Compositions and methods for detection of gene expression

Abstract

Provided herein are methods for enriching tumor derived EVs or brain-derived EVs from a biological sample, e.g. a biofluid, using a lectin or glycan-binding fragment thereof and an isolation component (e.g., a solid media). The present disclosure also provides compositions and kits for isolating brain-derived EVs or tumor derived EVs, and compositions comprising an isolated population of brain-derived EVs or tumor derived EVs.

Description

ATTORNEY DOCKET NO.: 14842-001-228COMPOSITIONS AND METHODS FOR DETECTION OF GENE EXPRESSIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Serial No. 63 / 686,914 filed August 26, 2024, the disclosure of which is incorporated by reference herein in its entirety.1. FIELD

[0002] This disclosure relates to the detection of gene expression that may or may not relate to the presence or absence of one or more cancers from various tissues using specific components found in whole blood or a segment thereof.2. BACKGROUND

[0003] Detection of a cancer, particularly early stage cancer, often involves complicated diagnostic techniques that are typically invasive and expensive. For example, for detection of nonsmall cell lung cancer (NSCLC) a biopsy is the most common method of detection. In a biopsy, a sample of tissue from the suspected tumor site is physically removed, often with the use of needle aspiration, by bronchoscopy, or other direct method of accessing and collecting a sample. The collecting of the sample for NSCLC involves directly accessing the lungs through the trachea or from outside the body directly through the skin. Either method is invasive and painful.

[0004] In some instances of lung cancer diagnosis, for example, a physician will perform a thoracentesis where a needle is inserted through the chest wall and used to collect lung fluid that is tested for the presence of cancer cells. Common and more invasive techniques include thoracoscopy, mediastinoscopy, or thoracotomy, each of which involve general anesthesia and an operating team to perform.

[0005] Detection of other cancer types also typically involve physically collecting a sample tissue and characterizing the tissue either as cancerous or benign. Often large sample volumes are required if additional testing is needed to determine cancer stage, invasiveness, or genotypic / phenotypic characterization. Large sample volume collection typically involves more invasive procedures.

[0006] Blood based disease detection is highly desirable the field of oncology and precision medicine. Such techniques could enable earlier detection of disease, diagnosis, patientNAI-5002577470vl 1ATTORNEY DOCKET NO.: 14842-001-228 stratification, and treatment monitoring without the need for invasive and painful diagnostic procedures. Circulating tumor cells (CTCs) and cell-free circulating tumor DNA (cfDNA) are the most common liquid biopsy analytes used in clinical settings. However, several limitations impede these approaches, including low quantity, unfavorable signal to noise ratios, and minimal detailed information on disease subtype or mechanisms driving disease.

[0007] As such, a need exists for compositions and methods that can be used to detect and / or characterize expression of genes, such as but not limited to genes that may be altered due to the presence or absence of one or more cancers, including brain cancers, where the compositions and methods are less invasive than direct tissue biopsy. A need also exists for compositions and methods that can be used to detect and / or characterize expression of brain / neuro genes, where the compositions and methods are less invasive than direct tissue biopsy.3. SUMMARY

[0008] In one aspect, provided herein is a composition comprising an isolated population of brain-derived extracellular vesicles (EVs), wherein the isolated population of brain-derived EVs are obtained by a method comprising: isolating EVs from a biofluid under conditions sufficient to preserve EV glycocalyx, wherein the biofluid is plasma or serum; contacting the isolated EVs with a lectin or glycan-binding fragment thereof conjugated to a solid media, wherein the lectin or glycan-binding fragment thereof specifically binds a biantennary galactosylated N-glycan with bisecting N-acetylglucosamine (GlcNAc); and removing unbound EVs from the solid media, thereby obtaining the isolated population of brain-derived EVs. In some embodiments, isolating does not comprise ultracentrifugation or addition of a polymer to the biofluid.

[0009] In some embodiments, removing unbound EVs comprises washing the solid media with a saline buffer. In some embodiments, the saline buffer is selected from the group consisting of phosphate buffered saline (PBS), HEPES buffered saline (HBS), and Dulbecco's phosphate buffered saline (DPBS). In some embodiments, the saline buffer comprises PBS, pH 7.4. The composition of claim 5, wherein the buffer further comprise about 0.1 mM CaCk and about 0.1 mM MnCh buffer.

[0010] In some embodiments, the lectin comprises phaseolus vulgaris erythroagglutinin (PHA-E) or a glycan-binding fragment thereof.NAI-5002577470vl 2ATTORNEY DOCKET NO.: 14842-001-228

[0011] In some embodiments, isolating comprises a positive charge-based isolation. In some embodiments, isolating comprises a membrane-based affinity column.

[0012] In some embodiments, the biofluid is from a subject having or suspected of having a neurological disease.

[0013] In some embodiments, the solid media comprises a magnetic particle, an agarose particle, or a hydrogel polymer. In some embodiments, the solid media comprises a magnetic particle. In some embodiments, the lectin is conjugated to the solid media via tosylation.

[0014] In some embodiments, contacting comprises rotating for about 12 to about 24 hours. In some embodiments, contacting is at about 2-8 °C.

[0015] In one aspect, provided herein is a composition comprising: (a) a heterogenous plurality of isolated EVs, where the isolated EVs comprise: (i) a first population of EVs; and (ii) a second population of EVs; and (b) a lectin or glycan-binding fragment thereof conjugated to a solid media, wherein the lectin or fragment thereof specifically binds a biantennary galactosylated N-glycan with bisecting GlcNAc; and (c) a buffer, wherein the first population of EVs are brain- derived and the second population of EVs are not brain-derived, and wherein the first population of EVs are specifically bound to the solid media and the second population of EVs are unbound.

[0016] In some embodiments, the lectin comprises PHA-E or a glycan-binding fragment thereof.

[0017] In some embodiments, the solid media comprises a magnetic particle, an agarose particle, or a hydrogel polymer. In some embodiments, the solid media comprises a magnetic particle. In some embodiments, the lectin is conjugated to the solid media via tosylation.

[0018] In some embodiments, the buffer comprises equal parts: a first solution comprising 3M ammonium sulfate, pH 7.9; and a second solution comprising 0.1 M sodium phosphate buffer, 0.1 mM CaCh, 0.1 mM MnCl2.

[0019] In another aspect, provided herein is a method for enriching brain-derived EVs from a biofluid, the method comprising: (a) isolating EVs from a biofluid under conditions sufficient to preserve EV glycocalyx, wherein the biofluid is plasma or serum; (b) contacting the isolated EVs with a lectin or glycan-binding fragment thereof conjugated to a solid media, wherein the lectin or glycan-binding fragment thereof specifically binds a biantennary galactosylated N-glycan withNAI-5002577470vl 3ATTORNEY DOCKET NO.: 14842-001-228 bisecting GlcNAc; and (c) removing unbound EVs from the solid media, thereby enriching brain- derived EVs from a biofluid. In some embodiments, isolating does not comprise ultracentrifugation or addition of a polymer to the biofluid.

[0020] In some embodiments, removing unbound EVs comprises washing the solid media with a saline buffer. In some embodiments, the saline buffer is selected from the group consisting of phosphate buffered saline (PBS), HEPES buffered saline (UBS), and Dulbecco's phosphate buffered saline (DPBS). In some embodiments, the saline buffer comprises PBS, pH 7.4. In some embodiments, the buffer further comprise about 0.1 mM CaCh and about 0.1 mM MnCh buffer.

[0021] In some embodiments, the lectin comprises PHA-E or a glycan-binding fragment thereof.

[0022] In some embodiments, isolating comprises a positive charge-based isolation. In some embodiments, isolating comprises a membrane-based affinity column.

[0023] In some embodiments, the biofluid is from a subject having or suspected of having a neurological disease.

[0024] In some embodiments, the solid media comprises a magnetic particle, an agarose particle, or a hydrogel polymer. In some embodiments, the solid media comprises a magnetic particle. In some embodiments, the lectin is conjugated to the solid media via tosylation.

[0025] In some embodiments, contacting comprises rotating for about 12 to about 24 hours. In some embodiments, contacting is at about 2-8 °C.

[0026] In another aspect, provided herein is a kit comprising: a lectin or glycan-binding fragment thereof conjugated to a solid media, wherein the lectin or glycan-binding fragment thereof specifically binds a biantennary galactosylated N-glycan with bisecting GlcNAc; a buffer suitable for preserving the EV glycocalyx; and instructions for use.

[0027] In some embodiments, the lectin comprises PHA-E or a glycan-binding fragment thereof.

[0028] In some embodiments, the pH of the buffer is about pH 7.0 to pH 8.0. In some embodiments, the pH of the buffer is about pH 7.4.NAI-5002577470vl 4ATTORNEY DOCKET NO.: 14842-001-228

[0029] Tn some embodiments, the solid media comprises a magnetic particle, an agarose particle, or a hydrogel polymer. In some embodiments, the solid media comprises a magnetic particle. In some embodiments, the lectin is conjugated to the solid media via tosylation.

[0030] In one aspect, provided herein is a composition comprising: (a) a solid media; (b) a lectin-mediated means for specifically binding a biantennary galactosylated N-glycan with bisecting GlcNAc; (c) a heterogenous plurality of isolated EVs, where the isolated EVs comprise: (i) a first population of EVs; and (ii) a second population of EVs; and (d) a buffer, wherein the first population of EVs are brain-derived and the second population of EVs are not brain-derived, and wherein the first population of EVs are specifically bound to the solid media and the second population of EVs are unbound.

[0031] In some embodiments, the lectin-mediated means comprises PHA-E or a glycan- binding fragment thereof.

[0032] In some embodiments, the solid media comprises a magnetic particle, an agarose particle, or a hydrogel polymer. In some embodiments, the solid media comprises a magnetic particle. In some embodiments, the lectin-mediated means is conjugated to the solid media via tosylation.

[0033] In some embodiments, the buffer comprises equal parts: a first solution comprising 3M ammonium sulfate, pH 7.9; and a second solution comprising 0.1 M sodium phosphate buffer, 0.1 mM CaCh, 0.1 mM MnCl2.

[0034] In another aspect, provided herein is a method for enriching brain-derived EVs from a biofluid, the method comprising: (a) isolating EVs from a biofluid under conditions sufficient to preserve EV glycocalyx, wherein the biofluid is substantially free or devoid of red-blood cells; (b) contacting the isolated EVs with a lectin-mediated means for specifically binding a biantennary galactosylated N-glycan with bisecting GlcNAc conjugated to a solid media; and (c) removing unbound EVs from the solid media, thereby enriching brain-derived EVs from a biofluid. In some embodiments, the biofluid is plasma or serum.

[0035] In some embodiments, removing unbound EVs comprises washing the solid media with a saline buffer.NAI-5002577470vl 5ATTORNEY DOCKET NO.: 14842-001-228

[0036] Tn some embodiments, the saline buffer is selected from the group consisting of phosphate buffered saline (PBS), HEPES buffered saline (HBS), and Dulbecco's phosphate buffered saline (DPBS). In some embodiments, the saline buffer comprises PBS, pH 7.4. In some embodiments, the buffer further comprise about 0.1 mM CaCh and about 0.1 mM MnCh buffer.

[0037] In some embodiments, the lectin-mediated means comprises PHA-E or a glycan- binding fragment thereof.

[0038] In some embodiments, isolating comprises a positive charge-based isolation. In some embodiments, isolating comprises a membrane-based affinity column.

[0039] Tn some embodiments, the biofluid is from a subject having or suspected of having a neurological disease.

[0040] In some embodiments, the solid media comprises a magnetic particle, an agarose particle, or a hydrogel polymer. In some embodiments, the solid media comprises a magnetic particle. In some embodiments, the lectin is conjugated to the solid media via tosylation.

[0041] In some embodiments, contacting comprises rotating for about 12 to about 24 hours. In some embodiments, contacting is at about 2-8 °C.

[0042] In another aspect, provided herein is a composition comprising an isolated population of tumor derived EVs, wherein the isolated population of tumor derived EVs are obtained by a method comprising: isolating EVs from a biofluid under conditions sufficient to preserve the EV glycocalyx; contacting the isolated EVs with two or more lectins or a gly can-binding fragment thereof conjugated to a solid media, wherein the lectins or glycan-binding fragment thereof are selected from the group consisting of AAL Aleuria Aurantia Lectin), SNA (Sambucus Nigra Lectin), GNL (Galunthus Nivalis Lectin), MAE I (Maackia Amurensis Lectin I), and M AL II (Maackia Anrurensis Lectin II), and combinations thereof; and (c) removing unbound EVs from the solid media, thereby obtaining the isolated population of tumor derived EVs.

[0043] In some embodiments, the biofluid is selected from the group consisting of plasma, serum, and blood.

[0044] In some embodiments, removing unbound EVs comprises washing the solid media with a saline buffer.NAI-5002577470vl 6ATTORNEY DOCKET NO.: 14842-001-228

[0045] Tn some embodiments, the saline buffer is selected from the group consisting of phosphate buffered saline (PBS), HEPES buffered saline (HBS), and Dulbecco's phosphate buffered saline (DPBS). In some embodiments, the saline buffer comprises PBS, pH 7.4. In some embodiments, the buffer further comprise about 0.1 mM CaCh and about 0.1 mM MnCh buffer.

[0046] In some embodiments, the two or more lectins or glycan-binding fragments thereof comprise three or more lectins selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II and combinations thereof. In some embodiments, the two or more lectins or glycan- binding fragments thereof comprise four or more lectins selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II and combinations thereof. In some embodiments, the two or more lectins or glycan-binding fragments thereof comprise five or more lectins selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II and combinations thereof. In some embodiments, the lectins or glycan-binding fragments thereof comprise AAL, SNA, GNL, MAL I, and MAL II. In some embodiments, the lectins or glycan-binding fragments thereof consist of AAL, SNA, GNL, MAL I, and MAL II.

[0047] In some embodiments, isolating comprises a positive charge-based isolation. In some embodiments, isolating comprises a membrane-based affinity column.

[0048] In some embodiments, the biofluid is from a subject having or suspected of having cancer.

[0049] In some embodiments, the solid media comprises a magnetic particle, an agarose particle, or a hydrogel polymer. In some embodiments, the solid media comprises a magnetic particle. In some embodiments, the two or more lectins are conjugated to the solid media via tosylation.

[0050] In some embodiments, contacting comprises rotating for about 12 to about 24 hours. In some embodiments, contacting is at about 2-8 °C.

[0051] In another aspect, provided herein is a composition comprising: (a) a heterogenous plurality of isolated EVs comprising tumor derived EVs; (b) two or more lectins or glycan-binding fragments thereof conjugated to a solid media, wherein the lectins or glycan-binding fragment thereof is selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II, andNAI-5002577470vl 7ATTORNEY DOCKET NO.: 14842-001-228 combinations thereof; and (c) a buffer. In some embodiments, the solid media has higher affinity for the TDEV, as compared to non-tumor derived EVs.

[0052] In some embodiments, the two or more lectins or glycan-binding fragments thereof comprise three or more lectins selected from the group consisting of AAL, SNA, GNL, MAE I, and MAL II and combinations thereof. In some embodiments, the two or more lectins or glycan- binding fragments thereof comprise four or more lectins selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II and combinations thereof. In some embodiments, the two or more lectins or glycan-binding fragments thereof comprise five or more lectins selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II and combinations thereof. In some embodiments, the lectins or glycan-binding fragments thereof comprise AAL, SNA, GNL, MAL I, and MAL II. In some embodiments, the lectins or glycan-binding fragments thereof consist of AAL, SNA, GNL, MAL I, and MAL II.

[0053] In some embodiments, the solid media comprises a magnetic particle, an agarose particle, or a hydrogel polymer. In some embodiments, the solid media comprises a magnetic particle. In some embodiments, the two or more lectins are conjugated to the solid media via tosylation.

[0054] In some embodiments, the buffer comprises equal parts: a first solution comprising 3M ammonium sulfate, pH 7.9; and a second solution comprising 0.1 M sodium phosphate buffer, 0.1 mM CaCh, 0.1 mM MnCh.

[0055] In yet another aspect, provided herein is a method for enriching tumor derived EVs from a biofluid, the method comprising: (a) isolating EVs from a biofluid under conditions sufficient to preserve the EV glycocalyx; (b) contacting the isolated EVs with two or more lectins or a glycan-binding fragment thereof conjugated to a solid media, wherein the lectins or glycan- binding fragment thereof are selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II, and combinations thereof; and (c) removing unbound EVs from the solid media, thereby enriching tumor derived EVs from a biofluid. In some embodiments, the biofluid is selected from the group consisting of plasma, serum, or blood.

[0056] In some embodiments, removing unbound EVs comprises washing the solid media with a saline buffer. In some embodiments, the saline buffer is selected from the group consisting of phosphate buffered saline (PBS), HEPES buffered saline (HBS), and Dulbecco's phosphateNAI-5002577470vl 8ATTORNEY DOCKET NO.: 14842-001-228 buffered saline (DPBS). Tn some embodiments, the saline buffer comprises PBS, pH 7.4. In some embodiments, the buffer further comprise about 0.1 mM CaCh and about 0.1 mM MnCh buffer.

[0057] In some embodiments, the two or more lectins or glycan-binding fragments thereof comprise three or more lectins selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II and combinations thereof. In some embodiments, the two or more lectins or glycan- binding fragments thereof comprise four or more lectins selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II and combinations thereof. In some embodiments, the two or more lectins or glycan-binding fragments thereof comprise five or more lectins selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II and combinations thereof. In some embodiments, the lectins or glycan-binding fragments thereof comprise AAL, SNA, GNL, MALI, and MAL II . In some embodiments, the lectins or glycan-binding fragments thereof consist of AAL, SNA, GNL, MAL I, and MAL II.

[0058] In some embodiments, isolating comprises a positive charge-based isolation. In some embodiments, isolating comprises a membrane-based affinity column.

[0059] In some embodiments, the biofluid is from a subject having or suspected of having a cancer.

[0060] In some embodiments, the solid media comprises a magnetic particle, an agarose particle, or a hydrogel polymer. Tn some embodiments, the solid media comprises a magnetic particle. In some embodiments, the two or more lectins are conjugated to the solid media via tosylation.

[0061] In some embodiments, contacting comprises rotating for about 12 to about 24 hours. In some embodiments, contacting is at about 2-8 °C.

[0062] In another aspect, provided herein is a kit comprising: two or more lectins or glycan- binding fragments thereof conjugated to a solid media, wherein the lectins or glycan-binding fragment thereof are selected from the group consisting of AAL, SNA, GNL, MAL I, and MALII, and combinations thereof; and instructions for use. In some embodiments, the solid media has higher affinity for the TDEV, as compared to non-tumor derived EVs.

[0063] In some embodiments, the two or more lectins or glycan-binding fragments thereof comprise three or more lectins selected from the group consisting of AAL, SNA, GNL, MAL I,NAI-5002577470vl 9ATTORNEY DOCKET NO.: 14842-001-228 and MAL II and combinations thereof. In some embodiments, the two or more lectins or glycan- binding fragments thereof comprise four or more lectins selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II and combinations thereof. In some embodiments, the two or more lectins or glycan-binding fragments thereof comprise five or more lectins selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II and combinations thereof. In some embodiments, the lectins or glycan-binding fragments thereof comprise AAL, SNA, GNL, MAL I, and MAL II. In some embodiments, the lectins or glycan-binding fragments thereof consist of AAL, SNA, GNL, MAL I, and MAL II.

[0064] In some embodiments, the kit further comprises a buffer suitable for preserving the EV glycocalyx. In some embodiments, the pH of the buffer is about pH 7.0 to pH 8.0. In some embodiments, the pH of the buffer is about pH 7.4.

[0065] In some embodiments, the solid media comprises a magnetic particle, an agarose particle, or a hydrogel polymer. In some embodiments, the solid media comprises a magnetic particle. In some embodiments, the two or more lectins are conjugated to the solid media via tosylation.

[0066] In one aspect, provided herein is a process for detecting the presence or absence of a cancer in a subject comprising: obtaining a biological sample from the subject; contacting at least a portion of said biological sample with one or more detection agents, said one or more detection agents capable of specifically binding a glycoform on or within said portion; isolating said extracellular vesicles to form enriched extracellular vesicles; subjecting said enriched extracellular vesicles to analysis of protein and / or nucleic acid content within or on said enriched extracellular vesicles; and detecting the presence or absence of said cancer in said subject when said enriched extracellular vesicles contain one or more targets at a level that differs from a healthy control, wherein said target is a protein, nucleic acid encoding said protein, or a combination thereof.

[0067] In some embodiments, the process further comprises subjecting said biological sample to fractionation to obtain isolated extracellular vesicles wherein said portion of said biological sample is said isolated extracellular vesicles. In some embodiments, the process further comprises identifying said cancer based on the identity of the one or more targets.

[0068] In some embodiments, said cancer is non-small cell lung cancer, ovarian cancer, colorectal cancer, or a glioma. In some embodiments, said target proteins are a known oncoprotein.NAI-5002577470vl 10ATTORNEY DOCKET NO.: 14842-001-228In some embodiments, said cancer is non-small cell lung cancer and said protein is PHB1, STIM1 , or both. In some embodiments, said cancer is ovarian cancer and said is MUC1, MUC16, or both. In some embodiments, said cancer is colorectal cancer. In some embodiments, said cancer is a glioma.

[0069] In some embodiments, said biological sample is whole blood, plasma, or serum.

[0070] In some embodiments, said detection agent comprises one or more lectins. In some embodiments, at least one of said lectins is selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II, and PHA-E. In some embodiments, at least one of said lectins is PHA-E, optionally wherein said detection agent consists of PHA-E. In some embodiments, at least two of said lectins is selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II. In some embodiments, at least three of said lectins is selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II. In some embodiments, said detection agent comprises five lectins, optionally AAL, SNA, GNL, MAL I, and MAL II. In some embodiments, said detection agent further comprises an isolation component.

[0071] In some embodiments, said isolation component comprises a magnetic bead, a fluorescent agent, avidin, biotin, a protein sequence tag, an antibody or fragment thereof. In some embodiments, said isolation component comprises a magnetic bead.

[0072] In another aspect, provided herein is a process for measuring efficacy of a cancer therapy in a subject comprising: obtaining a first biological sample from the subject prior to administration of a therapy; contacting at least a portion of said first biological sample with one or more detection agents, said one or more detection agents capable of specifically binding a glycoform, isolating first extracellular vesicles from said portion of said first biological sample to form first enriched extracellular vesicles; subjecting said first enriched extracellular vesicles to analysis of protein or nucleic acid content within or on said first enriched extracellular vesicles; determining the presence or absence of a one or more target proteins or target nucleic acids encoding said target proteins in said first enriched extracellular vesicles; obtaining a second biological sample from the subject following administration of said therapy; contacting at least a portion of said second biological sample with one or more of said detection agents, isolating second enriched extracellular vesicles from said portion of said second biological sample; subjecting said second enriched extracellular vesicles to analysis of protein or nucleic acid content within or onNAI-5002577470vl 11ATTORNEY DOCKET NO.: 14842-001-228 said second enriched extracellular vesicles; determining the presence or absence of the one or more of said target proteins or said target nucleic acids encoding said target proteins in said second enriched extracellular vesicles; determining efficacy of said therapeutic based on a difference in identity or amount of said target proteins in said first enriched extracellular vesicles and said second enriched extracellular vesicles.

[0073] In some embodiments, said cancer is a lung cancer, ovarian cancer, colorectal cancer, or a glioma. In some embodiments, said cancer is non-small cell lung cancer and said protein is PHB1, STIM1, or both. In some embodiments, said wherein said cancer is ovarian cancer and said is MUC1, MUC16, or both. In some embodiments, said cancer is colorectal cancer. In some embodiments, said cancer is a glioma.

[0074] In some embodiments, said biological sample is whole blood, plasma, or serum.

[0075] In some embodiments, said detection agent comprises one or more lectins. In some embodiments, at least one of said lectins is selected from the group consisting of AAL, SNA, GNL, MAL I, MAL II, and PHA-E. In some embodiments, at least one of said lectins is PHA-E, optionally wherein said detection agent consists of PHA-E. In some embodiments, at least two of said lectins is selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II. In some embodiments, at least three of said lectins is selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II. In some embodiments, said detection agent comprises five lectins.

[0076] In some embodiments, said detection agent further comprises an isolation component. In some embodiments, said isolation component comprises a magnetic bead, a fluorescent agent, avidin, biotin, a protein sequence tag, an antibody or fragment thereof. In some embodiments, said isolation component comprises a magnetic bead.

[0077] In yet another aspect, provided herein is a composition for detecting the presence of absence of a cancer in a subject, comprising one or more detection agents comprising one or more lectins selected from the group consisting of AAL, SNA, GNL, MAL I, MAL II, and PHA-E. In some embodiments, the composition comprises three or more lectins. In some embodiments, the composition comprises five of said lectins, optionally AAL, SNA, GNL, MAL I, and MAL II.

[0078] In some embodiments, said detection agent further comprises an isolation component.NAI-5002577470vl 12ATTORNEY DOCKET NO.: 14842-001-228

[0079] Tn some embodiments, said isolation component comprises a magnetic bead, a fluorescent agent, avidin, biotin, a protein sequence tag, an antibody or fragment thereof. In some embodiments, said isolation component comprises a magnetic bead.

[0080] In another aspect, provided herein is a process for detecting the presence or absence of a target in a biological sample comprising: obtaining a biological sample from a subject; contacting said at least a portion of said biological sample with one or more detection agents capable of specifically binding a glycoform, isolating enriched extracellular vesicles from said biological sample, said enriched extracellular vesicles bound to said detection agent; and subjecting said enriched extracellular vesicles or a component thereof to mass spectrometry to detect the presence or absence of the target.

[0081] In some embodiments, said target is a protein or a nucleic acid. In some embodiments, said target is a protein related to a disease or condition.

[0082] In some embodiments, said disease or condition is an inflammatory disease, a cancer, an allergic disease, an autoimmune disease, an infectious disease, a transplantation related disease, a degenerative disease, an injury associated with inflammation, a disease associated with a hypersensitivity, a cardiovascular disease, a glandular disease, a hepatic disease, a neurological disease, an a musculo-skeletal disease, a renal disease, a reproductive disease, a connective tissue disease, a neurodegenerative disease, necrosis, an inflammatory disease associated with an implant, a hematological disease, an eye disease, or a respiratory disease. In some embodiments, said disease or condition is a cancer.4. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] The aspects set forth in the drawings are illustrative and exemplary in nature and not intended to limit the subject matter defined by the claims. The following detailed description of the illustrative aspects can be understood when read in conjunction with the following drawings.

[0084] FIG. 1 illustrates the average number of MISEV category 1 and MISEV category 2 markers identified per sample from plasma, isolated EVs and enriched EVs demonstrating dramatically more markers identifiable in enriched EV populations.NAI-5002577470vl 13ATTORNEY DOCKET NO.: 14842-001-228

[0085] FIG. 2A - FIG. 2E illustrate a comparison of protein abundances of non-EV bound plasma proteins typically detected via mass spectrometry in patient plasma showing reduced abundances of each protein when analyzing from enriched EVs. **** is p<0.0001.

[0086] FIG. 3A and FIG. 3B illustrate total protein (FIG. 3A) and glycoprotein (FIG. 3B) numbers individually identifiable in isolated EVs (EV) or enriched EVs from human glioblastoma samples made using detection agents as provided herein.

[0087] FIG. 4A - FIG. 4E illustrate the average total oncoprotein sequence coverage (FIG. 4A) or neuroprotein sequence coverage (FIG. 4B) for individual proteins in various human glioblastoma samples or sample EV subpopulations; detection of the percent amino acid coverage of an exemplary individual oncoprotein in an individual sample from isolated EVs (FIG. 4C), Neuro SPARCs (FIG. 4D), and Tumor SPARCs (FIG. 4E) are also shown. **** is p<0.0001.

[0088] FIG. 5A - FIG. 5G illustrate differentially enriched glioma associated proteins as detected in glioma patients relative to healthy controls using detection agents as provided herein. ** is p<0.01, * is p<0.05. Specific proteins are illustrated in FIG. 5B (epidermal growth factor receptor; EGFR), FIG. 5C (Kirsten Rat Sarcoma virus oncogene homolog; KRAS), FIG. 5D (programmed death ligand 1; PD-L1), FIG. 5E (platelet derived growth factor receptor alpha; PDGFRA), FIG. 5F (v-raf murine sarcoma viral oncogene homolog Bl; BRAF), and FIG. 5G (platelet derived growth factor receptor beta; PDGFRB).

[0089] FIG. 6A and FIG. 6B illustrate significant enrichment of well characterized ovarian cancer markers MUC1 (FIG. 6A) and MUC16 (FIG. 6B) in stage I and II ovarian cancer subjects relative to healthy controls. *** is p<0.001, ** is p<0.01, * is p<0.05.

[0090] FIG. 7A and FIG. 7B illustrate PHB1 (FIG. 7A) and STIM1 (FIG. 7B) to be significantly upregulated in NSCLC tissue and are significantly enriched in enriched EV subpopulations prepared using Tumor Selective Protein Affinity Reagent Chemistries (SPARCs) from Stage I and II NSCLC patients. *** is p<0.001, ** is p<0.01.

[0091] FIG. 8 illustrates the average number of detectable oncoproteins from subjects with various cancers (i.e., brain, colorectal, lung, or ovarian cancer) using Tumor SPARC detection agents or from subjects with brain cancer using Neuro SPARC, as provided herein.NAI-5002577470vl 14ATTORNEY DOCKET NO.: 14842-001-228

[0092] FIG. 9A and FIG. 9B illustrate SPARCs improve the signal-to-noise in mass spectrometry with a liquid biopsy as a result of (a) lower detection of abundant proteins, such as albumin (FIG. 9A), and (b) increased detection of total proteins (FIG. 9B), compared to both total EVs and total plasma in the same samples. * is p<0.05.

[0093] FIG. 10 illustrates that PHA-E has the highest affinity for neuro / CNS-derived EVs isolated from U87 glioblastoma (primary brain malignancy; U87 MG) and SK-N-SH neuroblastoma (arises from neural crest cells) cell lines.

[0094] FIG. 11A - FIG. 11C illustrate that proteomics comparison of different methods demonstrates Neuro SPARC enables better protein and CNS marker detection via LC-MS / MS, as compared to total EVs and plasma (FIG. 11 A); differentially expressed proteins (DEP) from EVs enriched with Neuro SPARCs partially overlap with DEPs from EVs enriched with Tumor SPARCs (FIG. 1 IB); and Neuro SPARCs-enrich EVs contain more brain-enriched CNS marker proteins than Tumor SP ARCs-enriched EVs (FIG. 11C).

[0095] FIG. 12A - FIG. 12C illustrate detection of CNS cell of origin according to neuron markers (NCAM1, CD90 / THY1, and CD166 / ALCAM, respectively) from EVs enriched using Neuro SPARCs in brain cancer, brain tumor (benign), and healthy samples.

[0096] FIG. 13A and FIG. 13B illustrate detection of CNS cell of origin according to neuron / astrocyte markers (NRAM, and CD44, respectively) from EVs enriched using Neuro SPARCs in brain cancer, brain tumor (benign), and healthy samples.

[0097] FIG. 14A - FIG. 14C illustrate detection of CNS cell of origin according to microglia markers (HLA-DR, CD163, and CD36, respectively) from EVs enriched using Neuro SPARCs in brain cancer, brain tumor (benign), and healthy samples.

[0098] FIG. 15A - FIG. 15C illustrate detection of cell of origin CNS markers (CD38, CD40, and GDla / ST8SIA5, respectively) from EVs enriched using Neuro SPARCs in brain cancer, brain tumor (benign), and healthy samples.

[0099] FIG. 16 illustrate unique genes detected across diseases in EVs enriched using individual Tumor SPARCs (left column = colorectal cancer; middle column = healthy control; right column = ovarian cancer).NAI-5002577470vl 15ATTORNEY DOCKET NO.: 14842-001-228

[0100] FIG. 17 illustrate a volcano plot showing that enrichment using Neuro SPARCS identified abundant differentially expressed genes.

[0101] FIG. 18A - FIG. 18C illustrate volcano plots showing that enrichment using Neuro SPARCs (FIG. 18C) identifies more DEPs, and the DEPs overall display larger average significance and fold change than plasma (FIG. 18 A) or total EV methods (FIG. 18B).5. DETAILED DESCRIPTION

[0102] While the use of biomarkers for the detection of cancers using minimally invasive techniques has been studied, the biomarkers and methods identified to date fall short either in lacking the ability to robustly and reliably detect a cancer, or lacking in sufficient correlation between a biomarker level and the actual presence or absence of a cancer.

[0103] Extracellular vesicle(s) (EV(s)) are a biofluid derived nanoparticle analyte comprising lipid bilayer encapsulated protein, nucleic acid, and metabolite cargos. EVs are derived from various cell types and organ systems throughout the body, functioning as local and systemic messengers. Unfortunately, the utility of EVs in biofluids has not been realized because EVs suffer from the same drawbacks as cfDNA and CTCs. Their low abundance, extremely small size, and the sheer diversity and heterogeneity of the EVs circulating in biofluids make it extremely difficult to discover, utilize, and robustly detect EV derived biomarkers.

[0104] It was found in identifying the aspects as provided herein that in oncology, tumor derived EVs (TDEVs) serve as a unique window of insight into the molecular phenotype of the diseased tissue of origin. Similarly, in neuro-oncology, neurology and neurodegenerative diseases, brain-derived EVs (BDEVs) provide insight into diseases of the brain and central nervous system. However, TDEVs and BDEVs make up a miniscule portion of all EVs circulating in blood plasma and are therefore challenging to capture and interrogate. Subpopulation isolation techniques utilizing immunoaffinity are feasible, but no universal tumor markers or tissue specific markers have been demonstrated to be reliably present and accessible on TDEVs or BDEVs. The results as provided herein demonstrate for the first time development of a clinically applicable, enrichment technology, for example a bead-based enrichment technology, dedicated to circumventing the signal to noise issues generally found in liquid biopsy so as to be able to much more reliably detect the presence of a target within a biological sample, particularly when the target is related to the presence or absence of a particular disease or condition.NAI-5002577470vl 16ATTORNEY DOCKET NO.: 14842-001-228

[0105] As provided herein, an “extracellular vesicle” or “EV” is a particle that is released from a cell, is delimited by a lipid bilayer, and cannot replicate on its own as defined by the minimal information for studies of extracellular vesicles (MISEV2023; Welsh JA, et al. J Extracell Vesicles. 2024 Feb;13(2):el2404), which is incorporated by reference herein in its entirety.

[0106] As provided herein, an “isolated EV” is an EV that is separated from one or more other components of a biological sample to some degree.

[0107] As provided herein, an “enriched EV” is an EV that is separated from one or more other components of a biological sample and / or a population of isolated EVs using a one or more detection agents as provided herein.

[0108] As such, the compositions and methods as provided herein utilize a wide ranging approach at detecting the presence or absence of a target and or a disease or condition such as but not limited to cancer that does not suffer drawbacks of low concentrations of a marker in an analyzed tissue, and, optionally, is not necessarily limited to correlations of one marker with any particular condition. This disclosure provides compositions and methods that isolate and often concentrate particular sets or types of extracellular vesicles where these subsets of enriched extracellular vesicles much more reliably and robustly correlate with the presence or absence of a disease or condition in a subject and / or are able to be more readily detected relative to the biological sample from which the enriched EVs were obtained.

[0109] As such, provided herein are processes for detecting the presence or absence of a disease or condition such as but not limited to a cancer in a subject that includes: obtaining a biological sample from the subject; contacting said biological sample with one or more detection agents, the one or more detection agents optionally capable of specifically binding a glycoform on or within an extracellular vesicle within the biological sample; isolating enriched extracellular vesicle(s) from at least a portion of the biological sample using the detection agent; subjecting the enriched extracellular vesicle(s) to analysis of protein or nucleic acid content within or on the extracellular vesicle(s); and optionally detecting the presence or absence of the disease or condition in the subject when the enriched extracellular vesicle(s) contain(s) one or more target proteins or one or more target nucleic acids, such as but not limited to nucleic acids encoding the one or more target proteins and / or noncoding RNA molecules (e. ., microRNAs, long noncoding RNAs).NAI-5002577470vl 17ATTORNEY DOCKET NO.: 14842-001-228

[0110] Also provided herein are methods for enriching tumor derived EVs or brain-derived EVs (such as a method described in Section 5.5) from a biological sample, e.g. a biofluid (such as a biological sample described in Section 5.1) using an isolation component (e.g., a solid media) (such as an isolation component described in Section 5.3) comprising a lectin or glycan-binding fragment thereof (such as a lectin described in Section 5.4). The present disclosure also provides compositions (such as a composition described in Section 5.8) and kits (such as a kit described in Section 5.9) for isolating brain-derived EVs or tumor derived EVs, and compositions comprising an isolated population of brain-derived EVs or tumor derived EVs (such as a composition described in 5.8).5.1. Biological Sample

[0111] A process as provided herein includes obtaining a biological sample (e.g., a biofluid) from a subject. Any extracellular portion of any organism or cell may be used as a biological sample. Illustrative non-limiting examples of a biological sample include a whole blood, plasma, serum, urine, feces, saliva, mucus, lymph fluid, cerebrospinal fluid, pericardial fluid, semen, milk, amniotic fluid, peritoneal fluid, sweat, synovial fluid, tears, or the like. In some aspects, a biological sample is whole blood. In some aspects, a biological sample is plasma.

[0112] A biological sample may be obtained from a subject. A subject as used herein is optionally a human, non-human primate, equine, bovine, canine, porcine, feline, or other mammal or non-mammal. In some aspects, a subject is a human. In some aspects, a subject is a human, and a biological sample is whole blood, plasma or serum.

[0113] Optionally, a biological sample is whole blood, plasma, or serum. In some embodiments, a biological sample is whole blood. Whole blood may be obtained by venipuncture or other collection type, optionally into one or more anticoagulants illustratively but not limited to EDTA, potassium oxalate, sodium citrate, heparin, acid citrate dextrose, and the like. In some embodiments, a biological sample is not whole blood.

[0114] In some embodiments, a biological sample is plasma. Plasma may be obtained from the whole blood such as by differential centrifugation of the whole blood separating out the red blood cells and leaving the plasma. In some embodiments, a biological sample is not plasma.NAI-5002577470vl 18ATTORNEY DOCKET NO.: 14842-001-228

[0115] In some embodiments, a biological sample is serum. Serum may be obtained by known techniques such as by inducing clotting in a whole blood or plasma sample optionally by collecting the sample into a tube with no anticoagulant to produce the serum. In some embodiments, a biological sample is not serum.

[0116] Optionally, a biological sample is a bulk collection of EVs, optionally a non-specific collection of EVs meaning that the EVs may be representative of the EV population within a larger biological sample, illustratively plasma or whole blood. A process of obtaining an EV enriched biological sample, optionally includes subjecting a biological sample to size exclusion or membrane based affinity methods as are known in the art for the separation of EVs from a sample. In some embodiments, the biological sample is a bulk collection of EVs obtained from serum. In some embodiments, the biological sample is a bulk collection of EVs obtained from plasma. In some embodiments, the biological sample is a bulk collection of EVs obtained from blood. In some embodiments, the biological sample is a bulk collection of EVs not obtained from urine.

[0117] In some embodiments, the biological sample is a biofluid. In some embodiments, the biofluid is selected from the group consisting of plasma, serum, and blood. In some embodiments, the biofluid is selected from the group consisting of plasma, and serum. In specific embodiments, the biofluid is plasma. In other embodiments, the biofluid is serum. In some embodiments, the biofluid is from a subject having or suspected of having a neurological disease. In some embodiments, the biofluid is from a subject having or suspected of having a neurodegenerative disease (e.g., Alzheimer’s disease). In some embodiments, the biofluid is from a subject having or suspected of having cancer. In some embodiments, the biofluid is blood. In some embodiments, the biofluid is not blood and is substantially free or devoid of red-blood cells. In some embodiments, the biofluid is not urine.5.2. Glycoforms

[0118] The present disclosure provides isolation components, e.g., a solid media (such as an isolation component described in Section 5.3) comprising a detection agent (e.g., a glycan binding protein or glycan-binding fragment thereof) that is capable of capturing EVs based on interaction with a glycoform on the EV. In some embodiments, a detection agent is optionally a compound capable of binding, optionally selectively binding a glycoform. A glycoform is optionally an O- linked glycoform, an N-linked glycoform, mannosylation, or a lipid linked glycoform. An N-NAI-5002577470vl 19ATTORNEY DOCKET NO.: 14842-001-228 linked glycoform includes an N-acetylglucosamine (GlcNAc) residue linked to an asparagine or arginine and may be part of the consensus sequence NX(S / T), where X can be any amino acid except proline. It is noted that the presence of such a consensus sequence does not require the presence of an N-linked glycoform, but such N-linked glycoforms are localized to such a consensus sequence. An O-linked glycoform is bound to a serine or threonine residue. O-linked glycoforms have no single common core structure and do not require a consensus protein sequence. Lipid linked glycoforms may be phosphoglycan linked through the phosphate of a phosphoserine. Glycoforms are typically formed by competing glycosyl transferases that may produce significant heterogeneity in the types of glycoforms found in a particular pool such as a protein pool. Glycoforms are optionally groupings of modified or unmodified P-glucose, P-mannose, and / or P- galactose. Illustrative examples of glycoforms may be found in Lowe and Marth, Essentials in Glycobiology, Chapter 16, Varki A, Cummings R, Esko J, et al., editors. Cold Spring Harbor (NY): 1999.

[0119] In some aspects, a glycoform is a fucose linked (a -1,6) to N-acetylglucosamine or a fucose linked (a -1,3) to N-acetyllactosamine structure. Such structures are preferentially bound by the lectin AAL (Aleuria Aurantia Lectin). In some aspects, a glycoform includes a sialic acid attached to terminal galactose in ot-2,6 or a-2,3 linkage such as are preferentially bound by the lectin SNA (Sambucus Nigra Lectin). Optionally, a glycoform is a structure containing (a-1,3) mannose residues such as are preferentially bound by GNL (Galunthus Nivalis Lectin). Optionally, a glycoform includes a gal (P-1,4) glcNAc that may or may not be substituted by a sialic acid at the 3 position of galactose such as is bound by the lectin MAL I (Maackia Amurensis Lectin I). Optionally, a glycoform includes a sialic acid in an (a-2,3) linkage such as is preferentially bound by the lectin MAL II (Maackia Amurensis Lectin II). Optionally, a glycoform includes a biantennary galactosylated N-glycan with bisecting N-acetylglucosamine (GlcNAc) such as is preferentially bound by the lectin PHA-E (Phaseolus Vulgaris erythroagglutinin). Optionally, a glycoform is a combination of erythroagglutinin (E-subunit) and leucoagglutinin (L-subunit) in a family of four subunits that may combine to form as many as five isolectins. It is appreciated that other such glycoforms or modifications thereof may be utilized as well. Optionally, a lectin is not only AAL such that if AAL is used more than one lectin is used in addition. Optionally, a lectin is not only SNA such that if SNA is used more than one lectin is used in addition. Optionally, a lectin is not only AAL and SNA such that if AAL and SNA are used at least one additional lectin is used.NAI-5002577470vl 20ATTORNEY DOCKET NO.: 14842-001-228In some embodiments, a lectin provided herein does not comprise SNA. In some embodiments, a lectin provided herein does not comprise AAL. In some embodiments, a lectin provided herein does not comprise concanavalin A (Con A). In some embodiments, a lectin provided herein does not comprise OAA ( scillatoria Agardhii Lectin).5.3. Isolation component

[0120] A detection agent optionally includes a binding element, such as a lectin described in Section 5.4) and an isolation component. A binding element is optionally a compound that may be selective for a glycoform. Optionally, such binding elements include but are not limited to an antibody, a lectin, or other molecule. A lectin is optionally a lectin such as those available from Vector Laboratories. Inc. (Newark, CA). In some embodiments, the binding element is a lectin or glycan binding fragment thereof (such as a lectin described in Section 5.4) and the lectin is conjugated to the isolation component via tosylation.

[0121] In some embodiments, the isolation component is a solid media. In specific embodiments, the solid media comprises a magnetic particle, an agarose particle, or a hydrogel polymer. In some embodiments, the solid media comprises a magnetic particle. In some embodiments, the solid media comprises an agarose particle. In some embodiments, the solid media comprises a hydrogel polymer. In some embodiments, the solid media is a bead. In some embodiments, the solid media is a magnetic bead. In some embodiments, the solid media is not a magnetic bead. In some embodiments, the solid media is not a planar structure. In some embodiments, the solid media is not a slide. In certain embodiments, the solid media is not glass, for example, is not a glass slide. In certain embodiments, the solid media is not a well, for example, is not a well of a well plate. In certain embodiments, the solid media is not part of an array or blot, for example, a lectin array, e.g., lectin microarray, or lectin blot. In certain embodiments, the solid media is not a Janus nanoparticle.

[0122] In some embodiments, the binding element is a lectin (such as a lectin described in Section 5.4). In some embodiments, the lectin is conjugated to the solid media via tosylation. In some embodiments, the lectin is conjugated to a magnetic particle via tosylation. In some embodiments, the lectin is conjugated to a bead, e.g., a magnetic bead, such as a DYNABEAD, available from Thermo Fisher Scientific, Waltham, MA.NAI-5002577470vl 21ATTORNEY DOCKET NO.: 14842-001-228

[0123] A detection agent optionally further includes one or more isolation elements. An isolation element allows for selective separation of a detection agent, optionally when bound to a target, for purification, detection, analysis or other purpose. Optionally, an isolation component is a bead, a fluorescent agent, avidin, biotin, a protein sequence tag, or an antibody or fragment thereof. In some embodiments, the isolation component (e.g., solid media) does not comprise a biotin. In some embodiments, the isolation component (e.g, solid media) does not comprise an antibody. In some embodiments, the isolation component (e.g, solid media) does not comprise a labeled moiety, e.g., does not comprise a FITC-labeled moiety, a magnetic label moiety or an antibody label moiety. Optionally, an isolation component is a bead, optionally a resin bead, magnetic bead, or other. Illustrative resin beads include optionally antibody or lectin bound beads. Optionally, a bead is bound to a lectin and also to a protein or an antibody where the lectin is used for selective binding of a target and the protein or antibody is used for isolation of that target. In some aspects, a bead is a magnetic bead such as a DYNABEAD, available from Thermo Fisher Scientific, Waltham, MA. A magnetic bead may be bound to a binding element. In some aspects, a detection agent is a magnetic bead bound to one or more lectins.

[0124] A detection agent may be used to isolate one or more extracellular vesicles or extracellular vesicle types from the biological sample or a portion thereof. The term “isolate” may but does not necessarily require complete isolation. Optionally, isolation includes purification relative to other components of the biological sample of about 2-fold, about 5-fold, about 10-fold, optionally about 100-fold, optionally about 1000-fold or greater. In some embodiments, isolation includes reduction and / or removal of ubiquitous or particularly abundant proteins found in biofluids, e.g., prior to EV isolation or enrichment, such as albumin, alpha-1 antitrypsin, a-l-acid glycoprotein, haptoglobin and transferrin. In some embodiments, detection of albumin is reduced by more than about 50%, as compared to a level of albumin in the biofluid prior to EV isolation and / or enrichment. In some embodiments, detection of albumin is reduced by more than about 80%, as compared to a level of albumin in the biofluid prior to EV isolation and / or enrichment. In some embodiments, detection of albumin in a biofluid, for example, in blood, plasma or serum, is reduced by more than about 90%, as compared to a level of albumin in the biofluid prior to EV isolation and / or enrichment. In some embodiments, detection of albumin is reduced by more than about 95%, as compared to a level of albumin in the biofluid prior to EV isolation and / or enrichment. In some embodiments, detection of albumin is reduced by more than about 96%, asNAI-5002577470vl 22ATTORNEY DOCKET NO.: 14842-001-228 compared to a level of albumin in the biofluid prior to EV isolation and / or enrichment. In some embodiments, detection of albumin is reduced by more than about 97%, as compared to a level of albumin in the biofluid prior to EV isolation and / or enrichment. In some embodiments, detection of albumin is reduced by more than about 98%, as compared to a level of albumin in the biofluid prior to EV isolation and / or enrichment. In some embodiments, detection of albumin is reduced by more than about 99%, as compared to a level of albumin in the biofluid prior to EV isolation and / or enrichment. In some embodiments, detection of transferrin is reduced by more than about 50%, as compared to a level of albumin in the biofluid prior to EV isolation and / or enrichment. In some embodiments, detection of transferrin is reduced by more than about 80%, as compared to a level of transferrin in the biofluid prior to EV isolation and / or enrichment. In some embodiments, detection of transferrin is reduced by more than about 90%, as compared to a level of transferrin in the biofluid prior to EV isolation and / or enrichment. In some embodiments, detection of transferrin is reduced by more than about 95%, as compared to a level of transferrin in the biofluid prior to EV isolation and / or enrichment. In some embodiments, detection of transferrin is reduced by more than about 96%, as compared to a level of transferrin in the biofluid prior to EV isolation and / or enrichment. In some embodiments, detection of transferrin is reduced by more than about 97%, as compared to a level of transferrin in the biofluid prior to EV isolation and / or enrichment. In some embodiments, detection of transferrin is reduced by more than about 98%, as compared to a level of transferrin in the biofluid prior to EV isolation and / or enrichment. In some embodiments, detection of transferrin is reduced by more than about 99%, as compared to a level of transferrin in the biofluid prior to EV isolation and / or enrichment. In some embodiments, detection of alpha- 1 antitrypsin is reduced by more than about 50%, as compared to a level of alpha- 1 antitrypsin in the biofluid prior to EV isolation and / or enrichment. In some embodiments, detection of alpha- 1 antitrypsin is reduced by more than about 80%, as compared to a level of alpha- 1 antitrypsin in the biofluid prior to EV isolation and / or enrichment. In some embodiments, detection of alpha- 1 antitrypsin is reduced by more than about 90%, as compared to a level of alpha- 1 antitrypsin in the biofluid prior to EV isolation and / or enrichment. In some embodiments, detection of alpha- 1 antitrypsin is reduced by more than about 95%, as compared to a level of alpha- 1 antitrypsin in the biofluid prior to EV isolation and / or enrichment. In some embodiments, detection of alpha- 1 antitrypsin is reduced by more than about 96%, as compared to a level of alpha- 1 antitrypsin in the biofluid prior to EV isolation and / or enrichment. In some embodiments,NAI-5002577470vl 23ATTORNEY DOCKET NO.: 14842-001-228 detection of alpha-1 antitrypsin is reduced by more than about 97%, as compared to a level of alpha- 1 antitrypsin in the biofluid prior to EV isolation and / or enrichment. In some embodiments, detection of alpha- 1 antitrypsin is reduced by more than about 98%, as compared to a level of alpha- 1 antitrypsin in the biofluid prior to EV isolation and / or enrichment. In some embodiments, detection of alpha- 1 antitrypsin is reduced by more than about 99%, as compared to a level of alpha- 1 antitrypsin in the biofluid prior to EV isolation and / or enrichment. In some embodiments, detection of a-l-acid glycoprotein is reduced by more than about 50%, as compared to a level of a- 1 -acid glycoprotein in the biofluid prior to EV isolation and / or enrichment. In some embodiments, detection of a-l-acid glycoprotein is reduced by more than about 80%, as compared to a level of a-l-acid glycoprotein in the biofluid prior to EV isolation and / or enrichment. In some embodiments, detection of a-l-acid glycoprotein is reduced by more than about 90%, as compared to a level of a-l-acid glycoprotein in the biofluid prior to EV isolation and / or enrichment. In some embodiments, detection of a-l-acid glycoprotein is reduced by more than about 95%, as compared to a level of a-l-acid glycoprotein in the biofluid prior to EV isolation and / or enrichment. In some embodiments, detection of a-l-acid glycoprotein is reduced by more than about 96%, as compared to a level of a-l-acid glycoprotein in the biofluid prior to EV isolation and / or enrichment. In some embodiments, detection of a-l-acid glycoprotein is reduced by more than about 97%, as compared to a level of a-l-acid glycoprotein in the biofluid prior to EV isolation and / or enrichment. In some embodiments, detection of a-l-acid glycoprotein is reduced by more than about 98%, as compared to a level of a-l-acid glycoprotein in the biofluid prior to EV isolation and / or enrichment. In some embodiments, detection of a-l-acid glycoprotein is reduced by more than about 99%, as compared to a level of a-l-acid glycoprotein in the biofluid prior to EV isolation and / or enrichment. In some embodiments, detection of haptoglobin is reduced by more than about 50%, as compared to a level of haptoglobin in the biofluid prior to EV isolation and / or enrichment. In some embodiments, detection of haptoglobin is reduced by more than about 80%, as compared to a level of haptoglobin in the biofluid prior to EV isolation and / or enrichment. In some embodiments, detection of haptoglobin is reduced by more than about 90%, as compared to a level of haptoglobin in the biofluid prior to EV isolation and / or enrichment. In some embodiments, detection of haptoglobin is reduced by more than about 95%, as compared to a level of haptoglobin in the biofluid prior to EV isolation and / or enrichment. In some embodiments, detection of haptoglobin is reduced by more than about 96%, as compared to a level of haptoglobin in the biofluid prior to EV isolationNAI-5002577470vl 24ATTORNEY DOCKET NO.: 14842-001-228 and / or enrichment. Tn some embodiments, detection of haptoglobin is reduced by more than about 97%, as compared to a level of haptoglobin in the biofluid prior to EV isolation and / or enrichment. In some embodiments, detection of haptoglobin is reduced by more than about 98%, as compared to a level of haptoglobin in the biofluid prior to EV isolation and / or enrichment. In some embodiments, detection of haptoglobin is reduced by more than about 99%, as compared to a level of haptoglobin in the biofluid prior to EV isolation and / or enrichment.[00125J Optionally, some level of remaining contaminant remains in the sample following isolation. Optionally, an EV or sub-population of EVs are substantially isolated meaning that the EVs are isolated to a degree that is or near that which is achievable using the technique of isolation performed.5.4. Lectin

[0126] The present disclosure provides one or more lectins or a glycan-binding fragment thereof capable of capturing EVs based on interaction with a glycoform on the EV (such as a glycoform described in Section 5.2) and embodiments wherein the one or more lectins are conjugated to one or more isolation components, e.g., a solid media (such as an isolation component described in Section 5.3). In some embodiments, the one or more lectins are selected from the group consisting of AAL, SNA, GNL, MAL I, MAL II, and PHA-E, and combinations thereof. In some embodiments, the one or more lectins are selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II and combinations thereof. In some embodiments, the one or more lectins are selected from the group consisting of GNL, MAL I, and MAL II and combinations thereof. In some embodiments, one lectin is conjugated to one or more isolation components, for example only PHA-E. In some embodiments, two or more lectins are conjugated to one or more isolation components, e.g., a solid media (such as an isolation component described in Section 5.3), and the two or more lectins are selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II. In some embodiments, three or more lectins are conjugated to one or more isolation components, e.g., a solid media (such as an isolation component described in Section 5.3), and the three or more lectins are selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II. In some embodiments, four or more lectins are conjugated to one or more isolation components, e.g., a solid media (such as an isolation component described in Section 5.3), and the four or more lectins are selected from the group consisting of AAL, SNA,NAI-5002577470vl 25ATTORNEY DOCKET NO.: 14842-001-228GNL, MAL I, and MAL II. In some embodiments, five or more lectins are conjugated to one or more isolation components, e.g., a solid media (such as an isolation component described in Section 5.3), and the five or more lectins are selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II and combinations thereof. Optionally, in some embodiments the solid media includes three or more lectins. In some embodiments, the solid media includes four or more lectins. In some embodiments, the solid media includes five or more lectins, optionally five lectins. Optionally, the solid media includes the lectins AAL, SNA, GNL, MAL I, and MAL II. In some embodiments, the solid media comprises a magnetic particle, an agarose particle, or a hydrogel polymer. In some embodiments, the solid media comprises a magnetic particle. In some embodiments, the solid media comprises an agarose particle. In some embodiments, the solid media comprises a hydrogel polymer. In some embodiments, the lectin is conjugated to the solid media via tosylation. In some embodiments, the lectin is conjugated to a magnetic particle via tosylation. In some embodiments, the lectin is conjugated to a magnetic bead, such as a DYNABEAD, available from Thermo Fisher Scientific, Waltham, MA. In some embodiments, the solid media is not a magnetic bead. In some embodiments, the solid media is not a planar structure. In some embodiments, the solid media is not a slide. In certain embodiments, the solid media is not glass, for example, is not a glass slide. In certain embodiments, the solid media is not a well, for example, is not a well of a well plate. In certain embodiments, the solid media is not part of an array or blot, for example, a lectin array, e.g., lectin microarray, or lectin blot. In certain embodiments, the solid media is not a Janus nanoparticle.

[0127] In some embodiments, a lectin or glycan-binding fragment thereof is conjugated to the solid media. In some embodiments, the lectin or glycan-binding fragment thereof comprises AAL. In some embodiments, the lectin or glycan-binding fragment thereof does not comprise AAL. In some embodiments, the lectin or glycan-binding fragment thereof comprises SNA. In some embodiments, the lectin or glycan-binding fragment thereof does not comprise AAL. In some embodiments, the lectin or glycan-binding fragment thereof comprises GNL. In some embodiments, the lectin or glycan-binding fragment thereof does not comprise GNL. In some embodiments, the lectin or glycan-binding fragment thereof comprises MAL I. In some embodiments, the lectin or glycan-binding fragment thereof does not comprise MAL I. In some embodiments, the lectin or glycan-binding fragment thereof comprises MAL II. In some embodiments, the lectin or glycan-binding fragment thereof does not comprise MAL II. In someNAI-5002577470vl 26ATTORNEY DOCKET NO.: 14842-001-228 embodiments, the lectin or glycan-binding fragment thereof comprises PHA-E. Tn some embodiments, the lectin or glycan-binding fragment thereof does not comprise PHA-E. In some embodiments, the lectin or glycan-binding fragment thereof comprises AAL, and SNA. In some embodiments, the lectin or glycan-binding fragment thereof does not comprise AAL, and SNA. In some embodiments, the lectin or glycan-binding fragment thereof comprises AAL, and GNL. In some embodiments, the lectin or glycan-binding fragment thereof comprises AAL, and MAE I. In some embodiments, the lectin or glycan-binding fragment thereof comprises AAL, and MAL II. In some embodiments, the lectin or glycan-binding fragment thereof comprises SNA, and GNL. In some embodiments, the lectin or glycan-binding fragment thereof comprises SNA, and MAL I. In some embodiments, the lectin or glycan-binding fragment thereof comprises SNA, and MAL II. In some embodiments, the lectin or glycan-binding fragment thereof comprises GNL, and MALI. In some embodiments, the lectin or glycan-binding fragment thereof comprises GNL, and MALII. In some embodiments, the lectin or glycan-binding fragment thereof comprises MAL I, and MAL II. In some embodiments, the lectin or glycan-binding fragment thereof comprises AAL, SNA, and GNL. In some embodiments, the lectin or glycan-binding fragment thereof comprises AAL, SNA, and MAL I. In some embodiments, the lectin or glycan-binding fragment thereof comprises AAL, SNA, and MAL II. In some embodiments, the lectin or glycan-binding fragment thereof comprises AAL, GNL, and MAL I. In some embodiments, the lectin or glycan-binding fragment thereof comprises AAL, GNL, and MAL II. In some embodiments, the lectin or glycan- binding fragment thereof comprises AAL, MAL I, and MAL II. In some embodiments, the lectin or glycan-binding fragment thereof comprises SNA, GNL, and MAL I. In some embodiments, the lectin or glycan-binding fragment thereof comprises SNA, GNL, and MAL II. In some embodiments, the lectin or glycan-binding fragment thereof comprises SNA, MAL I, and MAL II. In some embodiments, the lectin or glycan-binding fragment thereof comprises GNL, MAL I, and MAL II. In some embodiments, the lectin or glycan-binding fragment thereof comprises AAL, SNA, GNL, and MAL I. In some embodiments, the lectin or glycan-binding fragment thereof comprises AAL, SNA, GNL, and MAL II. In some embodiments, the lectin or glycan-binding fragment thereof comprises AAL, SNA, MAL I, and MAL II. In some embodiments, the lectin or glycan-binding fragment thereof comprises AAL, GNL, MAL I, and MAL II. In some embodiments, the lectin or glycan-binding fragment thereof comprises SNA, GNL, MAL I, and MAL II. In some embodiments, the lectin or glycan-binding fragment thereof comprises AAL,NAI-5002577470vl 27ATTORNEY DOCKET NO.: 14842-001-228SNA, GNL, MAL T, and MAL II. In some embodiments, the solid media comprises a magnetic particle, an agarose particle, or a hydrogel polymer. In some embodiments, the solid media comprises a magnetic particle. In some embodiments, the solid media comprises an agarose particle. In some embodiments, the solid media comprises a hydrogel polymer. In some embodiments, the lectin is conjugated to the solid media via tosylation. In some embodiments, the lectin is conjugated to a magnetic particle via tosylation. In some embodiments, the lectin is conjugated to a magnetic bead, such as a DYNABEAD, available from Thermo Fisher Scientific, Waltham, MA. In some embodiments, the solid media is not a magnetic bead. In some embodiments, the solid media is not a planar structure. In some embodiments, the solid media is not a slide. In certain embodiments, the solid media is not glass, for example, is not a glass slide. In certain embodiments, the solid media is not a well, for example, is not a well of a well plate. In certain embodiments, the solid media is not part of an array or blot, for example, a lectin array, e.g., lectin microarray, or lectin biot in certain embodiments, the solid media is not a Janus nanoparticle.

[0128] In some embodiments, a lectin or glycan-binding fragment thereof is conjugated to the solid media and the lectin or glycan-binding fragment thereof consists of AAL. In some embodiments, the lectin or glycan-binding fragment thereof does not consists of AAL. In some embodiments, the lectin or glycan-binding fragment thereof consists of SNA. In some embodiments, the lectin or glycan-binding fragment thereof does not consists of SNA. In some embodiments, the lectin or glycan-binding fragment thereof consists of GNL. In some embodiments, the lectin or glycan-binding fragment thereof consists of MAL I. In some embodiments, the lectin or glycan-binding fragment thereof consists of MAL II. In some embodiments, the lectin or glycan-binding fragment thereof consists of PHA-E. In some embodiments, the lectin or glycan-binding fragment thereof consists of AAL, and SNA. In some embodiments, the lectin or glycan-binding fragment thereof does not consist of AAL, and SNA. In some embodiments, the lectin or glycan-binding fragment thereof consists of AAL, and GNL. In some embodiments, the lectin or glycan-binding fragment thereof consists of AAL, and MALI. In some embodiments, the lectin or glycan-binding fragment thereof consists of AAL, and MALII. In some embodiments, the lectin or glycan-binding fragment thereof consists of SNA, and GNL. In some embodiments, the lectin or glycan-binding fragment thereof consists of SNA, and MAL I. In some embodiments, the lectin or glycan-binding fragment thereof consists of SNA, and MALNAI-5002577470vl 28ATTORNEY DOCKET NO.: 14842-001-228II. In some embodiments, the lectin or glycan-binding fragment thereof consists of GNL, and MALI. In some embodiments, the lectin or glycan-binding fragment thereof consists of GNL, and MALII. In some embodiments, the lectin or glycan-binding fragment thereof consists of MAL I, and MAL II. In some embodiments, the lectin or glycan-binding fragment thereof consists of AAL, SNA, and GNL. In some embodiments, the lectin or glycan-binding fragment thereof consists of AAL, SNA, and MAL I. In some embodiments, the lectin or glycan-binding fragment thereof consists of AAL, SNA, and MAL II. In some embodiments, the lectin or glycan-binding fragment thereof consists of AAL, GNL, and MAL I. In some embodiments, the lectin or glycan-binding fragment thereof consists of AAL, GNL, and MAL II. In some embodiments, the lectin or glycan- binding fragment thereof consists of AAL, MAL I, and MAL II. In some embodiments, the lectin or glycan-binding fragment thereof consists of SNA, GNL, and MAL I. In some embodiments, the lectin or glycan-binding fragment thereof consists of SNA, GNL, and MAL II. In some embodiments, the lectin or glycan-binding fragment thereof consists of SNA, MAL I, and MAL II. In some embodiments, the lectin or glycan-binding fragment thereof consists of GNL, MAL I, and MAL II. In some embodiments, the lectin or glycan-binding fragment thereof consists of AAL, SNA, GNL, and MAL I. In some embodiments, the lectin or glycan-binding fragment thereof consists of AAL, SNA, GNL, and MAL II. In some embodiments, the lectin or glycan-binding fragment thereof consists of AAL, SNA, MAL I, and MAL II. In some embodiments, the lectin or glycan-binding fragment thereof consists of AAL, GNL, MAL I, and MAL II. In some embodiments, the lectin or glycan-binding fragment thereof consists of SNA, GNL, MAL I, and MAL II. In some embodiments, the lectin or glycan-binding fragment thereof consists of AAL, SNA, GNL, MAL I, and MAL II. In some embodiments, the solid media comprises a magnetic particle, an agarose particle, or a hydrogel polymer. In some embodiments, the solid media comprises a magnetic particle. In some embodiments, the solid media comprises an agarose particle. In some embodiments, the solid media comprises a hydrogel polymer. In some embodiments, the lectin is conjugated to the solid media via tosylation. In some embodiments, the lectin is conjugated to a magnetic particle via tosylation. In some embodiments, the lectin is conjugated to a magnetic bead, such as a DYNABEAD, available from Thermo Fisher Scientific, Waltham, MA. In some embodiments, the solid media is not a magnetic bead. In some embodiments, the solid media is not a planar structure. In some embodiments, the solid media is not a slide. In certain embodiments, the solid media is not glass, for example, is not a glass slide.NAI-5002577470vl 29ATTORNEY DOCKET NO.: 14842-001-228In certain embodiments, the solid media is not a well, for example, is not a well of a well plate. In certain embodiments, the solid media is not part of an array or blot, for example, a lectin array, e.g., lectin microarray, or lectin blot. In certain embodiments, the solid media is not a Janus nanoparticle.

[0129] In some embodiments, provide herein is a plurality of isolation components e.g., a solid media (such as an isolation component described in Section 5.3), comprising a first isolation component and at least a second isolation component. In some embodiments, one or more lectins are conjugated to the first isolation component, e.g., a solid media (such as an isolation component described in Section 5.3), and one or more lectins are conjugated to the second isolation component, e.g., a solid media (such as an isolation component described in Section 5.3). In some embodiments, the one or more lectins conjugated to the first isolation component are identical to the one or more lectins conjugated to the second isolation component. In some embodiments, the one or more lectins conjugated to the first isolation component are nonidentical to the one or more lectins conjugated to the second isolation component. By way of example, and without limitation, in some embodiments SNA is conjugated to a first isolation component, and GNL, MAL I, and MAL II are conjugated to a second isolation component. As a further non-limiting example, in some embodiments the plurality of isolation components can include (a) SNA conjugated to a first isolation component, (b) GNL conjugated to a second isolation component; (c) MAL I conjugated to a third isolation component; (d) MAL II conjugated to a fourth isolation component; and (e) AAL conjugated to a fifth isolation component, where a unique lectin is conjugated to each isolation component.5.5. Methods and Processes

[0130] In one aspect, provided herein is a method for enriching brain-derived EVs from a biofluid, the method comprising: (a) isolating EVs from a biofluid (such as a biofluid described in Section 5.1) under conditions sufficient to preserve the EV glycocalyx, wherein the biofluid is substantially free or devoid of red-blood cells; (b) contacting the isolated EVs with a lectin (such as a lectin described in section 5.4)or glycan-binding fragment thereof conjugated to a solid media (such as a solid media described in Section 5.3), wherein the lectin or glycan-binding fragment thereof specifically binds a biantennary galactosylated N-glycan with bisecting GlcNAc; and (c) removing unbound EVs from the solid media, thereby enriching brain-derivedNAI-5002577470vl 30ATTORNEY DOCKET NO.: 14842-001-228EVs from a biofluid. In some embodiments, the method further comprises determining the presence or absence of a neuro-specific protein and / or nucleic acid. In some embodiments, the biofluid is serum or plasma. In some embodiments, the biofluid is serum. In some embodiments, the biofluid is plasma. In some embodiments, the biofluid is not blood. In specific embodiments, the biofluid is not urine. In some embodiments, the lectin is PHA-E or a glycan-binding fragment thereof. In some embodiments, the biofluid is from a subject having or suspected of having a neurological disease. In some embodiments, the biofluid is from a subject having or suspected of having a neurodegenerative disease (e.g., Alzheimer’s disease). In some embodiments, the biofluid is from a subject having or suspected of having a brain cancer. In some embodiments, the biofluid is from a subject having or suspected of having glioma. In some embodiments, the biofluid is from a subject having or suspected of having a meningioma or benign brain tumor.

[0131] In another aspect, provided herein is a method for enriching for EVs comprising biantennary galactosylated N-glycan with bisecting GlcNAc, the method comprising: (a) contacting isolated EVs with a lectin (such as a lectin described in section 5.4) or glycan-binding fragment thereof conjugated to a solid media (such as a solid media described in Section 5.3), wherein the lectin or glycan-binding fragment thereof specifically binds a biantennary galactosylated N-glycan with bisecting GlcNAc; and (b) removing unbound EVs from the solid media, thereby enriching for EVs comprising biantennary galactosylated N-glycan with bisecting GlcNAc. In some embodiments, the method further comprises determining the presence or absence of a neuro-specific protein and / or nucleic acid. In some embodiments, the isolated EVs are isolated from serum or plasma. In some embodiments, the isolated EVs are isolated from serum. In some embodiments, the isolated EVs are isolated from plasma. In some embodiments, the isolated EVs are not isolated from blood. In specific embodiments, the isolated EVs are not isolated from urine. In some embodiments, the lectin is PHA-E or a glycan-binding fragment thereof. In some embodiments, the isolated EVs are isolated from a subject having or suspected of having a neurological disease. In some embodiments, the biofluid is from a subject having or suspected of having a neurodegenerative disease (e.g., Alzheimer’s disease). In some embodiments, the isolated EVs are isolated from a subject having or suspected of having a brain cancer. In some embodiments, the isolated EVs are isolated from a subject having or suspected of having glioma. In some embodiments, the isolated EVs are isolated from a subject having or suspected of having a meningioma or benign brain tumor.NAI-5002577470vl 31ATTORNEY DOCKET NO.: 14842-001-228

[0132] In one aspect, provided herein is a method for enriching EVs comprising biantennary galactosylated N-glycan with bisecting GlcNAc from a biofluid, the method comprising: (a) isolating EVs from the biofluid under conditions sufficient to preserve the EV glycocalyx; (b) contacting the isolated EVs with a lectin or glycan-binding fragment thereof conjugated to a solid media (such as a solid media described in Section 5.3), wherein the lectin comprises PHA- E; and (c) removing unbound EVs from the solid media, thereby enriching EVs comprising biantennary galactosylated N-glycan with bisecting GlcNAc from the biofluid, wherein the biofluid is plasma or serum. In some embodiments, the biofluid is serum. In some embodiments, the biofluid is plasma. In some embodiments, the method further comprises determining the presence or absence of a neuro-specific protein and / or nucleic acid. In some embodiments, the biofluid is from a subject having or suspected of having a neurological disease. In some embodiments, the biofluid is from a subject having or suspected of having a neurodegenerative disease (e.g., Alzheimer’s disease). In some embodiments, the biofluid is from a subject having or suspected of having a brain cancer. In some embodiments, the biofluid is from a subject having or suspected of having glioma. In some embodiments, the biofluid is from a subject having or suspected of having a meningioma or benign brain tumor.

[0133] In yet another aspect, provided herein is a method for enriching tumor derived EVs from a biofluid, the method comprising: (a) isolating EVs from a biofluid (such as a biofluid described in Section 5.1) under conditions sufficient to preserve the EV glycocalyx; (b) contacting the isolated EVs with two or more lectins or glycan-binding fragment thereof conjugated to a solid media (such as a solid media described in Section 5.3), wherein the two or more lectins are selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II; and (c) removing unbound EVs from the solid media, thereby enriching tumor derived EVs from a biofluid. In some embodiments, the biofluid is serum, plasma, or blood. In some embodiments, the biofluid is serum. In some embodiments, the biofluid is plasma. In some embodiments, the biofluid is blood. In specific embodiments, the biofluid is not urine. In some embodiments, the two or more lectins comprises three lectins selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II. In some embodiments, the two or more lectins comprise four lectins selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II. In some embodiments, the two or more lectins comprise AAL, SNA, GNL, MAL I, and MAL II. In some embodiments, the two or more lectins consists of AAL, SNA, GNL, MAL I, and MAL II. InNAI-5002577470vl 32ATTORNEY DOCKET NO.: 14842-001-228 some embodiments, the biofluid is from a subject having or suspected of having cancer. Tn some embodiments, the biofluid is from a subject having or suspected of having colorectal cancer. In some embodiments, the biofluid is from a subject having or suspected of having glioma. In some embodiments, the biofluid is from a subject having or suspected of having a meningioma or benign brain tumor. In some embodiments, the biofluid is from a subject having or suspected of having ovarian cancer. In some embodiments, the biofluid is from a subject having or suspected of having non-small cell lung cancer. In some embodiments, the biofluid is not from a subject having or suspected of having prostate cancer.

[0134] In another aspect, provided herein is a method for enriching tumor derived EVs, the method comprising: (a) contacting isolated EVs with two or more lectins or glycan-binding fragment thereof conjugated to a solid media (such as a solid media described in Section 5.3), wherein the two or more lectins are selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II; and (c) removing unbound EVs from the solid media, thereby enriching tumor derived EVs. In some embodiments, the isolated EVs are from serum, plasma, or blood. In some embodiments, the isolated EVs are isolated from serum. In some embodiments, the isolated EVs are isolated from plasma. In some embodiments, the isolated EVs are isolated from blood. In some embodiments, the isolated EVs are not isolated from blood. In specific embodiments, the isolated EVs are not isolated from urine. In some embodiments, the two or more lectins comprises three lectins selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II. In some embodiments, the two or more lectins comprise four lectins selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II. In some embodiments, the two or more lectins comprise AAL, SNA, GNL, MAL I, and MAL II. In some embodiments, the two or more lectins consists of AAL, SNA, GNL, MAL I, and MAL II. In some embodiments, the isolated EVs are isolated from a subject having or suspected of having cancer. In some embodiments, the isolated EVs are isolated from a subject having or suspected of having colorectal cancer. In some embodiments, the isolated EVs are isolated from a subject having or suspected of having glioma. In some embodiments, the isolated EVs are isolated from a subject having or suspected of having a meningioma or benign brain tumor. In some embodiments, the isolated EVs are isolated from a subject having or suspected of having ovarian cancer. Tn some embodiments, the isolated EVs are isolated from a subject having or suspected of having non-NAI-5002577470vl 33ATTORNEY DOCKET NO.: 14842-001-228 small cell lung cancer. In some embodiments, the isolated EVs are not isolated from a subject having or suspected of having prostate cancer.

[0135] Various methods for removing EVs from solid media are known in the art. Nonlimiting examples include removing unbound EVs comprises by washing the solid media with a buffer, e.g.. a saline buffer. Exemplary saline buffers include phosphate buffered (PBS), HEPES buffered saline (HBS), and Dulbecco's phosphate buffered saline (DPBS). In some embodiments, the saline buffer comprises PBS. In some embodiments, the saline buffer comprises HBS. In some embodiments, the saline buffer comprises DPBS. In some embodiments, the saline buffer comprises PBS, pH 7.4. In some embodiments, the saline buffer further comprise about 0.1 mM CaCk and about 0.1 mM MnCh buffer.

[0136] Bulk EVs (also termed total EVs) can be isolated and concentrated from a biofluid using various methods known in the art, wherein the purification methods do not, or only minimally, disrupt the EV glycocalyx - the corona or pericellular matrix that surrounds EVs. In some embodiments, isolating comprises ultracentrifugation. In some embodiments, isolating comprises size-exclusion chromatography (SEC). In some embodiments, isolating comprises magnetic bead-based isolation. In some embodiments, isolating comprises a positive chargebased isolation to capture negatively charged EV. In some embodiments, isolating comprises a membrane-based affinity column. In some embodiments, isolating comprises an exoEasy™ column. In some embodiments, isolating does not comprise ultracentrifugation. In some embodiments, isolating does not comprise SEC. In some embodiments, isolating does not comprise heparin-affinity purification.

[0137] The resulting population of isolated EVs obtained after isolating and concentrating from a biofluid (z.e., the total EVs) can, for example, be about 1 x 1010to about 1 x 1012particles / mL. In some embodiments, the concentration of isolated total EVs from a biofluid prior to enrichment with a Tumor SPARC or Neuro SPARC is at least about 1 x 1010particles / mL. In some embodiments, the concentration of isolated total EVs from a biofluid prior to enrichment with a Tumor SPARC or Neuro SPARC is at least about 1 x 1011particles / mL. In some embodiments, the concentration of isolated total EVs from a biofluid prior to enrichment with a Tumor SPARC or Neuro SPARC is at least about 1 x 1012particles / mL. In some embodiments, the concentration of isolated total EVs from a biofluid prior to enrichment with a Tumor SPARCNAI-5002577470vl 34ATTORNEY DOCKET NO.: 14842-001-228 or Neuro SPARC is at least about 1 x 1013particles / mL. In some embodiments, the concentration of isolated total EVs from a biofluid prior to enrichment with a Tumor SPARC or Neuro SPARC is more than about 1 x IO10particles / mL. In some embodiments, the concentration of isolated total EVs from a biofluid prior to enrichment with a Tumor SPARC or Neuro SPARC is more than about 1 x 1011particles / mL. In some embodiments, the concentration of isolated total EVs from a biofluid prior to enrichment with a Tumor SPARC or Neuro SPARC is more than about 1 x 1012parti cles / mL.

[0138] A process includes contacting said biological sample or EVs obtained from the biological sample with one or more detection agents. In some embodiments, contacting is for about 12 to about 24 hours, e.g., comprises rotating for about 12 to about 24 hours. In some embodiments, contacting is for about 16 to about 24 hours, e.g., comprises rotating for about 16 to about 24 hours. In some embodiments, contacting is for about 12 to about 16 hours, e.g., comprises rotating for about 12 to about 16 hours. In some embodiments, contacting is for about 12 to about 24 hours at about 2-8 °C, e.g., comprises rotating for about 12 to about 24 hours at about 2-8 °C. In some embodiments, contacting is for about 16 to about 24 hours at about 2-8 °C, e.g., comprises rotating for about 16 to about 24 hours at about 2-8 °C. In some embodiments, contacting is for about 12 to about 16 hours at about 2-8 °C, e.g., comprises rotating for about 12 to about 16 hours at about 2-8 °C. In some embodiments, contacting is for less than 12 hours at about 20-22°C, e.g., comprises rotating for less than 12 hours at about 20-22°C. In some embodiments, contacting comprises rotating for about 2 to about 6 hours at about 20-22°C. In some embodiments, contacting for about 4 to about 6 hours at about 20-22°C, e.g., comprises rotating for about 4 to about 6 hours at about 20-22°C. In some embodiments, contacting is for about 2 to about 4 hours at about 20-22°C comprises rotating for about 2 to about 4 hours at about 20-22°C.

[0139] Also provided are methods detecting one or more target proteins or genes by mass spectrometry wherein the method includes obtaining a biological sample from a subject; contacting the biological sample or a portion thereof with one or more detection agents capable of specifically binding a glycoform on or within extracellular vesicles within the biological sample, isolating the extracellular vesicles bound to said detection agent to form isolated extracellular vesicles; and subjecting the isolated extracellular vesicles or a component thereof to mass spectrometry to detect the presence or absence of the target. While the method may be used with a detection agent asNAI-5002577470vl 35ATTORNEY DOCKET NO.: 14842-001-228 described herein, any detection agent that is able to selectively bind any extracellular vesicle glycoform may be used in a process. It was found that by subjecting a set of extracellular vesicles to isolation using one or more detection agents that is specific for one or more glycoforms on an extracellular vesicle, that the ability to detect many different target proteins or nucleic acids is dramatically improved. For example, the number of unique proteins detectable by the Orbitrap Astral mass spectrometer from ThermoFisher Scientific (Waltham, MA) using a standard preparation of EVs is about 3354. When the EVs are subjected to further isolation using a detection agent specific for a glycoform, optionally a lectin, the number of unique proteins that may be detected by the same platform increases by as much as 1.5-fold representing dramatically greater detectability of proteins that are at concentrations too low to be detected in whole blood, plasma, or even isolated EVs without further isolation using a detection agent as provided herein. Optionally, the number of unique proteins shows a fold-improvement of 1.05 or greater, optionally 1.1 or greater, optionally 1.15 or greater, optionally 1.2 or greater, optionally 1.25 or greater, optionally 1.3 or greater, optionally 1.35 or greater, optionally 1.4 or greater, optionally 1.45 or greater, optionally 1.5 or greater. Optionally, a method is able to detect protein or nucleic acid that is below the lower limit of detection (LLOD) of the same protein or nucleic acid when present in a biological sample from which enriched EVs are obtained using the same analytical technique, optionally mass spectrometry. Optionally, the process is able to detect protein or nucleic acid that are 99 percent or less of the LLOD of the same protein or nucleic acid when present in a biological sample from which enriched EVs are obtained using the same analytical technique, optionally mass spectrometry, optionally 95%, optionally 90%, optionally 80%, optionally 70%, optionally 60%, optionally 50%, optionally 40%, optionally 30%, optionally 20%, optionally 10%, optionally 5%, optionally 1%, optionally 0.5%, optionally 0.1%, optionally 0.01%, optionally 0.001%, or less.

[0140] The target proteins or nucleic acids that may be detectable using the present methods (such as target proteins or nucleic acids that may not be detectable using prior mass spectrometry methods using biological samples, or EVs isolated from biological samples using prior techniques, or may be more readily or repeatably detected by the present methods) may be related to a disease or condition. A target that is related to a disease or condition is one that is elevated or depressed in expression level or activity relative to a normal population. Optionally statistically significantly elevated or depressed in expression level or activity relative to a normal population. IllustrativeNAI-5002577470vl 36ATTORNEY DOCKET NO.: 14842-001-228 diseases or conditions detectable by the present methods include, but is not limited to, an inflammatory disease, a cancer, an allergic disease, an autoimmune disease, an infectious disease, a transplantation related disease, a degenerative disease, an injury associated with inflammation, a disease associated with a hypersensitivity, a cardiovascular disease, a glandular disease, a hepatic disease, a neurological disease, an a musculoskeletal disease, a renal disease, a reproductive disease, a connective tissue disease, a neurodegenerative disease, necrosis, an inflammatory disease associated with an implant, a hematological disease, an eye disease, or a respiratory disease. Optionally, a disease or condition is a cancer. In some embodiments, the cancer is brain cancer. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is not prostate cancer. In some embodiments, the disease or condition is a neurodegenerative disease. Optionally, disease or condition is an inflammatory disease, a cancer, an allergic disease, an autoimmune disease, an infectious disease, a transplantation related disease, a degenerative disease, an injury associated with inflammation, a disease associated with a hypersensitivity, a cardiovascular disease, a glandular disease, a hepatic disease, a neurological disease, an a musculoskeletal disease, a renal disease, a reproductive disease, a connective tissue disease, a neurodegenerative disease, necrosis, an inflammatory disease associated with an implant, a hematological disease, an eye disease, or a respiratory disease and a detection agent includes a lectin, optionally AAL, SNA, GNL, MAL I, MAL II, or PHA-E, or a combination thereof. Optionally, the lectin is only PHA-E. Optionally, the detection agents include three or more lectins, optionally from the group AAL, SNA, GNL, MAL I, and MAL II. Optionally, the detection agents include five lectins, optionally from the group AAL, SNA, GNL, MAL I, and MAL II.5.6. Characterization of EVs

[0141] Following isolating the extracellular vesicle using the detection agent, the isolated and / or enriched extracellular vesicles may be subjected to characterization analysis. Characterization analysis is optionally characterization of the components of the isolated and / or enriched EVs optionally, lipid composition, glycoform composition, protein composition and / or nucleic acid composition. Optionally, an isolated and / or enriched EVs are subjected to characterization of the protein and / or nucleic acid content, and optionally quantification relative to a standard.NAI-5002577470vl 37ATTORNEY DOCKET NO.: 14842-001-228

[0142] Various techniques for EV protein detection are known in the art, and are suitable for use with the present disclosure. In some embodiments, the protein components found on and / or within the isolated and / or enriched EVs are detected using ELISA. In some embodiments, the protein components found on and / or within the isolated and / or enriched EVs are detected using flow cytometry. In some embodiments, the protein components found on and / or within the isolated and / or enriched EVs are detected using an immunoassay. Optionally, an EV is subjected to a proteomics characterization for the identification of the protein components found on and / or within the isolated and / or enriched EVs. Illustrative methods of proteomic analyses may be found in Mallia, et al., Diagnostics (Basel), 2020; 10(10): 843. Illustratively, EVs may be subjected to lysis and / or isolation of a protein component of the material. The proteins may be then detected and / or quantified (optionally relatively quantified to a control (e.g. fold change) by a process such as mass spectrometry, or other desired method. Mass spectrometry is optionally using a mass spectrometer that operates by matrix-assisted laser desorption ionization (MALDI), quadrupole, linear ion trap, or other technology, or a combination thereof. Determination of the presence or absence of a protein may be achieved by comparison to a database relevant to the subject, such as a human proteome database.

[0143] Optionally, an EV is subjected to characterization of nucleic acid content. Nucleic acid is optionally deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Optionally, nucleic acid is RNA. In some embodiments, the RNA is microRNA (miRNA). In some embodiments, the RNA is messenger RNA (mRNA). In some embodiments, the RNA is long non-coding RNA (IncRNA). Nucleic acid may be obtained from isolated EVs and / or enriched EVs using one or more nucleic acid isolation methods known in the art. EVs are optionally lysed and separation of genomic DNA and / or protein. The remaining RNA is optionally quantified and is subjected to preparation of RNA libraries. The resulting RNA library(ies) is sequenced by PCR or NGS methods for identification and quantification of nucleic acid within the isolated EVs and / or enriched EVs. In some embodiments, an EV is subjected to characterization of nucleic acid content by targeted detection of a particular sequence and / or mutation. In some embodiments, an EV is subjected to characterization of nucleic acid content via PCR. an EV is subjected to characterization of nucleic acid content via microarray. In some embodiments, an EV is not subjected to characterization of nucleic acid content via microarray.NAI-5002577470vl 38ATTORNEY DOCKET NO.: 14842-001-2285.7. Uses

[0144] In some aspects, the protein and / or nucleic acid content of the isolated and enriched population of EVs generated by the methods provided herein (such as the methods described in Section 5.5) can be analyzed. In some embodiments, the protein and / or nucleic acid content can be analyzed for mutations, alterations, fusions, and / or post-translational modifications, relative to a healthy control reference. In some embodiments, the protein content can be analyzed for alterations, fusions, and / or post-translational modifications, relative to a healthy control reference. In some embodiments, the nucleic acid content can be analyzed for mutations, relative to a healthy control reference sequence. The resulting analyzed protein or nucleic acid content of the isolated EVs and / or enriched EVs are then optionally compared to a known healthy control for the presence or absence of one or more target proteins or target nucleic acids or a difference in a level or presence of one or more target proteins or target nucleic acids relative to a heathy control or control population. It was found that by comparing particular sets of proteins / nucleic acids relative to control that differences in the amounts of expression of particular proteins or nucleic acids in the isolated EVs and / or enriched EVs allow for detection of the presence or absence of a cancer in a subject. Of interest, the cancer need not be a cancer particular to the tissue type used in the biological sample. For example, several solid or other tumor types can be readily detected by the relative abundance of one or more target proteins or nucleic acids relative to the expression levels of those genes or nucleic acids in a healthy control known to be free of the cancer. Optionally, the presence of a cancer is detected by one or more of the target proteins or nucleic acids being at a level that is statistically different than in a healthy control. In some embodiments, the presence of a cancer is detected by one or more of the target proteins or nucleic acids being at a level that is statistically different than in a benign tumor. In some embodiments, the presence of a cancer is detected by one or more of the target proteins or nucleic acids being at a level that is statistically different than in a different cancer from the same tissue. In some embodiments, the presence of a cancer subtype is detected by one or more of the target proteins or nucleic acids being at a level that is statistically different than another cancer subtype of the same cancer. Statistically different is optionally an increase in expression level of the protein or nucleic acid that differs in an amount that is statistically outside the normal level of the protein or nucleic acid expression level. Statistically different is optionally a decrease in expression level of the protein or nucleic acid thatNAI-5002577470vl 39ATTORNEY DOCKET NO.: 14842-001-228 differs in an amount that is statistically outside the normal level of the protein or nucleic acid expression level.

[0145] In some aspects, more than one target protein or target nucleic acid is used in the detection of the presence or absence of a cancer. Optionally, 2 targets are detected. Optionally, the number of targets is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more. The number and the identity of the targets is optionally specific for the detection of the presence or absence of any particular cancer. Optionally, a first cancer type has a different target or set of targets than a second type of cancer.

[0146] As provided herein the processes disclosed herein may optionally be able to further stratify the cancer on a severity scale as is recognized in the art. In some aspects, a process as provided herein can distinguish between a Stage 0 cancer and a more severe stage of cancer. A stage 0 cancer is one that includes abnormal cells, but the abnormality has not spread to nearby or distant tissue. A stage 1 cancer is one where a cancer is present. Stage 2 and stage 3 cancer is defined by the size of the tumor and the degree to which it has spread to nearby tissues. A stage 4 cancer is one in which the cancer has spread to distant (non-neighboring) tissue. The staging of stage 0, stage 1, stage 2, stage 3, and stage 4 cancers is performed by processes known to those of ordinary skill in the art. A process as provided herein is optionally able to stratify a cancer as stage relative to other stages. Optionally, a process as provided herein is used to stratify a cancer as either stage 0, stage 1, stage 2, stage 3, or stage 4.

[0147] In some aspects, a process as provided herein can be used to demonstrate a worsening or improving prognosis or disease status over time whereby the target(s) are measured at a first time and a second time and a change in the detecting as provided herein indicates a worsening or improving of the cancer at the second time relative to the first time. For example, a cancer may be present as a stage 0 cancer at a first time. A second biological sample is optionally obtained at a second time where the second time is after the first time, and a change that is more or less toward the levels of the targets in a normal indicates an improving or worsening of the cancer stage or stratification respectively. Optionally, the target(s) is closer to a normal control at a second time indicates an improvement in cancer stage or severity. Optionally, the target(s) is of increasedNAI-5002577470vl 40ATTORNEY DOCKET NO.: 14842-001-228 difference relative to a normal control at a second time indicates a worsening (higher number) in cancer stage or severity.

[0148] In some aspects, a first and a second biological sample are obtained from a subject at a time that differs in one day or more, one week or more, one month or more, or one year or more. The choice of time between obtaining a first and a second biological sample is performed as understood as acceptable and used by a practicing physician in the field of the cancer that is being detected or studied.

[0149] In some aspects, a subject is administered one or more treatments for the cancer during or between obtaining a first biological sample and a second biological sample. Optionally, a first disease state score is used to quantify the stage or severity of the cancer at a first time by a process as used herein. A disease state score is an overall value of a difference in the expression level of one or more targets in a biological sample.

[0150] For example, a disease state score is an overall measurement of fold-change of the one or more targets in the biological sample relative to a control. As a non-limiting example, a first disease state score is optionally an absolute value of fold change of each target relative to a control biological sample where a first target protein is expressed at 1.2 fold higher level relative to control and a second target protein is expressed at a 1.2 fold lower level relative to a control leading to a disease state score of 2.4. the exponential value of a normalized expression of a target multiplied by the coefficient of importance for that target. In some aspects, a disease state score is measured using only fold-changes of each target that differs from control in a statistically significant manner. If a change in any particular target protein is not statistically significantly different from control (optionally p<0.05), the fold change is considered zero. In some embodiments, a disease state score is an overall measurement of normalized expression of the one or more targets in the biological sample. As a further non-limiting example, a first disease state score is optionally the exponential value of a normalized expression of a target multiplied by the coefficient of importance for that target. Optionally a disease state score is obtained by a logistic regression analysis where the log odds of disease may increase by expA(coef x value) (e.g., coefl x valuel + coef2 x value2 + . . . , where each protein has its own coefficient and values) where the coef is the gene’s / proteins weight / coefficient and value is the sample’s measured quantity (normalized). A disease state score at a second time that is lower than a first time indicates improving of the cancer. A disease stateNAI-5002577470vl 41ATTORNEY DOCKET NO.: 14842-001-228 score at a second time that is higher than a first time indicates equivalent or worsening of the cancer.

[0151] Optionally, between obtaining a first biological sample and a second (or subsequent) biological sample, a therapy is administered to the subject. A therapy is optionally any compound that is known to, is desired to, or is being tested to improve the condition of the subject. Any compound that is in use or being tested for efficacy toward any particular cancer may be used as a therapeutic in the provided processes. Optionally, a therapy is an antibody, a hormone, a small molecule therapeutic (i.e. molecular weight lower than that of an antibody, optionally lower than 1000 Da), radiation, stem cell administration, hyperthermia, chemotherapy, surgery, or other therapeutic, or a combination thereof. The therapy may be administered for any treatment time as desired in the art, optionally one administration or more, optionally administration continuously or discontinuously for 1 day or more, one week or more, one month or more, or one year or more.

[0152] The processes as provided herein are optionally used to detect and / or optionally determine a disease state score, and / or stratify any cancer type. Optionally, a cancer type is an acquired immune deficiency syndrome (AIDS)-associated cancer (e.g. cancer resulting from AIDS), Acoustic Neuroma (Vestibular Schwannoma), Adrenal Tumor, Anal Cancer, Appendiceal Cancer, Basal Cell Carcinoma, Benign Blood Disorders, Bile Duct Cancer (Cholangiocarcinoma), Bladder Cancer, Bone Cancer, Brain Metastases, Brain Tumors & Brain Cancer, Breast Cancer, Cancer of Unknown Primary Origin, Central Nervous System (CNS), Lymphoma, Cervical Cancer, Colon Cancer, Colorectal Cancer, Esophageal Cancer, Gallbladder Cancer, Gastrointestinal Neuroendocrine Tumors, Genitourinary Cancer, Gestational, Trophoblastic Disease, Glioma, Meningioma, Benign Brain Tumor, Graft-Versus-Host Disease (GVHD), Gynecologic Cancer, Head & Neck Cancers, Histiocytosis, Kaposi Sarcoma, Kidney Cancer (Renal Cell Cancer), Leukemia and Other Blood Cancers, Liver Cancer, Liver Metastases (Secondary Liver Cancer), Lung Cancer (e.g. non-small cell lung cancer (NSCLC), Lymphoma, Male Breast Cancer, Melanoma, Merkel Cell Carcinoma, Mouth (Oral) Cancer, Multiple Myeloma, Myelodysplastic Syndrome (MDS), Neurofibromatosis, Ovarian Cancer, Pancreatic Cancer, Pancreatic Cysts, Peritoneal & Pleural Mesothelioma Cancers, Pituitary Gland Tumors, Primary Brain Tumors, Prostate Cancer, Rare Blood Disorders, Rectal Cancer, Salivary Gland Cancer, Skin Cancer, Skull Base Tumors, Soft Tissue Sarcoma, Spine Tumors & Spinal Cancer, Squamous Cell Carcinoma, Stomach (Gastric) Cancer, Testicular Cancer (Germ Cell Tumors),NAI-5002577470vl 42ATTORNEY DOCKET NO.: 14842-001-228Throat Cancer, Thymoma & Other Thymic Tumors, Thyroid Cancer, Tracheal Diseases, Uterine (Endometrial) Cancer, Uterine Sarcoma, or a combination thereof. In some embodiments, the cancer is not prostate cancer.

[0153] A target is optionally used to detect the presence or absence of a disease state score or disease severity score, or stratify a cancer. A cancer is optionally a lung cancer, optionally nonsmall cell lung cancer. A target for NSCLC is optionally one or more of prohibitin 1 (PHB1), stromal interaction molecule 1 (STIM1), or combinations thereof. Optionally, the number of targets is any number between 1 and 100 or more targets. Optionally, the number of targets used for detection of NSCLC is equal to or greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34, 36, 37, 38, 39, 40, 41 ,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 14, 115, 116, 117, or more of the targets.A cancer is optionally an ovarian cancer. A target for ovarian cancer is optionally one or more of mucin 1 (MUC1), mucin 16 (MUC16) or combinations thereof. Optionally, the number of targets for ovarian cancer is any number between 1 and 100 or more targets. Optionally, the number of targets used for detection of ovarian is equal to or greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 14, 115, 116, 117, or more of the targets.5.8. Compositions

[0154] In one aspect, provided herein are compositions comprising an isolation component covalently attached to one or more lectins.

[0155] For example, provided herein are compositions that may be used for detecting the presence or absence of a cancer in a subject, wherein the composition includes one or more isolation components and one or more lectins selected from the group consisting of AAL, SNA, GNL, MAL I, MAL II, and PHA-E and combinations thereof. In some embodiments, theNAI-5002577470vl 43ATTORNEY DOCKET NO.: 14842-001-228 composition includes one or more isolation components, and one or more lectins selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II and combinations thereof. In some embodiments, the composition comprises two lectins selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II. In some embodiments, the composition comprises three lectins selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II. In some embodiments, the composition comprises four lectins selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II. In some embodiments, the composition comprises five lectins selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II and combinations thereof. Optionally, in some embodiments the composition includes three or more lectins. In some embodiments, the composition includes four or more lectins. In some embodiments, the composition includes five or more lectins optionally five lectins. Optionally, the composition includes the lectins AAL, SNA, GNL, MAL I, and MAL II.

[0156] In some embodiments, the composition comprises AAL. In some embodiments, the composition comprises SNA. In some embodiments, the composition comprises GNL. In some embodiments, the composition comprises MAL I. In some embodiments, the composition comprises MAL II. In some embodiments, the composition comprises PHA-E. In some embodiments, the composition comprises AAL, and SNA. In some embodiments, the composition comprises AAL, and GNL. In some embodiments, the composition comprises AAL, and MAL I. In some embodiments, the composition comprises AAL, and MAL II. In some embodiments, the composition comprises SNA, and GNL. In some embodiments, the composition comprises SNA, and MAL I. In some embodiments, the composition comprises SNA, and MAL II. In some embodiments, the composition comprises GNL, and MAL I. In some embodiments, the composition comprises GNL, and MAL II. In some embodiments, the composition comprises MAL I, and MAL II. In some embodiments, the composition comprises AAL, SNA, and GNL. In some embodiments, the composition comprises AAL, SNA, and MAL I. In some embodiments, the composition comprises AAL, SNA, and MAL II. In some embodiments, the composition comprises AAL, GNL, and MAL I. In some embodiments, the composition comprises AAL, GNL, and MAL II. In some embodiments, the composition comprises AAL, MAL I, and MAL II. In some embodiments, the composition comprises SNA, GNL, and MAL I. In some embodiments, the composition comprises SNA, GNL, and MAL II. In some embodiments, the composition comprises SNA, MAL I, and MAL II. In some embodiments, the composition comprises GNL,NAI-5002577470vl 44ATTORNEY DOCKET NO.: 14842-001-228MAL I, and MAL II. In some embodiments, the composition comprises A AL, SNA, GNL, and MAL I. In some embodiments, the composition comprises AAL, SNA, GNL, and MAL II. In some embodiments, the composition comprises AAL, SNA, MAL I, and MAL II. In some embodiments, the composition comprises AAL, GNL, MAL I, and MAL II. In some embodiments, the composition comprises SNA, GNL, MAL I, and MAL II. In some embodiments, the composition comprises AAL, SNA, GNL, MAL I, and MAL II.[00157J In some embodiments, the composition comprises one lectin, consisting of AAL. In some embodiments, the composition comprises one lectin, consisting of SNA. In some embodiments, the composition comprises one lectin, consisting of GNL. In some embodiments, the composition comprises one lectin, consisting of MAL I. In some embodiments, the composition comprises one lectin, consisting of MAL II. In some embodiments, the composition comprises one lectin, consisting of PHA-E. In some embodiments, the composition comprises two lectins, consisting of AAL, and SNA. In some embodiments, the composition comprises two lectin, consisting of AAL, and GNL. In some embodiments, the composition comprises two lectins, consisting of AAL, and MAL I. In some embodiments, the composition comprises two lectins, consisting of AAL, and MAL II. In some embodiments, the composition comprises two lectins, consisting of SNA, and GNL. In some embodiments, the composition comprises two lectins, consisting of SNA, and MAL I. In some embodiments, the composition comprises two lectins, consisting of SNA, and MAL II. In some embodiments, the composition comprises two lectins, consisting of GNL, and MAL I. In some embodiments, the composition comprises two lectins, consisting of GNL, and MAL II. In some embodiments, the composition comprises two lectins, consisting of MAL I, and MAL II. In some embodiments, the composition comprises a lectin consisting of AAL, SNA, and GNL. In some embodiments, the composition comprises three lectins, consisting of AAL, SNA, and MAL I. In some embodiments, the composition comprises three lectins, consisting of AAL, SNA, and MAL II. In some embodiments, the composition comprises three lectins, consisting of AAL, GNL, and MAL 1. In some embodiments, the composition comprises three lectins, consisting of AAL, GNL, and MAL II. In some embodiments, the composition comprises three lectins, consisting of AAL, MAL I, and MAL II. In some embodiments, the composition comprises three lectins, consisting of SNA, GNL, and MAL I. In some embodiments, the composition comprises three lectins, consisting of SNA, GNL, and MAL II. In some embodiments, the composition comprises three lectins, consisting of SNA, MAL I, andNAI-5002577470vl 45ATTORNEY DOCKET NO.: 14842-001-228MAL II. In some embodiments, the composition comprises three lectins, consisting of GNL, MAL I, and MAL II. In some embodiments, the composition comprises four lectins, consisting of AAL, SNA, GNL, and MAL I. In some embodiments, the composition comprises four lectins, consisting of AAL, SNA, GNL, and MAL II. In some embodiments, the composition comprises four lectins, consisting of AAL, SNA, MAL I, and MAL II. In some embodiments, the composition comprises four lectins, consisting of AAL, GNL, MAL I, and MAL II. In some embodiments, the composition comprises four lectins, consisting of SNA, GNL, MAL I, and MAL II. In some embodiments, the composition comprises five lectins, consisting of AAL, SNA, GNL, MAL I, and MAL II.

[0158] Also provided are compositions that may be used for detecting the presence or absence of a neurological disease in a subject, wherein the composition includes one or more isolation components and one or more lectins comprising PHA-E. In some embodiments, the composition includes one or more isolation components and one lectin, wherein the lectin is PHA-E. In some embodiments, the neurological disease is a neurodegenerative disease (e.g., Alzheimer’s disease).

[0159] The isolation component of the composition as provided herein is optionally any isolation component as described herein, optionally a magnetic bead, a fluorescent agent, avidin, biotin, a protein sequence tag, an antibody or fragment thereof. In some embodiments, the isolation component is a solid media. In specific embodiments, the solid media comprises a magnetic particle, an agarose particle, or a hydrogel polymer. In some embodiments, the solid media comprises a magnetic particle. In some embodiments, the binding element is a lectin (such as a lectin described in Section 5.4). In some embodiments, the solid media comprises an agarose particle. In some embodiments, the solid media comprises a hydrogel polymer. In some embodiments, the lectin is conjugated to the solid media via tosylation. In some embodiments, the lectin is conjugated to a magnetic particle via tosylation. In some embodiments, the lectin is conjugated to magnetic beads, such as a DYNABEAD, available from Thermo Fisher Scientific, Waltham, MA. In some embodiments, the solid media is not a magnetic bead. In some embodiments, the solid media is not a planar structure. In some embodiments, the solid media is not a slide. In certain embodiments, the solid media is not glass, for example, is not a glass slide. In certain embodiments, the solid media is not a well, for example, is not a well of a well plate. In certain embodiments, the solid media is not part of an array or blot, for example, a lectin array, e.g., lectin microarray, or lectin blot. In certain embodiments, the solid media is not a Janus nanoparticle.NAI-5002577470vl 46ATTORNEY DOCKET NO.: 14842-001-228

[0160] In one aspect, provided herein is a composition comprising an isolated population of brain-derived extracellular vesicles (EVs), wherein the isolated population of brain-derived EVs are obtained by a method provided herein (such as a method described in Section 5.5). In another aspect, provided herein is a composition comprising an isolated population of tumor derived extracellular vesicles (EVs), wherein the isolated population of tumor derived EVs are obtained by a method provided herein (such as a method described in Section 5.5).

[0161] In another aspect, provided herein is a composition comprising one or more lectins (such as a lectin described in section 5.4) or gly can-binding fragment thereof conjugated to a solid media (such as a solid media described in Section 5.3), and an isolated population of EVs bound to the lectin-conjugated solid media. In some embodiments, the lectin comprises PHA-E or a glycan-binding fragment thereof. In some embodiments, the lectin consists of PHA-E or a glycan- binding fragment thereof. In some embodiments, the one or more lectins is selected from the group consisting of AAL, SNA, GNL, MAL I, MAE II, and PHA-E. In some embodiments, the lectin is conjugated to the solid media via tosylation. In some embodiments, the lectin is conjugated to a magnetic particle via tosylation. In some embodiments, the lectin is conjugated to magnetic beads, such as a DYNABEAD, available from Thermo Fisher Scientific, Waltham, MA. In some embodiments, the solid media is not a magnetic bead. In some embodiments, the solid media is not a planar structure. In some embodiments, the solid media is not a slide. In certain embodiments, the solid media is not glass, for example, is not a glass slide. In certain embodiments, the solid media is not a well, for example, is not a well of a well plate. In certain embodiments, the solid media is not part of an array or blot, for example, a lectin array, e.g., lectin microarray, or lectin blot. In certain embodiments, the solid media is not a Janus nanoparticle.

[0162] In a further aspect, provided herein is a composition comprising: (a) a heterogenous plurality of isolated EVs, where the isolated EVs comprise: (i) a first population of EVs; and (ii) a second population of EVs; and (b) a lectin (such as a lectin described in section 5.4) or glycan- binding fragment thereof conjugated to a solid media (such as a solid media described in Section 5.3), wherein the lectin or fragment thereof specifically binds a biantennary galactosylated N- glycan with bisecting GlcNAc; and (c) a buffer, wherein the first population of EVs are brain- derived and the second population of EVs are not brain-derived, and wherein the first population of EVs are specifically bound to the solid media and the second population of EVs are unbound. In some embodiments, the lectin comprises PHA-E or a glycan-binding fragment thereof. InNAI-5002577470vl 47ATTORNEY DOCKET NO.: 14842-001-228 some embodiments, the lectin consists of PHA-E or a glycan-binding fragment thereof In some embodiments, the buffer comprises equal parts: a first solution comprising 3M ammonium sulfate, pH 7.9; and a second solution comprising 0.1 M sodium phosphate buffer, 0.1 mM CaCh, 0.1 mM MnCh. In some embodiments, the lectin is conjugated to the solid media via tosylation.

[0163] In yet another aspect, provided herein is a composition comprising: (a) a heterogenous plurality of isolated EVs comprising tumor derived EVs; (b) two or more lectins or glycan-binding fragments thereof conjugated to a solid media, wherein the lectins or glycan-binding fragment thereof is selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II, and combinations thereof; and (c) a buffer. In some embodiments, the two or more lectins or glycan- binding fragments thereof comprise three or more lectins selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II and combinations thereof. In some embodiments, the two or more lectins or glycan-binding fragments thereof comprise four or more lectins selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II and combinations thereof. In some embodiments, the two or more lectins or glycan-binding fragments thereof comprise five or more lectins selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II and combinations thereof. In some embodiments, the lectins or glycan-binding fragments thereof comprise AAL, SNA, GNL, MAL I, and MAL II. In some embodiments, the lectins or glycan- binding fragments thereof consist of AAL, SNA, GNL, MAL I, and MAL II. In some embodiments, the lectins are conjugated to the solid media via tosylation. In some embodiments, the buffer comprises equal parts: a first solution comprising 3M ammonium sulfate, pH 7.9; and a second solution comprising 0.1 M sodium phosphate buffer, 0.1 mM Ca Ch, 0.1 mM Mn Ch.5.9. Kits

[0164] In one aspect, provided herein is a kit comprising a lectin or glycan-binding fragment thereof conjugated to a solid media, wherein the lectin or glycan-binding fragment thereof specifically binds a biantennary galactosylated N-glycan with bisecting GlcNAc; a buffer suitable for preserving the EV glycocalyx; and instructions for use. In some embodiments, wherein the lectin comprises PHA-E or a glycan-binding fragment thereof. In some embodiments, the lectin is conjugated to the solid media via tosylation.NAI-5002577470vl 48ATTORNEY DOCKET NO.: 14842-001-228

[0165] In another aspect, provided herein is a kit comprising two or more lectins or glycan- binding fragments thereof conjugated to a solid media, wherein the lectins or glycan-binding fragment thereof are selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II, and combinations thereof; and instructions for use. In some embodiments, the two or more lectins or glycan-binding fragments thereof comprise three or more lectins selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II and combinations thereof. In some embodiments, the two or more lectins or glycan-binding fragments thereof comprise four or more lectins selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II and combinations thereof. In some embodiments, the two or more lectins or glycan-binding fragments thereof comprise five or more lectins selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II and combinations thereof. In some embodiments, the lectins or glycan-binding fragments thereof comprise AAL, SNA, GNL, MAL I, and MAL II. In some embodiments, the lectins or glycan-binding fragments thereof consist of AAL, SNA, GNL, MAL I, and MAL II. In some embodiments, the lectins are conjugated to the solid media via tosylation.

[0166] In some embodiments, the pH of the buffer is about pH 7.0 to pH 8.0. In some embodiments, the pH of the buffer is about pH 7.4.

[0167] In some embodiments, the solid media comprises a magnetic particle, an agarose particle, or a hydrogel polymer. In some embodiments, the solid media comprises a magnetic particle. In some embodiments, the solid media is not a magnetic bead. In some embodiments, the solid media is not a planar structure. In some embodiments, the solid media is not a slide. In certain embodiments, the solid media is not glass, for example, is not a glass slide. In certain embodiments, the solid media is not a well, for example, is not a well of a well plate. In certain embodiments, the solid media is not part of an array or blot, for example, a lectin array, e.g., lectin microarray, or lectin blot. In certain embodiments, the solid media is not a Janus nanoparticle.

[0168] Various aspects of the present invention are illustrated by the following non-limiting examples. The examples are for illustrative purposes and are not a limitation on any practice of the present invention. It will be understood that variations and modifications can be made without departing from the spirit and scope of the invention. While the examples are generally directed to the use of plasma from humans, a person having ordinary skill in the art recognizes that similar techniques and other techniques known in the art readily translate the examples to other organismsNAI-5002577470vl 49ATTORNEY DOCKET NO.: 14842-001-228 and other biological materials. Reagents are commercially available, and a person of ordinary skill in the art readily understands where such reagents may be obtained.6. EXAMPLES

[0169] Example 1: Detection of EV characterization MISEV markers.

[0170] Category 1 and Category 2 MISEV markers were detected in various preparations where markers are as defined by Welsh, et al., Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches, MISEV Organizing Committee, 07 February 2024, doi.org / 10.1002 / jev2.12404, MISEV Category 1 markers are typically transmembrane or GPI-anchored proteins including tetraspanins (CD9, CD81, CD63) and major histocompatibility complex class 1 (MHC1) molecules. MISEV Category 2 markers include proteins that are incorporated from the cytosol into EVs, largely due to lipid- or membrane proteinbinding capacity. Examples of MISEV category 2 markers include tumour susceptibility gene 101 (TSG101), heat shock 70 kDa proteins and flotillins -1 & -2. Definitions of such markers can be found in Newman, et al. Addressing MISEV guidance using targeted LC-MS / MS: A method for the detection and quantification of extracellular vesicle-enriched and contaminant protein markers from blood. J Extracell Biol, 2022 Sep 5; 1 (9):e56, doi: 10.1002 / jex2.56 and references cited therein as all are specifically incorporated herein by reference. The specific markers analyzed in this study are as in Tables 1 and 2 as follows.Table 1: EV Characterization MISEV markers - MISEV Category 1 Markers.NAI-5002577470vl 50ATTORNEY DOCKET NO.: 14842-001-228Table 2: EV Characterization MISEV markers - MISEV Category 2 MarkersNAI-5002577470vl 51ATTORNEY DOCKET NO.: 14842-001-228

[0171] Plasma samples were obtained from commercial sources of healthy or cancer (glioma, NSCLC, colorectal cancer, etc.) or from healthy internal donors on-site. Blood was collected in either Streck or K2EDTA sample collection tubes. Samples of internally collected blood were inverted several times (8-10 times for EDTA tubes and 2-3 times for Streck tubes) immediately after collection. Plasma was processed within 2 hours and maintained at room temperature throughout the spin protocol. Blood was pooled and centrifuged for 15 minutes at 2500 x g at room temperature. The plasma was then transferred to unused tubes and centrifuged a second time at 2500 x g for 15 minutes at room temperature. Once received and / or processed, all plasma samples were stored at -80 °C until use.

[0172] EVs were separated from plasma using membrane-based affinity columns from the exoEasy Maxi kit (Qiagen). The protocol was performed as per the manufacturer’s recommendations, using 2 milliliters (mL) of plasma per exoEasy column. Briefly, plasma samples were thawed at room temperature and transferred in 1 mL aliquots to 1.5 mL tubes for a pre-clear spin. Plasma was centrifuged at room temperature at 10,000 x g for 5 minutes to pellet particulate matter. Supernatant was collected, applied at 2 mL per column, and eluted in 400 pL of Qiagen buffer XE, for a five-fold concentration of EVs. Eluates from the same biological sample were pooled prior to distribution for downstream analyses. Aliquots of all EV preparations were removed for EV characterization via CE Western, and Nanoparticle Tracking (NTA) analyses. MISEV (Minimal Information for studies of extracellular vesicles), recommends that EVs be characterized by their size, concentration and protein composition. To determine size and concentration, EVs were diluted to 1 : 10,000 in particle-free water and 1 mL was injected into the ZetaView Mono instrument (ParticleMetrix) that had been calibrated per the manufacturer'sNAI-5002577470vl 52ATTORNEY DOCKET NO.: 14842-001-228 recommendations. A measurement was taken 3 times at 1 1 positions with sensitivity and shutter values at 80 and 150, respectively. The sample’s concentration, the average particle size, and its Zeta potential were recorded. For protein composition, EVs were lysed in RIPA buffer, then diluted 1 :5 in RIPA buffer. Samples were then run on the Jess Automated Western Blot system (ProteinSimple) against primary antibodies for EV markers CD63 and Beta-actin, and contamination markers albumin, and CD41a. For all samples, the remaining EVs were stored at 4 °C until Intact Extracellular Vesicle (IEV) subpopulation enrichment. Protein Lo-Bind tubes (Eppendorf) were used throughout the entire protocol. In general, EVs were processed within 48 hours of separation from plasma.

[0173] Panels comprised of lectins from Vector Laboratories, Inc. (Newark, CA) that recognize different glycosylation modifications were prepared individually and / or mixed to give the desired EV selectivity. A tumor panel containing lectins selected for their ability to interact with cancer-associated aberrant glycosylation to enrich cancer derived EVs was generated from a mixture of equal volumes of AAL, SNA, GNL, MAL I, MAL II all conjugated to magnetic beads (DYNABEADs) to form detection agents. Each of the foregoing detection agents is termed a “Tumor SPARC” as provided herein. A neuropanel consisting of a single lectin, PHA-E, was also used to enrich EVs. PHA-E lectin was also conjugated to magnetic beads (DYNABEADs) to form detection agents. The PHA-E detection agent is termed a “Neuro SPARC” as provided herein.

[0174] Lectin coupling to tosyl -activated magnetic beads was performed according to the protocol as provided by Dutt M, et al., Semi-Automated Lectin Magnetic Bead Array (LeMBA) for Translational Serum Glycoprotein Biomarker Discovery and Validation, Methods Mol Biol. 2023;2628:395-411, doi: 10.1007 / 978-1 -0716-2978-9_25, PMID: 36781799. Briefly, magnetic beads were washed 3 times with lx volume of lectin coating buffer (0.1 molar (M) sodium phosphate pH 7.4 / 0.1 millimolar (mM) CaCh / 0.1 mM MnCh). Beads were activated with 3 molar (M) ammonium sulfate (pH 7.9) and a stock solution of the lectin was added directly to the bead- ammonium sulfate mixture. The beads and lectin solution were rotated end-over-end at 37 °C for 24 hours to conjugate the lectin to the beads. After incubation, 10 pl of the bead solution was retained for quality control by SDS-PAGE analysis to confirm successful conjugation. The lectin solution was removed, and 2 weight percent (wt%) glycine solution was added to the beads to block unreacted tosyl groups. This mixture was rotated end-over-end at 37 °C for 16 hours. After blocking, the beads were washed three times with storage buffer (20 mM TrisHCl pH 7.4 / 150 mMNAI-5002577470vl 53ATTORNEY DOCKET NO.: 14842-001-228NaCl / 1 mM CaCh / l mM MnCh / 0.5 wt% Triton X-100 / lx Protease inhibitor cocktail). Lectin- coated beads and bead mixtures were stored in storage buffer for up to 6 months at 4 °C.

[0175] Extracellular vesicles were subjected to subpopulation enrichment to form enriched EVs by combining the detection agents as described above that had been washed 3 times with lx volume PBS with added metal ions (PBS pH 7.4 / 0.1 mM CaCh / O.l mM MnCh). The EV-bead mixture was rotated end-over-end at 2-8 °C for 16 hours to pulldown target EVs. After the supernatant was removed, nonspecifically associated EVs and proteins were removed by washing the beads with the PBS / metal ion buffer. The EV-bound beads were split in half for protein and RNA analyses. For protein, the beads were resuspended in PBS / lx Protease inhibitor cocktail and stored at -20 °C until analysis.

[0176] RNA analyses

[0177] Total RNA from EV and enriched EV preparations was extracted using the Norgen RNA / Protein Purification kit (Norgen Biotek Ontario, Canada) with minor modifications. Within two hours of isolation, 100-200 pl of EV beads suspended in PBS / lx protease inhibitor cocktail were magnetized and the supernatant was taken off. 300 pL of Norgen buffer SKP was added to the beads and the mixture was heated at 55 °C for 10 minutes to enhance lysis. Lysates were then processed according to manufacturer’s recommendations. gDNA was removed using included gDNA removal columns and EV proteins were purified and retained using the optional protein workflow. RNA was eluted in 0.25X of Norgen buffer A (nuclease-free water, 50 pl), immediately concentrated 2.5X via evaporation and aliquoted / stored at -80 °C. Protein was eluted in an equal volume of buffer C and stored at -20 °C.

[0178] One microliter of extracted, concentrated total RNA was quantified using the Promega Quantifluor RNA assay on a Tecan Infinite 200 Pro plate reader (ex 492 nm / em 540 nm) per manufacturer’s recommended protocol. For qualitative assessment, 1 pl of RNA was run on an Agilent RNA 6000 Pico chip (Bioanalyzer 2100, Agilent Technologies Inc., Santa Clara, CA USA).

[0179] EV RNA libraries were prepared using the Tecan Ovation SoLo RNA-Seq library kit for Illumina (Tecan, Mannedorf, Switzerland) to capture longer RNAs and the Qiagen QIAseq miRNA library kit for targeted miRNA capture (Qiagen, Hilden Germany) following manufacturer’s standard recommendations for generating indexed libraries with UMIs. FiveNAI-5002577470vl 54ATTORNEY DOCKET NO.: 14842-001-228 nanograms (ng) of purified, concentrated total RNA was used as input for the Tecan protocol, while 3 ng of the same was used to generate Qiagen libraries. Purified libraries were quantified using the Qubit IX dsDNA high-sensitivity assay (ThermoFisher, Waltham, MA, USA) and library size distribution was assessed using the Agilent Bioanalyzer 2100 high-sensitivity DNA kit.

[0180] EV libraries were sequenced on an Element Biosciences AVITI system (San Diego, CA, USA) following the Freestyle protocol. Between 4 (FFPE) and 30 (miRNA) indexed libraries were pooled and 5-17 picomolar (pM) total library was sequenced using the AVITI 2x150 Cloudbreak Freestyle high-output kit with a 2-5% phiX spike-in. On average, 30M (miRNA), 130M (long RNA) and 150-200M (FFPE) reads were collected for each sample. For RNA expression analyses, reads were trimmed, deduplicated, mapped to the human GRCh38 reference genome, normalized and quantified using nf-core pipelines for maseq (version 3.14.0) and smrnaseq analysis (version 2.2.2). (Philip Ewels, Alexander Peltzer, Sven Fillinger, Harshil Patel, Johannes Alneberg, Andreas Wilm, Maxime Ulysse Garcia, Paolo Di Tommaso & Sven Nahnsen. Nat Biotechnol. 2020 Feb 13. doi: 10.1038 / s41587-020-0439-x.) Matched healthy controls were used as comparators for relative gene or miRNA expression analyses using DESeq2 (Love MI, Huber W, Anders S (2014). “Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.” Genome Biology, 15, 550. doi: 10.1186 / sl3059-014-0550-8.).

[0181] Protein analyses

[0182] Plasma, isolated EV, and enriched EV preparations were assayed for total protein content using the nanodrop BCA assay. Aliquots of these samples were acetone precipitated for mass spectrometry by the addition of 0.1 M NaCl and 4 parts acetone. The resulting protein-solvent solution was chilled overnight or incubated at room temperature for 30 minutes and centrifuged at 17,000 x g for 5 minutes to pellet precipitated proteins and the resultant pellet air dried. Plasma, EVs and EV / protein-bound beads were assayed for EV markers and contamination markers in accordance with ISEV guidelines by automated capillary immunoblot (ProteinSimple).

[0183] Acetone precipitated pellets and EV / protein-bound beads were sent for protein identification and quantification by mass spectrometry. Proteins were digested with trypsin / LysC followed by LC / MS / MS analysis. Briefly, tryptic peptides were loaded onto a reverse phase column (ThermoScientific PepMap RSLC, Cl 8, 2 pM 100 A, 75 pm x 20 mm) and separatedNAI-5002577470vl 55ATTORNEY DOCKET NO.: 14842-001-228 using a linear gradient of 3-36 wt% acetonitrile in 1 wt% formic acid. The total elution duration was 110 min at a flow rate of 0.3 pl / min. Eluted peptides were ionized and sprayed into the Exploris 240 Orbitrap Mass Spectrometer (ThermoScientific) via the Nano Easy-Spray Ion Source. Proteins were identified and quantified against the human sequence database using Proteome Discoverer 2.5 (ThermoScientific) based on the SEQUEST algorithm. Carbamidomethylation (+57.021 Da) of cysteines was set as fixed modification, and Oxidation / +15.995 Da (M), Phospho / +79.966 Da (S, T, Y), Methylation / +14.016 Da (R, K), Ubiquitination / +114.043 Da (K), and K acetylation (+42.011 Da) were set as dynamic modifications. The minimum peptide length was specified to 5 amino acids. The precursor mall tolerance was set to 15 ppm whereas fragment mass tolerance was set to 0.05 Da. Healthy control samples were analyzed in the same batch as the disease samples to enable fold change calculations.

[0184] The overall protein results are analyzed for the numbers of category 1 MISEV markers and category 2 MISEV markers observed in each sample type. As is plotted in FIG. 1, both category 1 and category 2 MISEV markers show increased detection in isolated EVs compared to as are detected in plasma. When the tumor panel enriched EVs or the neuro panel enriched EVs are used, the numbers of detectable category 1 and category 2 MISEV markers in each sample on average are much higher than plasma or isolated EVs. For MISEV 1 the comparisons resulted in the following pvalues: Plasma was significantly different from all other methods (pvalues .006, .004, and .00004 for EV, Neuro SPARC and Tumor SPARC, respectively). EV was significantly different (pvalues .015 and .001 for Neuro SPARC and Tumor SPARC, respectively). Neuro SPARC and Tumor SPARC were not significantly different from each other (pvalue = .686). For MISEV 2: Plasma was significantly different from all other methods (pvalues .0001, .0008, and .00005 for EV, Neuro SPARC and Tumor SPARC, respectively). EV was significantly different (pvalues .025 and .024 for Neuro SPARC and Tumor SPARC, respectively). Neuro SPARC and Tumor SPARC were not significantly different from each other (pvalue = .75).

[0185] Plasma specific proteins are also analyzed in the various samples. Samples from plasma, isolated EVs and enriched EVs are analyzed for the abundance of albumin (FIG. 2E), alpha- 1 antitrypsin (FIG. 2D), a- 1 -acid glycoprotein (FIG. 2C), haptoglobin (FIG. 2B) and transferrin (FIG. 2A). As is illustrated in FIG. 2A-FIG. 2E, the levels of each protein are reduced by general EV isolation as illustrated by the reduced abundance in samples using isolated EVs. For example, albumin - the most abundant protein in the blood - is reduced by more than 80%NAI-5002577470vl 56ATTORNEY DOCKET NO.: 14842-001-228 following isolation of total EVs, and by more than 97% after the EVs are enriched using Tumor SPARC or Neuro SPARC (FIG. 9A).

[0186] As a result of the removal of background, there is an improved resolution and depth of data. In particular, when EVs are then enriched using either the Tumor SPARCs or the Neuro SPARCs, there is a significant increase in the number of total proteins identified by mass spectrometry compared to both total EVs and plasma in the same samples (FIG. 9B).

[0187] Accordingly, when EVs are then enriched using either the tumor detection agents or the neuro detection agents the amount of each protein abundance is dramatically reduced relative to both plasma and isolated EVs. Thus, SPARCs improve the signal -to-noise, compared to both total EVs and total plasma in the same samples.

[0188] Example 2: Detection of total protein and glycoproteins

[0189] EVs are isolated from glioblastoma samples and enriched using the five Tumor SPARCs as a panel or the Neuro SPARC, as provided in Example 1, and subjected to isolation and characterization of total protein or glycoprotein. Briefly, plasma and EV preparations were assayed for total protein content using the nanodrop BCA assay. Aliquots of these samples were acetone precipitated for mass spectrometry by the addition of 0.1 MNaCl and 4 parts acetone. The resulting protein-solvent solution was chilled overnight or incubated at room temperature for 30 minutes and centrifuged at 17,000 x g for 5 minutes to pellet precipitated proteins and the resultant pellet air dried. Acetone precipitated pellets and EV / EV protein-bound beads were sent for protein identification and quantification by mass spectrometry. Proteins were digested with trypsin / LysC followed by LC / MS / MS analysis.

[0190] Tryptic peptides were loaded onto a reverse phase column (lonopticks, TS60cm, C18 60 cm x 75 pm, 1.7 pm 100A) and separated using a gradient of 5-6%B in 0.5 min; 6-8%B in 1.5 min; 8-8.5% in 1 min; 8.5-28% in 45 min; 28-50% in 13 min, 99%B wash in 7 min (Mobile phases: [A] 0.1% FA in H2O, [B] 0.1% FA in 80% ACN). The total elution duration was 68 min at a flow rate of 0.25 pL / min with 0.4 pl / min front-loading. Eluted peptides were ionized and sprayed into the Orbitrap Astral Mass Spectrometer (ThermoSci entific) using DIA scan (200 windows, 80k rez, 3m / z windows 380-980m / z, 25HCD, 500% AGC, 7ms IT). Proteins were identified and quantified against the human sequence database using Proteome Discoverer 3.1 (ThermoScientific).NAI-5002577470vl 57ATTORNEY DOCKET NO.: 14842-001-228

[0191] As is illustrated in FIG. 3A, the number of total proteins increases from about 3000 individually identified proteins in a general EV population to over 4000 individually identified proteins in each enriched EV preparation. Similarly, as illustrated in FIG. 3B, the number of glycoproteins individually identified in an isolated EV population was about 2600, but further enrichment using the detection agents as provided herein allows for identification of a much larger number of proteins.[00192J As is illustrated in FIG. 4A, when the total proteins identified are analyzed for a subpopulation of oncoproteins specifically (see OncoKB™ Database of Cancer Genes), the average percent coverage of all individual oncoprotein amino acid sequences identifiable in plasma is low, but is improved using an isolated EV population. When the percent amino acid sequence coverage of known oncoproteins is reviewed in enriched EVs using either Tumor SPARCs or Neuro SPARCs the total percentage of identifiable oncoprotein coverage is dramatically increased (FIG. 4A). Similarly, when the total proteins identified are analyzed for a subpopulation of neurospecific protein sequence coverage specifically (see Human Protein Atlas (proteinatlas. org / humanproteome / brain / human+brain) “tissue enriched” in brain marker list), the percent coverage of all neuro sequences individually identifiable in plasma is low, but is improved using an isolated EV population. Conversely, when the percent sequence coverage of known neuroproteins is reviewed in enriched EVs using Neuro SPARCs the total percentage of identifiable neuroprotein coverage is dramatically increased as is observed in FIG. 4B. As illustrated in FIG. 4C - FIG. 4E, when specifically looking at the percent amino acid coverage of an individual oncoprotein in an individual sample, the percent coverage of the protein utilizing either Tumor SPARCs or Neuro SPARCs enriched EVs is substantially higher compared to total EVs. Specifically, for an exemplary oncoprotein only 6% of the oncoprotein’s amino acid sequence coverage was identified in an isolated EV population, and undetectable in plasma. Detection of the oncoprotein’s amino acid sequence coverage increased to 40% and 53% of when using Neuro SPARCs or Tumor SPARCs, respectively (FIG. 4C - FIG. 4E).

[0193] Example 3: Detection of proteins in enriched EVs from patients with brain cancer.

[0194] Plasma samples from subjects with brain specific glioma were obtained. The presence of total brain cancer specific proteins are detected and quantified as in Example 2 from plasmaNAI-5002577470vl 58ATTORNEY DOCKET NO.: 14842-001-228 samples, isolated EVs, and enriched EVs prepared using either Tumor SPARCs or Neuro SPARCs. Proteins are quantified relative to healthy controls and each proteins amount and p-value are determined over the total number of samples. As is observed in FIG. 5A a specific subset of proteins show statistically significant changes in amount relative to controls and stratify to between a 2-5 fold increase in detection relative to enriched EVs from healthy controls. In addition, the studies show the detectability of low abundance proteins that are associated with cancers, specifically a glioma in FIGs. 5B-5G showing results specifically for epidermal growth factor receptor (EGFR), Kirsten Rat Sarcoma virus oncogene homolog (KRAS), programmed death ligand 1 (PD-L1), platelet derived growth factor receptor alpha (PDGFRA), v-raf murine sarcoma viral oncogene homolog B 1 (BRAF), and platelet derived growth factor receptor beta (PDGFRB), respectively. These studies demonstrate that enriched EVs are able to detect proteins that are statistically quantifiable from healthy controls and is able to detect the presence of a glioma in a subject using Tumor SPARCs.

[0195] Example 4: Detection of ovarian cancer (OVC).

[0196] Plasma samples from normal controls and known I and stage II malignant OVC are obtained and analyzed. Frozen EV enriched beads were sequentially defrosted at -20 °C and then 0 °C. Phosphate-Buffered Saline (PBS) was removed and EV enriched beads were digested on Biomek i7 automated workstation (Beckman Coulter) using the single-pot, solid-phase enhanced (SP3) method according to Hughes, Christopher S et al., Nature protocols, 2019. Briefly, samples are denatured with the addition of dithiolthreitol then alkylated with iodoacetamide. Proteins are then precipitated on beads by solvent addition to 50% ACN. Beads with bound proteins are then washed by 80% ethanol then 2x ACN. The beads are resuspended in the digestion buffer for overnight digest with trypsin. For the 24-minute gradient, data was acquired on a NeoVanquish liquid chromatography (LC) system coupled to an Orbitrap Astral mass spectrometer. The LC separation was achieved through the NeoVanquish 45 samples per day (SPD), 24-min gradient. Approximately 1 pg of depleted plasma digest was injected per analysis. Data from the Orbitrap Astral was then collected with a DIA method and peptides quantified using: 1] DIA-NN 1.8.1 (Demichev et al., Nature Methods, 2020) and 2] MSstats 4.0 (Kohler et al., J. Proteom Research, 2023).NAI-5002577470vl 59ATTORNEY DOCKET NO.: 14842-001-228

[0197] As illustrated in FIG. 6A and FIG. 6B, enriched EVs obtained with the panel of Tumor SPARCs show statistically significant increases in either MUC1 protein (FIG. 6A) or MUC 16 protein (FIG. 6B) demonstrating that Tumor SPARC enriched EVs are able to differentially demonstrate and correlate the amount of known OVC markers sufficient to detect the presence or absence of an ovarian cancer in a subject.

[0198] Example 5: Detection of non-small cell lung cancer (NSCLC).

[0199] Plasma samples from normal controls and known early stage NSCLC patients are obtained and analyzed as in Example 4 using the same detection agents and methods. As illustrated in FIG. 7A and FIG. 7B enriched EVs obtained with the panel of Tumor SPARCs show statistically significant increases in either PHB1 protein (FIG. 7A) or STIM1 protein (FIG. 7B) demonstrating that Tumor SPARC enriched EVs are able to differentially demonstrate and correlate the amount of known NSCLC markers sufficient to detect the presence or absence of an NSCLC in a subject.

[0200] Example 6: Detection of low abundance proteins and nucleic acids.

[0201] Plasma samples from normal controls or patients with diagnosed glioma (brain cancer), colorectal cancer, lung cancer or ovarian cancer were obtained and analyzed by the methods of Example 1. The average number of oncoproteins per sample are illustrated in FIG. 8. As can readily be observed, the number of identified oncoproteins when subjected to detection from EVs alone is much larger than the number detected from plasma demonstrating that enriching a sample with EVs dramatically enriches low abundance proteins and nucleic acids for enhanced detectability relative to a neat biological sample. When a detection agent is used, as provided herein, however, the number of proteins or nucleic acids detectable are much further improved showing that the claimed processes have the ability to dramatically bolster the ability to detect the presence of low abundance proteins relative to what can be detected in plasma or other biological samples not subjected to enrichment of EVs prior to detection. The results hold for proteins that are below the lower limit of detection in both plasma and from isolated EVs for subjects with glioma. In particular, the presence of KRAS, EGFR and PD-L1 proteins were detected in enriched EVs, which were undetectable in plasma or isolated EV samples from the same subjects when using the method described in Example 1. This is consistent with the results illustrated in FIG. 5B-G for detection of KRAS, EGFR, PD-L1, PDGFRA, BRAF, and PDGFRB proteins, where enriched EVs using detection agent(s) as provided herein in Example 3 allow for observation of each of theseNAI-5002577470vl 60ATTORNEY DOCKET NO.: 14842-001-228 oncoproteins specifically in enriched EVs, but where the levels are below or slightly above the LLOD in both plasma and isolated EVs.

[0202] Example 7 : N euro SPARC Identification as highest affinity binder of brain / neuro cell line EVs

[0203] This example demonstrates that PHA-E has the highest affinity for neuro / CNS-derived EVs. Briefly, lyophilized EV cell line standards from Hansa BioMed (Tallinn, Estonia) were resuspended in IxRIPA to known constant final protein concentration. Lysate was split in half and treated or not treated with glycosidase PNGaseF. Glycosidase treated and glycosidase untreated EV protein populations were assayed for lectin affinity using Lectin Blot with biotinylated lectins as probes with 18 different lectins spanning various glycan / sugar specificities. Unique bands that are present in glycosidase untreated lane that disappear in glycosidase treated lane indicate specific lectin - EV glycoprotein interactions. Each experiment was completed twice. The change in total area under the curve for all peaks called between glycosidase treated and untreated EVs is used as a measure of overall lectin - EV N-linked glycan bearing glycoprotein affinity.

[0204] Quantification of the difference in total peak area for EVs from U87 glioblastoma (primary brain malignancy) and SK-N-SH neuroblastoma (arises from neural crest cells) cells revealed that PHA-E was the lectin with the highest affinity for brain / neuro cell line EVs among the 18 different lectins assayed (FIG. 10).

[0205] Example 8: CNS Markers are Enriched in Neuro SPARCs Differentially Expressed Proteins

[0206] This example demonstrates that Neuro SPARCs enable improved total protein identification and enrich for more CNS markers than EV or total plasma inputs.

[0207] Briefly, Neuro SPARCs enrichment was performed as described in Example 1 using samples from patients in Table 3. Following Neuro SPARCs enrichment, the samples were analyzed by mass spectrometry according as described in Example 4. Proteomics comparison of brain enriched proteins detected from plasma, total EVs, or after Neuro SPARC enrichment demonstrates Neuro SPARC enables better protein and CNS marker detection via LC-MS / MS (FIG. 11 A).NAI-5002577470vl 61ATTORNEY DOCKET NO.: 14842-001-228

[0208] Table 3 Patient Characteristics

[0209] Moreover, the Neuro SPARCs enrich unique EV subpopulations. Comparison of the proteins differentially expressed between brain tumor / benign samples and healthy controls using Tumor SPARCs and Neuro SPARCs identified partial overlap between the EV subpopulations (FIG. 1 IB).NAI-5002577470vl 62ATTORNEY DOCKET NO.: 14842-001-228

[0210] Tn particular, Neuro SPARCs enrich more brain enriched CNS marker proteins from glioma samples, defined as markers having overlap with the Human Protein Atlas tissue enriched in brain marker list, as compared to Tumor SPARCs (FIG. 11C). 4099 proteins total were obtained from raw data for Neuro SPARCs enrichments in glioma and healthy patients. Of the 4099 proteins, 255 were classified as brain enriched markers, and Neuro SPARCs identified more brain enriched CNS marker proteins.[00211J Example 9: CNS cell of origin markers detected with Neuro SPARCs

[0212] This example demonstrates that Neuro SPARCs enable improved total protein identification and enrich for more CNS markers than EV or total plasma inputs.

[0213] Briefly, Neuro SPARCs enrichment was performed as described in Example 1 using samples from patients in Table 3. Following Neuro SPARCs enrichment, the samples were analyzed by mass spectrometry according as described in Example 4. Following enrichment using Neuro SPARCs CNS cell of origin markers according to neuron markers (NCAM1, CD90 / THY1, and CD166 / ALCAM) were detectable in brain cancer, brain tumor (benign), and healthy samples (FIG. 12A - FIG. 12C, respectively). Notably, THY1 neuronal marker was only detected using Neuro SPARCs, and ALCAM abundance was highest with Neuro SPARCs, lower in EVs and plasma (data not shown).

[0214] Similarly, CNS cell of origin markers according to neuron / astrocyte markers (NRAM, and CD44, respectively) were detectable in brain cancer, brain tumor (benign), and healthy samples (FIG. 13A and FIG. 13B, respectively). Comparison with total EVs and plasma also demonstrated that NRCAM abundance was highest with Neuro SPARCs, lower in EVs, and not detected in plasma, while CD44 abundance was highest with Neuro SPARCs, and lower in EVs and plasma (data not shown).

[0215] Detection of CNS cell of origin markers according to microglia markers (HLA-DR, CD163, and CD36, respectively) were also detectable in brain cancer, brain tumor (benign), and healthy samples (FIG. 14A - FIG. 14C, respectively). HLA-DRA and HLA-DRB1 abundances was highest with Neuro SPARCs, lower in EVs, and not detected in plasma, while. CD163 and CD36 abundance were highest with Neuro SPARCs, and lower in EVs and plasma (data not shown).NAI-5002577470vl 63ATTORNEY DOCKET NO.: 14842-001-228

[0216] Additionally, CNS cell of origin markers were detected with Neuro SPARCs, as measured by CD38, CD40, and GDla / ST8SIA5 in in brain cancer, brain tumor (benign), and healthy samples (FIG. 15A - FIG. 15C, respectively). Comparison with total EVs and plasma also demonstrated that CD40 microglial marker was only detected using Neuro SPARCs, and that ST8SIA5, responsible for ganglioside synthesis in the brain, was detected at higher abundance with Neuro SPARCs than in total EVs, not detected in plasma (data not shown).[00217J Example 10: Individual Lectins from Tumor SPARCs Identify Unique Genes

[0218] A tumor panel containing lectins selected for their ability to interact with cancer- associated aberrant glycosylation to enrich cancer derived EVs was generated from a mixture of equal volumes of AAL, SNA, GNL, MAL I, and MAL II all conjugated to magnetic beads (DYNABEADs) to form detection agents. Each of the foregoing detection agents is termed a “Tumor SPARC” as provided herein.

[0219] To determine whether the individual lectins identified unique subpopulations of EVs, single Tumor SPARCs were generated using each of the five individual lectins (i.e., AAL, SNA, GNL, MAL I, and MAL II). Unique genes were measured in some disease samples more than others, reliant on the individual SPARC used. For example, 14 genes were only detected in colorectal cancer samples using GNL as the individual lectin, which are not detected in any other disease or control sample (FIG. 16). Similarly, 8 genes are only detected in ovarian cancer samples using MAL II (FIG. 16). The other individual SPARCs each measure 3-8 unique genes for the tested disease samples, which were not detected in the other disease or control samples.

[0220] Taken together, the results indicate that the combination of lectins, such as on the Tumor SPARCs, provide nonredundant information about the EVs, which could not be obtained from using a single lectin.

[0221] Example 11: Transcriptomics highlights abundant differential expression in Neuro SPARCs enrichments.

[0222] Briefly, samples from Alzheimer’s Disease (AD) patients (n = 7) or healthy controls (n = 4) were processed with Neuro SPARCs according to the methods of Example 1. Transcriptomics analysis comparing Neuro SPARCs enriched EVs from Alzheimer’s Disease (AD) patients (n = 7) vs healthy controls (n = 4) yielded 1595 differentially expressed / enrichedNAI-5002577470vl 64ATTORNEY DOCKET NO.: 14842-001-228 genes (DEGs) using threshold of -logl O(FDR) > 1.3 and log2(foldchange) >0.6 or <-0.6 (FIG. 17). Among the DEGs, there were 297 Upregulated DEGs and 1298 Downregulated DEGs.

[0223] Characterization of the transcripts indicated that synaptic and neuronal transcripts are decreased in EVs isolated directly from AD vs Healthy brain tissue. In particular, various markers were downregulated in AD and were annotated to the GO terms Neurogenesis G0:0022008, and Synapse G00045202.

[0224] Example 12: Neuro SPARC enables better protein and differentially expressed protein detection

[0225] Samples from AD patients or healthy controls were processed with Neuro SPARCs according to the methods of Example 1, and proteins were detected using LC-MS / MS. As shown in FIG. 18A - FIG. 18C Neuro SPARCs decrease noise to improve protein detection via LC- MS / MS. In particular, proteomics analysis comparison of Neuro SPARCs enriched EVs (FIG. 18C) vs. total EV (FIG. 18B) and plasma (FIG. 18 A) shows more total proteins and DEPs identified with Neuro SPARCs than other methods. Quantification of the results are shown in Table 4 below.

[0226] Table 4: Proteomics analysis comparison of Neuro SPARCs enriched EVs vsTotal EV and Plasma

[0227] Taken together, this example demonstrates that Neuro SPARCs decrease noise to improve protein detection via LC-MS / MS, and identify more total proteins and DEPs with Neuro SPARCs than with total EVs or plasma.

[0228] The foregoing description of particular embodiment s) is merely exemplary in nature and is in no way intended to limit the scope of the invention, its application, or uses, which may, of course, vary. The invention is described with relation to the non-limiting definitions and terminology included herein. These definitions and terminology are not designed to function as a limitation on the scope or practice of the invention but are presented for illustrative and descriptiveNAI-5002577470vl 65ATTORNEY DOCKET NO.: 14842-001-228 purposes only. While the processes or compositions are described as an order of individual steps or using specific materials, it is appreciated that steps or materials may be interchangeable such that the description of the invention may include multiple parts or steps arranged in many ways as is readily appreciated by one of skill in the art.

[0229] It will be understood that, although the terms “first,” “second,” “third” etc. may be used herein to describe various elements, components, regions, layers, and / or sections, these elements, components, regions, layers, and / or sections should not be limited by these terms. These terms are only used to distinguish one element, component, region, layer, or section from another element, component, region, layer, or section. Thus, “a first element,” “component,” “region,” “layer,” or “section” discussed below could be termed a second (or other) element, component, region, layer, or section without departing from the teachings herein.

[0230] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms, including “at least one,” unless the content clearly indicates otherwise. “Or” means “and / or.” As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items. It will be further understood that the terms “comprises” and / or “comprising,” or “includes” and / or “including” when used in this specification, specify the presence of stated features, regions, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and / or groups thereof. The term “or a combination thereof’ means a combination including at least one of the foregoing elements.

[0231] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. It will be further understood that terms such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the present disclosure, and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0232] Scientific and technical terms used herein are intended to have the meanings commonly understood by those of ordinary skill in the art. Such terms are found defined and used in context in various standard references illustratively including J. Sambrook and D.W. Russell,NAI-5002577470vl 66ATTORNEY DOCKET NO.: 14842-001-228Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 3rd Ed., 2001 ; F.M. Ausubel, Ed., Short Protocols in Molecular Biology, Current Protocols; 5th Ed., 2002; B. Alberts et al., Molecular Biology of the Cell, 4th Ed., Garland, 2002; D.L. Nelson and M.M. Cox, Lehninger Principles of Biochemistry, 4th Ed., W.H. Freeman & Company, 2004; Wild, D., The Immunoassay Handbook, 3rd Ed., Elsevier Science, 2005; Gosling, J. P., Immunoassays: A Practical Approach, Practical Approach Series, Oxford University Press, 2005;Antibody Engineering, Kontermann, R. and Diibel, S. (Eds.), Springer, 2001; Harlow, E. and Lane, D., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988; Ausubel, F. et al., (Eds.), Short Protocols in Molecular Biology, Wiley, 2002; J. D. Pound (Ed.) Immunochemical Protocols, Methods in Molecular Biology, Humana Press; 2nd ed., 1998; B.K.C. Lo (Ed.), Antibody Engineering: Methods and Protocols, Methods in Molecular Biology, Humana Press, 2003; and Kohler, G. and Milstein, C., Nature, 256:495-497 (1975); the contents of each of which are incorporated herein by reference.

[0233] Methods involving conventional biological techniques are described herein. Such techniques are generally known in the art and are described in detail in methodology treatises such as Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992 (with periodic updates); and Short Protocols in Molecular Biology, ed. Ausubel et al., 52 ed., Wiley- Interscience, New York, 2002. Immunological methods (e.g, preparation of antigen-specific antibodies, immunoprecipitation, and immunoblotting) are described, e.g, in Current Protocols in Immunology, ed. Coligan et al., John Wiley & Sons, New York, 1991; and Methods of Immunological Analysis, ed. Masseyeff et al., John Wiley & Sons, New York, 1992.

[0234] Additional protocols such as PCR Protocols can be found in A Guide to Methods and Applications Academic Press, NY. Methods for protein purification include such methods as ammonium sulfate precipitation, column chromatography, electrophoresis, centrifugation, crystallization, and others. See, e.g., Ausubel, et al. (1987 and periodic supplements); Deutscher (1990) “Guide to Protein Purification,” Methods in Enzymology vol. 182, and other volumes in this series; Current Protocols in Protein Science, John Wiley and Sons, New York, NY; and manufacturer's literature on use of protein purification products known to those of skill in the art.NAI-5002577470vl 67ATTORNEY DOCKET NO.: 14842-001-228

[0235] Various modifications of the present invention, in addition to those shown and described herein, will be apparent to those skilled in the art of the above description. Such modifications are also intended to fall within the scope of the appended claims.

[0236] It is appreciated that all reagents are obtainable by sources known in the art unless otherwise specified. Methods of nucleotide amplification, cell transfection, and protein expression and purification are similarly within the level of skill in the art.

[0237] Patents, publications, and applications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. These patents, publications, and applications are incorporated herein by reference to the same extent as if each individual patent, publication, or application was specifically and individually incorporated herein by reference.

[0238] The foregoing description is illustrative of particular embodiments of the invention, but is not meant to be a limitation upon the practice thereof. The following claims, including all equivalents thereof, are intended to define the scope of the invention.NAI-5002577470vl 68

Claims

ATTORNEY DOCKET NO.: 14842-001-228WHAT IS CLAIMED IS1. A composition comprising an isolated population of brain-derived extracellular vesicles (EVs), wherein the isolated population of brain-derived EVs are obtained by a method comprising:(a) isolating EVs from a biofluid under conditions sufficient to preserve EV glycocalyx, wherein the biofluid is plasma or serum;(b) contacting the isolated EVs with a lectin or glycan-binding fragment thereof conjugated to a solid media, wherein the lectin or glycan-binding fragment thereof specifically binds a biantennary galactosylated N-glycan with bisecting N- acetylglucosamine (GlcNAc); and(c) removing unbound EVs from the solid media, thereby obtaining the isolated population of brain-derived EVs.

2. The composition of claim 1, wherein isolating does not comprise ultracentrifugation or addition of a polymer to the biofluid.

3. The composition of claim 1 or 2, wherein removing unbound EVs comprises washing the solid media with a saline buffer.

4. The composition of claim 3, wherein the saline buffer is selected from the group consisting of phosphate buffered saline (PBS), HEPES buffered saline (EIBS), and Dulbecco's phosphate buffered saline (DPBS).

5. The composition of claim 4, wherein the saline buffer comprises PBS, pH 7.4.

6. The composition of claim 5, wherein the buffer further comprise about 0.1 mM Ca Ch and about 0.1 mM Mn Ch buffer.

7. The composition of any one of claims 1 to 6, wherein the lectin comprises phaseolus vulgaris erythroagglutinin (PHA-E) or a glycan-binding fragment thereof.

8. The composition of any one of claims 1 to 7, wherein isolating comprises a positive charge-based isolation.NAI-5002577470vl 69ATTORNEY DOCKET NO.: 14842-001-2289. The composition of claim 8, wherein isolating comprises a membrane-based affinity column.

10. The composition of any one of claims 1 to 9, wherein the biofluid is from a subject having or suspected of having a neurological disease.

11. The composition of any one of claims 1 to 10, wherein the solid media comprises a magnetic particle, an agarose particle, or a hydrogel polymer.

12. The composition of claim 11, wherein the solid media comprises a magnetic particle.

13. The composition of claim 12, wherein the lectin is conjugated to the solid media via tosyl ati on.

14. The composition of any one of claims 1 to 13, wherein contacting comprises rotating for about 12 to about 24 hours.

15. The composition of claim 14, wherein contacting is at about 2-8 °C.

16. A composition comprising:(a) a heterogenous plurality of isolated EVs, where the isolated EVs comprise:(i) a first population of EVs; and(ii) a second population of EVs; and(b) a lectin or glycan-binding fragment thereof conjugated to a solid media, wherein the lectin or fragment thereof specifically binds a biantennary galactosylated N- glycan with bisecting GlcNAc; and(c) a buffer, wherein the first population of EVs are brain-derived and the second population of EVs are not brain-derived, andNAI-5002577470vl 70ATTORNEY DOCKET NO.: 14842-001-228 wherein the first population of EVs are specifically bound to the solid media and the second population of EVs are unbound.

17. The composition of claim 16, wherein the lectin comprises PHA-E or a glycan-binding fragment thereof.

18. The composition of claim 16 or 17, wherein the solid media comprises a magnetic particle, an agarose particle, or a hydrogel polymer.

19. The composition of claim 18, wherein the solid media comprises a magnetic particle.

20. The composition of claim 19, wherein the lectin is conjugated to the solid media via tosyl ati on.

21. The composition of any one of claims 16 to 20, wherein the buffer comprises equal parts:(a) a first solution comprising 3M ammonium sulfate, pH 7.9; and(b) a second solution comprising 0.1 M sodium phosphate buffer, 0.1 mM Ca Ch, 0.1 rnM Mn Ch.

22. A method for enriching brain-derived EVs from a biofluid, the method comprising:(a) isolating EVs from a biofluid under conditions sufficient to preserve EV glycocalyx, wherein the biofluid is plasma or serum;(b) contacting the isolated EVs with a lectin or glycan-binding fragment thereof conjugated to a solid media, wherein the lectin or glycan-binding fragment thereof specifically binds a biantennary galactosylated N-glycan with bisecting GlcNAc; and(c) removing unbound EVs from the solid media, thereby enriching brain-derived EVs from a biofluid.

23. The method of claim 22, wherein isolating does not comprise ultracentrifugation or addition of a polymer to the biofluid.NAI-5002577470vl 71ATTORNEY DOCKET NO.: 14842-001-22824. The method of claim 22 or 23, wherein removing unbound EVs comprises washing the solid media with a saline buffer.

25. The method of claim 24, wherein the saline buffer is selected from the group consisting of phosphate buffered saline (PBS), HEPES buffered saline (HBS), and Dulbecco's phosphate buffered saline (DPBS).

26. The method of claim 25, wherein the saline buffer comprises PBS, pH 7.4.

27. The method of claim 26, wherein the buffer further comprise about 0.1 mM Ca Ch and about 0.1 mM Mn Ch buffer.

28. The method of any one of claims 22 to 27, wherein the lectin comprises PHA-E or a glycan-binding fragment thereof.

29. The method of any one of claims 22 to 28, wherein isolating comprises a positive chargebased isolation.

30. The method of claim 29, wherein isolating comprises a membrane-based affinity column.

31. The method of any one of claims 22 to 30, wherein the biofluid is from a subject having or suspected of having a neurological disease.

32. The method of any one of claims 22 to 31, wherein the solid media comprises a magnetic particle, an agarose particle, or a hydrogel polymer.

33. The method of claim 32, wherein the solid media comprises a magnetic particle.

34. The method of claim 33, wherein the lectin is conjugated to the solid media via tosyl ati on.

35. The method of any one of claims 22 to 34, wherein contacting comprises rotating for about 12 to about 24 hours.

36. The method of claim 35, wherein contacting is at about 2-8 °C.

37. A kit compri sing :NAI-5002577470vl 72ATTORNEY DOCKET NO.: 14842-001-228(a) a lectin or glycan-binding fragment thereof conjugated to a solid media, wherein the lectin or glycan-binding fragment thereof specifically binds a biantennary galactosylated N-glycan with bisecting GlcNAc;(b) a buffer suitable for preserving the EV glycocalyx; and(c) instructions for use.

38. The kit of claim 37, wherein the lectin comprises PHA-E or a glycan-binding fragment thereof.

39. The kit of claim 37 or 38, wherein the pH of the buffer is about pH 7.0 to pH 8.0.

40. The kit of claim 39, wherein the pH of the buffer is about pH 7.4.4E The kit of any one of claims 37 to 40, wherein the solid media comprises a magnetic particle, an agarose particle, or a hydrogel polymer.

42. The kit of claim 41, wherein the solid media comprises a magnetic particle.

43. The kit of claim 42, wherein the lectin is conjugated to the solid media via tosylation.

44. A composition comprising:(a) a solid media;(b) a lectin-mediated means for specifically binding a biantennary galactosylated N- glycan with bisecting GlcNAc;(c) a heterogenous plurality of isolated EVs, where the isolated EVs comprise:(i) a first population of EVs; and(ii) a second population of EVs; and(d) a buffer,NAI-5002577470vl 73ATTORNEY DOCKET NO.: 14842-001-228 wherein the first population of EVs are brain-derived and the second population of EVs are not brain-derived, and wherein the first population of EVs are specifically bound to the solid media and the second population of EVs are unbound.

45. The composition of claim 44, wherein the lectin-mediated means comprises PHA-E or a glycan-binding fragment thereof.

46. The composition of claim 44 or 45, wherein the solid media comprises a magnetic particle, an agarose particle, or a hydrogel polymer.

47. The composition of claim 46, wherein the solid media comprises a magnetic particle.

48. The composition of claim 47, wherein the lectin-mediated means is conjugated to the solid media via tosylation.

49. The composition of any one of claims 44 to 48, wherein the buffer comprises equal parts:(a) a first solution comprising 3M ammonium sulfate, pH 7.9; and(b) a second solution comprising 0.1 M sodium phosphate buffer, 0.1 mM CaCh, 0.1 mM MnCh.

50. A method for enriching brain-derived EVs from a biofluid, the method comprising:(a) isolating EVs from a biofluid under conditions sufficient to preserve EV glycocalyx, wherein the biofluid is substantially free or devoid of red-blood cells;(b) contacting the isolated EVs with a lectin-mediated means for specifically binding a biantennary galactosylated N-glycan with bisecting GlcNAc conjugated to a solid media; and(c) removing unbound EVs from the solid media, thereby enriching brain-derived EVs from a biofluid.

51. The method of claim 50, wherein the biofluid is plasma or serum.NAI-5002577470vl 74ATTORNEY DOCKET NO.: 14842-001-22852. The method of claim 50 or 51 , wherein removing unbound EVs comprises washing the solid media with a saline buffer.

53. The method of claim 52, wherein the saline buffer is selected from the group consisting of phosphate buffered saline (PBS), HEPES buffered saline (HBS), and Dulbecco's phosphate buffered saline (DPBS).

54. The method of claim 53, wherein the saline buffer comprises PBS, pH 7.4.

55. The method of claim 54, wherein the buffer further comprise about 0.1 mM Ca Ch and about 0.1 mM Mn Ch buffer.

56. The method of any one of claims 50 to 55, wherein the lectin-mediated means comprises PHA-E or a glycan-binding fragment thereof.

57. The method of any one of claims 50 to 56, wherein isolating comprises a positive chargebased isolation.

58. The method of claim 57, wherein isolating comprises a membrane-based affinity column.

59. The method of any one of claims 50 to 58, wherein the biofluid is from a subject having or suspected of having a neurological disease.

60. The method of any one of claims 50 to 59, wherein the solid media comprises a magnetic particle, an agarose particle, or a hydrogel polymer.

61. The method of claim 60, wherein the solid media comprises a magnetic particle.

62. The method of claim 61, wherein the lectin is conjugated to the solid media via tosyl ati on.

63. The method of any one of claims 50 to 62, wherein contacting comprises rotating for about 12 to about 24 hours.

64. The method of claim 63, wherein contacting is at about 2-8 °C.NAI-5002577470vl 75ATTORNEY DOCKET NO.: 14842-001-22865. A composition comprising an isolated population of tumor derived EVs, wherein the isolated population of tumor derived EVs are obtained by a method comprising:(a) isolating EVs from a biofluid under conditions sufficient to preserve the EV glycocalyx;(b) contacting the isolated EVs with two or more lectins or a glycan-binding fragment thereof conjugated to a solid media, wherein the lectins or glycan-binding fragment thereof are selected from the group consisting of AAL (Aleuria Aurantia Lectin), SNA (Sambucus Nigra Lectin), GNL (Galunthus Nivalis Lectin), MAL I Maackia Amurensis Lectin I), and MAL II Maackia Amurensis Lectin IL), and combinations thereof; and(c) removing unbound EVs from the solid media, thereby obtaining the isolated population of tumor derived EVs.

66. The composition of claim 65, wherein the biofluid is selected from the group consisting of plasma, serum, and blood.

67. The composition of claim 65 or 66, wherein removing unbound EVs comprises washing the solid media with a saline buffer.

68. The composition of claim 67, wherein the saline buffer is selected from the group consisting of phosphate buffered saline (PBS), HEPES buffered saline (HBS), and Dulbecco's phosphate buffered saline (DPBS).

69. The composition of claim 68, wherein the saline buffer comprises PBS, pH 7.4.

70. The composition of claim 69, wherein the buffer further comprise about 0.1 mM Ca CI2 and about 0.1 mM Mn CI2 buffer.

71. The composition of any one of claims 65 to 70, wherein the two or more lectins or glycan-binding fragments thereof comprise three or more lectins selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II and combinations thereof.NAI-5002577470vl 76ATTORNEY DOCKET NO.: 14842-001-22872. The composition of any one of claims 65 to 70, wherein the two or more lectins or glycan-binding fragments thereof comprise four or more lectins selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II and combinations thereof.

73. The composition of any one of claims 65 to 70, wherein the two or more lectins or glycan-binding fragments thereof comprise five or more lectins selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II and combinations thereof.

74. The composition of any one of claims 65 to 70, wherein the lectins or glycan-binding fragments thereof comprise AAL, SNA, GNL, MAL I, and MAL II.

75. The composition of any one of claims 65 to 70, wherein the lectins or glycan-binding fragments thereof consist of AAL, SNA, GNL, MAL I, and MAL II.

76. The composition of any one of claims 65 to 75, wherein isolating comprises a positive charge-based isolation.

77. The composition of claim 76, wherein isolating comprises a membrane-based affinity column.

78. The composition of any one of claims 65 to 77, wherein the biofluid is from a subject having or suspected of having cancer.

79. The composition of any one of claims 65 to 78, wherein the solid media comprises a magnetic particle, an agarose particle, or a hydrogel polymer.

80. The composition of claim 79, wherein the solid media comprises a magnetic particle.

81. The composition of claim 80, wherein the two or more lectins are conjugated to the solid media via tosylation.

82. The composition of any one of claims 65 to 81, wherein contacting comprises rotating for about 12 to about 24 hours.

83. The composition of claim 82, wherein contacting is at about 2-8 °C.NAI-5002577470vl 77ATTORNEY DOCKET NO.: 14842-001-22884. A composition comprising:(a) a heterogenous plurality of isolated EVs comprising tumor derived EVs;(b) two or more lectins or glycan-binding fragments thereof conjugated to a solid media, wherein the lectins or glycan-binding fragment thereof is selected from the group consisting of AAL, SNA, GNL, MAL I, and MAE II, and combinations thereof; and(c) a buffer.

85. The composition of claim 84, wherein the solid media has higher affinity for the TDEV, as compared to non-tumor derived EVs.

86. The composition of claim 84 or 85, wherein the two or more lectins or glycan-binding fragments thereof comprise three or more lectins selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II and combinations thereof.

87. The composition of claim 84 or 85, wherein the two or more lectins or glycan-binding fragments thereof comprise four or more lectins selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II and combinations thereof.

88. The composition of claim 84 or 85, wherein the two or more lectins or glycan-binding fragments thereof comprise five or more lectins selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II and combinations thereof.

89. The composition of claim 84 or 85, wherein the lectins or glycan-binding fragments thereof comprise AAL, SNA, GNL, MAL I, and MAL II.

90. The composition of claim 84 or 85, wherein the lectins or glycan-binding fragments thereof consist of AAL, SNA, GNL, MAL I, and MAL II.

91. The composition of claim 84 or 90, wherein the solid media comprises a magnetic particle, an agarose particle, or a hydrogel polymer.

92. The composition of claim 85, wherein the solid media comprises a magnetic particle.NAI-5002577470vl 78ATTORNEY DOCKET NO.: 14842-001-22893. The composition of claim 92, wherein the two or more lectins are conjugated to the solid media via tosylation.

94. The composition of any one of claims 84 to 93, wherein the buffer comprises equal parts:(a) a first solution comprising 3M ammonium sulfate, pH 7.9; and(b) a second solution comprising 0.1 M sodium phosphate buffer, 0.1 mM Ca Ch, 0.1 mM Mn Ch.

95. A method for enriching tumor derived EVs from a biofluid, the method comprising:(a) isolating EVs from a biofluid under conditions sufficient to preserve the EV glycocalyx;(b) contacting the isolated EVs with two or more lectins or a glycan-binding fragment thereof conjugated to a solid media, wherein the lectins or glycan-binding fragment thereof are selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II, and combinations thereof; and(c) removing unbound EVs from the solid media, thereby enriching tumor derived EVs from a biofluid.

96. The method of claim 95, wherein the biofluid is selected from the group consisting of plasma, serum, or blood.

97. The method of claim 95 or 96, wherein removing unbound EVs comprises washing the solid media with a saline buffer.

98. The method of claim 97, wherein the saline buffer is selected from the group consisting of phosphate buffered saline (PBS), HEPES buffered saline (HBS), and Dulbecco's phosphate buffered saline (DPBS).

99. The method of claim 98, wherein the saline buffer comprises PBS, pH 7.4.

100. The method of claim 99, wherein the buffer further comprise about 0.1 mM Ca Ch and about 0.1 mM Mn Ch buffer.NAI-5002577470vl 79ATTORNEY DOCKET NO.: 14842-001-228101. The method of any one of claims 95 to 100, wherein the two or more lectins or glycan- binding fragments thereof comprise three or more lectins selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II and combinations thereof.

102. The method of any one of claims 95 to 100, wherein the two or more lectins or glycan- binding fragments thereof comprise four or more lectins selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II and combinations thereof.

103. The method of any one of claims 95 to 100, wherein the two or more lectins or glycan- binding fragments thereof comprise five or more lectins selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II and combinations thereof.

104. The method of any one of claims 95 to 100, wherein the lectins or glycan-binding fragments thereof comprise AAL, SNA, GNL, MAL I, and MAL II .

105. The method of any one of claims 95 to 100, wherein the lectins or glycan-binding fragments thereof consist of AAL, SNA, GNL, MAL I, and MAL II.

106. The method of any one of claims 95 to 105, wherein isolating comprises a positive charge-based isolation.

107. The method of claim 106, wherein isolating comprises a membrane-based affinity column.

108. The method of any one of claims 95 to 107, wherein the biofluid is from a subject having or suspected of having a cancer.

109. The method of any one of claims 95 to 108, wherein the solid media comprises a magnetic particle, an agarose particle, or a hydrogel polymer.

110. The method of claim 109, wherein the solid media comprises a magnetic particle.

111. The method of claim 110, wherein the two or more lectins are conjugated to the solid media via tosylation.NAI-5002577470vl 80ATTORNEY DOCKET NO.: 14842-001-228112. The method of any one of claims 95 to 11 1, wherein contacting comprises rotating for about 12 to about 24 hours.

113. The method of claim 112, wherein contacting is at about 2-8 °C.

114. A kit compri sing :(a) two or more lectins or glycan-binding fragments thereof conjugated to a solid media, wherein the lectins or glycan-binding fragment thereof are selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II, and combinations thereof; and(b) instructions for use.

115. The kit of claim 114, wherein the solid media has higher affinity for the TDEV, as compared to non-tumor derived EVs.

116. The kit of claim 114, wherein the two or more lectins or glycan-binding fragments thereof comprise three or more lectins selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II and combinations thereof.

117. The kit of claim 114, wherein the two or more lectins or glycan-binding fragments thereof comprise four or more lectins selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II and combinations thereof.

118. The kit of claim 114, wherein the two or more lectins or glycan-binding fragments thereof comprise five or more lectins selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II and combinations thereof.

119. The kit of claim 114, wherein the lectins or glycan-binding fragments thereof comprise AAL, SNA, GNL, MAL I, and MAL II.

120. The kit of claim 114, wherein the lectins or glycan-binding fragments thereof consist of AAL, SNA, GNL, MAL I, and MAL II.NAI-5002577470vl 81ATTORNEY DOCKET NO.: 14842-001-228121 . The kit of any one of claims 114 to 120, further comprising a buffer suitable for preserving the EV glycocalyx.

122. The kit of claim 121, wherein the pH of the buffer is about pH 7.0 to pH 8.0.

123. The kit of claim 121, wherein the pH of the buffer is about pH 7.4.

124. The kit of any one of claims 114 to 123, the solid media comprises a magnetic particle, an agarose particle, or a hydrogel polymer.

125. The kit of claim 124, wherein the solid media comprises a magnetic particle.

126. The kit of claim 125, wherein the two or more lectins are conjugated to the solid media via tosylation.

127. A process for detecting the presence or absence of a cancer in a subject comprising: obtaining a biological sample from the subject; contacting at least a portion of said biological sample with one or more detection agents, said one or more detection agents capable of specifically binding a glycoform on or within said portion; isolating said extracellular vesicles to form enriched extracellular vesicles; subjecting said enriched extracellular vesicles to analysis of protein and / or nucleic acid content within or on said enriched extracellular vesicles; and detecting the presence or absence of said cancer in said subject when said enriched extracellular vesicles contain one or more targets at a level that differs from a healthy control, wherein said target is a protein, nucleic acid encoding said protein, or a combination thereof.

128. The process of claim 127, further comprising subjecting said biological sample to fractionation to obtain isolated extracellular vesicles wherein said portion of said biological sample is said isolated extracellular vesicles.NAI-5002577470vl 82ATTORNEY DOCKET NO.: 14842-001-228129. The process of claims 127 or 128, further comprising identifying said cancer based on the identity of the one or more targets.

130. The process of any of claims 127-129, wherein said cancer is non-small cell lung cancer, ovarian cancer, colorectal cancer, or a glioma.

131. The process of claim 128, wherein said target proteins are a known oncoprotein.

132. The process of claims 127 or 128, wherein said cancer is non-small cell lung cancer and said protein is PHB1, STIM1, or both.

133. The process of claims 127 or 128, wherein said cancer is ovarian cancer and said is MUC1, MUC16, or both.

134. The process of claims 127 or 128, wherein said cancer is colorectal cancer.

135. The process of claims 127 or 128, wherein said cancer is a glioma.

136. The process of any of claims 127-135, wherein said biological sample is whole blood, plasma, or serum.

137. The process of any of claims 127-136, wherein said detection agent comprises one or more lectins.

138. The process of claim 137, wherein at least one of said lectins is selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II, and PHA-E.

139. The process of claim 138, wherein at least one of said lectins is PHA-E, optionally wherein said detection agent consists of PHA-E.

140. The process of claim 137, wherein at least two of said lectins is selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II.

141. The process of claim 137, wherein at least three of said lectins is selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II.NAI-5002577470vl 83ATTORNEY DOCKET NO.: 14842-001-228142. The process of claim 137, wherein said detection agent comprises five lectins, optionally AAL, SNA, GNL, M AL I, and M AL II.

143. The process of any of claims 127-142, wherein said detection agent further comprises an isolation component.

144. The process of claim 143, wherein said isolation component comprises a magnetic bead, a fluorescent agent, avidin, biotin, a protein sequence tag, an antibody or fragment thereof.

145. The process of claim 143, wherein said isolation component comprises a magnetic bead.

146. A process for measuring efficacy of a cancer therapy in a subject comprising: obtaining a first biological sample from the subject prior to administration of a therapy; contacting at least a portion of said first biological sample with one or more detection agents, said one or more detection agents capable of specifically binding a glycoform, isolating first extracellular vesicles from said portion of said first biological sample to form first enriched extracellular vesicles; subjecting said first enriched extracellular vesicles to analysis of protein or nucleic acid content within or on said first enriched extracellular vesicles; determining the presence or absence of a one or more target proteins or target nucleic acids encoding said target proteins in said first enriched extracellular vesicles; obtaining a second biological sample from the subject following administration of said therapy; contacting at least a portion of said second biological sample with one or more of said detection agents, isolating second enriched extracellular vesicles from said portion of said second biological sample;NAI-5002577470vl 84ATTORNEY DOCKET NO.: 14842-001-228 subjecting said second enriched extracellular vesicles to analysis of protein or nucleic acid content within or on said second enriched extracellular vesicles; determining the presence or absence of the one or more of said target proteins or said target nucleic acids encoding said target proteins in said second enriched extracellular vesicles; determining efficacy of said therapeutic based on a difference in identity or amount of said target proteins in said first enriched extracellular vesicles and said second enriched extracellular vesicles.

147. The process of claim 146, wherein said cancer is a lung cancer, ovarian cancer, colorectal cancer, or a glioma.

148. The process of claims 146 or 147, wherein said cancer is non-small cell lung cancer and said protein is PHB1, STIM1, or both.

149. The process of claims 146 or 147, wherein said wherein said cancer is ovarian cancer and said is MUC1, MUC16, or both.

150. The process of claims 146 or 147, wherein said cancer is colorectal cancer.

151. The process of claims 146 or 147, wherein said cancer is a glioma.

152. The process of any of claims 146-151, wherein said biological sample is whole blood, plasma, or serum.

153. The process of any of claims 146-152, wherein said detection agent comprises one or more lectins.

154. The process of claim 153, wherein at least one of said lectins is selected from the group consisting of AAL, SNA, GNL, MAL I, MAL II, and PHA-E.

155. The process of claim 153, wherein at least one of said lectins is PHA-E, optionally wherein said detection agent consists of PHA-E.NAI-5002577470vl 85ATTORNEY DOCKET NO.: 14842-001-228156. The process of claim 153, wherein at least two of said lectins is selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II.

157. The process of claim 153, wherein at least three of said lectins is selected from the group consisting of AAL, SNA, GNL, MAL I, and MAL II.

158. The process of claim 153, wherein said detection agent comprises five lectins.

159. The process of any of claims 153 to 158, wherein said detection agent further comprises an isolation component.

160. The process of claim 159, wherein said isolation component comprises a magnetic bead, a fluorescent agent, avidin, biotin, a protein sequence tag, an antibody or fragment thereof.

161. The process of claim 159, wherein said isolation component comprises a magnetic bead.

162. A composition for detecting the presence of absence of a cancer in a subject, comprising one or more detection agents comprising one or more lectins selected from the group consisting of AAL, SNA, GNL, MAL I, MAL II, and PHA-E.

163. The composition of claim 162, comprising 3 or more lectins.

164. The composition of claim 162 comprising five of said lectins, optionally AAL, SNA, GNL, MAL I, and MAL II.

165. The composition of any of claims 162-164, wherein said detection agent further comprises an isolation component.

166. The composition of claim 165, wherein said isolation component comprises a magnetic bead, a fluorescent agent, avidin, biotin, a protein sequence tag, an antibody or fragment thereof.

167. The composition of claim 165, wherein said isolation component comprises a magnetic bead.NAI-5002577470vl 86ATTORNEY DOCKET NO.: 14842-001-228168. A process for detecting the presence or absence of a target in a biological sample comprising: obtaining a biological sample from a subject; contacting said at least a portion of said biological sample with one or more detection agents capable of specifically binding a glycoform, isolating enriched extracellular vesicles from said biological sample, said enriched extracellular vesicles bound to said detection agent; and subjecting said enriched extracellular vesicles or a component thereof to mass spectrometry to detect the presence or absence of the target.

169. The process of claim 168, wherein said target is a protein or a nucleic acid.

170. The process of claim 168, wherein said target is a protein related to a disease or condition.

171. The process of claim 170, wherein said disease or condition is an inflammatory disease, a cancer, an allergic disease, an autoimmune disease, an infectious disease, a transplantation related disease, a degenerative disease, an injury associated with inflammation, a disease associated with a hypersensitivity, a cardiovascular disease, a glandular disease, a hepatic disease, a neurological disease, an a musculo-skeletal disease, a renal disease, a reproductive disease, a connective tissue disease, a neurodegenerative disease, necrosis, an inflammatory disease associated with an implant, a hematological disease, an eye disease, or a respiratory disease.

172. A process of claim 170, wherein said disease or condition is a cancer.NAI-5002577470vl 87

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