pH 7.4) at room temperature for 15 minutes, and then transferred to the sample loading port of a lateral flow device. After the samples were allowed to flow at room temperature for 15 minutes, visual readings were scored on a scale of 1 to 10. As shown in Figure 8C, both the buffer alone and H9 exosomes scored 3. H9MN FI exosomes scored 6, indicating successful capture of p24 from the lysed exosome preparation.

[0145] Other lysis buffers were also tested, and the results of visual line testing are shown in Table 3. The addition of surfactants and detergents increased the amount of detectable p24 in purified exosomes. Furthermore, sequential addition of EV lysis buffers was important for background reduction in buffer-only and H9 exosomes. Specifically, lysis buffer #2 (RPIA buffer containing 10% protease inhibitor) was added to purified exosomes for 15 minutes to promote lysis. This was diluted with an equal volume of HEPES and added to a lateral flow device. This reduced the background signal and maintained the detection of p24 in H9MN FI EV (Table 3, lysis buffer #2).

[0146] [Table 3]

[0147] A two-stage lateral flow device was assembled to capture and lysate EVs at a location separated from the p24 test line. This prevented lysis buffer components from interfering with p24 capture. The EV capture regions (anti-CD9, anti-CD63, anti-CD81) were positioned on a nitrocellulose membrane as shown in Figure 9A. The anti-p24 test line was positioned on another nitrocellulose membrane downstream. By adding EVs isolated from H9(p24-) and H9MN FI(p24+) cells with running buffer, the EVs were captured in the test region. Next, EV lysis buffer was added to release the EV contents, which were then passed through the anti-p24 gold conjugate region and captured in the p24 test region. A total of 30 μg of EVs isolated from H9 cells showed a visual score of 0, while 5 μg of EVs from H9MN FI cells showed a score of 4, demonstrating that p24 can be detected from EVs from the HIV protein-expressing H9 MNFI cell line after lysing the immunocaptured EVs.

[0148] As shown in Figure 10, the amount of p24 detectable from EVs may increase when different detergents or EV permeabilization reagents are used. Figure 10 shows images of strips using PBS (buffer only), 20 μg of EVs from H9 cells, and 20 μg of EVs from H9 MNFI cells under each condition. Below each image, the numerical values ​​obtained from the concentration measurement analysis of the strips are indicated. Compared to EVs in PBS buffer (undissolved), it is observed that the amount of detectable p24 from H9 MNFI-derived EVs increases when detergents or membrane disruptors are used.

[0149] Any method used for permeabilization can be incorporated into a lateral flow device to release the EV's cargo. This includes, but is not limited to, chemical methods using cations such as calcium phosphate, physical methods such as detergents and surfactants, ultrasound, electroporation, and photoporation (Zhang et al., Innovative Methodology 10 Jan 2017 https: / / doi.org / 10.1152 / ajplung.00423.2016; Guido et al., Microelectronic Engineering Volume 98, October 2012, Pages 707-710).

[0150] Extracellular vesicles containing exosomes can be immunoprecipitated on a lateral flow device, and their internal cargo can be detected by a lateral flow assay.

[0151] Example 2: Prostate cancer PSA lateral flow This example describes the detection of prostate-specific antigen (PSA) in extravasation plasma (EV).

[0152] Figures 11 and 12 are schematic diagrams showing one embodiment of the EV-PSA test.

[0153] material: 40nm gold colloid CN140 Nitrocellulose Membrane Polyester conjugate pad, Sample pad, Anti-PSA capture antibody Anti-PSA detection antibody HEPES Running Buffer (10mM HEPES, pH 7.4, 150mM NaCl, 0.05% Tween20, 1% BSA) PBS-T running buffer (using PBS and 0.1% Tween20 buffer) Glass fiber conjugate pad, Freeze-dried exosomes, various origins BBS Sucrose Recombinant PSA Triton X-100 saponin RIPA cell lysis buffer (25 mM Tris HCl (pH 7.6), 150 mM NaCl, 1% Tergitol NP 40, 1% sodium deoxycholic acid, 0.1% SDS) M-PER (trademark) Mammalian Protein Extraction Reagent, 78501 HEPES buffer (10mM HEPES (pH 7.4), 150mM NaCl, 0.05% Tween 20, 1% BSA) Running buffer (2mM Tris-HCl pH 7.6, 0.1% NP-40, 0.1% sodium deoxycholic acid, 0.01% SDS, 9mM HEPES pH 7.4, 150mM NaCl, 0.045% Tween20, 0.9% BSA) HALT 0.1X (AEBSF 0.1mM, aprotinin 80nM, bestatin 5μM, E64 1.5μM, leupeptin 2μM, pepstatin A 1μM, DMSO)

[0154] method: Both anti-PSA antibodies were diluted in sucrose borate buffer. The detection anti-PSA antibody was adsorbed onto gold colloid. 30 GU of 53 M gold conjugate at pH 7.0 was spotted onto the conjugate pad and dried at 37°C for 30 minutes. The other anti-PSA antibody was stripped at a concentration of 0.1 μL / mm onto a CN140 layered nitrocellulose membrane using a benchtop Isoflow device (Arista Biologicals, Pennsylvania). The stripped membrane was dried overnight at 37°C and then stored in a dry state. The prototype lateral flow tests were assembled into square cassettes and sealed with a cassette press. Exosomes purified from healthy plasma, PC3 (PSA-) cells, and LNCaP (PSA+) cells were diluted in HEPES running buffer with or without detergent or surfactant. For each condition, 100 μL of sample was added to each test and allowed to flow for 15 minutes. Evaluation was then performed using a visual scorecard, or image quantification was performed by concentration measurement analysis using ImageJ. Schneider, CA, Rasband, WS, & Eliceiri, KW (2012). NIH Image to ImageJ: 25 years of image analysis. Nature Methods, 9(7), 671-675.

[0155] Prostate-specific antigen (PSA) is detected using the EV capture LF format. In short, anti-CD63 antibody and anti-CD81 tetraspanin-capturing antibody were coated in stripes on nitrocellulose lateral flow strips, and EVs were captured upstream of the test area (Figures 13A and B). As shown in Figure 13, the LOD of the strips was measured using solutions containing purified PSA at different concentrations. The signal intensity associated with the test line was recorded 15 minutes after imaging. The detection limit was calculated to be 7.8 pg / mL using signal intensity measured with a portable lateral flow reader (DPP® Micro Reader, Chembio Diagnostics, Medford, New York) (Figures 13C and D).

[0156] To verify EV capture from prostate cancer cell lines, high-purity exosomes purified from PC3 cells (a prostate cancer cell line that does not express PSA) were compared with exosomes derived from LNCaP cells (a prostate cancer cell line that expresses PSA). Equimolar mixtures of anti-CD9, anti-CD63, and anti-CD81 were applied to the EV capture region. Gold-labeled detection antibodies corresponding to CD9, CD63, and CD81 were used as detection reagents. Visual score values ​​for each EV type using each detection antibody are shown. As shown in Figure 14, all three tetraspanins are present in LNCaP-derived EVs. In contrast, CD81 is hardly detectable in PC3-derived EVs. Furthermore, EVs from healthy human plasma are not detected with the anti-CD81 gold-labeled antibody. This data suggests that PSA-containing EVs can be enriched by using different combinations of tetraspanin-capturing antibodies. This allows for customization of the EV capture region for lysis by selective immunoprecipitation, enabling further enrichment. In this example, by using anti-CD81 in the capture region, PSA-containing EV can be selectively concentrated from the input sample.

[0157] PSA-containing extracellular viable cells (EVs) are detected in PCa cell lines. Extracellular proteins (EVs) isolated from LNCaP (PSA+) cells were compared with EVs isolated from "normal" plasma (low PSA). The LNCaP cell line is an androgen-sensitive PSA-expressing cell line derived from metastatic prostate cancer patients. Purified EVs were analyzed for total protein content by BCA and the number of EV particles per mL was measured by NTA. Purified EVs were added at various concentrations ranging from 3.125 to 25 μg / mL and measured using total PSA ELISA (Alpco, Salem, New Hampshire). At all input concentrations, the detected total PSA increased 2 to 20 times in LNCaP cell-derived EVs. The amount of total PSA measured in LNCaP-derived EVs increased with incubation in 25% RIPA buffer. This is consistent with the hypothesis that EV lysis promotes the detection of EV transport proteins. Total PSA exceeding 2 ng / mL was detected in purified EVs at 3.25 μg / mL (Figure 15).

[0158] The two-phase EV capture lateral flow technology enables highly sensitive detection of PSA from EVs. Using purified LNCaP(PSA+)-derived extravasation veneers (EVs), the PSA detection sensitivity was improved by adding a lysis buffer. As shown in Figure 16, the addition of 0.25X RIPA enhanced the visible detection of PSA on the test line. The signal also increased with increasing EV levels. This demonstrates that EV dissolution increases the detectable amount of PSA.

Claims

1. A method for detecting the presence of an analyte in a sample derived from the target, the following: Collect a patient sample containing extracellular vesicles; To capture extracellular vesicles derived from the patient sample; Lysizing the extracellular vesicles and releasing the proteins contained therein; Contacting the protein with one or more analyte-specific binders and / or one or more detection reagents suitable for detecting the analyte; and To determine whether the extracellular vesicles contain the subject of analysis, Methods that include...

2. The method according to claim 1, wherein the object of analysis is a protein.

3. The method according to claim 1 or 2, wherein the object of analysis is a cancer antigen or a cancer-specific protein.

4. The method according to claim 3, wherein the object of analysis is prostate-specific antigen (PSA).

5. The method according to claim 2, wherein the protein is derived from an infectious pathogen, and the presence of the analyte in the sample indicates infection of the analyte in the subject.

6. The method according to claim 5, wherein the infectious pathogen is a virus or a bacterium.

7. The method according to claim 1, wherein the object to be analyzed is nucleic acid.

8. The method according to claim 1 or 2, wherein the analyte is HIV protein p24, and optionally comprises at least one additional HIV protein selected from the group consisting of Nef, Tat, Vpu, and gp120.

9. A method for diagnosing human immunodeficiency virus (HIV) infection in a subject, the following: Collect a patient sample containing extracellular vesicles; To capture extracellular vesicles derived from the patient sample; Lysizing the extracellular vesicles and releasing the proteins contained therein; Contacting the protein with one or more HIV protein binders and / or one or more detection reagents suitable for detecting one or more HIV proteins; and To determine whether the extracellular vesicle contains HIV protein p24 and at least one additional HIV protein optionally selected from the group consisting of Nef, Tat, Vpu, and gp120. A method comprising determining the presence of p24 and at least one additional HIV protein, which indicates HIV infection in the subject.

10. The method of claim 9, wherein the at least one additional HIV protein is Nef.

11. The method of claim 9, wherein the at least one additional HIV protein is Tat.

12. The method of claim 9, wherein the at least one additional HIV protein is Vpu.

13. The method of claim 9, wherein the at least one additional HIV protein is gp120.

14. The method of claim 9, wherein the subject is a person who has received a vaccine against HIV infection.

15. The method of claim 9, wherein the subject has received or is receiving antiretroviral therapy.

16. The method according to any one of claims 1 to 15, wherein the extracellular vesicles are captured on a chromatography test strip.

17. The method according to claim 16, wherein the chromatography strip is located within a lateral flow assay device.

18. The method according to any one of claims 1 to 17, wherein the extracellular vesicle is captured by tetraspanin.

19. The method according to claim 18, wherein the tetraspanin is selected from the group consisting of CD9, CD63, and CD81.

20. The method according to any one of claims 1 to 19, wherein the extracellular vesicles are dissolved on a chromatography test strip.

21. The method according to claim 20, wherein the captured extracellular vesicles are dissolved by adding a lysis buffer or chase buffer to the chromatography test strip.

22. The method according to claim 21, wherein the dissolution buffer or chase buffer comprises a solvent selected from the group consisting of saponins, surfactants, detergents, and combinations thereof.

23. The method according to any one of claims 1 to 22, wherein the patient sample is a blood or serum sample.

24. The method according to any one of claims 1 to 23, wherein the detection reagent comprises one or more antigen-binding molecules.

25. The method according to claim 24, wherein the antigen-binding molecule is an antibody or an antibody fragment.

26. The method according to any one of claims 1 to 25, wherein the one or more analyte-specific binders and / or one or more detection reagents suitable for detecting the analyte are labeled antibodies or functional fragments thereof.

27. The method according to claim 26, wherein the labeled antibody or a functional fragment thereof is labeled with colloidal gold or colored microspheres.

28. The method according to any one of claims 1 to 25, wherein the one or more analyte-specific binders and / or one or more detection reagents suitable for detecting the analyte comprises one or more nucleic acid molecules.

29. The method according to claim 28, wherein the labeled nucleic acid is labeled with colloidal gold or colored microspheres.

30. The method according to any one of claims 1 to 29, wherein the extracellular vesicle is an exosome.

31. A chromatography test strip comprising multiple regions, wherein the chromatography test strip allows a fluid test sample to flow from a first end to a second end of the chromatography strip; The chromatography strip comprises a first region containing an extracellular vesicle binder, a first binding partner that binds to a first analyte, and optionally a second region containing at least a second binding partner that binds to at least a second analyte; and Reagents for visualizing the binding of analytes to labeled binding partners. Assay devices including...

32. The assay device according to claim 31, wherein the extracellular vesicle binding agent in the first region is tetraspanin.

33. The assay device according to claim 32, wherein the tetraspanin is selected from the group consisting of CD9, CD63, CD81 and combinations thereof.

34. The assay device according to any one of claims 31 to 33, wherein the first target of analysis is HIV p24.

35. The assay device according to any one of claims 31 to 33, wherein the at least second analyte is selected from the group consisting of HIV proteins selected from Nef, Tat, Vpu, gp120 and combinations thereof.

36. The assay device according to any one of claims 31 to 33, wherein the first analyte is PSA.

37. The assay device according to any one of claims 31 to 33, wherein the object to be analyzed is a protein or a fragment thereof.

38. The assay device according to any one of claims 31 to 37, wherein the first binding partner that binds to the first target of analysis is an antibody or a functional fragment thereof.

39. The assay device according to any one of claims 31 to 38, wherein the at least second binding partner that binds to the at least second analyte is an antibody or a functional fragment thereof.

40. The assay device according to any one of claims 31 to 33, wherein the object to be analyzed is nucleic acid.

41. The assay device according to claim 40, wherein the first binding partner that binds to the first target of analysis is a nucleic acid.

42. The assay device according to any one of claims 40 to 41, wherein the at least second binding partner that binds to the at least second analyte is a nucleic acid.

43. The assay device according to any one of claims 31 to 42, wherein the chromatography strip is a nitrocellulose strip.

44. The assay device according to any one of claims 31 to 43, wherein the chromatography strip is in fluid communication with the sample pad.

45. The assay device according to any one of claims 31 to 44, wherein the chromatography strip is in fluid communication with the conjugate pad.

46. The assay device according to claim 45, wherein the conjugate pad includes a first labeled binding partner that binds to the first analyte.

47. The assay device according to claim 46, wherein the first labeled binding partner is an antibody or a functional fragment thereof.

48. The assay device according to claim 46, wherein the first labeled binding partner is a nucleic acid.

49. The assay device according to any one of claims 46 to 48, wherein the first labeled binding partner is labeled with colloidal gold or a colored microsphere.

50. The assay device according to any one of claims 45 to 49, wherein the conjugate pad includes at least a second labeled binding partner that binds to the at least second analyte.

51. The assay device according to claim 50, wherein the at least one second labeled binding partner is an antibody or a functional fragment thereof.

52. The assay device according to any one of claims 50 to 51, wherein the first labeled binding partner is labeled with colloidal gold or a colored microsphere.

53. The assay device according to any one of claims 31 to 52, further comprising an absorbent pad that is in fluid communication with the chromatography test strip.

54. The assay device according to any one of claims 31 to 53, wherein the chromatography test strip further includes a control region.

55. The assay device according to any one of claims 31 to 54, wherein the extracellular vesicle binding agent is an exosome binding agent.

56. The following kits are included for detecting analytes in extracellular vesicles: a) The device according to any one of claims 31 to 55; and b) A container for the lysis buffer or chase buffer.

57. A kit according to claim 56, used for detecting an analyte in an extracellular vesicle.

58. The use according to claim 57, wherein the extracellular vesicle is an exosome.

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