Compositions and methods for detecting analytes in exosomes

Abstract

Provided herein are compositions and methods for detecting analytes in exosomes. In particular, the present invention relates to the capture and lysis of exosomes and detection of analytes in the exosomes using an immunoassay.

Description

COMPOSITIONS AND METHODS FOR DETECTING ANALYTES IN EXOSOMESCROSS-REFERENCE TO RELATED APPLICATIONSThe present application claims priority to U.S. Provisional Application No. 63 / 524,517, filed June 30, 2023, and U.S. Provisional Application No. 63 / 571,733, filed March 29, 2024, which are incorporated herein by reference in their entireties.FIELD OF THE INVENTIONProvided herein are compositions and methods for detecting analytes in exosomes. In particular, the present invention relates to the capture and lysis of exosomes and detection of analytes in the exosomes using an immunoassay.BACKGOUND OF THE INVENTIONAt home or point of care testing has become more prevalent after the COVID-19 pandemic. Lateral flow tests in particular are an easy-to-use platform to detect analytes in liquid samples, which makes at home testing possible for vulnerable populations. Generally, lateral flow tests have reduced sensitivity when compared to the more complicated and expensive clinical testing. However, the trade-offs between sensitivity and simplicity can be reduced by concentrating the analyte to be detected on the lateral flow test.Exosomes are secreted by cells and are characterized as vesicles ranging in size from 40- 200 nm and are a type of extracellular vesicle (EV). These EVs are a novel source of biomarkers indicative of disease, from their unique surface proteins to their cell origin- specific cargo. Not only are EVs prevalent in the blood but can be of tissue origin and circulate throughout the body and be detected in many different body fluids. EVs contain certain membrane proteins like integrins and tetraspanins that are key factors in membrane transport and fusion. These membrane proteins have been utilized as EV “markers” to enrich samples for EVs, including exosomes. EVs from body fluids like blood and urine are a non-invasive alternative to many current diagnostic tests.For some infectious diseases, like HIV, proteins from the pathogen are sequestered within EVs. In the example of HIV, the vims shares many properties, like the presence of tetraspanins in the outer membrane, with EVs. See, e.g., Jing et al., (2021) Exosomes in HIV infection,Current Opinion in HIV and AIDS 16(5):p 262-270. In the example of prostate cancer, prostatespecific antigen (PSA) in found both in the blood and within extracellular vesicles. In this example, the levels of PSA in blood are easily measured but have poor correlation with true incidence of disease. However, EV-associated PSA levels not only distinguish prostate cancer from healthy individuals, but can also distinguish prostate cancer from benign prostatic hyperplasia. See, e.g., 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.Prior work has demonstrated the ability to capture EV s from bodily fluids using antibodies against tetraspanins on a lateral flow test. See, e.g., 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 work only measured total EVs captured and did not further probe surface or cargo proteins of the captured EVs. The invention described here Not only captures EVs from the liquid sample, but also lyses the captured EVs to release cargo and detect that cargo on the test line of the lateral flow assay.SUMMARY OF THE INVENTIONProvided herein are 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 and detection of analytes in the extracellular vesicles using an immunoassay.Experiments described herein describe a sensitive test for an analyte (e.g., Human Immunodeficiency Virus (HIV) or PSA) using enrichment and capture of EVs to probe EV cargo for HIV specific antigens or PSA. Such methods provide needed improved detection of a variety of analytes.For example, in some embodiments, provided herein is a method of detecting the presence of an analyte in a sample from a subject, comprising: obtaining a patient sample comprising exosomes; capturing exosomes from the patient sample; lysing the exosomes to release proteins contained therein; contacting the proteins with one or more analyte- specific binding agents and / or one or more detection reagents suitable for detecting the analyte; and determining whether the exosomes comprise the analyte.Also provided is a method for diagnosing human immunodeficiency vims (HIV) infection in a subject, comprising: obtaining a patient sample comprising exosomes; capturing exosomes from the patient sample; lysing the exosomes to release proteins contained therein; contacting the proteins with one or more HIV protein binding agents and / or one or more detection reagents suitable for detecting one or more HIV proteins; and determining whether the exosomes comprise the HIV protein p24 and at least one additional HIV protein selected from, for example, Nef, Tat, Vpu, and / or gp!20; wherein a determination of the presence of p24 and the at least one additional HIV protein is indicative of HIV infection in the subject. In some embodiments, the least one additional HIV protein is Nef. In some embodiments, the least one additional HIV protein is Tat. In some embodiments, the least one additional HIV protein is Vpu. In some embodiments, the least one additional HIV protein is gpl20.Also provided is a method for diagnosing prostate cancer and / or distinguishing between prostate cancer and benign prostate hyperplasia (BPH) in a subject, comprising: obtaining a patient sample comprising exosomes; capturing exosomes from the patient sample; lysing the exosomes to release proteins contained therein; contacting the proteins with one or more PSA binding agents and / or one or more detection reagents suitable for detecting PSA; and determining whether the level of PSA in the exosomes as compared to a reference levels and / or control; wherein increased levels of PSA in comparison to the reference level and / or control is indicative of prostate cancer or BPA or allows distinguishment between the presence of prostate cancer and BPA.Further provided are devices, systems, and kits for use in the detection of analytes in exosomes. The analytes may be, for example, protein or fragments thereof or nucleic acids.The present disclosure is not limited to particular analytes. In some embodiments, the analyte is a protein. In some embodiments, the protein is from an infectious agent and the presence of the analyte in the sample is indicative of infection of the analyte in the subject. In some embodiments, the analyte is HIV protein p24 and at least one additional HIV protein is one or more of Nef, Tat, Vpu, and / or gpl20. 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, e.g., DNA, RNA, mRNA, rRNA, cell-free DNA and other nucleic acids.In some embodiments, the subject has been vaccinated against HIV infection. In some embodiments, the subject has is on antiretroviral therapy.In certain embodiments, the exosomes are captured on a chromatographic test strip (e.g., in a lateral flow assay device). In some embodiments, the exosomes arc captured with a tetraspanin (e.g., CD9, CD63, or CD81). In some embodiments, the exosomes are lysed on a chromatographic test strip (e.g., in a lateral flow device).The present disclosure is not limited to a particular sample type. Examples include but are not limited to a blood or serum sample.The present disclosure is not limited to a particular detection reagent or analyte binding molecule. Examples include but are not limited to antibodies, antibody fragments, and nucleic acids.Accordingly, in some aspects, the present invention provides methods of detecting the presence of an analyte in a sample from a subject, comprising: obtaining a patient sample comprising extracellular vesicles; capturing extracellular vesicles from the patient sample; lysing the extracellular vesicles to release proteins contained therein; contacting the proteins with one or more analyte- specific binding agents and / or one or more detection reagents suitable for detecting the analyte; and determining whether the extracellular vesicles comprise the analyte.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 and, optionally, at least one additional HIV protein selected from the group consisting of Nef, Tat, Vpu, and gpl20. In some embodiments, the analyte is prostate specific antigen (PSA). In some embodiments, the protein is from an infectious agent and the presence of the analyte in the sample is indicative of infection of the analyte in the subject. In some embodiments, the infectious agent is a virus or a bacteria. In some embodiments, the analyte is a nucleic acid.In another aspect, the present invention provides methods for diagnosing human immunodeficiency virus (HIV) infection in a subject, comprising: obtaining a patient sample comprising extracellular vesicles; capturing extracellular vesicles from the patient sample; lysing the extracellular vesicles to release proteins contained therein; contacting the proteins with one or more HIV protein binding agents and / or one or more detection reagents suitable for detecting one or more HIV proteins; and determining whether the extracellular vesicles comprise the HIV protein p24 and optionally at least one additional HIV protein selected from the group consisting of Nef, Tat, Vpu, and gpl20; wherein a determination of the presence of p24 and the at least one additional HIV protein is indicative of HIV infection in the subject. In some embodiments, the atleast 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 gpl20. In some embodiments, the subject has been vaccinated against HIV infection. In some embodiments, the subject has or is on antiretroviral therapy.In some embodiments of the foregoing aspects, the extracellular vesicles are captured on a chromatographic test strip. In some embodiments, the chromatographic strip is in a lateral flow assay device. In some embodiments, the extracellular vesicles are captured with a tetraspanin. In some embodiments, the tetraspanin is selected from the group consisting of CD9, CD63, and CD81. In some embodiments, the extracellular vesicles are lysed on a chromatographic test strip. In some embodiments, the captured extracellular vesicles are lysed by adding a lysis or chase buffer to the chromatographic test strip. In some embodiments, the lysis or chase buffer comprises a lysis agent selected from the group consisting of a saponin, a surfactant and a detergent and combinations thereof. In some embodiments, the patient sample is a blood or serum sample. In some embodiments, the detection reagents comprise one or more antigen binding molecules. In some embodiments, the antigen binding molecules are antibodies or antibody fragments. In some embodiments, the one or more analyte- specific binding agents and / or one or more detection reagents suitable for detecting the analyte are labelled antibodies or functional fragments thereof. In some embodiments, the labelled antibodies or functional fragments thereof are labelled with colloidal gold or colored microspheres. In some embodiments, the detection reagents comprise one or more nucleic acid molecules. In some embodiments, the one or more analyte- specific binding agents and / or one or more detection reagents suitable for detecting the analyte are labelled nucleic acids. In some embodiments, the labelled nucleic acids are labelled with colloidal gold or colored microspheres. In some embodiments, the extracellular vesicles are exosomes.In another aspect, the present invention provides an assay device comprising: a chromatographic test strip comprising a plurality of zones, the chromatographic test strip allowing flow of a fluidic test sample from a first end of the chromatographic strip to a second end of the chromatographic test strip; wherein the chromatographic strip comprises a first zone comprising an extracellular vesicle binding agent, a second zone comprising a first binding partner that binds to a first analyte and optionally at least a second binding partner that binds toat least a second analyte; and reagents for visualizing binding of the analytes to the labeled binding partners.In some embodiments, the extracellular vesicle binding agent in the first zone is a tetraspanin. In some embodiments, the tetraspanin is selected from the group consisting of CD9, CD63, and CD81 and combinations thereof. In some embodiments, the first analyte is HIV p24. In some embodiments, the at least a second analyte is selected from the group consisting of an HIV protein selected from Nef, Tat, Vpu, and gp!20 and combinations thereof. In some embodiments, the first analyte is PSA. In some embodiments, the analyte is a protein or fragment thereof. In some embodiments, the first binding partner that binds to a first analyte is an antibody or functional fragment thereof. In some embodiments, the at least a second binding partner that binds to at least a second analyte is an antibody or functional fragment thereof. In some embodiments, the analyte is a nucleic acid. In some embodiments, the first binding partner that binds to a first analyte is a nucleic acid. In some embodiments, the at least a second binding partner that binds to at least a second analyte is nucleic acid. In some embodiments, the chromatographic strip is a nitrocellulose strip. In some embodiments, the chromatographic strip is in fluidic communication with a sample pad. In some embodiments, the chromatographic strip is in fluidic communication with a conjugate pad. In some embodiments, the conjugate pad comprises a first labelled binding partner that binds to the first analyte. In some embodiments, the first labelled binding partner is an antibody or functional fragment thereof. In some embodiments, the first labelled binding partner is a nucleic acid. In some embodiments, the first labelled binding partner is labelled with colloidal gold or colored microspheres. In some embodiments, the conjugate pad comprises at least a second labelled binding partner that binds to the at least a second analyte. In some embodiments, the at least a second labelled binding partner is an antibody or functional fragment thereof. In some embodiments, the first labelled binding partner is labelled with colloidal gold or colored microspheres. In some embodiments, the assay devices further comprise an absorption pad in fluid communication with the chromatographic test strip. In some embodiments, the chromatographic test strip further comprises a control zone. In some embodiments, the extracellular vesicle binding agent is an exosome binding agent.In another aspect, the present invention provides a kit for detecting an analyte in an extracellular vesicle, comprising: a) the assay device as described above in any of the various embodiments; and b) a container of lysis or chase buffer.In another aspect, the kits are provided for use in detecting an analyte in an extracellular vesicle. In some embodiments, the extracellular vesicle is an exosome.Additional aspects of the invention are described herein.DESCRIPTION OF THE FIGURESFIG. 1A-B shows an overview of the EV-HIV Test. (A) Whole blood or plasma contains antibodies, virus particles, viral proteins, and extracellular vesicles (EV). (B) The blood / plasma sample is added to the Sample Pad and wicked across the nitrocellulose strip, allowing the immune capture of EVs via binding to tetraspanin proteins (CD 9, CD63, CD81).FIG. 2 shows Western blot analysis of H9 and H9MN Full-length Intact (H9MN FI) cells for the presence of p24.FIG. 3 shows a quantitative ELISA for HIV p24 protein. Exosomes purified by the ExoQuick or MagCapture method from normal cells (H9, black bars) or chronically infected with MN strain of HIV (H9MN FI, grey bars). Error bars represent standard deviation of the mean of triplicate measurements.FIG. 4A-B shows NS300 analysis of exosomes purified using the ExoQuick method. A. Extracellular vesicle size distribution in chronically infected H9MN FI cells. Mean size 191 nm. B. Extracellular vesicle size distribution in chronically infected H9MN FI cells. Mean size 157 nm.FIG. 5 shows an automated western analysis of purified exosomes from human plasma collected from HIV- and HIV+ subjects. A. Detection of HIV p24, measured as Area Under the Curve (AUC) for the ~24 kDa peak. B. Detection of HIV Tat, measured as AUC for the ~14 kDa peak. C. Detection of HIV Mef, measured as AUC of the ~27 kDa peak. Approximately 0.4 mg / mL of exosomes purifed from plasma from HIV negative or positive subjects were analyzed. HIV- n=10, HIV+ n=40.FIG. 6A-D shows immunocapture of exosomes on a lateral flow strip. A. Diagram of the tetraspanins capture lateral flow strips. Purified exosomes are added to the sample well. B. Diagram of the lateral flow capture device and tetraspanins capture zone. C. Running buffer onlywas tested on the exosome capture strips “Buffer.” A total of 10 pg / mL purified exosomes were captured by the test line. B. Score card used to provide a numerical value to the test strip intensity on a scale of 1-10.FIG. 7 shows limit of detection of purified exosomes using anti-CD63+anti-CD81 striped lateral flow strips. The y-axis shows the Cube Reader score of test line color, the x-axis shows exosome concentration in pg / mL.FIG. 8A-C shows lateral flow detection of p24 from purified exosomes. A. Diagram of p24 detection by lateral flow using lysed extracellular' vesicles (EVs). B. Diagram of nitrocellulose strip with capture and detection of p24 from purified EVs. C. Measurement of p24 in EV Lysis Buffer. Buffer alone (score 3 of 10). EVs from H9 cells lysed in EV Lysis Buffer, p24- (Score 3 of 10). EVs from H9MN FI cells lysed in EV Lysis Buffer, p24+ (score 6 of 10).FIG. 9A-B shows lateral flow detection of p24 from exosomes. A. Diagram of lateral flow device. B. Photographs of negative controls (first two panels) and detection of p24 on lateral flow device (last panel). Enriches EVs from each sample type were incubated with EV lysis buffer and run on the p24 lateral flow test. P24 is detected on the test line and anti-species is detected on the control line. Number below panels indicate visual scores.FIG. 10 shows the effects of different lysis buffer components on the release of p24 cargo from 20 ug of input EVs. PBS shows background of the system without any input EVs, H9 shows non-specific background from a p24- cell line, and H9 MNFI shows specific p24 signal from a p24-expressing cell line.FIG. 11 A-B shows an overview of the EV-PSA Test. (A) Whole blood or plasma contains antibodies, immune cells, cellular proteins, and extracellular vesicles (EV). (B) The blood / plasma sample is added to the Sample Pad and wicked across the nitrocellulose strip, allowing the immune capture of EVs via binding to tetraspanin proteins (CD 9, CD63, CD81). After lysis buffer or chase buffer is added, EV cargo is released and captured on the anti-PSA test line and detected with anti-PSA detection antibodies.FIG. 12 shows an overview of the EV-PSA test. A. Whole blood, urine, or plasma contains EVs with disease- specific biomarkers. B. Sequential EV capture and PSA detection.FIG. 13A-E shows PSA biomarker detection in the EV-PSA test. A. Detection of less than 0.5 ng / mL of PSA in the EV-PSA test. B. Design of EV-PSA test with multiple-stripe of anti-tetraspanin capture lines in EV capture zone. C. Further testing of lower concentrations ofPSA to pg levels. D. Scores read by densitometry using the Cube reader for increased sensitivity. E. Scores read visually using the 1-10 scale.FIG. 14 shows that different types of EVs have a different proportion of tetraspanins. Capture line is a 1:1:1 mix of anti-CD9, anti-CD63, and anti-CD81. Detection antibodies are labelled in the graph with the corresponding result for EV s purified from human plasma, EV s from PC3 (PSA-) cells, and EVs from LnCap (PSA+) cells.FIG. 15 shows the results from a PSA ELISA from intact and lysed EVs that PSA is detected in EVs from prostate cancer but not healthy controls. Purified EVs from Normal (no prostate cancer) and LNCaP (prostate cancer) were tested at various concentrations of total protein in either PBS or in lysis buffer.FIG. 16 shows PSA detection from lysis of purified exosomes from LNCaP cells (PSA+). Visual score (1-10) for triplicate strips are graphed with the standard error bars shown.DEFINITIONSTo facilitate an understanding of the present invention, a number of terms and phrases are defined below:As used herein, the term “exosome” refers to a membrane-bound extracellular vesicle (EVs) that is produced in the endosomal compartment of most eukaryotic cells. In multicellular organisms, exosomes are found, for example, in biological fluids including saliva, blood, urine and cerebrospinal fluid.As used herein, the term “analyte” refers to any molecule detected by the device and methods described herein. In some non-limiting example, the analyte is a protein or other biological molecule.The terms “detecting” or “detection” or “determining the level” refer to quantitatively or non-quantitatively determining the presence of the analyte(s) under investigation (e.g., HIV). "Detecting Formation of a Complex" refers to detecting a complex comprising a detector reagent by any method suitable for observing the particular label associated with the detector reagent; for instance, visual observation of a colored (or otherwise visible) label, measurement or visual detection of a fluorescent, chemiluminescent or radioactive label.As used herein, the term "sample" is used in its broadest sense. In one sense, it is meant to include a specimen or culture obtained from any source, as well as biological andenvironmental samples. Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples include urine, saliva, cerebrospinal fluid, and blood products, such as whole blood, plasma, serum and the like. Such examples are not however to be construed as limiting the sample types applicable to the present invention.The term “specific binding partner (or binding partner)” refers to a member of a pair of molecules that interact by means of specific, noncovalent interactions that depend on the three- dimensional structures of the molecules involved. Typical pairs of specific binding partners include antigen / antibody, hapten / antibody, hormone / receptor, nucleic acid strand / complementary nucleic acid strand, substrate / enzyme, inhibitor / enzyme, carbohydrate / lectin, biotin / (strept)avidin, and virus / cellular receptor.As used herein, the terms "immunoglobulin" or "antibody" refer to proteins that bind a specific antigen. Immunoglobulins include, but are not limited to, polyclonal, monoclonal, chimeric, and humanized antibodies, Fab fragments, F(ab')2 fragments, and includes immunoglobulins of the following classes: IgG, IgA, IgM, IgD, IbE, and secreted immunoglobulins (sig). Immunoglobulins generally comprise two identical heavy chains and two light chains. However, the terms "antibody" and "immunoglobulin" also encompass single chain antibodies and two chain antibodies.The term “label” refers to a molecule or composition bound to an analyte, analyte analog, detector reagent, antibody, or binding partner that is detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Examples of labels, including enzymes, colloidal gold particles, colored latex particles (e.g., microspheres), have been disclosed (U.S. Pat. Nos. 4,275,149; 4,313,734; 4,373,932; and 4,954,452, each incorporated by reference herein). Additional examples of useful labels include, without limitation, radioactive isotopes, co-factors, 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-coupled dye sacs. The attachment of a compound (e.g., a detector reagent) to a label can be through covalent bonds, adsorption processes, hydrophobic and / or electrostatic bonds, as in chelates and the like, or combinations of these bonds and interactions and / or may involve a linking group.The phrase "specifically binds to an analyte" or "specifically immunoreactive with," when referring to an antibody, refers to a binding reaction which is determinative of the presence of the analyte in the presence of a heterogeneous population of molecules such as proteins and other biologic molecules. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular analyte and do not bind in a significant amount to other analytes present in the sample. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular analyte. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow and Lane, Antibodies, A Laboratory Manual, CSHP, New York (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.The term “capture agent” refers to an unlabeled specific binding partner that is specific for (i) an extracellular vesicle (e.g., exosome), (ii) an analyte, as in a sandwich assay, or (iii) a detector reagent or an analyte, as in a competitive assay, or for (iv) an ancillary specific binding partner, which itself is specific for the analyte, as in an indirect assay. As used herein, an "ancillary specific binding partner" is a specific binding partner that binds to the specific binding partner of an analyte. For example, an ancillary specific binding partner may include an antibody specific for another antibody, for example, goat anti-human antibody. A "capture area" is a region of a lateral flow device where the capture reagent is immobilized. A lateral flow device may have more than one capture area, for example, a "primary capture area," a "secondary capture area," and so on. Often a different capture reagent will be immobilized in the primary, secondary, or other capture areas. Multiple capture areas may have any orientation with respect to each other on the lateral flow substrate; for example, a primary capture area may be distal or proximal to a secondary (or other) capture area and vice versa. Alternatively, a primary capture area and a secondary (or other) capture area may be oriented perpendicularly to each other such that the two (or more) capture areas form a cross or a plus sign or other symbol.The term “detector reagent” refers to a specific binding partner that is conjugated to a label. Detector reagents include, for example, labeled analyte- specific binding members or labeled ancillary specific binding members (such as enzyme-conjugate, goat anti-human antibodies).The term “lateral flow device” refers to an analytical device in the form of a test strip used in lateral flow chromatography, in which a test sample fluid, suspected of containing an analyte, flows (for example by capillary action) through the strip (which is frequently made of bibulous materials such as paper, nitrocellulose, and cellulose). The test fluid and any suspended analyte can flow along the strip to a detection zone in which the analyte (if present) interacts with a detection agent to indicate a presence, absence and / or quantity of the analyte.The term “sample application area” refers to an area where a fluid sample is introduced to a immunochromatographic test strip, such as an immunochromatographic test strip present in a lateral flow device. In one example, the sample may be introduced to the sample application area by external application, as with a dropper or other applicator. In another example, the sample application area may be directly immersed in the sample, such as when a test strip is dipped into a container holding a sample. In yet another example, the sample may be poured or expressed onto the sample application area.The term “solid support” or “substrate” means material which is insoluble, or can be made insoluble by a subsequent reaction. Numerous and varied solid supports are known to those in the art and include, without limitation, nitrocellulose, the walls of wells of a reaction tray, multi-well plates, test tubes, polystyrene beads, magnetic beads, membranes, microparticles (such as latex particles), and sheep (or other animal) red blood cells. Any suitable porous material with sufficient porosity to allow access by detector reagents and a suitable surface affinity to immobilize capture reagents is contemplated by this term. For example, the porous structure of nitrocellulose has excellent absorption and adsorption qualities for a wide variety of reagents, for instance, capture reagents. Nylon possesses similar characteristics and is also suitable. Microporous structures are useful, as are materials with gel structure in the hydrated state.Further examples of useful solid supports include: natural polymeric carbohydrates and their synthetically modified, cross-linked or substituted derivatives, such as agar, agarose, crosslinked alginic acid, substituted and cross-linked guar gums, cellulose esters, especially with nitric acid and carboxylic acids, mixed cellulose esters, and cellulose ethers; natural polymers containing nitrogen, such as proteins and derivatives, including cross-linked or modified gelatins; natural hydrocarbon polymers, such as latex and rubber; synthetic polymers which may be prepared with suitably porous structures, such as vinyl polymers, including polyethylene,polypropylene, polystyrene, polyvinylchloride, polyvinylacetate and its partially hydrolyzed derivatives, polyacrylamides, polymcthacrylatcs, copolymers and terpolymers of the above polycondensates, such as polyesters, polyamides, and other polymers, such as polyurethanes or polyepoxides; porous inorganic materials such as sulfates or carbonates of alkaline earth metals and magnesium, including barium sulfate, calcium sulfate, calcium carbonate, silicates of alkali and alkaline earth metals, aluminum and magnesium; and aluminum or silicon oxides or hydrates, such as clays, alumina, talc, kaolin, zeolite, silica gel, or glass (these materials may be used as filters with the above polymeric materials); and mixtures or copolymers of the above classes, such as graft copolymers obtained by initializing polymerization of synthetic polymers on a pre-existing natural polymer.It is contemplated that porous solid supports, such as nitrocellulose, described hereinabove are preferably in the form of sheets or strips. The thickness of such sheets or strips may vary within wide limits, for example, from about 0.01 to 0.5 mm, from about 0.02 to 0.45 mm, from about 0.05 to 0.3 mm, from about 0.075 to 0.25 mm, from about 0.1 to 0.2 mm, or from about 0.11 to 0.15 mm. The pore size of such sheets or strips may similarly vary within wide limits, for example from about 0.025 to 15 microns, or more specifically from about 0.1 to 3 microns; however, pore size is not intended to be a limiting factor in selection of the solid support. The flow rate of a solid support, where applicable, can also vary within wide limits, for example from about 12.5 to 90 sec / cm (i.e., 50 to 300 sec / 4 cm), about 22.5 to 62.5 sec / cm (i.e., 90 to 250 sec / 4 cm), about 25 to 62.5 sec / cm (i.e., 100 to 250 sec / 4 cm), about 37.5 to 62.5 sec / cm (i.e., 150 to 250 sec / 4 cm), or about 50 to 62.5 sec / cm (i.e., 200 to 250 sec / 4 cm). In specific embodiments of devices described herein, the flow rate is about 62.5 sec / cm (i.e., 250 sec / 4 cm). In other specific embodiments of devices described herein, the flow rate is about 37.5 sec / cm (i.e., 150 sec / 4 cm).The surface of a solid support may be activated by chemical processes that cause covalent linkage of an agent (e.g., a capture reagent) to the support. However, any other suitable method may be used for immobilizing an agent (e.g., a capture reagent) to a solid support including, without limitation, ionic interactions, hydrophobic interactions, covalent interactions and the like. The particular forces that result in immobilization of an agent on a solid phase are not important for the methods and devices described herein.Except as otherwise physically constrained, a solid support may be used in any suitable shapes, such as films, sheets, strips, or plates, or it may be coated onto or bonded or laminated to appropriate inert carriers, such as paper, glass, plastic films, or fabrics.A "lateral flow substrate" is any solid support or substrate that is useful in a lateral flow device.DESCRIPTION OF THE INVENTIONProvided herein are 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 detection of analytes in the EVs using an immunoassay.Experiments described herein developed a method to screen extracellular vesicles, including exosomes, for disease- specific biomarkers. Examples 1 describes HIV- 1 / 2 (HIV) proteins only. While the results described herein are exemplified with detection of HIV proteins, the devices, compositions, kits, and methods described herein find use in the detection of any number of analytes. Non-limiting examples of suitable analytes are described below.Current “4thGeneration” HIV- 1 / 2 combo tests screen for both the presence of antigen (p24) and antibodies to the envelope protein (gp!60, gp41, gpl20). The assays described herein test for antigen only, include detection of p24, and add at least one additional biomarker (e.g., Nef, Tat, Vpu, and / or gpl20). These additional viral proteins Nef, Tat, Vpu, and gpl20 were previously thought to be expressed only during a short window of acute viral infection. However, recent work has shown that these proteins can be expressed during the chronic stage of infection and even in patients on antiretroviral therapy (ART) with undetectable levels of viral genome. 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(l):e0191613.In order to develop a diagnostic looking at HIV- 1 / 2 antigens only, additional concentration of these antigens was used to enable detection of low-level expression in the majority of HIV- infected individuals. See, e.g., 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. demonstrated that HIV proteins Nef and Vpu were persistently upregulated and detected in plasma extracellular vesicles (EV). Other work supportsthe presence of Nef in EV or in exosomes, and Nef is a known modulator of endosomal trafficking (See, c.g., FIG. 5; McNamara ct al., Nef secretion into Extracellular Vesicles or Exosomes Is Conserved across Human and Simian Immunodeficiency Viruses. mBio. 2018 (9)e02344-17, Roeth et al., Human Immunodeficieny virus type 1 Nef: Adapting to Intracellular Trafficking Path ways. Microbiol Mol Biol Rev 70:548-563).Extracellular vesicles are thought to have 3 major subgroups: apoptotic bodies, microvesicles, and exosomes. All three have enriched surface membrane proteins and can be distinguished both by size and by the membrane protein markers. EVs can be purified through the tetraspanin cell surface markers including CD9, CD63, and CD81. See, e.g., 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.In some embodiments, the present disclosure utilized an immunocapture of EVs though the tetraspanins CD9, CD63, and CD81 on a lateral flow immunoassay test to enrich for EVs prior to lysing the EVs and liberating the proteins to be captured on multiplex test lines for p24, Nef, Tat, and gpl20.Accordingly, the present invention provides devices, kits and methods for detecting the presence of an analyte in an EV or exosome. Exemplary devices and methods are described below.I. Isolation of ExosomesExtracellular vesicles (EVs), which include exosomes, are small plasma membrane- enclosed bodies of cellular origin with a diameter range of 30-150 nm (Doyle et al, Cells. 2019 Jul; 8(7): 727). They are released from the cellular plasma membrane through direct outward budding after incorporation of proteins, nucleic acids, and lipids. Exosomes are considered to be a method of cell-to-cell communication by transfer of important cargo molecules. The membrane and luminal proteins contained in EVs can be used as valuable biomarkers of diseases, including cancer and infectious disease (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 mostbody fluids (blood plasma, serum, urine, saliva, breast milk, bronchial lavage fluid, amniotic fluid, cerebrospinal fluid, and malignant ascites) in high concentrations (Raimondo ct al., Proteomics . 2011 Feb;l l(4):709-20. doi: 10.1002 / pmic.201000422. Epub 2011 Jan 17).HIV proteins Nef and Vpu are persistently upregulated and detected in plasma extracellular vesicles (EV) from HIV infected individuals. Other work supports the presence of Nef in EV or in exosomes, and Nef is a known modulator of endosomal trafficking. Extracellular vesicles are thought to have 3 major subgroups: apoptotic bodies, microvesicles, and exosomes. All three have enriched surface membrane proteins and can be distinguished both by size and by the membrane protein markers. In some embodiments, EVs are purified through exosome- specific cell-surface markers (e.g., the tetraspanin cell surface markers including CD9, CD63, and CD81). In HIV, EVs containing CD81 have been shown to contain Nef, gpl20(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)).The present disclosure is not limited to particular methods for isolation of EVs. Several methods for isolation of EVs have been developed. Three main categories for exosome isolation are: ultracentrifugation and ultrafiltration to separate by size, and immunoaffinity to separate by membrane proteins exclusive to exosomes (e.g., tetraspanins) (Chen et al., Front. Bioeng. Biotechnol., 05 January 2022). In some embodiments, the present disclosure uses immunocapture method that utilize tetraspanin expression on exosomes. The tetraspanin superfamily defined by the four transmembrane domains and post-translational modifications (Stipp et al., Volume 28, ISSUE 2, P106-112, February 2003). The tetraspanins CD9, CD37, CD63, CD81, and CD82 have been used as biomarkers for exosomes because they are enriched in EVs (Andreuet al., Front. Immunol., 16 September 2014 Sec. Vaccines and Molecular Therapeutics Volume 5 - 2014).In certain embodiments, immunocapture and enrichment of EVs from biofluids utilizes the immunocapture of tetraspanins on a lateral flow strip (e.g., in a lateral flow assay device). The concentrated EVs on the lateral flow strip can then be lysed on lateral flow strip and the internal cargo liberated and probed in downstream test strips on the device. Exemplary devices are described below.II. Assay Devices and MethodsThe present invention provides devices, kits and methods for detecting analytes in EVs. Such devices, kits, and methods find use in a variety of research, screening, and diagnostic applications.In some embodiments of the present invention, assays are suitable for point-of-care use such as at a doctor’s office or at home. In other embodiments, the assays are suitable for use in a clinical setting. In either embodiment, the assays are 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 analyte in the test sample is compared to a standard, such as an control sample or standard curve.The present disclosure is not limited to particular analytes. In some embodiments, the analyte is a protein or other biological molecule. In some embodiments, the analyte is a protein from an infectious agent (e.g., HIV or other viral, bacterial, or fungal pathogen) or a cancerspecific or related protein. In some embodiments, the analyte is a nucleic acid. Detectable nucleic acids include, put are not limited to DNA, RNA, mRNA, rRNA, cell free DNA and the like. Lateral flow assays of nucleic acids include those for amplified nucleic acids, such as amplification via PCR, isothermal amplification methods such as LAMP and other amplification technologies as well as detection that incorporates endonucleases such as the Cas9 endonuclease. See, e.g., 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 et al., Biomedicines 11(9):2344 (2023); Saxena et al., IJMS 23(21): 13105 (2022); all of which are incorporated herein by reference in their entirety. In embodiments, where the analyte is a nucleic acid, the processes of the present invention may further comprise an amplification step where the nucleic sequences to be detected are amplified, for example by thermal or isothermal amplification.In some embodiments, the analyte is detected by binding to a capture molecule specific for the analyte (for example, an aptamer, or an antibody in an immunoassay). The present invention is not limited to a particular capture molecule or antibody. Any capture molecule orantibody or functional fragment thereof (e.g., monoclonal or polyclonal antibodies, Fab fragments, scFv, etc.) that detects the analyte may be utilized. Exemplary methods for the generation of antibodies are described below. Antibody-analyte complex resulting from antibody binding is detected by techniques known in the art. In some embodiments, a detection reagent is utilized. In some embodiments, the detection reagent is a labeled antibody or functional fragment thereof that binds to the analyte. The present invention is not limited to a particular detection format. A variety of detection formats are contemplated, including, but not limited to, radio-immunoassay, ELISA (enzyme linked immunosorbant assay), "sandwich" immunoassay, immunoradiometric assay, gel diffusion precipitation reaction, immunodiffusion assay, precipitation reaction, agglutination assay (e.g., gel agglutination assay, hemagglutination assay, etc.), complement fixation assay, immunofluorescence assay, protein A assay, and immunoelectrophoresis assay.In some embodiments, the immunoassay devices of the present invention permit the performance of relatively inexpensive, disposable, membrane-based assays for the visual identification of the presence (or absence) of an analyte in a liquid sample. Such devices are usually formatted as freestanding dipsticks (e.g., test strips) or as devices having some sort of housing. Typically, an immunoassay device of the present invention can be used with as little as about 200 pl of liquid sample, and detection of an analyte in the sample can (but need not) be complete within 2-5 minutes. In preferred embodiments, no ancillary instrumentation is required to perform such tests, and such devices easily can be used in clinics, laboratories, field locations, and the home even by inexperienced persons.Immunoassay devices have been developed for the routine identification or monitoring of physiological and pathological conditions (e.g., infectious diseases, pregnancy, cancer, endocrine disorders) using different biological samples (e.g., urine, serum, plasma, blood, saliva), and for analysis of environmental samples (e.g., natural fluids and industrial plant effluents) for instance for contamination. Many of these tests are based on the highly specific interactions between specific binding pairs. Examples of such binding pairs include antigen / antibody, hapten / antibody, lectin / carbohydrate, apoprotein / cofactor and biotin / (strept)avidin. Furthermore, many of these tests involve devices (e.g., solid phase, lateral flow test strips, flow-through tests) with one or more of the members of a binding pair attached to a mobile or immobile solid phasematerial such as latex beads, glass fibers, glass beads, cellulose strips or nitrocellulose membranes (U.S. Pat. Nos. 4,703,017; 4,743,560; 5,073,484).In some embodiments, the assay device is lateral flow assay device. There are a number of commercially available lateral flow type tests and patents disclosing methods for the detection of analytes. See, e.g., U.S. Pat. No. 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; WO 97 / 06439; and WO 98 / 36278, all of which are incorporated herein by reference.The lateral flow assay devices of the present invention include a strip of absorbent or porous material (such as a microporous membrane (e.g., nitrocellulose)), which, in some instances, can be made of different substances each joined to the other in zones, which may be abutted and / or overlapped. In some examples, the absorbent strip can be fixed on a supporting non-interactive material (such as nonwoven polyester), for example, to provide increased rigidity to the strip. Zones within each strip may differentially contain the specific binding partner(s) and / or other reagents required for the detection and / or quantification of the particular analyte being tested for. Thus these zones can be viewed as functional sectors or functional regions within the test device.In some embodiments, assay strips of the present disclosure comprise an EV capture zone and one or more analyte capture zones.In some embodiments, a fluid sample (or a sample suspended in a fluid) is introduced to the strip at the proximal end of the strip, for instance by dipping or spotting. A sample is collected or obtained using methods well known to those skilled in the art. The sample containing the analyte to be detected may be obtained from any biological source. Examples of biological sources include blood serum, blood plasma, urine, spinal fluid, saliva, fermentation fluid, lymph fluid, tissue culture fluid and ascites fluid of a human or animal. The sample may be diluted, purified, concentrated, filtered, dissolved, suspended or otherwise manipulated prior to immunoassay to optimize the immunoassay results. The fluid migrates distally through all the functional regions of the strip. The final distribution of the fluid in the individual functional regions depends on the adsorptive capacity and the dimensions of the materials used.In some embodiments, porous solid supports, such as nitrocellulose, described hereinabove are preferably in the form of sheets or strips. The thickness of such sheets or stripsmay vary within wide limits, for example, from about 0.01 to 0.5 mm, from about 0.02 to 0.45 mm, from about 0.05 to 0.3 mm, from about 0.075 to 0.25 mm, from about 0.1 to 0.2 mm, or from about 0.11 to 0.15 mm. The pore size of such sheets or strips may similarly vary within wide limits, for example from about 0.025 to 15 microns, or more specifically from about 0.1 to 3 microns; however, pore size is not intended to be a limiting factor in selection of the solid support. The flow rate of a solid support, where applicable, can also vary within wide limits, for example from about 12.5 to 90 sec / cm (i.e., 50 to 300 sec / 4 cm), about 22.5 to 62.5 sec / cm (i.e., 90 to 250 sec / 4 cm), about 25 to 62.5 sec / cm (i.e., 100 to 250 sec / 4 cm), about 37.5 to 62.5 sec / cm (i.e., 150 to 250 sec / 4 cm), or about 50 to 62.5 sec / cm (i.e., 200 to 250 sec / 4 cm). In specific embodiments of devices described herein, the flow rate is about 62.5 sec / cm (i.e., 250 sec / 4 cm). In other specific embodiments of devices described herein, the flow rate is about 37.5 sec / cm (i.e., 150 sec / 4 cm).In some embodiments, the assay devices include a detector reagent. The detector reagent provides a means to detect the formation of a complex between an analyte and a capture reagent (such as a first antibody specific for the analyte). A detector may be integrated into an immunoassay device (for example included in a conjugate pad, as described below), or may be applied to the device from an external source.A detector may be a single reagent or a series of reagents that collectively serve the detection purpose. In some instances, a detector reagent is a labeled binding partner specific for the analyte. In other instances, a detector reagent collectively includes an unlabeled first binding partner specific for the analyte and a labeled second binding partner specific for the first binding partner and so forth. In each instance, a detector reagent specifically detects bound analyte of an analyte-capture reagent complex and, therefore, a detector reagent preferably does not substantially bind to or react with the capture reagent or other components localized in the analyte capture area. Such non-specific binding or reaction of a detector may provide a false positive result. Optionally, a detector reagent can specifically recognize a positive control molecule (such as a non-specific human IgG for a labeled Protein A detector, or a labeled Protein G detector, or a labeled anti-human Ab(Fc)) that is present in a secondary capture area. In some preferred embodiments, the detector may be conjugated to or otherwise associated with a label. Examples of labels, including enzymes, colloidal gold particles, colored latex particles (e.g., microspheres), have been disclosed (U.S. Pat. Nos. 4,275,149; 4,313,734; 4,373,932; and4,954,452, each incorporated by reference herein). Additional examples of useful labels include, without limitation, radioactive isotopes, co-factors, 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-coupled dye sacs. The attachment of a compound (e.g., a detector reagent) to a label can be through covalent bonds, adsorption processes, hydrophobic and / or electrostatic bonds, as in chelates and the like, or combinations of these bonds and interactions and / or may involve a linking group.A lateral flow device is an analytical device comprising a test strip, through which flows a test sample fluid that is suspected of containing an analyte of interest. The test fluid and any suspended analyte can flow along the strip to a detection zone in which the analyte (if present) interacts with a capture agent and a detection agent to indicate a presence, absence and / or quantity of the analyte. Many lateral flow devices are one-step lateral flow assays in which a biological fluid is placed in a sample area on a strip and allowed to migrate along the strip until the liquid comes into contact with a specific binding partner that interacts with an analyte in the liquid. Once the analyte interacts with the binding partner, a signal (such as a fluorescent or otherwise visible dye) indicates that the interaction has occurred. Multiple discrete binding partners can be placed on the strip (for example in parallel lines) to detect multiple analytes in the liquid. The test strips can also incorporate control indicators, which provide a signal that the test has adequately been performed, even if a positive signal indicating the presence (or absence) of an analyte is not seen on the strip.The construction and design of lateral flow devices is described, for example, in 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, N.H. 03431, (603) 352-3810; both of which are incorporated herein by reference. Lateral flow devices have a wide variety of physical formats. Any physical format that supports and / or houses the basic components of a lateral flow device in the proper function relationship is contemplated by this disclosure.In some embodiments, lateral flow devices of the present invention comprise an elongated housing containing a lateral flow strip that extends substantially the entire length ofhousing. In some embodiments, the lateral flow strip is divided into a proximal sample application pad positioned below a sample introduction port, an intermediate test result membrane, and a distal absorbent pad. The flow strip is interrupted by a conjugate pad that contains labeled conjugate. A flow path along the strip passes from the proximal pad, through conjugate pad, into a test result membrane, for eventual collection in absorbent pad. Selective binding agents (such as exosome or analyte specific antibody) are positioned on a proximal test line in the test result membrane. A control line is provided in the test result membrane slightly distal to the test line. A fluid sample containing an analyte of interest, is applied to the sample pad through the sample introduction port. In some embodiments, the sample may be applied to the sample introduction port dropwise or by dipping the end of the device containing the sample introduction port into the sample. From the sample pad, the sample passes, for instance by capillary action, to the conjugate pad. In the conjugate pad, exosomes in the sample bind to a first capture zone. Following binding, a buffer that results in lysis or perforation of the exosomes in then added to the assay device. Next, the analyte of interest may bind (or be bound by) a mobilized or mobilizable detector reagent in a second and optionally third (or more) capture zone. For example, an analyte may bind to a labeled (e.g., gold-conjugated) antibody detector reagent contained in the conjugate pad. The analyte complexed with the detector reagent may subsequently flow to the test result membrane where the complex may further interact with a capture reagent, such as analyte specific antibody, which is immobilized at the proximal test line. The formation of the immunocomplex between analyte, labeled (e.g., gold-conjugated) detector reagent, and immobilized antibody can be detected by the appearance of a visible line at the proximal test line, which results from the accumulation of the label (e.g., gold) in the localized region of the proximal test line. The control line may contain an immobilized, detector-reagent- specific binding partner, which can bind the detector reagent in the presence or absence of the analyte. Such binding at the control line indicates proper performance of the test, even in the absence of the analyte of interest.The particular materials used in a particular lateral flow device will depend on a number of variables, including, for example, the analyte to be detected, the sample volume, the desired flow rate and others. In some embodiments, the sample pad receives the sample, and may serve to remove particulates from the sample. In some embodiments, the sample pad is cellulose. Sample pads may be treated with one or more release agents, such as buffers, salts, proteins,detergents, and surfactants. Such release agents may be useful, for example, to promote rcsolubilization of conjugatc-pad constituents, and to block non-specific binding sites in other components of a lateral flow device, such as a nitrocellulose membrane. Representative release agents include, for example, trehalose or glucose (l%-5%), PVP or PVA (0.5%- 2%), Tween 20 or Triton X-100 (0.1%- 1 %), casein (1 %-2%), SDS (0.02%-5%), and PEG (0.02%-5%).The conjugate pad holds a detector reagent. In some embodiments, a detector reagent may be applied externally, for example, from a developer bottle, in which case a lateral flow device need not contain a conjugate pad (see, for example, U.S. Pat. No. 4,740,468). Detector reagent(s) contained in a conjugate pad is typically released into solution upon application of the test sample. A conjugate pad may be treated with various substances to influence release of the detector reagent into 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, without limitation, hydroxypropylmethyl cellulose, SDS, Brij and 0-lactose.The absorbent pad acts to increase the total volume of sample that enters the device. This increased volume can be useful, for example, to wash away unbound analyte from the membrane. Any of a variety of materials is useful to prepare an absorbent pad. In some device embodiments, an absorbent pad can be paper (i.e., cellulosic fibers). One of skill in the art may select a paper absorbent pad on the basis of, for example, its thickness, compressibility, manufacturability, and uniformity of bed volume. The volume uptake of an absorbent made may be adjusted by changing the dimensions (usually the length) of an absorbent pad.Figure 1 provides an illustration of a preferred embodiment of a lateral flow device of the present invention. As can be seen, Fig. 1 depicts an assay device comprising: a chromatographic test strip comprising a plurality of zones, the chromatographic test strip allowing flow of a fluidic test sample from a first end of the chromatographic strip to a second end of the chromatographic test strip; wherein the chromatographic strip comprises a first zone comprising an exosome binding agent, a second zone 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 reagents for visualizing binding of the analytes to the labeled binding partners, wherein the reagents for visualizing binding of the analytes are preferably located in a conjugate pad.In some preferred embodiments, the exosome binding agent in the first zone is a tctraspanin. In some particularly preferred embodiments, the tetraspanin is selected from the group consisting of CD9, CD63, and CD81 and combinations thereof.In some preferred embodiments, the second zone comprises one or more binding partners for at least one analyte that serve to capture analytes released from exosomes that are bound to the exosome capture zone and lysed via contact with a chase or lysis buffer. In some preferred embodiments, the one or more binding partners are antibodies or functional fragments thereof that specifically bind the desired analyte. In some embodiments, the first analyte is HIV p24. In some preferred embodiments the binding partner is an antibody or functional fragment thereof of that binds p24. In some embodiments, the at least a second analyte is selected from the group consisting of an HIV protein selected from Nef, Tat, Vpu, and gpl20 and combinations thereof. In some preferred embodiments, the at least a second binding partner is an antibody or functional fragment thereof that binds to Nef, Tat, Vpu, or gpl20.In other embodiments, the analyte is prostate serum antigen (PSA). In some preferred embodiments the assays of the present invention provide for detection of PSA in EVs. In some preferred embodiments, elevated levels of PSA in EVs compared to a control are indicative of prostate cancer and the measured levels of PSA may be utilized to distinguish between patients with healthy prostates or that have benign prostate hyperplasia (BPH) and prostate cancer.In some preferred embodiments, the chromatographic test strip is a nitrocellulose strip, although other materials may be used as described above.In some preferred embodiments, the chromatographic strip is in fluidic communication with a sample pad. In some embodiments, a sample such as a biological sample that comprises exosomes, is applied to the sample pad when the device is being used. In some embodiments, the biological sample may be diluted with a running buffer.In some preferred embodiments, the chromatographic strip is in fluidic communication with the conjugate pad. As described above, the conjugate pad preferably comprises at least a first labelled binding partner that binds to the first analyte (e.g., p24 or PSA). In some preferred embodiments, the first labelled binding partner is an antibody or functional fragment thereof. Suitable labels are described above in detail. In some preferred embodiments, the first labelled binding partner is labelled with colloidal gold. In some preferred embodiments, the conjugate pad further comprises at least a second labelled binding partner that binds to the at least a secondanalyte (e.g., Nef, Tat, Vpu, or gp 120). In some preferred embodiments, the at least a second labelled binding partner is an antibody or functional fragment thereof. In some preferred embodiments, the at least a second labelled binding partner is labelled with colloidal gold.In some preferred embodiments, the device further comprises an absorption pad in fluid communication with the chromatographic test strip.The absorption pad, conjugate pad and sample pad may be formed from any suitable material. In preferred embodiments, the pads are formed from an absorbent material (e.g., glass fiber, cellulose, etc.).In some preferred embodiments, the chromatographic test strip further comprises a control zone.In some preferred embodiments, a user applies a biological sample that comprises extracellular vesicles, including exosomes (e.g., whole blood or plasma) to the sample pad. The biological sample may be pre-diluted with a running buffer, or running buffer may be applied to the sample pad before or preferably after application of the sample-to-sample pad. The biological sample in the buffer then flows from the sample pad to the chromatographic strip and then to the absorption pad. Extracellular vesicles, including exosomes, contained within the biological sample are captured as the buffer flows across and through the exosome capture zone.Next, the user applies a chase or lysis buffer to the conjugate pad. The chase or lysis buffer flows from the conjugate pad to the chromatographic test strip. The chase buffer carries the labelled binding partner(s) in the conjugate pad to the chromatographic test strip and also contain components that lyse or permeabilize the EVs captured in the EV capture zone. In some preferred embodiments, the lysis or chase buffer comprises one or more reagents that lyse or permeabilize Evs. In some preferred embodiments, the reagents are selected form the group consisting of saponins, surfactants and detergents, for example, Triton, Triton X-100, Tween 20, sodium dodecyl sulphate, and / or deoxycholate.Upon contact with the lysis or chase buffer, analytes are released from the EVs so that they can be bound by the labelled binding partners (i.e., detection reagents) and captured in the second zone (which can also be termed a detection zone). The user is then able to visualize detection of analytes by visual analysis of the second zone.In some embodiments, the present invention provides kits for use in detecting an analyte in a sample (such as, a biological sample). Such kits can be used, for example, to determine thepresence of an infectious agent in the sample. Certain embodiments of the disclosed kits are generally portable and provide a simple, rapid, and / or cost-effective way to determine the presence or absence of an analyte without the need for laboratory facilities, such as in a point-of- care facility.In some embodiments, the kits of the present invention include one or more immunoassay devices as disclosed herein, one or more antibodies and / or detection reagents, and a carrier means, such as a box, a bag, a satchel, plastic carton (such as molded plastic or other clear packaging), wrapper (such as, a sealed or sealable plastic, paper, or metallic wrapper), or other container. In some examples, kit components will be enclosed in a single packaging unit, such as a box or other container, which packaging unit may have compartments into which one or more components of the kit can be placed. In other examples, a kit includes one or more containers, for instance vials, tubes, and the like that can retain, for example, one or more biological samples to be tested, positive and / or negative control samples or solutions (such as, a positive control serum containing analyte), diluents (such as, phosphate buffers, or saline buffers), detector reagents (e.g., for external application to a kit device), substrate reagents for visualization of detector reagent enzymes (such as, 5-bromo-4-chloro-3- indolyl phosphate, nitroblue tetrazolium in dimethyl formamide), and / or wash solutions (such as, Tris buffers, saline buffer, or distilled water).Other kit embodiments include syringes, finger-prick devices, alcohol swabs, gauze squares, cotton balls, bandages, latex gloves, incubation trays with variable numbers of troughs, adhesive plate sealers, data reporting sheets, which may be useful for handling, collecting and / or processing a biological sample. Kits may also optionally contain implements useful for introducing samples into a sample chamber of an immunoassay device, including, for example, droppers, Dispo-pipettes, capillary tubes, rubber bulbs (e.g., for capillary tubes), and the like. Still other kit embodiments may include disposal means for discarding a used immunoassay device and / or other items used with the device (such as patient samples, etc.). Such disposal means can include, without limitation, containers that are capable of containing leakage from discarded materials, such as plastic, metal or other impermeable bags, boxes or containers.In some embodiments, a kit of the present invention will include instructions for the use of an immunoassay device or antigen-coated plate. The instructions may provide direction on how to apply sample to the test device or plate, the amount of time necessary or advisable to waitfor results to develop, and details on how to read and interpret the results of the test. Such instructions may also include standards, such as standard tables, graphs, or pictures for comparison of the results of a test. These standards may optionally include the information necessary to quantify analyte using the test device, such as a standard curve relating intensity of signal or number of signal lines to an amount of analyte therefore present in the sample.III. AntibodiesIn some embodiments, the devices, kits and methods of the present invention, which are described in detail above, utilize antibodies that bind to an analyte (e.g., HIV proteins) or other reagents so that the level of the analyte in a sample can be determined.An antibody of the present invention may be any monoclonal or polyclonal antibody raised against the analyte, as long as it can recognize the analyte. Antibodies can be produced by using the specific analyte or analytes as the antigen according to a conventional antibody or antiserum preparation process.The present invention contemplates the use of both monoclonal and polyclonal antibodies and functional fragments thereof. Any suitable method may be used to generate the antibodies used in the methods and compositions of the present invention, including but not limited to, those disclosed herein. For example, for preparation of a monoclonal antibody, protein, as such, or together with a suitable carrier or diluent is administered to an animal (e.g., a mammal) under conditions that permit the production of antibodies. For enhancing the antibody production capability, complete or incomplete Freund's adjuvant may be administered. Normally, the protein is administered once every 2 weeks to 6 weeks, in total, about 2 times to about 10 times. Animals suitable for use in such methods include, but are not limited to, primates, rabbits, dogs, guinea pigs, mice, rats, sheep, goats, etc.For preparing monoclonal antibody producing cells, an individual animal whose antibody titer has been confirmed (e.g., a mouse) is selected, and 2 days to 5 days after the final immunization, its spleen or lymph node is harvested and antibody producing cells contained therein are fused with myeloma cells to prepare the desired monoclonal antibody producer hybridoma. Measurement of the antibody titer in antiserum can be carried out, for example, by reacting the labeled protein, as described hereinafter and antiserum and then measuring the activity of the labeling agent bound to the antibody. The cell fusion can be carried out accordingto known methods, for example, the method described by Koehler and Milstein (Nature 256:495

[1975] ). As a fusion promoter, for example, polyethylene glycol (PEG) or Sendai virus (HVJ), preferably PEG is used.Examples of myeloma cells include NS 1, P3U1, SP2 / 0, AP 1 and the like. The proportion of the number of antibody producer cells (spleen cells) and the number of myeloma cells to be used is preferably about 1:1 to about 20:1. PEG (preferably PEG 1000 PEG 6000) is preferably added in a concentration of about 10% to about 80%. Cell fusion can be carried out efficiently by incubating a mixture of both cells at about 20°C to about 40°C, preferably about 30°C to about 37 °C for about 1 minute to 10 minutes.Various methods may be used for screening for a hybridoma producing the antibody (e.g., against a specific analyte). For example, where a supernatant of the hybridoma is added to a solid phase (e.g., microplate) to which antibody is adsorbed directly or together with a carrier and then an anti-immunoglobulin antibody (if mouse cells are used in cell fusion, anti-mouse immunoglobulin antibody is used) or Protein A labeled with a radioactive substance or an enzyme is added to detect the monoclonal antibody against the protein bound to the solid phase. Alternately, a supernatant of the hybridoma is added to a solid phase to which an antiimmunoglobulin antibody or Protein A is adsorbed and then the protein labeled with a radioactive substance or an enzyme is added to detect the monoclonal antibody against the protein bound to the solid phase.Selection of the monoclonal antibody can be carried out according to any known method or its modification. Normally, a medium for animal cells to which HAT (hypoxanthine, aminopterin, thymidine) are added is employed. Any selection and growth medium can be employed as long as the hybridoma 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, a serum free medium for cultivation of a hybridoma (SFM 101, Nissui Seiyaku) and the like can be used. Normally, the cultivation is carried out at 20°C to 40°C, preferably 37°C for about 5 days to 3 weeks, preferably 1 week to 2 weeks under about 5% CO2 gas. The antibody titer of the supernatant of a hybridoma culture can be measured according to the same manner as described above with respect to the antibody titer of the anti-protein in the antiserum.Separation and purification of a monoclonal antibody can be carried out according to the same manner as those of conventional polyclonal antibodies such as separation and purificationof immunoglobulins, for example, salting out, alcoholic precipitation, isoelectric point precipitation, electrophoresis, adsorption and desorption with ion exchangers (c.g., DEAE), ultracentrifugation, gel filtration, or a specific purification method wherein only an antibody is collected with an active adsorbent such as an antigen binding solid phase, Protein A or Protein G and dissociating the binding to obtain the antibody.Polyclonal antibodies may be prepared by any known method or modifications of these methods including obtaining antibodies from patients. For example, a complex of an immunogen (an antigen against the protein) and a carrier protein is prepared, and an animal is immunized by the complex according to the same manner as that described with respect to the above monoclonal antibody preparation. A material containing the antibody is recovered from the immunized animal and the antibody is separated and purified.As to the complex of the immunogen and the carrier protein to be used for immunization of an animal, any carrier protein and any mixing proportion of the carrier and a hapten can be employed as long as an antibody against the hapten, which is crosslinked on the carrier and used for immunization, is produced efficiently. For example, bovine serum albumin, bovine cycloglobulin, keyhole limpet hemocyanin, etc. may be coupled to a hapten in a weight ratio of about 0.1 part to about 20 parts, preferably, about 1 part to about 5 parts per 1 part of the hapten.In addition, various condensing agents can be used for coupling of a hapten and a carrier. For example, glutaraldehyde, carbodiimide, maleimide-activated ester, activated ester reagents containing thiol group or dithiopyridyl group, and the like find use with the present invention. The condensation product as such or together with a suitable carrier or diluent is administered to a site of an animal that permits the antibody production. For enhancing the antibody production capability, complete or incomplete Freund's adjuvant may be administered. Normally, the protein is administered once every 2 weeks to 6 weeks, in total, about 3 times to about 10 times.The polyclonal antibody is recovered from blood, ascites and the like, of an animal immunized by the above method. The antibody titer in the antiserum can be measured according to the same manner as that described above with respect to the supernatant of the hybridoma culture. Separation and purification of the antibody can be carried out according to the same separation and purification method of immunoglobulin as that described with respect to the above monoclonal antibody.In addition to polyclonal and monoclonal antibodies, any other antigen binding protein that binds to the analyte may be utilized. Examples of other antigen binding proteins include chimeric and humanized antibodies, Fab fragments, F(ab')2 fragments, and single chain antibodies (scFv).EXPERIMENTALExample 1 - HIV DetectionThis Example described a sensitive test for Human Immunodeficiency Virus (HIV) using enrichment and capture of EVs to probe EV cargo for HIV specific antigens. HIV viral proteins are present within EVs (i.e., “EV cargo”) isolated from the blood 20,24-27 and urine 28 of both viremic and non-viremic (<20 viral copies / mL) HIV patients. See, e.g., 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 Api- 42 secretion in SH-SY5Y neural cells. J. Neurovirol. 22, 179-190 (2016). 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 EVs has fueled basic research into the mechanisms by which EV s contribute to HIV pathogenesis and disease progression. See, e.g., 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).This example described the use of the presence or absence of HIV proteins in EV s for diagnostic purposes.Materials and Methods:FIG. 1 shows an overview of the EV-HIV assay.Western BlotsMaterials:4-12% Bis-Tris gradient gels, Fisher Scientific NP0321BOX20X MOPS SDS Running buffer4X NuPage LDS Sample BufferDTT recombinant p24Novex Sharp Pre- stained ladderNuPage Transfer bufferMethanol, Fisher ScientificMouse anti-p24 monoclonal antibodyGoat anti-mouse-HRP conjugateGoat anti-mouse- Alkaline Phosphatase conjugate10X Washing bufferDry MilkNovex PVDF membraneSuperSignal West PicoPlus HRP substrateBCIP / NBT Alkaline Phosphatase substrateMethods:Briefly, samples were run on 4-12% gradient gels at 180V for 1 hour, then transferred to the PVDF membrane at 30V for 1 hour. Blots were blocked for at least 1 hour in 5% Milk blotting buffer. Blots were washed three times, then probed overnight with the target antibody diluted in blotting buffer. Blots were washed three times, then probed for one hour with detection antibody diluted in blotting buffer. Blots were washed, then incubated with detection substrate as directed by the substrate manufacturer.ELIS AsMaterials:HIV-1 p24 ELISA kit, SinobiologicalMethods:The p24 ELISA was run as directed by the manufacturer’s instructions. Briefly, a standard curve was prepared. Samples were diluted as appropriate for the sample (generally 1:100). Standards and samples were added to the 96 well plate and incubated for 2 hours. Thewells were washed three times and 100 pL of diluted detection antibody added to each well. The plate was incubated for 1 hour, then each well was washed three times. 100 pL of substrate was added to each well and the plate was incubated for 30 minutes. The reaction was stopped and each well read at 450 nm on a Tecan GeniOS SpectraFluor plus plate reader (Mannedorf, Switzerland).Exosome Purification methodsMaterials:ExoQuick-TC, System Biosciences EXOTC10A-1ExoQuick, System Biosciences EXOQ A-1Thrombin Plasma Prep Kit, System Biosciences TMEXO-1 MagCapture Exosome Isolation Kit V2, FujiFilm 294-84101 IX PBS, Intuitive Bioscience Bi l lSodium Heparin, Fisher Scientific AC411210010Methods:For Exosome purification two different methods were used, a magnetic bead-based affinity isolation method and a polymer precipitation method. Both methods were performed per the manufacturer instructions, see brief descriptions below. Both methods used different materials or processes for tissue culture and human plasma. All are described below.Affinity isolation, MagCapture Exosome Isolation Kit V2 (for tissue culture):Prepare the magnetic beads by equilibrating in Washing buffer. Spin down the cell and cell debris by sequential centrifuge spins of increasing forces. Mix the supernatant with Exosome Binding Enhancer, 5 U / mL sodium heparin, and the previously equilibrated beads and incubate for 1 hour. Wash beads and then remove the bound exosomes by using Exosome Elution Buffer.Affinity isolation, MagCapture Exosome Isolation Kit V2 (for human plasma):Prepare the magnetic beads by equilibrating in Washing buffer. Spin down the cell and cell debris by sequential centrifuge spins of increasing forces. Mix the supernatant with Exosome Binding Enhancer and the previously equilibrated beads and incubate for 1 hour. Wash beads and then remove the bound exosomes by using Exosome Elution Buffer.Precipitation isolation, ExoQuick-TC (for tissue culture):Collect cell culture media and centrifuge at 3,000 x g for 15 minutes. Transfer supernatant to a fresh tube and add 200 pL of ExoQuick-TC per 1 mL of media supernatant. Mix the tubes and incubate overnight at 2-8°C. Centrifuge at 1,500 x g for 30 minutes. Aspirate the supernatant and re-suspend the pellet in 500 pL of IX PBS.Precipitation isolation, ExoQuick (for human plasma);Pre-treat the human plasma with thrombin plasma prep to increase the exosome yield by adding 2 pL of Thrombin Plasma Prep to 250 pL of each human plasma. Incubate for 5 minutes. Centrifuge samples at 10,000 x g for 5 minutes, then transfer the supernatant to a fresh tube. Add 63 pL of ExoQuick to each sample and incubate for 30 minutes at 2-8°C. After the incubation, centrifuge the samples for 30 minutes at 1,500 x g. Aspirate the supernatant and then re-suspend the pellet in 200 pL of IX PBS.Exosome isolation from plasmaThe exosomes were isolated using Norgen resin purification (Norgen Biotek 57400) according to the manufacturer’s instructions. A standard volume of 250 pL of each plasma sample was pre-cleared of cell debris by centrifugation at 2,500 x g and the pellet was discarded. The samples were brought to 1 mL using 750 pL ultrapure water. Next, 3 mL of nuclease-free water, 100 pL ExoC buffer, and 200 pL Slurry E were added to each sample and allowed to stand at room temperature for 5 minutes. The samples were then vortexed for 10 seconds and centrifuged at 2,000 RPM for 2 minutes. After discarding the supernatant, 200 pL ExoR buffer was added to the sample pellets and allowed to stand at room temperature for 5 minutes. The samples were vortexed for 10 seconds and centrifuged at 500 RPM for 2 minutes. The supernatant of each sample was transferred to a mini filter spin column assembled with a 2 mL tube and centrifuged at 6,000 RPM for 1 minute. The exosomes were contained in the sample collected in the 2 mL tube.Cells and serumMaterials:H9MN Full Length Intact (FI) cells; generous gift from Dr. Cliff Lane, Division of Intramural Research of the National Institute of Allergy and Infectious Diseases, NIH H9 cells, ATCC HTB-176HIV- human plasma, Precision for Medicine various lotsHIV+ human plasma, Precision for Medicine various lotsRPMI 1640, Gibco 1187-085Fetal Bovine Scram, Fisher Scientific 10437028Glutamine, Fisher Scientific 25030081Gentamicin, Gibco 15710-064HEPES, Gibco 15630-080Exosome depleted fetal bovine serum, Fisher Scientific A27208-03Methods:Cell lines were maintained as needed using RPMI modified with Fetal Bovine Serum, Glutamine, HEPES and Gentamicin at 37°C in 5% CO2. Prior to exosome purification, cells were split into media prepared with exosome depleted FBS for a minimum of 36 hours.Cell lysate preparationMaterials:BCA kit, Fisher Scientific 23225RIPA buffer, Fisher Scientific 89900HALT Protease Inhibitor, Fisher Scientific 87785Methods:Tissue culture cell lysis was performed as per the RIPA Buffer manufacturer’s instructions. Briefly, lysis buffer was prepared by adding HALT Protease Inhibitor to RIPA buffer. The cells were pelleted, resuspended in the prepared lysis buffer, and lysed at 2-8° for 15 minutes. The lysis reaction was centrifuged at 14,000 x g for 15 minutes to pellet insoluble debris and the supernatant transferred to a fresh tube. Protein concentrations of the lysed cell pellets were determined using the BCA kit as directed by the manufacturer.Nanoparticle Tracking AnalysisMaterials:Purified exosomesPBSMethods:Purified exosomes were analyzed on a NanoSight NS300 (Malvern Panalytical, Malvern, UK). Briefly, the particles are re-suspended by vortexing, then diluted in PBS and injected into the NS300 for size analysis and particle counts.Measurement of exosome protein concentration. The concentration was measured using a micro-BCA assay. The standards were prepared according to the manufacturer’s instructions. Two pL of sample or standard were added to a 96-well plate, followed by 40 L of BCA Working Reagent (prepared fresh). The samples were incubated at 37° C for 30 minutes, cooled to room temperature, and then read on a Tecan Infinite M Nano using a NanoQuant plate or on an Eppendorf Biophotometer using a pCuvette.Abby™ Automated Chemiluminescence Western Protein Analyzer. The Abby automated Western protein analyzer platform (ProteinSimple, Bio-Techne) uses small- volume capillaries to separate proteins by size followed by immunodetection. Separated proteins are immobilized to the capillaries by ultraviolet light, probed with antibodies and HRP conjugated secondary antibodies, and then detected by chemiluminescence. A molecular weight ladder is run with each full cartridge of 25 capillaries, and additional molecular weight markers are included with the loading dye in each cartridge to adjust for variations in the calculated molecular weight across the capillaries. The general process includes preparing samples at appropriate protein concentrations, mixing those samples with loading dye, denaturing them, and then loading them into the appropriate row of a specialized plate. The antibody of interest is then diluted and placed in the appropriate row of 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 and started. Because samples and antibodies are loaded into individual wells,Exosome Immunocapture Lateral Flow Materials:Anti-human CD9 antibody,40 nm gold colloid,CN140 nitrocellulose membranePolyester conjugate padSample pad,Anti-human CD63 antibodyAnti-human CD 81 antibodyHEPES running buffer (lOmM HEPES, pH7.4, 150mM NaCl, 0.05% Tween20, 1% BSA)PBS-T running buffer (using PBS, 0.1% Tween20 buffer)Methods:The CD9 antibody used for detection was adsorbed to gold colloid. Anti-human CD63 and anti-human CD81 antibodies were striped onto the CN140 laminated nitrocellulose membrane at 0.1 pL / mm using a benchtop Isoflow instrument (Arista Biologicals, PA). Striped membranes were dried overnight at 37°C before storing under desiccation until use. Purified exosomes were thawed and diluted in HEPES running buffer to a concentration of 10 pg / mL. In a microtiter plate, 100 pL of diluted exosomes or HEPES buffer alone were incubated with 30 GU of anti-CD9 gold conjugate. Nitrocellulose strips with the tetraspanin test line and sample adsorption pad were added to the microtiter wells and were allowed to run for 15 minutes.HIV p24 Lateral Flow and Testing EV Lysis BuffersMaterials:40 nm gold colloidCN140 nitrocellulose membranePolyester conjugate padSample padAnti-p24 antibody, used for capture reagentAnti-p24 antibody, used for detection reagentHEPES running buffer (lOmM HEPES, pH7.4, 150mM NaCl, 0.05% Tween20, 1% BSA)PBS-T running buffer (using PBS, 0.1% Tween20 buffer)Glass fiber conjugate padLyophilized exosomes, various sourcesBBS sucrose,Recombinant p24Triton X-100SaponinRIP A cell lysis buffer (25mM Tris HC1 (pH7.6), 150mM NaCl, 1%, Tergitol NP 40, 1% sodium deoxycholate, 0.1% SDS)M-PER™ Mammalian Protein Extraction Reagent, 78501HEPES buffer (lOmM HEPES (pH7.4), 150mM NaCl, 0.05%, Tween20, 1% BSA)Running Buffer (2mM Tris-HCl pH 7.6, 0.1 % NP-40, 0. 1 % sodium deoxycholate, 0.01 % SDS, 9 mM HEPES pH 7.4, 150 mM NaCl, 0.045% Tween 20 and 0.9% BSA)HALT 0.1X(AEBSF 0.1 mM, Aprotinin 80 nM, Bestatin 5 pM, E64 1.5 pM, Leupeptin 2 pM, Pepstatin A 1 pM, and DMSO) Methods:Both anti-p24 antibodies were diluted in borate sucrose buffer. The anti-p24 antibody used for detection was adsorbed to gold colloid. Conjugate pads were spotted with 30 GU of 53M gold conjugate at pH 7.0 and dried at 37°C for 30 minutes. The capture anti-p24 antibody was striped onto the CN140 laminated nitrocellulose membrane at 0.1 pL / mm using a benchtop Isoflow instrument (Arista Biologicals, PA). Striped membranes were dried overnight at 37°C before storing under desiccation until use. Prototype lateral flow tests were assembled in square cassettes and closed using a cassette press. Purified exosomes from H9 (p24-) or H9MN FI (p24+) cells were diluted in HEPES running buffer with and without detergents or surfactants. For each condition, 100 pL of sample were added to each test and allowed to run for 15 minutes before scoring with a visual score card or image quantified by densitometry analysis using ImageJ. Schneider, C. A., Rasband, W. S., & Eliceiri, K. W. (2012). NIH Image to Imagel: 25 years of image analysis. Nature Methods, 9(7), 671-675.Automated western analysis was performed on the Abby (Simple Western) instrument using the 12-200kDa separation module. Depending on the primary antibody, an anti-mouse or anti-rabbit detection module was used. Values for the peaks corresponding to the protein of interest were quantified in the Compass software to measure the Area Under the Curve (AUC) for each band in each lane.ResultsChronically infected H9MN FI cells express p24 in whole cell lysate and in purified exosomes.The H9 (parental, HIV- cell line) and H9MN FI (stable provirus, HIV+ cell line) cells were grown in growth media supplemented with 10% FBS. At least 24 hours prior to collection, cells were washed and growth media replaced with exosome-free media. After 24 hours, cells were removed and centrifuged to pellet the cells. The pellet was treated with RIPA buffer to lyse the cells. The supernatant from the centrifugation was used to purify exosomes (EV) using the MagCapture method. Lysates were run at 2.5 pg per lane, and 5 pg of the purified exosomes wasrun per lane (FIG. 2). Detection of p24 with the mouse anti-p24 Gag monoclonal antibody was confirmed by running 0.5 pg recombinant p24 protein in a separate lane.Exosomes from H9MN FI cell lines enriched by 2 different methods contain p24 protein in high quantities.To confirm that the p24 detected was from exosomes, exosomes purified from H9 and H9MN FI cells were characterized to confirm quantify and size distribution. The concentration and size distribution of EVs was determined using the NanoSight NS300 (Malvern Panalytical, Malvern UK). H9MN FI had 3.5 x 109EV particles / mL while H9 had 1.0 x 109particles / mL, confirming that the material purified is in the size range of exosomes (30-150 nm) and were of similar number of particles (FIGs. 3-4).Exosomes purified from HIV+ plasma contain HIV p24.Exosome purification was performed by ExoQuick and MagCapture method from human plasma samples collected from HIV- and HIV+ subjects. Purified exosomes from each sample were analyzed for size and quantity using the NanoSight 300. The results are summarized in Table 1. Both exosome purification methods result in similar amount of particles per mL. When tested on the p24 ELISA, the H9 derived exosomes had signal below the limit of detection. The exosomes derived from H9MN FI cells, despite the purification method, had similar levels of HIV p24 protein in ng / mL quantities.Table 1. Comparison of EV concentration, protein concentration, and measured p24 between H9 and H9MN FI purified exosomes and cell lysates.In addition to performing exosome characterization by NanoSight, a CD63 ELISA was performed to confirm that the exosomes contained the tetraspanins markers that will be used by lateral flow. From the same samples, the p24 ELISA was performed to measure the amount of HIV specific protein found. In the HIV- plasma samples, exosomes were detected by bothNanoSight and CD63 ELISA, but no measurable amounts of p24. In the HIV+ plasma, NanoSight and CD63 ELISA confirm presence of exosomes and the p24 ELISA confirms presence of HIV protein in the exosome preparation.Table 2. Characterization of ExoQuick purified exosomes from plasma samples of HIV- and HIV+ individuals. Data from CD63 ELISA and HIV p24 ELISA arc shown. <LOD indicates below the limit of detection of the assay.Confirmation of detection of HIV proteins in purified EVs.To further demonstrate the presence of HIV proteins in extracellular vesicles, including exosomes, from human plasma EVs were enriched using the silicone carbide resin method from plasma samples. Extracellular vesicles were enriched from 0.2 mL plasma from HIV- subjects and HIV+ subjects and run on an automated western analysis system. A total of 0.4 mg of enriched EVs were run per samples. The abundance of HIV proteins p24, Nef, and Tat were measured in samples using specific antibodies. As shown in Figure 5, EVs enriched from HIV+ samples have significant levels of p24, Nef, and Tat present when compared to HIV- samples. This confirms the presence of HIV proteins in EVs, and demonstrates their utility as diagnostic biomarkers for HIV infection.Demonstrate EV immunoprecipitation by lateral flow.To perform exosome purification and concentration in a point-of-care method, a lateral flow assay for immunocapture of exosomes was developed following the method of Oliveria- Rodriguez et al. (J Extracell Vesicles. 2016; 5: 10.3402 / jev.v5.31803. ) Briefly, CD63 and CD81 capture antibodies were striped onto a nitrocellulose lateral flow strip. An anti-CD9 goldconjugate was used as a detection antibody to determine exosome capture. As shown in FIG. 6, 10 pg / mL of exosome protein can be capture by the tetraspanins lateral flow, with a calculated LOD of between 2.5 and 5 g / mL or approximately 5 x 107EV particles (Fig. 7). The LOD was calculated using signal intensities determined using a DPP® Micro Reader, a portable lateral flow reader (Chembio Diagnostics, Medford NY). This demonstrates successful capture of exosomes by CD63 / CD81 immunocapture on a lateral flow device with low nonspecific binding and visible specific signal.Demonstrate detection of HIV p24 by lateral flow from purified exosomes using lysis buffers to release cargo.If p24 is isolated in exosomes, it would be contained in the exosome interior as a cargo protein. To better detect HIV cargo proteins in exosomes, different buffers were tested to identify a formulation that can release exosomes cargo without interfering with the function of capture and detection antibodies. To test these formulations, a standard p24 lateral flow strip was developed using anti-p24 capture and gold conjugated detection antibodies.Demonstrate EVs can be lysed and cargo captured on lateral flow.Exosomes isolated from H9 (HIV-) and H9MN FI (HIV+) cells were used to determine if the amount of p24 contained in exosomes could be detected on a lateral flow assay. Lateral flow technology is not considered a sensitive method of detection of biomarkers; however, the isolation of extracellular vesicles should concentrate p24 and increase the amount for detection. To test this several buffers with properties that disrupt lipid raft, which are highly concentrated in extracellular vesicles and exosomes, were screened to identify formulations that would both release EV cargo and be compatible with lateral flow technology.Purified exosomes were incubated with different buffers to include detergents and chemicals known to break apart membranes. One component, saponin, has been specifically used in methods for loading exosomes because of its ability to perforate exosomes (Chen et al., Front. Cell Dev. Biol., 08 October 2021). The key properties of saponin are that it interacts with the membrane-bound cholesterol, creating pores in the exosomal membranes and 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, treatment with saponin can permeabilize the virus membrane and release virus content. Triton also interacts with the membrane and allows permeabilization, resulting in release of cargo available for detection. Other detergents, includingTriton X- 100, Tween 20, sodium dodecyl sulphate, and deoxycholate may also be used (Xabier Ostcikoctxca ct al., Org Biomol Chcm. 2015 Oct 14; 13(38):9775-82. doi: 10.1039 / c5ob01451d.).Enriched exosomes from H9 (p24-) and H9MN FI (p24+) cells were incubated with a variety of buffers to test the effectiveness of exosome lysis. One example is shown in Fig. 8 on the standard p24 lateral flow test. Briefly, purified exosomes from H9 and H9MN FI cells were incubated with a lysis buffer (10% MPER in 2.5 mM bicine, 9 mM HEPES, 135 mM NaCl, 0.045% Tween 20 and 0.9% BSA at pH 7.4) in 1.5 mL tubes for 15 minutes at room temperature, then transferred to the sample loading port of the lateral flow device. Sample was allowed to flow for 15 minutes at room temperature before visual readout was scored on the 1-10 scale. As shown in Fig 8 C, both the buffer only and H9 exosomes had a score of 3. The H9MN FI exosomes had a score of 6, indicating successful capture of p24 from the lysed exosome preparation.Other lysis buffer were tested and the results of visual line inspection are shown in Table 3. The addition of detergents and surfactants increases the amount of detectable p24 in purified exosomes. Additionally, the sequential addition of EV lysis buffer was critical to reducing the background in the buffer only and H9 Evs. Specifically, Lysis Buffer #2 (RPIA buffer with 10% protease inhibitors) was added to purified exosomes for 15 minutes to promote lysis. This was diluted with an equal volume of HEPES and then added to the lateral flow device. This reduced the background signal and maintained the detection of p24 in the H9MN FI EVs (Table 3, Lysis Buffer #2).Table 3. Summary of LF scores for p24 LF assay using different lysis buffer conditions. Area Under the Curve (AUC) was measured by densitometry analysis of captured images using Image J.A two-step lateral flow device was assembled to allow capture of EVs and lysis in a separated location from the p24 test line to avoid lysis buffer components from interfering with p24 capture. The EV capture zone (anti-CD9, anti-CD63, and anti-CD81) was placed on the nitrocellulose membrane as shown in Fig. 9A. The anti-p24 test line is placed downstream on a separate nitrocellulose membrane. By adding EVs isolated from H9 (p24-) and H9MN FI (p24+) cells in running buffer, the EVs are captured in the test zone. Next, the EV Lysis Buffer is added to release EV contents and flow EV cargo through the anti-p24 gold conjugate zone and is captured on the p24 test zone. A total of 30 pg EVs isolated from H9 cells had a visual score of 0, while 5 pg of EVs from H9MN FI EVs had a score of 4, demonstrating the ability to detect p24 from EVs from H9 MNFI cell lines expressing HIV proteins after lysis of immunocaptured EVs.As shown in Fig. 10, different detergents and EV permeabilization reagents can result in an increased detection of p24 from EVs. Images from strips ran with PBS (buffer alone), 20 pg of EVs from H9 cells, and 20 pg of EVs from H9 MNFI cells are shown in Fig. 10 for each condition, with numerical values listed below each image from densitometry analysis of the strips. When compared to EVs in PBS buffer (no lysis), and increase in detectable p24 from H9 MNFI EVs is observed when detergents or membrane disrupting agents are used.Any method used for permeabilization could be incorporated into a lateral flow device to release EV cargo, including but not limited to: chemical methods using cations like calcium phosphate, detergents and surfactants, physical methods including ultrasound, electroporation, and photoporation (Zhang et al., Innovative Methodology 10 Jan2017https: / / doi.org / l 0.1 152 / ajplung.00423.2016; Guido et al., Microelectronic Engineering Volume 98, October 2012, Pages 707-710).Extracellular vesicles, including exosomes, can be immunoprecipitated on a lateral flow device and internal cargo detected by lateral flow assay.Example 2: Prostate Cancer PSA Lateral FlowThis example describes the detection of prostate specific antigen (PSA) in EVs.Figures 11 and 12 shows a schematic of one embodiments of an EV-PSA Test.Materials:40 nm gold colloidCN140 nitrocellulose membrane,Polyester conjugate pad,Sample pad,Anti-PSA capture antibodyAnti-PSA detection antibodyHEPES running buffer (lOmM HEPES, pH7.4, 150mM NaCl, 0.05% Tween20, 1%BSA)PBS-T running buffer (using PBS, 0.1% Tween20 buffer)Glass fiber conjugate pad,Lyophilized exosomes, various sourcesBBS sucroseRecombinant PSATriton X-100SaponinRIP A cell lysis buffer (25mM Tris HC1 (pH7.6), 150mM NaCl, 1%, Tergitol NP 40, 1% sodium deoxycholate, 0.1% SDS)M-PER™ Mammalian Protein Extraction Reagent, 78501HEPES buffer (lOmM HEPES (pH7.4), 150mM NaCl, 0.05%, Tween20, 1% BSA) Running Buffer (2mM Tris-HCl pH 7.6, 0.1% NP-40, 0.1% sodium deoxycholate, 0.01%SDS, 9 mM HEPES pH 7.4, 150 mM NaCl, 0.045% Tween 20 and 0.9% BSA)HALT 0. 1X(AEBSF 0.1 mM, Aprotinin 80 nM, Bestatin 5 pM, E64 1 .5 pM, Leupeptin 2 pM, Pcpstatin A 1 pM, and DMSO) Methods:Both anti-PSA antibodies were diluted in borate sucrose buffer. The anti-PSA antibody used for detection was adsorbed to gold colloid. Conjugate pads were spotted with 30 GU of 53M gold conjugate at pH 7.0 and dried at 37°C for 30 minutes. The other anti-PSA antibody was striped onto the CN140 laminated nitrocellulose membrane at 0.1 pL / mm using a benchtop Isoflow instrument (Arista Biologicals, PA). Striped membranes were dried overnight at 37°C before storing under desiccation until use. Prototype lateral flow tests were assembled in square cassettes and closed using a cassette press. Purified exosomes from healthy plasma, PC3 (PSA-) or LNCaP (PSA+) cells were diluted in HEPES running buffer with and without detergents or surfactants. For each condition, 100 pL of sample were added to each test and allowed to run for 15 minutes before scoring with a visual score card or image quantified by densitometry analysis using ImageJ. Schneider, C. A., Rasband, W. S., & Eliceiri, K. W. (2012). NIH Image to ImageJ: 25 years of image analysis. Nature Methods, 9(7), 671-675.Prostate Specific Antigen (PSA) is detected on a EV-capture LF format.Briefly, anti-CD63 and anti-CD81 tetraspanin capture antibodies were striped onto a nitrocellulose lateral flow strip to capture EVs upstream of the test zone (Fig. 13 A and B). A solution containing purified PSA a different concentrations, as shown in Fig. 13, was tested to determine the LOD of the strips. The intensity of the signal associated with the Test Line was recorded after 15 minutes, when images are captured. The limit of detection was calculated to be 7.8 pg / mL (Fig. 13 C and D), using signal intensities measured with a portable lateral flow reader (DPP® Micro Reader, Chembio Diagnostics, Medford, NY).To examine the capture of EVs from prostate cancer cell lines, highly purified exosomes isolated from PC3 (prostate cancer cell line that does not express PSA) was compared to exosomes from LNCaP cells (prostate cancer cell line that expresses PSA). A equimolar mix of anti-CD9, anti-CD63, and anti-CD81 was striped into the EV capture zone. Gold conjugated detection antibodies for each of CD9, CD63, and CD81 were used as detection reagents. Visual score values for each EV type are shown using the different detection antibodies. As shown in Fig. 14, LNCaP EVs have all three tetraspanins present on EVs. This is in contrast to PC3 EVs, where CD81 is barely detectable. Additionally, EVs from healthy human plasma are not detectedusing the anti-CD81 gold conjugate. This data suggests that using different combinations of tctraspanin capture antibodies can enrich samples for EVs containing PSA, allowing customization of the EV capture zone to further enrich EVs for lysis by selective immunoprecipitation. In this example, using anti-CD81 in the capture zone could selectively enrich for PSA-containing EVs from input sample.PSA-containing EVs are detected in PCa cell lines.EVs were isolated from LNCaP (PSA+) and compared to EVs isolated from “normal” plasma (low PSA). The LNCaP cell line is an androgen sensitive, PSA-expressing cell line derived from a patient with metastatic prostate cancer. Purified EVs were analyzed by BCA for total protein content and NTA to determine the number of EV particles per mL. Various amounts of purified EVs, ranging from 3.125-25 pg / mL were tested using a total PSA ELISA (Alpco, Salem NH). At all input concentrations of EVs tested, there was between a 2-20 fold increase in total PSA detected in EVs from LNCaP cells. The amount of total PSA measured in LNCaP derived EVs was increased by incubation in 25% RIPA buffer, consistent with the hypothesis that lysis of EVs enhances detection of EV cargo proteins. Greater than 2 ng / mL of total PSA was detected in 3.25 g / mL purified EVs (Fig 15).Two-phase EV capture lateral flow technology enables sensitive detection PSA from EVs.Using EVs purified LNCaP (PSA+), detection of PSA was increased by addition of lysis buffers. As shown in Fig. 16, using 0.25X RIPA increased the visual detection of PSA on the test line. Signal increased with increasing amounts of input EVs. This demonstrates that lysis of EVs increases the amount of detectable PSA from EVs.

Claims

CLAIMSWhat is claimed is:

1. A method of detecting the presence of an analyte in a sample from a subject, comprising: obtaining a patient sample comprising extracellular vesicles; capturing extracellular vesicles from the patient sample; lysing the extracellular vesicles to release proteins contained therein; contacting the proteins with one or more analyte- specific binding agents and / or one or more detection reagents suitable for detecting the analyte; and determining whether the extracellular vesicles comprise the analyte.

2. The method of claim 1, wherein the analyte is a protein.

3. The method of claims 1 or 2, wherein the analyte is a cancer antigen or cancer- specific protein.

4. The method of claim 3, wherein the analyte is prostate specific antigen (PSA).

5. The method of claim 2, wherein the protein is from an infectious agent and the presence of the analyte in the sample is indicative of infection of the analyte in the subject.

6. The method of claim 5, wherein the infectious agent is a virus or a bacteria.

7. The method of claim 1, wherein the analyte is a nucleic acid.

8. The method of any one of claims 1 to 2, wherein the analyte is HIV protein p24 and, optionally, at least one additional HIV protein selected from the group consisting of Nef, Tat, Vpu, and gpl20.

9. A method for diagnosing human immunodeficiency vims (HIV) infection in a subject, comprising: obtaining a patient sample comprising extracellular vesicles; capturing extracellular vesicles from the patient sample; lysing the extracellular vesicles to release proteins contained therein; contacting the proteins with one or more HIV protein binding agents and / or one or more detection reagents suitable for detecting one or more HIV proteins; and determining whether the extracellular vesicles comprise the HIV protein p24 and optionally at least one additional HIV protein selected from the group consisting of Nef, Tat, Vpu, and gpl20; wherein a determination of the presence of p24 and the at least one additional HIV protein is indicative of 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 gp!20.

14. The method of claim 9, wherein the subject has been vaccinated against HIV infection.

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

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

17. The method of claim 16, wherein the chromatographic strip is in a lateral flow assay device.

18. The method of any one of claims 1 to 17, wherein the extracellular vesicles are captured with a tctraspanin.

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

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

21. The method of claim 20, wherein the captured extracellular vesicles are lysed by adding a lysis or chase buffer to the chromatographic test strip.

22. The method of claim 21, wherein the lysis or chase buffer comprises a lysis agent selected from the group consisting of a saponin, a surfactant and a detergent and combinations thereof.

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

24. The method of any one of claims 1 to 23, wherein the detection reagents comprise one or more antigen binding molecules.

25. The method of claim 24, wherein the antigen binding molecules are antibodies or antibody fragments.

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

27. The method of claim 26, wherein the labelled antibodies or functional fragments thereof are labelled with colloidal gold or colored microspheres.

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

29. The method of claim 28, wherein the labelled nucleic acids are labelled with colloidal gold or colored microspheres.

30. The method of any one of claims 1 to 29, wherein the extracellular vesicles are exosomes.

31. An assay device comprising: a chromatographic test strip comprising a plurality of zones, the chromatographic test strip allowing flow of a fluidic test sample from a first end of the chromatographic strip to a second end of the chromatographic test strip; wherein the chromatographic strip comprises a first zone comprising an extracellular vesicle binding agent, a second zone 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 reagents for visualizing binding of the analytes to the labeled binding partners.

32. The assay device of claim 31, wherein the extracellular vesicle binding agent in the first zone is a tetraspanin.

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

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

35. The assay device of any one of claims 31 to 33, wherein the at least a second analyte is selected from the group consisting of an HIV protein selected from Nef, Tat, Vpu, and gpl20 and combinations thereof.

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

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

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

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

40. The assay device of any one of claims 31 to 33, wherein the analyte is a nucleic acid.

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

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

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

44. The assay device of any one of claims 31 to 43, wherein the chromatographic strip is in fluidic communication with a sample pad.

45. The assay device of any one of claims 31 to 44, wherein the chromatographic strip is in fluidic communication with a conjugate pad.

46. The assay device of claim 45, wherein the conjugate pad comprises a first labelled binding partner that binds to the first analyte.

47. The assay device of claim 46, wherein the first labelled binding partner is an antibody or functional fragment thereof.

48. The assay device of claim 46, wherein the first labelled binding partner is a nucleic acid.

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

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

51. The assay device of claim 50, wherein the at least a second labelled binding partner is an antibody or functional fragment thereof.

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

53. The assay device of any one of claims 31 to 52, further comprising an absorption pad in fluid communication with the chromatographic test strip.

54. The assay device of any one of claims 31 to 53, wherein the chromatographic test strip further comprises a control zone.

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

56. A kit for detecting an analyte in an extracellular vesicle, comprising: a) the device of any one of claims 31 to 55; and b) a container of lysis or chase buffer.

57. Kit of claim 56 for use in detecting an analyte in an extracellular vesicle.

58. Use of claim 57, wherein the extracellular vesicle is an exosome.

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