Methods, assays and compositions for measuring intraoperative brain damage or injury - Patents.com

Abstract

The present disclosure relates to methods and assays for quantifying brain-derived exosomes in biological samples from subjects before, during and after surgery.The present disclosure also provides methods and compositions for measuring and / or prognosing brain injury during surgery.The compositions and methods of the present disclosure are also useful in preventing and / or treating brain injury in subjects undergoing surgery and predicting postoperative cognitive dysfunction (POCD) and / or predicting postoperative delirium (POD).

Description

[Technical field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 144,985, filed February 3, 2021, which is incorporated by reference in its entirety.

[0002] FIELD OF THEINVENTION The present disclosure relates to methods and assays for quantifying brain-derived exosomes in biological samples from subjects before, during and after surgery.The present disclosure also provides methods and compositions for measuring and / or prognosing brain injury during surgery.The compositions and methods of the present disclosure are also useful for preventing and / or treating brain injury in subjects undergoing surgery, and predicting postoperative cognitive dysfunction (POCD) and / or predicting postoperative delirium (POD). [Background technology]

[0003] Postoperative cognitive dysfunction or decline (POCD) is defined as persistent cognitive impairment occurring within weeks to months after a surgical procedure. The rate of major complications (e.g., mortality) after cardiac surgery is currently lower due to improvements in medical and surgical techniques, but the incidence of POCD remains the most common postoperative complication. The reported incidence of POCD varies significantly depending on the definition, the examination method, and the timing of the postoperative evaluation. The incidence of POCD after cardiac surgery has been reported to be 30-80% in the first few weeks after surgery and 10-60% after 3-6 months. POCD is associated with significant adverse events, including prolonged hospitalization and rehabilitation, increased mortality, reduced quality of life, and more commonly work disability and early retirement, thereby resulting in a significant burden on the healthcare system.

[0004] Brain-derived exosomes / vesicles (e.g., neuron-derived, astrocyte-derived, oligodendrocyte-derived exosomes / vesicles), herein referred to as BDE, are present in biological samples, and their increased levels can be used to assess damage to brain tissue during or after surgery. Normal levels of BDE in biological samples after surgery have not been established. Thus, there is a need in the art for methods to quantify BDE in biological samples and assess changes in BDE levels as a result of surgery.

[0005] Postoperative delirium (POD) is a postoperative state in which subjects have alterations in consciousness, orientation, memory, perception, and behavior. POD is not uncommon and is one of the common problems experienced in various types of surgery. POD is associated with poor outcomes including decreased functional and cognitive function, longer hospital stay, institutionalization, increased costs, and increased mortality. In addition, POD also increases the workload of nursing staff. Therefore, prediction and prevention of POD is of great importance to anesthesiologists, surgeons, and hospitalists to reduce overall healthcare costs and maintain the quality of patient care. If it is predicted before surgery, surgeons can consider less invasive procedures, anesthesiologists can modify anesthesia protocols, and nursing staff can prepare for POD management. Furthermore, neurologists can be actively involved in the care of patients with various preventive treatments to prevent the more problematic postoperative cognitive decline (POCD). Therefore, prediction of POD is a substantial unmet need in anesthesiology.

[0006] Further, there is a need in the art for methods for quantifying BDE and compositions for measuring brain damage during and after surgery. Additionally, there is a need in the art for compositions for quantifying exosomes, and compositions and methods useful for treating neurological disorders associated with cardiac surgery and other cardiac procedures. The present disclosure meets this need by providing an accurate, non-invasive method for measuring changes in BDE levels and predicting and / or prognosing brain damage associated with surgery. The methods of the present disclosure are useful for predicting, diagnosing, or prognosing postoperative cognitive decline (POCD) and / or postoperative delirium (POD) in subjects undergoing or having undergone cardiac surgery. The present disclosure further provides novel methods, assays, kits, and compositions for quantifying exosomes in biological samples from subjects undergoing cardiac surgery. The present disclosure also provides compositions and methods useful for preventing and / or treating brain damage in subjects undergoing surgery. Summary of the Invention [Problem to be solved by the invention]

[0007] Summary of the Invention The present disclosure relates to novel methods, compositions and kits for detecting and quantitating exosomes in samples from subjects that have undergone surgery. [Means for solving the problem]

[0008] In some embodiments, the disclosure provides a method comprising: (i) obtaining a biological sample from a subject undergoing or having undergone surgery; and (ii) detecting brain-derived exosomes in the sample by contacting the sample with an antibody and detecting binding between the brain-derived exosomes and the antibody. In other embodiments, detecting binding between the brain-derived exosomes and the antibody further comprises contacting the brain-derived exosomes with a second antibody. In other embodiments, the antibody is an anti-CD171 antibody, an anti-EAAT1 antibody, and an anti-MOG antibody. In yet other embodiments, the second antibody is an anti-CD9 antibody, an anti-CD63 antibody, or an anti-CD81 antibody. In some embodiments, the method further comprises quantifying the level of brain-derived exosomes in the biological sample. In other embodiments, the exosomes are selected from the group consisting of neuron-derived exosomes, astrocyte-derived exosomes, oligodendrocyte-derived exosomes, and microglia-derived exosomes. In yet other embodiments, the subject is diagnosed with or suspected of having brain injury. In yet other embodiments, the brain injury is post-operative cognitive dysfunction or decline (POCD). In some embodiments, the brain injury is post-operative delirium (POD). In other embodiments, the biological sample is selected from the group consisting of whole blood, serum, plasma, urine, interstitial fluid, peritoneal fluid, cervical swab, tears, saliva, buccal swab, skin, brain tissue, and cerebrospinal fluid. In some embodiments, the surgery is cardiac surgery. In other embodiments, the surgery is open-chest surgery. In yet other embodiments, the surgery is transcatheter aortic valve replacement (TAVR) or total aortic valve replacement (TAR). In yet other embodiments, the surgery further comprises a transcatheter procedure, an endoscopic procedure, or a laparoscopic procedure.

[0009] The present disclosure provides novel methods, compositions and kits for detecting and quantifying brain-derived exosomes (BDEs) in a sample from a subject undergoing or having undergone surgery. In some embodiments, the disclosure provides a method comprising: (i) obtaining a biological sample from a subject undergoing or having undergone surgery; and (ii) detecting BDEs in the sample by contacting the sample with an antibody that binds to BDE and detecting binding between the BDE and the antibody. In other embodiments, detecting binding between the brain-derived exosomes and the antibody further comprises contacting the BDE with a second antibody. In other embodiments, the antibody is an anti-CD171 antibody, an anti-EAAT1 antibody, an anti-MOG antibody and / or a neuron-selective BDE antibody. In some embodiments, the exosomes are vesicles. In yet other embodiments, the second antibody is an anti-CD9 antibody, an anti-CD63 antibody and / or an anti-CD81 antibody. In some embodiments, the method further comprises quantifying the level of BDEs in the biological sample. In other embodiments, the exosomes are selected from the group consisting of neuron-derived exosomes, astrocyte-derived exosomes and oligodendrocyte-derived exosomes. In yet another embodiment, the subject has been diagnosed with or is suspected of having or developing postoperative brain injury or postoperative cognitive dysfunction or decline (POCD). In yet another embodiment, the subject has been diagnosed with or is suspected of having or developing postoperative delirium (POD). In another embodiment, the biological sample is selected from the group consisting of whole blood, serum, plasma, urine, interstitial fluid, peritoneal fluid, cervical swab, tears, saliva, buccal swab, skin, brain tissue, and cerebrospinal fluid. In some embodiments, the surgery is cardiac surgery. In some embodiments, the brain-derived exosomes comprise at least two exosomes from the group consisting of neuron-derived exosomes, astrocyte-derived exosomes, oligodendrocyte-derived exosomes, and microglia-derived exosomes. In another embodiment, the method further comprises determining a ratio of two exosomes from the group consisting of neuron-derived exosomes, astrocyte-derived exosomes, oligodendrocyte-derived exosomes, and microglia-derived exosomes.In some embodiments, the rate of exosomes is increased or decreased compared to a control or predetermined value. In other embodiments, a low rate of NDE / ADE and / or ODE / ADE pre-operatively indicates that the subject may have post-operative delirium.

[0010] In other embodiments, the present disclosure provides a method for measuring and / or detecting brain damage in a subject undergoing a surgical procedure, comprising: (i) obtaining one or more biological samples from the subject before, during, and / or after undergoing surgery, or any combination thereof; (ii) detecting BDE in the one or more samples by contacting the one or more samples with an antibody and detecting binding between brain-derived exosomes and the antibody; and (iii) comparing the level of brain-derived exosomes in the one or more samples with a control sample and / or a sample from the same subject obtained at different times during the surgical procedure. In some embodiments, the level of BDE increases or decreases compared to the control sample or the sample from the subject obtained at different times during the procedure. In other embodiments, the antibody is an anti-CD171 antibody, an anti-EAAT1 antibody, an anti-MOG antibody, and / or other neuroselective antibody. In yet other embodiments, the second antibody is an anti-CD9 antibody, an anti-CD63 antibody, and / or an anti-CD81 antibody, and / or other exosome-specific antibody. In some embodiments, the method further comprises quantifying the level of BDE in the biological sample. In other embodiments, the exosomes are selected from the group consisting of neuron-derived exosomes, astrocyte-derived exosomes, oligodendrocyte-derived exosomes, and microglia-derived exosomes. In still other embodiments, the subject has been diagnosed with or is suspected of having brain injury. In still other embodiments, the brain injury is post-operative cognitive dysfunction or decline (POCD). In still other embodiments, the subject has been diagnosed with or is suspected of having or developing post-operative delirium (POD). In other embodiments, the biological sample is selected from the group consisting of whole blood, serum, plasma, urine, interstitial fluid, peritoneal fluid, cervical swab, tears, saliva, buccal swab, skin, brain tissue, and cerebrospinal fluid. In some embodiments, the surgery is cardiac surgery. In other embodiments, the method further comprises determining a ratio of two exosomes from the group consisting of neuron-derived exosomes, astrocyte-derived exosomes, oligodendrocyte-derived exosomes, and microglia-derived exosomes. In some embodiments, the ratio of exosomes is increased or decreased compared to a control value or a predetermined value.In other embodiments, a low rate of pre-operative NDE / ADE and / or ODE / ADE indicates that the subject may have post-operative delirium.

[0011] In another embodiment, the present disclosure provides a method for identifying a subject for preventing, minimizing, or treating damage to the brain during surgery, comprising: (i) obtaining a biological sample from a subject undergoing or having undergone surgery; (ii) detecting whether brain-derived exosomes are present in the sample by contacting the sample with an antibody and detecting binding between the brain-derived exosomes and the antibody; and (iii) administering an effective amount of a compound to prevent, minimize, or treat damage to the brain. In some embodiments, the biological sample is selected from the group consisting of whole blood, serum, plasma, urine, interstitial fluid, peritoneal fluid, cervical swab, tears, saliva, buccal swab, skin, brain tissue, and cerebrospinal fluid. In other embodiments, the compound is selected from the group consisting of anti-inflammatory compounds, anti-oxidative stress compounds, free radical scavenger compounds, and neuronal growth and survival factor compounds. In some embodiments, the free radical scavenger compound is edaravone. In yet another embodiment, the compound is administered before, during, or after surgery. In yet another embodiment, the compound is administered when brain-derived exosomes are detected in the sample. In some embodiments, the compound is administered when brain-derived exosomes are increased or decreased in the sample compared to a control. In other embodiments, the exosomes are selected from the group consisting of neuron-derived exosomes, astrocyte-derived exosomes, oligodendrocyte-derived exosomes, and microglia-derived exosomes. In yet other embodiments, the biological sample is selected from the group consisting of whole blood, serum, plasma, urine, interstitial fluid, peritoneal fluid, cervical swab, tears, saliva, buccal swab, skin, brain tissue, and cerebrospinal fluid. In other embodiments, the method further comprises determining the ratio of two exosomes from the group consisting of neuron-derived exosomes, astrocyte-derived exosomes, oligodendrocyte-derived exosomes, and microglia-derived exosomes. In some embodiments, the ratio of exosomes is increased or decreased compared to a control value or a predetermined value. In other embodiments, the compound is administered when the level of brain-derived exosomes is increased or decreased compared to a control value or a predetermined value.In other embodiments, a low rate of pre-operative NDE / ADE and / or ODE / ADE indicates that the subject may have post-operative delirium.

[0012] In other embodiments, the present disclosure provides a method for predicting, diagnosing and / or prognosing post-operative cognitive dysfunction or decline (POCD) or post-operative delirium (POD) in a subject, comprising: (i) obtaining a biological sample from a subject undergoing or having undergone surgery; (ii) detecting one or more brain-derived exosomes in the sample; and (iii) comparing the level of brain-derived exosomes in the sample to a control sample or control value, thereby predicting, diagnosing and / or prognosing post-operative cognitive dysfunction or decline (POCD) or post-operative delirium (POD) in the subject. In some embodiments, the biological sample is selected from the group consisting of whole blood, serum, plasma, urine, interstitial fluid, peritoneal fluid, cervical swab, tears, saliva, buccal swab, skin, brain tissue and cerebrospinal fluid. In some embodiments, the exosomes are selected from the group consisting of neuron-derived exosomes, astrocyte-derived exosomes, oligodendrocyte-derived exosomes and microglia-derived exosomes. In yet other embodiments, the biological sample is selected from the group consisting of whole blood, serum, plasma, urine, interstitial fluid, peritoneal fluid, cervical swab, tears, saliva, buccal swab, skin, brain tissue, and cerebrospinal fluid. In some embodiments, the brain-derived exosomes comprise at least two exosomes from the group consisting of neuron-derived exosomes, astrocyte-derived exosomes, oligodendrocyte-derived exosomes, and microglia-derived exosomes. In other embodiments, the method further comprises determining a ratio of two exosomes from the group consisting of neuron-derived exosomes, astrocyte-derived exosomes, oligodendrocyte-derived exosomes, and microglia-derived exosomes. In some embodiments, the ratio of exosomes is increased or decreased compared to a control value or a predetermined value. In some embodiments, the subject has undergone or will undergo cardiac surgery. In other embodiments, the surgery is open-chest surgery. In still other embodiments, the surgery is transcatheter aortic valve replacement (TAVR) or total aortic valve replacement (TAR). In yet other embodiments, the procedure further comprises a transcatheter procedure, an endoscopic procedure, or a laparoscopic procedure.

[0013] In some embodiments, the present disclosure provides a kit for measuring brain damage in a subject undergoing or having undergone surgery, comprising one or more agents that specifically bind to brain-derived exosomes, one or more containers for collecting and / or holding biological samples, and instructions for use thereof. In some embodiments, the agents are anti-CD9 antibodies, anti-CD171 antibodies, anti-EAAT1 antibodies, anti-MOG antibodies, anti-CD63 antibodies, and / or anti-CD81 antibodies. In other embodiments, the exosomes are selected from the group consisting of neuron-derived exosomes, astrocyte-derived exosomes, oligodendrocyte-derived exosomes, and microglia-derived exosomes. In yet other embodiments, the surgery is cardiac surgery.

[0014] In some embodiments, the present disclosure provides a method for detecting surgical brain injury in a subject with a specificity of at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%. In some embodiments, the present disclosure provides a method for detecting surgical brain injury in a subject with a sensitivity of at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%. In other embodiments, the present disclosure provides a method for detecting surgical brain injury in a subject with an accuracy of at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%.

[0015] These and other embodiments of the present disclosure will readily suggest themselves to those of ordinary skill in the art in light of the disclosure herein, and all such embodiments are specifically contemplated.

[0016] Each limitation of the disclosure may encompass various embodiments of the disclosure. Thus, it is anticipated that each limitation of the disclosure relating to any one element or combination of elements may be included in each aspect of the disclosure. The disclosure is not limited in its application to the details of the configuration and arrangement of the components described in the following description. The disclosure is capable of other embodiments and can be practiced or carried out in various ways. Additionally, the words and terms used herein are for the purpose of explanation and should not be considered as limiting. The use of "including," "comprising," "having," "containing," "involving," and variations thereof herein means to encompass the items listed thereafter and equivalents thereof, as well as additional items. It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 shows data illustrating one embodiment of the assay of the present disclosure. [Figure 2A-2B] Figure 2 shows data demonstrating assay reproducibility in relative light units (RLU) for an exemplary ELISA of the present disclosure. Figure 2A shows replicate results of anti-CD171 NDE capture with multiple plasma samples. Figure 2B shows a similar lack of reproducible activity using an IgG control antibody. [Diagram 3] 3 shows data showing plasma levels of neuron-derived exosomes (NDE) in pre-operative subjects. The Y-axis shows U / mL using a plasma dilution curve of a standard plasma control arbitrarily assigned 100 U / mL. [Figure 4A-4B]Figure 4A shows data showing postoperative changes in neuron-derived exosome (NDE) plasma units / ml levels in transcatheter aortic valve implantation (TAVI) subjects and total arterial revascularization (TAR) subjects (Figure 4B). [Figure 5A-5I] Figures 5A-5I show data showing changes in plasma levels of NDE (neuron-derived exosomes), ADE (astrocyte-derived exosomes), and ODE (oligodendrocyte-derived exosomes) after TAR treatment. Blood samples were taken pre- and post-operatively, and on days 1, 2, and 5 post-operatively. U / mL and logU / mL are shown for NDE (Figure 5A,B), ADE (Figure 5D,E), and ODE (Figure 5G,H). Figure 5A shows subjects who did not develop POD. Figure 5B shows subjects who developed POD 5 days post-operatively. Figure 5C shows data when the Log(U / mL) of post-operative samples is subtracted from the log(U / mL) of pre-operative samples (Δlog). Δ represents the mean-standard deviation (n=11) of POD(-), and ● represents the mean+standard deviation (n=7) of POD(+) subjects. Figure 5D shows subjects who did not develop POD. FIG. 5E shows subjects who developed POD 5 days after surgery. FIG. 5F shows data when Log(U / mL) of post-op samples is subtracted from Log(U / mL) of pre-op samples (Δlog). Δ represents the mean minus standard deviation of POD(-) (n=11), and ● represents the mean plus standard deviation of POD(+) subjects (n=7). FIG. 5G shows subjects who did not develop POD. FIG. 5H shows subjects who developed POD 5 days after surgery. FIG. 5I shows data when Log(U / mL) of post-op samples is subtracted from Log(U / mL) of pre-op samples (Δlog). Δ represents the mean minus standard deviation of POD(-) (n=11), and ● represents the mean plus standard deviation of POD(+) subjects (n=7). [Figures 6A-6C]FIG. 6A shows data showing plasma levels of NDE in subjects who developed POD (+) and subjects who did not develop POD (-). The Y-axis shows U / mL using a plasma dilution curve of a standard plasma control arbitrarily assigned 100 U / mL. FIG. 6B shows data showing plasma levels of ADE in subjects who developed POD (+) and subjects who did not develop POD (-). The Y-axis shows U / mL using a plasma dilution curve of a standard plasma control arbitrarily assigned 100 U / mL. FIG. 6C shows data showing plasma levels of ODE in subjects who developed POD (+) and subjects who did not develop POD (-). The Y-axis shows U / mL using a plasma dilution curve of a standard plasma control arbitrarily assigned 100 U / mL. [Figure 7A-7E] FIG. 7A shows data showing the rate of NDE / ADE. FIG. 7B shows data showing the rate of ODE / NDE. FIG. 7C shows data showing the rate of ODE / ADE. FIG. 7D shows POD prediction in TAR subjects analyzed by ROC, with an AUC score of NDE / ADE of 81%. FIG. 7E shows POD prediction in TAR subjects analyzed by ROC, with an AUC score of ODE / ADE of 84%. [Figure 8A-8B] Figure 8A shows data showing assay validation. A: Capture antibody specificity. A mouse monoclonal antibody against human CD171 (IgG2a), control mouse IgG and control mouse IgG2a were immobilized in ELISA wells. Three different control human plasmas (light grey, medium grey and dark grey columns) and diluent only (white column, not visible) were applied to the indicated wells and ELISA was performed using an anti-human CD9 probe. Figure 8B shows data showing assay validation. B: Probe specificity. Three control plasmas (light grey, medium grey and dark grey columns) and buffer only (white column, not visible) were applied to mouse IgG and anti-CD171 immobilized ELISA wells, respectively. Each well was then reacted with biotinylated probes for mouse IgG, mouse IgG2b and anti-human CD9 (mouse monoclonal IgG2b). [Fig. 8C-8D]Figure 8C shows data showing assay validation. C. Exosome specificity I: Exosome reduction. Plasma samples were incubated with anti-CD81 immobilized paramagnetic beads (dark grey column) and control mouse IgG immobilized paramagnetic beads (light grey column), respectively, and then the supernatant plasma samples were applied to three different ELISA wells, where anti-CD171, anti-CD81 and control mouse IgG were pre-immobilized. After each well was thoroughly washed, each well was reacted with biotinylated anti-CD9 to complete the sandwich ELISA. Figure 8D shows data showing assay validation. D. Exosome specificity II: Spiked exosomes. Exosomes were prepared from control plasma by ExoQuick and suspended in PBS. Three different dilutions of exosomes and PBS were added to plasma or plasma diluent, and [CD171+CD9+] was measured. [Fig. 8E-8G] Figure 8E shows data illustrating assay validation. E. Plasma dilution study. Three control plasma samples were diluted in PBS (black and white symbols) and applied to mouse IgG (dotted line) and anti-CD171 immobilized ELISA wells (solid line). ELISA was then performed. Figure 8F shows data illustrating assay validation. F. Intra-assay reproducibility. Assays were always performed in duplicate and showed a variation of two replicates (RLU) (n=134 subjects). Each point represents one individual. Figure 8G shows data illustrating assay validation. G. Inter-assay reproducibility. Three different control plasma samples were analyzed 12 separate times. The first data was used as 100% and the % of variation in each experiment was shown. [Figure 9A-9E]FIG. 9A shows data showing exosome validation. A.SEM. Plasma samples were applied to anti-CD171 immobilized ELISA wells using exactly the same protocol as our ELISA. After each well was thoroughly washed with PBS, the bottom of each well was punched out, suspended in a fixing solution, and analyzed by SEM. FIG. 9B shows data showing exosome validation. B.SEM. Plasma samples were applied to anti-CD171 immobilized ELISA wells using exactly the same protocol as our ELISA. After each well was thoroughly washed with PBS, the bottom of each well was punched out, suspended in a fixing solution, and analyzed by SEM. FIG. 9C shows data showing exosome validation. C.SEM. Plasma samples were applied to anti-CD81 immobilized ELISA wells using exactly the same protocol as our ELISA. After each well was thoroughly washed with PBS, the bottom of each well was punched out, suspended in a fixing solution, and analyzed by SEM. FIG. 9D shows data showing exosome validation. D.SEM. Plasma samples were applied to anti-CD81 immobilized ELISA wells using exactly the same protocol as our ELISA. After each well was thoroughly washed with PBS, the bottom of each well was punched out, suspended in fixation solution, and analyzed by SEM. Figure 9E shows data showing exosome validation. E.NTA. Plasma samples were applied to anti-CD171 and control mouse IgG immobilized paramagnetic beads. The captured exosomes were eluted with low pH 2.5 solution, neutralized, and then analyzed for particle size distribution and concentration by NanoSight NS300. The solid line is the eluate from anti-CD171 immobilized paramagnetic beads, and the dotted line is the eluate from control IgG-bound paramagnetic beads. [Figure 10] Figure 10 shows the data showing the range of control values. A total of 192 subjects, ranging from teenagers to over 80 years old, were used to determine [CD171+CD9+], and the Y-axis is U / ml on a logarithmic scale. Bars are the median values ​​for each group, and statistical analysis (Student's t-test) was performed using log values. [Figures 11A-11C]Figures 11A-11C are data showing monitored plasma levels of [CD171+CD9+], where the Y axis is % change in U / ml from the first blood sample in each subject, and the X axis is the time period (hours to years) during which blood samples were taken. A: High school cross country runners (non-contact sport, n=9). Blood was taken monthly from July to November. B: High school American football players (contact sport, n=22). Blood was taken monthly from July to November. C: Adult soccer players (n=19). Blood samples were taken before and 2, 24, and 48 hours after intensive heading practice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Description of the Invention It is to be understood that this disclosure is not limited to the particular methodology, protocols, cell lines, assays, and reagents described herein, as these may vary. It is also to be understood that the terminology used herein is intended to describe particular embodiments of the disclosure, and is in no way intended to limit the scope of the disclosure as set forth in the appended claims.

[0019] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to a "fragment" includes a plurality of such fragments. A reference to an "antibody" is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, the preferred methods, devices, and materials are described herein. All publications cited herein are incorporated by reference in their entirety for the purpose of describing and disclosing the methodologies, reagents, and tools reported in the publications that may be used in connection with this disclosure. Nothing in this specification should be construed as an admission that the disclosure is not entitled to antedate such disclosure by prior disclosure.

[0021] The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, cell biology, genetics, immunology and pharmacology, within the skill of the art, and such techniques are fully explained in the literature. See, e.g., Gennaro, A.R., ed. (1990) Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co.; Colowick, S. et al., eds., Methods In Enzymology, Academic Press, Inc.; Handbook of Experimental Immunology, vols. I-IV (D.M. Weir and C.C. Blackwell, eds., 1986, Blackwell Scientific Publications); Maniatis, T. et al., eds. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., vols. I-III, Cold Spring Harbor Laboratory Press; Ausubel, F.M. et al., eds. (1999) Short Protocols in Molecular Biology, 4th ed., John Wiley & Sons; Ream et al., eds. (1998) Molecular Biology Techniques: An Intensive Laboratory Course, Academic Press; PCR (Introduction to Biotechniques Series), 2nd ed. (Newton & Graham, eds., 1997, Springer Verlag).

[0022] The present disclosure relates, in part, to the discovery that brain-derived exosomes can be assayed to identify subjects suffering from or who may suffer from surgical brain damage or injury and / or post-operative cognitive dysfunction or decline (POCD) or post-operative delirium (POD).

[0023] The present disclosure is based in part on the discovery of unexpected abnormal levels of certain brain-derived exosomes present in the circulation of subjects undergoing surgery (e.g., cardiac surgery). The present disclosure demonstrates that brain-derived exosome levels can be assayed to predict, diagnose, and / or prognose brain damage or injury and / or postoperative cognitive dysfunction or decline (POCD) or postoperative delirium (POD) in subjects undergoing surgery. The present disclosure further demonstrates that measurement of brain-derived exosomes from a subject can be used to predict the subsequent development of postoperative cognitive dysfunction or decline (POCD) (e.g., to identify subjects at risk of developing POCD) or the subsequent development of postoperative delirium (POD) (e.g., to identify subjects at risk of developing POD).

[0024] The present disclosure also provides compositions for use in the methods described herein. Such compositions can include small molecule compounds; peptides and proteins, including antibodies or their functionally active fragments; and polynucleotides, including small interfering ribonucleic acid (siRNA), microRNA (miRNA), ribozymes and antisense sequences. (See, for example, Zeng (2003) Proc Natl Acad Sci USA 100:9779-9784; and Kurreck (2003) Eur J Biochem 270:1628-1644.)

[0025] The present disclosure further provides kits for measuring brain damage or injury in a subject undergoing or having undergone surgery, in these embodiments, the kits include one or more antibodies that specifically bind to brain-derived exosomes, one or more containers for collecting and / or holding biological samples, and instructions for using the kit.

[0026] Section headings are used herein for organizational purposes only and are not to be construed as limiting the subject matter described herein in any way.

[0027] Biological samples The present disclosure provides methods and assays for quantifying brain-derived exosomes in biological samples from subjects before, during, and after surgery. The present disclosure also provides methods and compositions for measuring brain damage or injury during surgery. Brain-derived exosome levels are determined in biological samples obtained from subjects before, during, and / or after surgery, or combinations thereof. In some embodiments, the biological sample of the present disclosure is blood. In some embodiments, about 1-10 mL of blood is collected from a subject. In other embodiments, about 10-50 mL of blood is collected from a subject. Blood can be collected from any suitable area of ​​the body, including the arm, leg, or blood accessible via a central venous catheter. In some embodiments, blood is collected before, during, or after surgery. The timing of collection can also be adjusted to increase the number and / or composition of exosomes present in the sample. For example, blood can be collected after exercise or after a procedure that induces vasodilation.

[0028] Blood can be mixed with various components after collection to preserve or prepare the sample for subsequent procedures. For example, in some embodiments, blood is treated with anticoagulants, cell fixatives, protease inhibitors, phosphatase inhibitors, protein, DNA or RNA preservatives after collection. In some embodiments, blood is collected by venipuncture using evacuated blood collection tubes containing anticoagulants such as EDTA or heparin. Blood can also be collected using heparin-coated syringes and hypodermic needles. Blood can also be combined with components that are useful for cell culture. For example, in some embodiments, blood is combined with cell culture medium or supplemented cell culture medium (e.g., cytokines).

[0029] Biological samples may also be obtained from other sources known in the art, including whole blood, serum, plasma, urine, interstitial fluid, peritoneal fluid, cervical swabs, tears, saliva, buccal swabs, skin, cerebrospinal fluid, or other tissues, including, for example, brain tissue.

[0030] Exosome Enrichment or Isolation The sample can be enriched for exosomes by positive selection, negative selection, or a combination of positive and negative selection. In some embodiments, the exosomes are captured directly. In other embodiments, blood cells are captured and exosomes are harvested from the remaining biological sample. In some embodiments, the enriched brain-derived exosomes in the biological sample are neuron-derived exosomes, astrocyte-derived exosomes, oligodendrocyte-derived exosomes, and / or microglia-derived exosomes.

[0031] Samples can also be enriched for brain-derived exosomes based on differences in biochemical properties of exosomes. For example, samples can be enriched for exosomes based on antigen, nucleic acid, metabolic, gene expression, or epigenetic differences. In some embodiments based on antigenic differences, antibody-conjugated magnetic or paramagnetic beads in a magnetic field gradient or fluorescently labeled antibodies by flow cytometry are used. In some embodiments based on nucleic acid differences, flow cytometry is used. In some embodiments based on metabolic differences, dye uptake / exclusion measured by flow cytometry or another sorting technique is used. In some embodiments based on gene expression, cell culture with cytokines is used. Samples can also be enriched for exosomes based on other biochemical properties known in the art. For example, samples can be enriched for exosomes based on pH or motility. Furthermore, in some embodiments, multiple methods are used to enrich for exosomes. In other embodiments, samples are enriched for exosomes using antibodies, ligands, or soluble receptors.

[0032] In other embodiments, surface markers are used to positively enrich for exosomes in a sample. In other embodiments, NCAM, CD9, CD63, CD81, neuron-specific enolase, various neuron or astrocyte adhesion proteins, microglial CD18 / 11, or CD3 T cell membrane cell surface markers are used to enrich for exosomes. In other embodiments, SNAP25, CD171, EAAT1, OMGP, DR1, SR2A, SR2C, GABAB1, GluR-1, KOR, OR, or DAT are used to enrich for exosomes. In some embodiments, cell surface markers that are not found on the exosome population are used to negatively enrich for exosomes by depleting the cell population. Flow cytometry sorting can also be used to further enrich for exosomes using intracellular or extracellular markers linked to cell surface markers or fluorescent labels. Intracellular and extracellular markers can include nuclear stains or antibodies against intracellular or extracellular proteins preferentially expressed in exosomes. The cell surface marker may include an antibody against a cell surface antigen preferentially expressed on exosomes (e.g., NCAM). In some embodiments, the cell surface marker is a neuron-derived exosome surface marker, including, for example, NCAM or CD171, CD9, SNAP25, EAAT1, OMGP, DR1, SR2A, SR2C, GABAB1, GluR-1, KOR, OR, or DAT. In some embodiments, monoclonal NCAM, CD9, CD63, CD81, neuron-specific enolase, CD171, SNAP25, EAAT1, OMGP, DR1, SR2A, SR2C, GABAB1, GluR-1, KOR, OR, or DAT antibodies are used to enrich or isolate exosomes from a sample. In certain embodiments, the NCAM, CD9, CD63, CD81, neuron-specific enolase, CD171, SNAP25, EAAT1, OMGP, DR1, SR2A, SR2C, GABAB1, GluR-1, KOR, OR, or DAT antibody is biotinylated.In this embodiment, the biotinylated NCAM or CD171 antibody can form an antibody-exosome complex, which can then be isolated using streptavidin-agarose resin or beads. In other embodiments, the NCAM, CD9, CD63, CD81, neuron-specific enolase, CD171, SNAP25, EAAT1, OMGP, DR1, SR2A, SR2C, GABAB1, GluR-1, KOR, OR or DAT antibody is a monoclonal anti-human NCAM, CD9, CD63, CD81, neuron-specific enolase, CD171, SNAP25, EAAT1, OMGP, DR1, SR2A, SR2C, GABAB1, GluR-1, KOR, OR or DAT antibody.

[0033] In some embodiments, the enriched exosomes from the biological sample are then enriched for a particular type of brain-derived exosomes (e.g., a subpopulation of brain-derived exosomes). For example, the biological sample is enriched for exosomes, and then the enriched exosomes are then enriched for neural-derived exosomes. In some embodiments, the biological sample is enriched for an individual neural source of exosomes. In certain aspects, the neural source of exosomes is microglia, neurons, or astrocytes. In other embodiments, surface markers are used to enrich for a particular type of exosomes (e.g., neural-derived exosomes). In some embodiments, CD171, CD9, SNAP25, EAAT1, OMGP, DR1, SR2A, SR2C, GABAB1, GluR-1, KOR, OR, and / or DAT cell surface markers are used to enrich for a particular type of exosomes. In some embodiments, cell surface markers not found on the exosomes of interest are used to negatively enrich exosomes by depleting undesirable cell populations. Flow cytometry sorting can also be used to further enrich for specific types of exosomes using cell surface markers or intracellular or extracellular markers linked to fluorescent labels. Intracellular and extracellular markers can include nuclear stains or antibodies against intracellular or extracellular proteins preferentially expressed in or on the exosomes of interest. Cell surface markers can include antibodies against cell surface antigens preferentially expressed on exosomes (e.g., CD171, CD9, SNAP25, EAAT1, OMGP, DR1, SR2A, SR2C, GABAB1, GluR-1, KOR, OR, and DAT). In some embodiments, the cell surface marker is a neuron-derived exosome surface marker, including, for example, CD171. In some embodiments, the cell surface marker is an astrocyte-derived exosome surface marker, including, for example, EAAT1. In some embodiments, the cell surface marker is an oligodendrocyte-derived exosome surface marker, including, for example, OMG.In some embodiments, the cell surface marker is a receptor for dopamine, serotonin, GABA, glutamate, opioids, orexin, adrenaline, noradrenaline, acetylcholine, and / or dopamine transporter. In some embodiments, monoclonal CD171, CD9, SNAP25, EAAT1, OMG, DR1, SR2A, SR2C, GABAB1, GluR-1, KOR, OR, or DAT antibodies are used to enrich or isolate exosomes from a sample. In certain embodiments, the CD171, CD9, SNAP25, EAAT1, OMGP, DR1, SR2A, SR2C, GABAB1, GluR-1, KOR, OR, or DAT antibodies are biotinylated. In this embodiment, the biotinylated CD171, CD9, SNAP25, EAAT1, OMGP, DR1, SR2A, SR2C, GABAB1, GluR-1, KOR, OR or DAT antibody can form an antibody-exosome complex, which can then be isolated using streptavidin-agarose resin or beads. In another embodiment, the CD171, CD9, SNAP25, EAAT1, OMGP, DR1, SR2A, SR2C, GABAB1, GluR-1, KOR, OR or DAT antibody is a monoclonal anti-human CD171, CD9, SNAP25, EAAT1, OMGP, DR1, SR2A, SR2C, GABAB1, GluR-1, KOR, OR or DAT antibody.

[0034] In other embodiments, brain-derived exosomes are isolated or enriched from a biological sample, comprising contacting the biological sample with an agent under conditions in which exosomes present in the biological sample bind to the agent to form an exosome-agent complex, and isolating the exosomes from the exosome-agent complex to obtain a sample comprising exosomes, wherein the purity of the exosomes present in the sample is greater than the purity of the exosomes present in the biological sample. In certain embodiments, the agent is an antibody or a lectin. Lectins useful for forming exosome-lectin complexes are described in US Patent Application Publication No. 2012 / 0077263. In some embodiments, the brain-derived exosomes are neuron-derived exosomes, astrocyte-derived exosomes, oligodendrocyte-derived exosomes, or microglia-derived exosomes. In some embodiments, multiple isolation or enrichment steps are performed. In certain aspects of this embodiment, a first isolation step is performed to isolate exosomes from a blood sample, and a second isolation step is performed to isolate neural-derived exosomes from other exosomes. In other embodiments, the exosome portion of the exosome-drug complex is lysed using a lysis reagent and the protein levels of the lysed exosomes are assayed. In some embodiments, the antibody-exosome complex is fabricated on a solid phase. In still other embodiments, the method further comprises liberating the exosomes from the antibody-exosome complex. In certain embodiments, the solid phase is non-magnetic beads, magnetic beads, agarose, or sepharose. In other embodiments, the exosomes are liberated by exposing the antibody-exosome complex to a low pH of 3.5 to 1.5. In still other embodiments, the liberated exosomes are neutralized by adding a high pH solution. In other embodiments, the liberated exosomes are lysed by incubating the liberated exosomes with a lysis solution. In still other embodiments, the lysis solution contains a protease inhibitor and a phosphatase inhibitor.

[0035] Brain damage or injury The present disclosure provides methods for measuring, diagnosing and / or prognosing brain damage or injury in subjects undergoing or having undergone surgery. Brain damage is an injury that causes destruction or deterioration or deleterious changes to brain cells. Brain damage can occur during surgery if the patient's brain is deprived of oxygen for an extended period of time, or as a result of toxicity from anesthesia. When this occurs, it can be due to severe surgical trauma, the anesthetic itself, administration of an incorrect dose of anesthetic due to a medical record entry error, miscalculation of the patient's size and weight, incorrect selection of anesthetic, length of time under anesthesia, use and duration of cardiopulmonary bypass procedures or other invasive procedures, etc.

[0036] Delirium is an acute confusional state that results in inattention and cognitive impairment. Medical or surgical conditions can often cause delirium. Delirium in surgical subjects is associated with neurological tissue damage in the brain, which can lead to loss of cognitive abilities (e.g., postoperative delirium, POD).

[0037] Cognitive impairment is the most common clinical evidence of brain injury after cardiac surgery. It can be detected by neuropsychological testing by trained examiners. Impairments in memory, psychomotor speed, executive function, visuo-constructive ability, and concentration are characteristic of postoperative brain injury. Postoperative cognitive impairment (POCD) can be detected in 14%-48% of subjects before discharge from hospital after cardiac surgery. Moreover, impairments remain detectable in at least 30% at 6 weeks and in 25% at 6 months. (Seines et al. Lancet 1999;353:1601; and van Dijk et al. J Thorac Cardiovasc Surg. 2000;120:632.)

[0038] In some embodiments, the present disclosure allows a physician to measure, diagnose and / or prognose brain injury in a subject during and / or after surgery. In other embodiments, the present disclosure allows a physician to rule out or exclude brain injury as a possible diagnosis. In yet other embodiments, the present disclosure allows a physician to identify a subject at risk of developing brain injury after surgery. In other embodiments, the present disclosure allows a physician to predict whether a subject will later develop postoperative cognitive dysfunction or decline (POCD) or postoperative delirium (POD). In further embodiments, the present disclosure allows a physician to prescribe a treatment regimen or predict the benefit of treatment for a subject undergoing or having undergone surgery. In some embodiments, the surgery is a cardiac surgery (e.g., total arterial revascularization or transcatheter aortic valve replacement).

[0039] In some embodiments, an increase in circulating brain-derived exosomes during or after surgery indicates that brain injury is occurring or has occurred, and proportional changes in circulating brain-derived exosomes are associated with the severity of brain injury, including delirium and postoperative cognitive impairment or decline.

[0040] Brain-derived exosomes and antigens Brain-derived exosome levels are assayed in biological samples taken from subjects with or at risk of having brain injury during surgery. Brain-derived exosome antigens can be used to detect or measure the level of brain-derived exosomes in biological samples. In some embodiments, the antigen is a receptor for dopamine, serotonin, GABA, glutamate, opioids, orexin, adrenaline, noradrenaline, acetylcholine, and / or dopamine transporter. In some embodiments, the antigen is CD9, CD61, CD81, CD171, SNAP25, EAAT1, OMG, DR1, SR2A, SR2C, GABAB1, GluR-1, KOR, OR, and / or DAT. Other known antigens can be used in combination with the antigens of the present disclosure. Examples of such antigens are provided in US Patent Application Publication No. 2015 / 0119278, the contents of which are incorporated herein by reference.

[0041] Those skilled in the art have several methods and devices that can be used for the detection and analysis of brain-derived exosomes disclosed herein. For the level of exosomes in a patient's test sample, immunoassay devices and methods are often used. These devices and methods can utilize labeled molecules in various sandwich, competitive, or non-competitive assay formats to generate a signal related to the presence or amount of an analyte of interest. Furthermore, certain methods and devices, such as biosensors and optical immunoassays, can be used to determine the presence or amount of an analyte without the need for labeled molecules.

[0042] Preferably, the brain-derived exosomes are analyzed using immunoassays, although other methods are known to those skilled in the art. The presence or amount of brain-derived exosomes is generally determined by using antibodies specific for each marker and detecting specific binding. Any suitable immunoassay can be utilized, such as enzyme-linked immunoassay (ELISA), radioimmunoassay (RIA), competitive binding assay, planar waveguide technology, etc. Specific immunological binding of the antibody to the marker can be detected directly or indirectly. Direct labels include fluorescent or luminescent tags, metals, dyes, radionuclides, etc., attached to the antibody. Indirect labels include various enzymes known in the art, such as alkaline phosphatase, horseradish peroxidase, etc.

[0043] The use of immobilized antibodies specific for brain-derived exosomes is also contemplated by the present disclosure. Antibodies can be immobilized on a variety of solid supports, such as magnetic or chromatographic matrix particles, the surface of an assay location (such as a microtiter well), or pieces of solid substrate material (such as plastic, nylon, paper, etc.). An assay strip can be prepared by coating an antibody or multiple antibodies in an array on a solid support. The strip can then be dipped into a test sample and rapidly processed through washing and detection steps to generate a measurable signal, such as a colored spot.

[0044] The analysis of multiple brain-derived exosomes may be performed separately or simultaneously in one test sample. To efficiently process multiple samples, several markers can be combined in one test. Furthermore, those skilled in the art will recognize the value of testing multiple samples (e.g., at successive time points) from the same individual. By testing such successive samples, changes in brain-derived exosome levels over time can be identified. Increases or decreases in brain-derived exosome levels, as well as the lack of change in marker levels, can provide useful information regarding the state of the disease, including, but not limited to, the approximate time from the onset of an event, the presence and amount of recoverable tissue, the appropriateness of drug therapy, the effectiveness of various therapies, identifying the severity of the event, identifying the severity of the disease, and identifying the patient's outcome, including the risk of future events.

[0045] Assays consisting of combinations of brain-derived exosomes mentioned in this disclosure can be constructed to provide relevant information related to differential diagnosis. Such panels can be constructed using 1, 2, 3, 4, or more than 5 or individual brain-derived exosomes. Analysis of a subset of brain-derived exosomes, including a single brain-derived exosome or a larger panel of brain-derived exosomes, can be performed using the methods described in this disclosure to optimize clinical sensitivity or specificity in various clinical settings. In some embodiments, the brain-derived exosomes are selected from the group consisting of neuron-derived exosomes, astrocyte-derived exosomes, oligodendrocyte-derived exosomes, and microglia-derived exosomes. In some embodiments, the ratio of two exosomes from the group consisting of neuron-derived exosomes, astrocyte-derived exosomes, oligodendrocyte-derived exosomes, and microglia-derived exosomes is determined.

[0046] Analysis of brain-derived exosome levels can also be performed in a variety of physical formats. For example, the use of microtiter plates or automation can be used to facilitate the processing of multiple test samples. Alternatively, single sample formats can be developed to facilitate immediate treatment and diagnosis in a timely manner, for example, in an outpatient delivery or emergency room setting. Particularly useful physical formats include surfaces with multiple individual addressable locations for detecting multiple different analytes. Such formats include protein microarrays or "protein chips" and capillary devices.

[0047] The brain-derived exosomes of the present disclosure play an important role in early detection and monitoring of brain injury during and / or after surgery. Brain-derived exosomes associated with brain injury are typically identified in measurable body samples. The measured amount can be correlated with the pathophysiology of an underlying disease or disease, the presence or absence of brain injury, and the probability of future postoperative cognitive dysfunction or decline (POCD) or postoperative delirium (POD). When a subject is treated for symptoms, the measured amount also correlates with responsiveness to therapy. In some embodiments, an increase in the level of one or more brain-derived exosomes of the present disclosure is indicative of brain injury. Thus, the method of the present disclosure is useful in diagnosing brain injury.

[0048] In some embodiments, the level of brain-derived exosomes is measured by a method selected from the group consisting of immunohistochemistry, immunocytochemistry, immunofluorescence, immunoprecipitation, western blotting, and ELISA.

[0049] Clinical Assay Performance The disclosed method can be used in clinical assays to measure, diagnose and / or prognose brain injury in subjects undergoing or having undergone surgery. The performance of clinical assays can be evaluated by determining the sensitivity, specificity, area under the ROC curve (AUC), accuracy, positive predictive value (PPV) and negative predictive value (NPV) of the assay. Described herein are assays for measuring, diagnosing and / or prognosing brain injury in subjects undergoing or having undergone surgery.

[0050] The clinical performance of the assay may be based on sensitivity. The sensitivity of the assay of the present disclosure may be at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%. The clinical performance of the assay may be based on specificity. The specificity of the assay of the present disclosure may be at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%. The clinical performance of the assay may be based on area under the ROC curve (AUC). The AUC of the assay of the present disclosure may be at least about 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9 or 0.95. The clinical performance of the assay may be based on precision. The accuracy of the assays of the present disclosure may be at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%.

[0051] composition Compositions useful in the methods of the present disclosure include compositions that specifically recognize an antigen on brain-derived exosomes, where the antigen is CD9, CD63, CD81, or CD171, EAAT1, or OMG. In yet other embodiments, the composition is selected from the group consisting of a peptide, a nucleic acid, an antibody, and a small molecule.

[0052] In certain embodiments, the present disclosure relates to compositions that specifically detect brain-derived exosomes. As detailed elsewhere herein, the present disclosure is based on the discovery that CD9 and CD171 are specific antigens for a subpopulation of brain-derived exosomes that can be used to diagnose or predict the prognosis of surgical brain injury. In some embodiments, the compositions of the present disclosure specifically bind to and detect CD9, CD171, SNAP25, EAAT1, OMGP, DR1, SR2A, SR2C, GABAB1, GluR-1, KOR, OR, and DAT. In other embodiments, the compositions of the present disclosure specifically bind to and detect dopamine, serotonin, GABA, glutamate, opioid, orexin, adrenaline, noradrenaline, acetylcholine, and / or dopamine transporter receptors.

[0053] In some embodiments, the composition comprises an antibody, which specifically binds to an antigen found on brain-derived exosomes. The term "antibody" as used herein and discussed further below is intended to include fragments thereof that specifically react with brain-derived exosomes. Antibodies can be fragmented using conventional techniques, and the fragments can be screened for utility in the same manner as described above for whole antibodies. For example, F(ab)2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab)2 fragments can be treated to reduce disulfide bridges to generate Fab fragments. Antigen-binding portions can also be generated by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding portions include, among others, Fab, Fab', F(ab')2, Fv, dAb and complementarity determining region (CDR) fragments, single chain antibodies (scFv), single domain antibodies, bispecific antibodies, chimeric antibodies, humanized antibodies, diabodies, and polypeptides that contain at least a portion of an immunoglobulin sufficient to confer specific antigen binding to the polypeptide. In certain embodiments, the antibody further comprises a detectable label attached thereto (eg, the label can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor).

[0054] In certain embodiments, the antibodies of the present disclosure are monoclonal antibodies, and in certain embodiments, the present disclosure makes available methods for generating novel antibodies that specifically bind to the brain-derived exosomes of the present disclosure. For example, a method for generating monoclonal antibodies that specifically bind to brain-derived exosomes may include administering to a mouse an immunogenic composition comprising brain-derived exosomes or fragments thereof in an amount effective to stimulate a detectable immune response, obtaining antibody-producing cells from the mouse (e.g., cells from the spleen), fusing the antibody-producing cells with myeloma cells to obtain antibody-producing hybridomas, and testing the antibody-producing hybridomas to identify hybridomas that produce monoclonal antibodies that specifically bind to brain-derived exosomes. Once the hybridomas are obtained, they can be grown in cell culture, optionally under culture conditions in which the hybridoma-derived cells produce monoclonal antibodies that specifically bind to brain-derived exosomes. The monoclonal antibodies can be purified from the cell culture.

[0055] The term "specifically reacts with" when used in reference to an antibody is intended to mean that the antibody is sufficiently selective between the antigen of interest (e.g., brain-derived exosomes) and other antigens that are not of interest, as is generally understood in the art. In certain methods using antibodies, such as therapeutic applications, a higher degree of specificity in binding may be desired. Monoclonal antibodies generally have a stronger tendency (compared to polyclonal antibodies) to effectively discriminate between the desired antigen and cross-reacting polypeptides. One of the characteristics that influences the specificity of an antibody:antigen interaction is the affinity of the antibody for the antigen. The desired specificity can be achieved with a variety of affinities, but generally, preferred antibodies have a specificity of about 10 -6 , 10 -7 , 10 -8 , 10 -9 It has the following affinity (dissociation constant):

[0056] The antibodies can be generated to specifically bind to epitopes of exosomes of the present disclosure, including, for example, neuron-derived exosomes, astrocyte-derived exosomes, oligodendrocyte-derived exosomes, In some embodiments, the neuron-derived exosomes are presynaptic dopaminergic neuron-derived exosomes or postsynaptic dopaminergic, serotonergic, GABAergic, glutamatergic and opioid neuron-derived exosomes.

[0057] Furthermore, the techniques used to screen antibodies to identify desirable antibodies can affect the properties of the resulting antibodies. A variety of different techniques are available to test the interaction between antibodies and antigens to identify particularly desirable antibodies. Such techniques include ELISA, surface plasmon resonance binding assays (e.g., Biacore binding assays, Biacore AB, Uppsala, Sweden), sandwich assays (e.g., paramagnetic bead systems from IGEN International, Inc., Gaithersburg, Md.), Western blots, immunoprecipitation assays, immunocytochemistry, and immunohistochemistry.

[0058] In some embodiments, the present disclosure relates to compositions used to prevent and / or treat brain injury in a subject undergoing surgery. As detailed elsewhere herein, the present disclosure is based on the discovery that CD171, CD9, CD61, CD81, SNAP25, EAAT1, OMG, DR1, SR2A, SR2C, GABAB1, GluR-1, KOR, OR and DAT are cell surface markers for specific subpopulations of brain-derived exosomes.

[0059] Thus, in one embodiment, the present disclosure provides compositions useful for detecting and / or quantifying subpopulations of brain-derived exosomes during or after surgery.

[0060] Treatment Method The compositions and methods of the present disclosure are also useful for preventing, minimizing or treating damage to the brain during surgery. The present disclosure provides a method for preventing, minimizing or treating damage to the brain during surgery, comprising: (i) obtaining a biological sample from a patient who is undergoing or has undergone surgery; (ii) detecting whether brain-derived exosomes are present in the sample by contacting the sample with an antibody and detecting binding between the brain-derived exosomes and the antibody; and (iii) administering an effective amount of a compound to prevent, minimize or treat damage to the brain. In some embodiments, the biological sample is selected from the group consisting of whole blood, serum, plasma, urine, interstitial fluid, peritoneal fluid, cervical swab, tears, saliva, buccal swab, skin, brain tissue and cerebrospinal fluid. In other embodiments, the compound is selected from the group consisting of anti-inflammatory compounds, anti-oxidative stress compounds, free radical scavengers (e.g., edaravone), and neuronal proliferation and survival factor compounds. In yet other embodiments, the compound is administered before, during or after surgery. In yet other embodiments, the compound is administered if brain-derived exosomes are detected in the sample. In some embodiments, the compound is administered if brain-derived exosomes are increased or decreased in the sample compared to a control. In other embodiments, the exosomes are selected from the group consisting of neuron-derived exosomes, astrocyte-derived exosomes, oligodendrocyte-derived exosomes, and microglia-derived exosomes. In yet other embodiments, the biological sample is selected from the group consisting of whole blood, serum, plasma, urine, interstitial fluid, peritoneal fluid, cervical swab, tears, saliva, buccal swab, skin, brain tissue, and cerebrospinal fluid.

[0061] In some embodiments, a neuroprotective agent is used in the method of the present disclosure. Neuroprotective agents that can be used in the method of the present disclosure are described in Panahi et al. (2018) Journal of Pharmacopuncture; 21(4): 226-240. In some embodiments, the neuroprotective compound is a glutamate blocker (e.g., polygynine R18, NA-1), a statin (e.g., atorvastatin), a hormone (e.g., melatonin), a hematopoietic growth factor (e.g., erythropoietin), a free radical scavenger (e.g., tempol), an immunosuppressant (e.g., cyclosporine A), a mucolytic agent (e.g., NAC), a beta-adrenergic receptor blocker (e.g., carvedilol), a COX-2 inhibitor (e.g., celecoxib), and / or a herbal medicine (e.g., curcumin).

[0062] kit Another aspect of the present disclosure includes a kit for detecting or measuring brain damage in a subject undergoing or having undergone surgery. A variety of kits with different components are contemplated by the present disclosure. Generally speaking, the kit includes a means for quantifying one or more brain-derived exosomes in a biological sample obtained from a subject. In another embodiment, the kit includes a means for collecting a biological sample, a means for quantifying one or more brain-derived exosomes in the biological sample, and instructions for use of the kit contents. In certain embodiments, the kit includes a means for enriching or isolating brain-derived exosomes in a biological sample. In further aspects, the means for enriching or isolating exosomes includes reagents necessary for enriching or isolating brain-derived exosomes from a biological sample. In certain aspects, the kit includes a means for quantifying the amount of brain-derived exosomes or a specific type of brain-derived exosomes (e.g., neural-derived exosomes). In further aspects, the means for quantifying the amount of brain-derived exosomes includes reagents necessary for detecting the amount of brain-derived exosomes.

[0063] These and other embodiments of the present disclosure will readily occur to those of ordinary skill in the art in light of the disclosure herein. EXAMPLES

[0064] The present disclosure will be further understood by reference to the following examples, which are intended to be merely exemplary of the present disclosure. These examples are provided merely to illustrate the disclosure of the claims. The present disclosure is not limited in scope by the illustrated embodiments, which are intended only as illustrations of single aspects of the present disclosure. All methods that are functionally equivalent are within the scope of the present disclosure. In addition to those described herein, various modifications of the present disclosure will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to be included within the scope of the appended claims.

[0065] [Example 1] Isolation and quantification of brain-derived exosomes from biological samples from subjects undergoing cardiac surgery Brain-derived exosomes (BDE) were isolated and quantified from biological samples of subjects undergoing cardiac surgery as follows. Forty-seven subjects undergoing transcatheter aortic valve implantation (TAVI) and 18 subjects undergoing total arterial revascularization (TAR) were recruited in this study after Institutional Review Board (IRB) approval at the National Cerebral and Cardiovascular Center (NCCC), Japan. EDTA blood was collected pre- and post-operatively on the same day, followed by collection on days 1, 2, and 5, and plasma was separated and frozen in a -80°C freezer. These samples were sent to the laboratory in dry ice packages for neuron-derived exosome (NDE) analysis.

[0066] The principle of an exemplary assay is shown in Figure 1. Plasma samples (5 μL diluted with buffer to a final volume of 40 μL) were applied to enzyme-linked immunosorbent assay (ELISA) wells in which antibodies against neurons (CD171 = L1CAM) were previously immobilized. After CD171+ biomolecules were captured, unbound material was removed by extensive washing steps, followed by reaction with biotinylated anti-CD9 antibodies. CD171+CD9+ double positive biomolecules (neuron-derived exosomes, NDE) were then detected by chemiluminescence ELISA using horseradish peroxidase-conjugated streptavidin and biotin-tyramide amplification. Since exosomes are sticky and bind nonspecifically to IgG, plasma samples were also applied to ELISA wells in which control mouse IgG was immobilized. For CD9 reactions, an excess of control mouse IgG was included to exclude such nonspecific reactions. Mouse IgG was used as a control, since both anti-CD171 and CD9 were mouse monoclonal antibodies.

[0067] NDE in pre-surgery, post-surgery, and day 1-5 samples was analyzed on a single ELISA plate, and post-surgery changes (% change) were calculated by dividing each ELISA reading (relative light units (RLU)) by the RLU of the pre-surgery value. To combine data from multiple ELISA plates, an equivalent dilution of standard plasma control sample was assigned as 100 units / ml and used on each ELISA plate, and RLU was converted to U / mL.

[0068] As shown in Figure 2A, the replicate variation of CD171+CD9+ double positive values ​​(NDE) was r 2 = 0.9966. Nonspecific binding to IgG (Figure 2B, x-axis) was very small compared to the NDE values ​​(Figure 2B, y-axis). Therefore, in subsequent studies, NDE values ​​were analyzed without IgG.

[0069] As shown in Figure 3, plasma levels of NDE in preoperative samples did not differ between the TAVI and TAR groups.

[0070] As shown in Figure 4A, the postoperative NDE values ​​for 41 TAVI subjects were stable and less than 200%, while 6 subjects showed an increase of more than 200% (12.5%). Conversely, 16 TAR subjects showed an increase of more than 200% (88.9%), and only 2 subjects showed an increase of more than 200% (Figure 4B). The increase was substantially higher in TAR than in TAVI, with 4 TAR subjects showing an increase of more than 1000% (10-fold) (Figure 4B). Interestingly, such increases in both TAVI and TAR were transient and gradually decreased. However, even after 5 days, 5 of 38 TAVI subjects (13.2%) and 13 of 17 TAR subjects (76.5%) still showed an increase of more than 200%.

[0071] These results show that the plasma level of NDE is significantly increased in subjects after TAVI and TAR surgery.These results demonstrate that the disclosed method and composition can be used to quantify and detect brain-derived exosomes in samples from subjects after surgery.These results further suggest that the disclosed method and composition are useful for measuring brain damage associated with surgery.These results also suggest that the disclosed composition and method are useful for preventing and / or treating brain damage in subjects undergoing surgery.

[0072] [Example 2] Prediction of postoperative delirium by measuring brain-derived exosomes in peripheral blood Brain-derived exosomes (BDE) were isolated and quantified from biological samples of subjects undergoing cardiac surgery to predict postoperative delirium as follows. Plasma samples were collected preoperatively from subjects undergoing minimally invasive transcatheter aortic valve replacement (TAVI) (n=30) and invasive open-chest total aortic valve replacement (TAR) (n=29), respectively. Each patient was followed for at least 5 days to observe delirium. In the first set of studies, preoperative, postoperative, and postoperative days 1, 2, and 5 (5 time points per subject) from 11 TAR subjects who did not exhibit POD and 7 TAR subjects who developed POD were analyzed. In subsequent studies, preoperative plasma samples from all subjects (30 TAVI and 29 TAR) were analyzed.

[0073] EDTA plasma samples used for standards and assay controls were purchased from three different vendors (Innovative Research, Novi, MI; BioIVT, Westbury, NY; and Equitech Enterprise, Kerrville, TX). Antibodies against human CD171 (Thermo Fisher Scientific, Waltham MA), EAAT1 (excitatory amino acid transporter 1) (Abeam, Waltham, MA), MOG (myelin oligodendrocyte glycoprotein) (Thermo Fisher), CD9 (biotinylated, BD Biosciences, Franklin Lakes, NJ), and control mouse IgG (Equitech), 96-well plates for enzyme-linked immunosorbent assay (ELISA) (Sigma Aldrich, St Louis MO), ELISA coating buffer (BioLegend, San Diego, CA), and various reagents for ELISA (Thermo Fisher Scientific) were also purchased from the designated suppliers.

[0074] Anti-CD171, anti-EAAT1 and anti-MOG were immobilized on the ELISA wells. Plasma samples were diluted with plasma diluent to a final volume of 40 μL and then applied to the ELISA wells and incubated for 1 h at room temperature. After extensive washing steps with PBS, each well was reacted with biotinylated probes supplemented with 0.8% bovine serum albumin, 40 μg / mL mouse IgG, and the reaction continued for another hour. After washing each well twice with PBS, each well was reacted with a 1 / 4,000 dilution of poly-horseradish peroxidase (HRP)-conjugated streptavidin supplemented with 10% BSA and 30% blocker casein, and incubation continued for 20 min. After a washing step, each well was incubated with 0.0006% hydrogen peroxide (CVS Pharmacy, Irvine, CA) diluted in PBS:water (1:1) solution for 5 min to remove non-specifically bound HRP conjugates. After aspiration of hydrogen peroxide, each well was mixed with 1 / 3 diluted chemiluminescent substrate (Super Signal) for 4 min, and then relative light units (RLU) were determined by a luminometer (Active GLO, ANSH Labs, Webster, TX). Using a plasma standard arbitrarily assigned to 100 units / mL (U / mL), ELISA measurements of RLU were converted to U / mL by a four-parameter logistic equation.

[0075] Plasma levels of NDE were widely distributed among subjects, and such distribution was also seen in ODE (Figure 5A, 5B, 5G, 5G, y-axis is logarithmic scale). When the changes (Δlog) of NDE, ADE, and ODE were calculated (Figure 5C, 5F, and 5I), plasma levels of NDE and ODE, but not ADE, increased significantly after TAR surgery, and such increases in NDE and ODE were significantly higher in POD subjects than in non-POD subjects. When data from each patient were analyzed (Figure 5A, 5B, 5D, 5E, 5G, and 5H), postoperative levels of NDE and ODE were quite similar between the POD(-) and POD(+) groups, whereas preoperative values ​​were slightly lower in the POD(+) patient group compared to the POD(-) patient group, although statistical significance was not confirmed due to the limited number of subjects.

[0076] In a subsequent study, we focused on the preoperative values ​​of NDE, ADE, and ODE, increasing the number of TAR subjects (n=29) and including TAVI subjects (n=30). Approximately half of the TAR subjects (n=13) developed POD, while only two subjects with TAVI showed POD. As shown in Figures 6A-6C, the preoperative values ​​of NDE (Figure 6A) and ODE (Figure 6C) were lower in POD(+) subjects than in POD(-). However, after calculating the proportions of NDE / ADE, ODE / NDE, and ODE / ADE, the variance was eliminated (Figures 7A-7C, y-axis is linear scale). When POD(-) and (+) subjects were combined, the TAVI and TAR groups showed similar NDE / ADE, ODE / NDE, and ODE / ADE, respectively (Figures 7A-7C). However, in the TAR group, NDE / ADE (Figure 7A) and ODE / ADE (Figure 7C) were significantly lower in POD(+) subjects than in POD(-) subjects (p=0.0016 for NDE / ADE, p=0.001 for ODE / ADE). These results showed that subjects with low preoperative NDE / ADE and ODE / ADE levels were more likely to develop POD, with receiver operating characteristic (ROC) analysis showing an area under the curve (AUC) of more than 81% (Figures 7D-7E). Such low NDE / ADE and ODE / ADE subjects did not induce POD when minimally invasive TAVR was used.

[0077] These results showed that plasma levels of NDE and ODE were significantly increased in subjects after TAR surgery. NDE and ODE in POD(+) subjects were significantly higher than POD(-) subjects (Figures 5A-5I). These results demonstrated that the methods and compositions of the present disclosure can be used to quantify and detect brain-derived exosomes in samples from subjects after surgery. These results also revealed that the methods and compositions of the present disclosure are useful for predicting postoperative delirium. These results further suggested that the methods and compositions of the present disclosure are useful for measuring brain injury associated with surgery. These results also suggested that the compositions and methods of the present disclosure are useful for preventing and / or treating brain injury in subjects undergoing surgery.

[0078] [Example 3] Monitoring after brain injury using a plasma-based immunoassay between the neuronal marker CD171 and the exosomal marker CD9 Monitoring after brain injury using a plasma-based immunoassay between the neuronal marker CD171 and the exosomal marker CD9 was performed as follows.

[0079] Mouse monoclonal antibodies against human CD171 (IgG2a, Thermo Fisher Scientific, Waltham, MA) and CD81 (BD Biosciences, Franklin Lakes, NJ), control mouse IgG2a (BioLegend, San Diego, CA) and normal mouse IgG (Equitech-Bio, Kerrville, TX) were diluted in ELISA coating buffer (BioLegend) and 50 μL was applied to high volume white 8-well strips (Coming #3923, Sigma Aldrich, St Louis MO). After 1 h of incubation with constant shaking at 700 rpm (shaking was applied throughout the incubation), each well was washed once with phosphate buffered saline (PBS, Thermo Fisher) and incubated with undiluted blocker casein (Thermo Fisher) for an additional 1 h.

[0080] Plasma samples were diluted to a final volume of 40 μL with plasma diluent and then applied to ELISA wells pre-immobilized with the appropriate antibodies and control IgG. The ELISA wells were incubated for 1 h at room temperature. After an extensive washing step with PBS, each well was reacted with biotinylated probes supplemented with 0.8% bovine serum albumin (BSA, Thermo Fisher), 40 μg / mL mouse IgG (Equitech-Bio), and the reaction was continued for another hour. To keep the captured exosomes intact, no tween-20 was used at any point during the procedure. Biotinylation was performed using EZ link Sulfo-NHS-LC-Biotin (Thermo Fisher) according to the vendor's protocol, followed by removal of free biotin by spin columns. After washing each well twice with PBS, each well was reacted with 1 / 4,000 dilution of poly-horseradish peroxidase (HRP)-conjugated streptavidin (Thermo Fisher) supplemented with 10% BSA (Equitech-Bio) and 30% blocker casein (Thermo Fisher), and the incubation continued for 20 min. After a washing step, each well was incubated for 5 min with 0.0006% hydrogen peroxide (CVS pharmacy, Irvine, CA) diluted in PBS:water (1:1) solution to remove nonspecifically bound HRP conjugate. After aspiration of hydrogen peroxide, each well was mixed with 1 / 3 dilution of chemiluminescent substrate (Super Signal Thermo Fisher) for 4 min, and then the relative light units (RLU) were measured with a luminometer (Active GLO, ANSH Labs, Webster, TX). Using standard plasma arbitrarily assigned to 100 units / mL (U / mL), ELISA measurements of RLU were converted to U / mL by a four-parameter logistic equation.

[0081] After capturing and washing the exosomes, the bottom of each ELISA well was punched and suspended in 2% glutaraldehyde in 0.1 M sodium phosphate buffer (PBS), pH 7.4 (Sigma Aldrich). For conductive staining, the bottom of the well was treated with 1% tannic acid (Nacalai Tesque, Kyoto, Japan) in 0.1 M PBS for 1 h, rinsed with buffer for 1 h, and immersed in 1% osmium tetroxide (OsO4) in 0.1 M PBS for 1 h. The specimens were then dehydrated in a graded ethanol series, transferred to t-butyl alcohol, and dried in a freeze dryer (ID-2; Eiko Co., Tokyo, Japan). The dried specimens were then mounted on aluminum bases and coated with platinum-palladium in an ion-sputter coater (El 030; Hitachi, Tokyo, Japan). Finally, the specimens were observed under a field emission scanning electron microscope (S4100; Hitachi, Tokyo, Japan) with an accelerating voltage of 5 kV.

[0082] The present inventors first identified [CD171 + CD9 + We confirmed that the exosomes were not irreversible and could be dissociated by washing with an acidic solution of pH 2.5-2.0. We also found suitable paramagnetic beads (PM-50, Spherotech, Lake Forest, IL) with the same surface properties as the ELISA wells, allowing the procedure developed for ELISA to be immediately applied to the paramagnetic beads. After verifying the performance of the paramagnetic beads, the plasma sample was applied to the paramagnetic beads. The captured exosomes were then eluted with a pH 2.5 solution and immediately neutralized by adding an appropriate amount of 1 M Tris, pH 8.0. High-resolution particle size distribution and concentration analysis were performed using a NanoSight NS300 at Particle Technology Labs (Downers Grove, IL).

[0083] Control EDTA-plasma samples were purchased from three different vendors (Innovative Research, Novi, MI; BioIVT, Westbury, NY; and Equitech Enterprise, Kerrville, TX). EDTA plasma was collected in accordance with Institutional Review Board approval, and consent was obtained from each participant. Plasma samples from high school and adult soccer players were collected from Indiana University, and samples from collegiate rugby players were collected from Keio University (Japan). Plasma samples from subjects undergoing TAR were collected at five time points (preoperative, postoperative, and postoperative days 1, 2, and 5) at the National Cerebral and Cardiovascular Center (Japan). All plasma samples were frozen and stored in a -80°C freezer until analysis.

[0084] ELISA validation. Specificity of capture antibodies: A mouse monoclonal antibody against human CD171 (IgG2a), control mouse IgG and control mouse IgG2a were immobilized on ELISA wells. Three different control human plasmas and dilutions alone were applied to the given wells and ELISA was performed using an anti-human CD9 probe. As shown in Figure 8A, all three plasmas showed higher CD9 signals on the anti-CD171 immobilized wells than the control mouse IgG and isotype control mouse IgG wells. Plasma #3 showed limited signal in the IgG2a control wells, but the levels of anti-CD171 were much higher than those of IgG2a. Plasma #3 is a good example highlighting the problem of non-specific binding of IgG as well as the variability of plasma from different donors.

[0085] Probe specificity: Three control plasmas and buffer only were applied to ELISA wells immobilized with mouse IgG or anti-CD171 and reacted with biotinylated probes of mouse IgG, mouse IgG2b, or anti-human CD9 (mouse monoclonal IgG2b). As shown in Figure 8B, all three plasma samples showed high anti-CD9 signals in the anti-CD171 immobilized wells, while the signals from the control wells, control probes, and buffer controls were minimal, indicating CD9 specificity.

[0086] Exosome specificity I: Exosome reduction. Plasma samples were incubated with anti-CD81 or control mouse IgG immobilized paramagnetic beads for 1 h each to remove exosomes. The beads were removed by magnetic separation, and the supernatant plasma samples were applied to three different ELISA wells pre-immobilized with anti-CD171, anti-CD81, and control mouse IgG. After extensive washing, the wells were reacted with biotinylated anti-CD9 to complete the sandwich ELISA. As shown in Figure 8C, [CD81+CD9+] exosomes in anti-CD81-treated plasma (w / , dark gray column) were reduced by about 50% compared to control IgG-treated plasma (w / o, light gray column), suggesting that about half of the exosomes were removed by this procedure. [CD171+CD9+] exosomes were reduced to the same extent by exosome reduction treatment, suggesting that [CD171+CD9+] are exosomes.

[0087] Exosome specificity II: spiked exosomes. Exosomes were prepared from control plasma using the ExoQuick (System Biosciences, Palo Alto, CA) procedure and suspended in PBS. Three different dilutions of exosomes and PBS were added to plasma or plasma diluents, and [CD171+CD9+] was measured. As shown in Figure 8D (□), the prepared exosomes showed [CD171+CD9+] in a dose-dependent manner. The addition of exosomes to plasma samples also increased [CD171+CD9+] in a dose-dependent manner with a slope similar to that of exosomes alone (●). These data suggest that [CD171+CD9+] is exosome-dependent.

[0088] Plasma dilution test: As shown in Figure 8E, [CD171+CD9+] in three different plasmas showed a linear plasma volume-dependent increase in anti-CD171 immobilized wells (filled symbols; ●▲■), but the plasma volume-dependent increase was not observed when the samples were placed in the control mouse IgG immobilized ELISA wells (open symbols;

[0089] [ka] ), the signal remained very low. These data support not only the specificity of the assay, but also the ability to convert the ELISA data results for each sample to U / mL using a dilution curve of standard plasma.

[0090] Assay reproducibility: Assays were always performed in replicates and, as shown in Figure 8F, intra-assay replicate variation (n=134 subjects) was extremely small, whereas inter-assay (n=8 runs) variation (Figure 8G) remained within 90-158% variation from the first measurement.

[0091] Confirmation of exosomes. SEM: Since SEM requires a flat surface, we carefully removed the bottom of each ELISA well without damaging the surface. We tested two different exosomes as capture agents, NDE using anti-CD171 (Figure 9A-9B) and pan-exosomes using anti-CD81 (Figure 9C-9D), respectively. SEM images showed visualization of exosomes with distinct surface characteristics. Both were spherical and of similar size, between 100 and 200 nm. NDE was slightly smaller than pan-exosomes. Figures 9A-9D show a single typical exosome in each panel, but many similar figures were also identified.

[0092] NTA: Plasma samples were applied to anti-CD171 and control mouse IgG immobilized paramagnetic beads. Captured exosomes were eluted with low pH 2.5 solution, neutralized, and then applied to NTA as described in the methods. As shown in Figure 9E, the eluted solution from anti-CD171 immobilized paramagnetic beads showed particles of 150-300 nm, which is equivalent to the size of SEM, while the solution from control IgG immobilized beads showed no such particles. Anti-CD171 also showed small peaks around 380, 500, 580, and 720 nm, suggesting the presence of large molecules / aggregates in the sample.

[0093] Range of control values. We collected plasma samples from various sources across a wide age range from teenagers to over 80s (192 subjects in total). These samples were collected from three different commercial sources and from different academic collaborators in both the United States and Japan. All genders and ethnicities were represented. Surprisingly, [CD171 + CD9 + ] was widely distributed among individuals, with the variance being approximately 2-3 logarithmic range (Figure 10). However, the variation was not random, but showed a normal distribution on a logarithmic scale in each age group. Interestingly, this level decreased significantly with age (Figure 10).

[0094] [CD171 + CD9 + Monitoring the level of

[0095] Control :[CD171 + CD9 + ] varied significantly between individuals (Figure 10), but values ​​were stable within the same individual from July to November in high school cross country runners (non-contact sport, n=9), falling within + / - 50% of the value obtained in July, except for one individual who showed large fluctuations (Figure 11A), similar to results reported in our previous publication (7), even though the assay had been substantially improved over the course of the study.

[0096] Contact sports players: High school football players (n=22) were also stable from July to November (Figure 11B). No high school players sustained a concussion this season. Adult soccer players (n=19) also showed a small increase 2 hours after practice, but remained very stable for 3 days after intensive heading practice (Figure 11C).

[0097] These results revealed that the plasma levels of brain-derived exosomes were significantly increased in subjects after TAR surgery. Brain-derived exosomes in POD(+) subjects were significantly higher than those in POD(-) subjects. This result demonstrated that the disclosed method and composition can be used to quantify and detect brain-derived exosomes in samples from subjects after surgery. These results also revealed that the disclosed method and composition are useful for predicting postoperative delirium. These results further suggested that the disclosed method and composition may be useful for measuring brain injury associated with surgery. These results also suggested that the disclosed composition and method may be useful for preventing and / or treating brain injury in subjects undergoing surgery.

[0098] Various modifications of the present disclosure in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0099] All references cited herein are incorporated by reference in their entirety.

Claims

1. A method comprising: (i) obtaining a biological sample from a subject undergoing or having undergone surgery; and (ii) detecting brain-derived exosomes in the sample by contacting the sample with an antibody and detecting binding between the brain-derived exosomes and the antibody.

2. 2. The method of claim 1, wherein detecting binding between the brain-derived exosomes and the antibody further comprises contacting the brain-derived exosomes with a second antibody.

3. The method of claim 1 , wherein the antibody is an anti-CD171 antibody, an anti-EAAT1 antibody, or an anti-MOG antibody.

4. The method of claim 2 , wherein the second antibody is an anti-CD9 antibody, an anti-CD63 antibody or an anti-CD81 antibody.

5. 10. The method of claim 1, further comprising quantifying the level of brain-derived exosomes in the biological sample.

6. 2. The method of claim 1, wherein the exosomes are selected from the group consisting of neuron-derived exosomes, astrocyte-derived exosomes, oligodendrocyte-derived exosomes, and microglia-derived exosomes.

7. 2. The method of claim 1, further comprising determining a ratio of two exosomes selected from the group consisting of neuron-derived exosomes, astrocyte-derived exosomes, oligodendrocyte-derived exosomes and microglia-derived exosomes.

8. 8. The method of claim 7, wherein a low pre-operative NDE / ADE and / or ODE / ADE rate indicates that the subject has post-operative delirium.

9. 1. A method for measuring and / or detecting brain injury in a subject undergoing a surgical procedure, comprising: (i) obtaining one or more biological samples from the subject before, during, and / or after undergoing surgery, or any combination thereof; (ii) detecting brain-derived exosomes in the one or more samples by contacting the one or more samples with an antibody and detecting binding between the brain-derived exosomes and the antibody; and (iii) comparing the level of brain-derived exosomes in the one or more samples to a control.

10. 1. A method for preventing, minimizing or treating damage to the brain during surgery, comprising: (i) obtaining a biological sample from a subject undergoing or who has undergone surgery; (ii) detecting whether brain-derived exosomes are present in the sample by contacting the sample with an antibody and detecting binding between the brain-derived exosomes and the antibody; and (iii) administering an effective amount of a compound to prevent, minimize or treat damage to the brain.

11. A kit for measuring brain damage in a subject undergoing or having undergone surgery, comprising one or more agents that specifically bind to brain-derived exosomes, one or more containers for collecting and / or holding biological samples, and instructions for use thereof.

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