Systems and methods for determining UCH-L1, GFAP and other biomarkers in blood samples - Patents.com

Abstract

Disclosed herein are systems and methods for determining ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or any combination thereof, in a blood sample obtained from a subject. Also disclosed herein are systems and methods for determining CK-MB, β-hCG, thyroid stimulating hormone (TSH), homocysteine, free thyroxine (free T4), or any combination thereof, in a blood sample.

Description

[Technical Field]

[0001] This application is a continuation of U.S. Patent Application No. 63 / 291,287 filed December 17, 2021, U.S. Patent Application No. 63 / 308,287 filed February 9, 2022, U.S. Patent Application No. 63 / 309,031 filed February 11, 2022, U.S. Patent Application No. 63 / 309,033 filed February 11, 2022, U.S. Patent Application No. 63 / 333,836 filed April 22, 2022, and U.S. Patent Application No. 63 / 333,836 filed April 22, 2022. This application claims priority to U.S. Patent Application No. 63 / 333,841, filed April 22, 2020, U.S. Patent Application No. 63 / 402,115, filed August 30, 2022, U.S. Patent Application No. 63 / 402,132, filed August 30, 2022, and U.S. Patent Application No. 63 / 423,118, filed November 7, 2022, the contents of each of which are incorporated herein by reference.

[0002] The computer-readable nucleotide / amino acid sequence listing submitted herewith is incorporated by reference in its entirety into this specification and is identified as follows:

[0003] The present disclosure relates to systems and methods for determining the amount of ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof, in a blood sample (e.g., a capillary blood sample and / or a venous blood sample) obtained from a subject. The amount of UCH-L1, GFAP, or a combination thereof in a blood sample obtained from a subject can aid in the diagnosis and assessment of whether the subject has suffered, may have suffered, or is suspected of having suffered a head injury, such as a traumatic brain injury (TBI). The present disclosure further relates to systems and methods for determining the amount of CK-MB, β-hCG, thyroid-stimulating hormone (TSH), homocysteine, free thyroxine (free T4), or any combination thereof, in a blood sample (e.g., a capillary blood sample and / or a venous blood sample) obtained from a subject. [Background technology]

[0004] Biological samples for use in laboratory tests to determine the amount or presence of one or more analytes of interest are typically obtained by venipuncture by a trained phlebotomist or nurse, which typically involves inserting a needle or syringe into a subject's vein. Unfortunately, venipuncture can be difficult or impractical in certain situations, such as for newborns, the elderly, subjects who are afraid of needles and / or syringes, and / or locations without a nearby hospital or clinic. Once a venous blood sample is collected from a subject, the sample is typically packaged and transported to a processing center for analysis. Summary of the Invention [Problem to be solved by the invention]

[0005] Unfortunately, traditional sample collection and testing of venous blood samples has weaknesses. For example, most blood tests, except for the most basic of tests, require substantially larger volumes of venous blood from a subject. Due to the large sample volume required, extraction of blood from alternative sample sites on a subject, which may be less painful and / or less invasive, is often untenable because it does not produce the sample volumes required for traditional testing methodologies. Thus, there is a need in the art for a method for determining the amount and / or presence of one or more analytes of interest in a biological sample obtained from a subject that does not require obtaining a venous blood sample from the subject. [Means for solving the problem]

[0006] (Summary of the Invention) In one aspect, the present disclosure relates to a method. In one aspect, the present disclosure relates to a method, the method comprising: (A) performing at least one assay for (i) ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof; or (ii) CK-MB, β-hCG, thyroid-stimulating hormone (TSH), homocysteine, free thyroxine (free T4), or any combination thereof, in a capillary blood sample obtained from the subject to determine the amount of (i) UCH-L1, GFAP, or a combination thereof; or (ii) CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof; and (B) using a point-of-care device or a non-point-of-care device, communicating the determined amount of (i) UCH-L1, GFAP, or a combination thereof; or (ii) CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof in the sample; The sample is collected from a location on a subject other than a finger (1) in a decentralized setting; (2) without the use of a syringe, standard needle, or a combination thereof; (3) by a user who is not trained in collecting blood samples from subjects; (4) by a robot; (5) by a blood collection device that is self- or other-administered; or (6) by any combination of (1)-(5), Further, the method includes the steps of: (i) the assay being capable of being performed in less than about 30 minutes; (ii) the determined amount of (a) UCH-L1, GFAP, or a combination thereof; or (B) CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof in the sample being capable of being transmitted in less than about 30 minutes from the time the sample is collected; or (iii) a combination of (i) and (ii).

[0007] In some aspects of the above-described methods, the sample is collected using a microsampling device.

[0008] In other aspects of the above methods, the sample is processed prior to performing the assay, and further, the sample is whole blood and is processed into serum or plasma.

[0009] In some aspects, the microsampling device (a) comprises a plasma separation device; or (b) is operably linked to a plasma separation device.

[0010] In still further aspects of the above-described methods, the assay comprises subjecting the sample to: (I) (a) an anti-UCH-L1 antibody that binds to UCH-L1 to determine the amount of UCH-L1 in the sample; (b) an anti-GFAP antibody that binds to GFAP to determine the amount of GFAP in the sample; or (c) a combination of (a) and (b); or (II) (a) an anti-CK-MB antibody that binds to CK-MB to determine the amount of CK-MB in the sample; (b) an anti-β-hCG antibody that binds to β-hCG to determine the amount of β-hCG in the sample; (c) an anti-TSH antibody that binds to TSH to determine the amount of TSH in the sample; (d) an anti-homocysteine ​​antibody that binds to homocysteine ​​to determine the amount of homocysteine ​​in the sample; (e) an anti-free T4 antibody that binds to free T4 to determine the amount of free T4 in the sample; or (f) any combination of (a) through (e). The method includes contacting the

[0011] In still further aspects of the above-described methods, the assay comprises: (I) (a) if the assay is for GFAP, the GFAP assay comprises a scaling factor of GFAP in the capillary sample compared to GFAP in the venous sample of about 1.0:1.0; (b) if the assay is for UCH-L1, the UCH-L1 assay comprises a scaling factor of UCH-L1 in the capillary sample compared to UCH-L1 in the venous sample of about 2.5:1.0 to about 1.5:1.0; or (c) a combination of (a) and (b); or (II) (a) if the assay is for CK-MB, the CK-MB assay comprises a conversion factor of CK-MB in the capillary sample relative to CK-MB in the venous sample of about 0.5:1.0 to about 1:0:1.2; (b) if the assay is for β-hCG, the β-hCG assay comprises a conversion factor of β-hCG in the capillary sample relative to β-hCG in the venous sample of about 0.8:1.0 to about 1:0:1.4; (c) if the assay is for TSH, the TSH assay comprises a conversion factor of β-hCG in the capillary sample relative to β-hCG in the venous sample of about 0.75:1.0 to about 1.2:1.0. (c) if the assay is for homocysteine, the homocysteine ​​assay includes a conversion factor of homocysteine ​​in the capillary sample compared to homocysteine ​​in the venous sample of from about 1.2:1.0 to about 0.9:1.0; (e) if the assay is for free T4, the free T4 assay includes a conversion factor of free T4 in the capillary sample compared to free T4 in the venous sample of from about 0.8:1.0 to about 1.2:1.0; or (f) any combination of (a)-(e).

[0012] In still further aspects of the above-described methods, if the assay is (a) an assay for GFAP, UCH-L1, or GFAP and UCH-L1, the method is used to aid in the diagnosis and assessment of a subject who has suffered or may have suffered a head injury; (b) if the assay is for CK-MB, the method is used to diagnose myocardial infarction in a subject; (c) if the assay is for β-hCG, the method is used to determine whether the subject is pregnant; or (d) if the assay is for TSH, the method is used to assess thyroid function in a subject, diagnose thyroid disease in a subject, or determine whether thyroid function is impaired in a subject. (e) if the assay is for homocysteine, the method is used to diagnose hyperhomocysteinemia, homocystinuria, or hyperhomocysteinemia and homocystinuria in a subject, or to treat a subject with hyperhomocysteinemia, homocystinuria, or hyperhomocysteinemia and homocystinuria; or (f) if the assay is for free T4, the method is used to assess thyroid function in a subject, diagnose thyroid disease in a subject, treat thyroid disease in a subject, or any combination thereof.

[0013] In still further aspects of the above-described methods, the amount of (a) UCH-L1, GFAP, and UCH-L1 and UCH-L1 and GFAP; or (b) CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof, is transmitted within (a) about 25 minutes from the time the sample is collected; (b) less than about 20 minutes from the time the sample is collected; (c) about 4 to about 20 minutes from the time the sample is collected; (d) about 15 to about 18 minutes from the time the sample is collected; or (e) less than about 18 minutes from the time the sample is collected.

[0014] In still further aspects, communicating the determined amount of (i) UCH-L1, GFAP or a combination thereof; or (ii) CK-MB, β-hCG, TSH, homocysteine, free T4 or any combination thereof in the sample comprises communicating the level of (i) UCH-L1, GFAP or a combination thereof; or (ii) CK-MB, β-hCG, TSH, homocysteine, free T4 or any combination thereof in the sample.

[0015] In still further aspects of the above-described methods, the sample is processed using a plasma separation device.

[0016] In still further aspects of the above-described method, the plasma separation device comprises: a hydrophobic layer including at least one microchannel having first and second channels that define a path for capillary fluid flow; and an upper layer adjacent to the hydrophobic layer, the surface of the upper layer facing the hydrophobic layer being hydrophilic; wherein the sample comprises blood or a blood product.

[0017] In some aspects, the device further comprises a bottom layer adjacent to the hydrophobic layer, wherein the surface of the bottom layer facing the hydrophobic layer is hydrophilic.

[0018] In still other embodiments, the top layer, the hydrophobic layer, the bottom layer, or any combination thereof, are adhered to one another.

[0019] In yet another embodiment, at least one microchannel extends longitudinally along a portion of the hydrophobic layer to an opening at the second end.

[0020] In still yet a further embodiment, at least one microchannel is less than about 80 mm in length.

[0021] In yet another embodiment, at least one microchannel is less than about 5 mm wide.

[0022] In yet another aspect, the top layer, the bottom layer, or the top and bottom layers are entirely hydrophilic.

[0023] In still further aspects, the overall composition of the top layer, the bottom layer, or both the top and bottom layers, respectively, comprises the same or different materials.

[0024] In still further embodiments, the hydrophobic layer, the top layer, the bottom layer, or any combination thereof, has a combined thickness of from about 100 to about 600 microns.

[0025] In yet another embodiment, the hydrophobic layer has a thickness of about 100 to about 200 microns.

[0026] In still further embodiments, the top layer, the bottom layer, or each of the top and bottom layers has a thickness of from about 50 to about 200 microns.

[0027] In yet another embodiment, the top layer of the device includes a sample inlet. In yet another embodiment, when the top layer includes the sample inlet, the hydrophobic layer and optionally the bottom layer can each include an opening below the sample inlet. In yet a further embodiment, the sample inlet includes a separation membrane. In yet a further embodiment, the separation membrane is a plasma separation membrane. In yet another embodiment, the device further includes a hydrophilic mesh or film located above the separation membrane, below the separation membrane, or both above and below the separation membrane. In yet a further embodiment, the opening in the hydrophobic layer is connected to a first end of at least one microchannel.

[0028] In yet another embodiment, the device further comprises an agglutinating agent. In some embodiments, the agglutinating agent comprises a lectin (e.g., soybean lectin), Merquat-100, concanavalin A, DEAE-dextran, poly-L-lysine, polyvinylpyrrolidone, poly(2-(dimethylamino)ethyl methacrylate), or any combination thereof. In yet further embodiments, the agglutinating agent is coated on or incorporated within the separation membrane, the top layer, the hydrophobic layer, the bottom layer, or any combination thereof.

[0029] In a still further aspect, the plasma separation device comprises: a vessel or tube having an inlet and an outlet end; a blood holding chamber at the inlet end of the container or tube; a removable serum holding chamber at the outlet end of the container or tube; a material disposed within the container between the blood-holding chamber and the serum-holding chamber for separating components of the whole blood based on size; The device includes:

[0030] In still other aspects of the above-described methods, the materials used in the device used in plasma separation include glass or porous beads, membranes, filters, glass or other fibrous materials, or any combination thereof.

[0031] In still further embodiments, the filters described above allow the passage of particles or molecules smaller than about 0.7 microns, about 0.6 microns, about 0.5 microns, about 0.4 microns, or about 0.3 microns.

[0032] In still further aspects, in devices used during plasma separation, a pressure differential between the blood holding chamber and the serum holding chamber allows whole blood to move from the blood holding chamber through a filter to produce serum and / or plasma that is collected in the serum holding chamber.

[0033] In still further aspects of the above-described methods, the plasma separation device is operably linked to a microsampling device.

[0034] In still further aspects of the above-described methods, the microsampling device comprises a plasma separation device.

[0035] In still further aspects of the above-described methods, the plasma separation device comprises a filter, a membrane, a synthetic paper, or any combination thereof.

[0036] In still further aspects of the above-described methods, (a) the point-of-care device comprises a cartridge; or (b) the non-point-of-care device is a higher throughput assay analyzer.

[0037] In still further aspects of the above-described methods, the amount of UCH-L1, GFAP, CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof, is communicated (a) by a point-of-care device or a non-point-of-care device, in a document and / or spreadsheet, on a mobile device, on a computer, on a website, in an email, or any combination thereof; or (b) by display on a point-of-care device or a non-point-of-care device.

[0038] In still further aspects of the above-described methods, the assays used in the above-described methods can be analog assays, digital assays, or a combination of analog or digital assays.

[0039] In still further aspects of the above methods, the subject is a human.

[0040] In another aspect, the present disclosure relates to a system comprising: a microsampling device for collecting capillary blood samples from a subject; a reactor that receives a capillary blood sample and contains assays for (i) ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof; or (ii) CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof; and and analyzing the reactor to provide the amount of (i) ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof; or (ii) CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof, in the sample. may include:

[0041] In yet another aspect, the system further includes a plasma separation device for producing a processed capillary blood sample.

[0042] In some aspects, the plasma separation device comprises: a) a hydrophobic layer having first and second ends and including at least one microchannel defining a path for capillary fluid flow; and an upper layer adjacent to the hydrophobic layer, wherein a surface of the upper layer facing the hydrophobic layer is hydrophilic; or b) a pre-evacuated container or tube having an inlet and an outlet end; a blood-retaining chamber at the inlet end of the container or tube; a removable serum-retaining chamber at the outlet end of the container or tube; and a filter disposed within the container between the blood-retaining chamber and the serum-retaining chamber. The present invention includes an apparatus having:

[0043] In a still further embodiment, the device used during plasma separation in a) further comprises a bottom layer adjacent to the hydrophobic layer, the surface of the bottom layer facing the hydrophobic layer being hydrophilic.

[0044] In a further aspect, the top layer, the hydrophobic layer, the bottom layer, or any combination thereof, are adhered to one another.

[0045] In another embodiment of the above system, the at least one microchannel extends longitudinally along a portion of the hydrophobic layer to an opening at the second end.

[0046] In yet another embodiment of the above system, at least one microchannel is less than about 80 mm in length.

[0047] In yet another embodiment of the above system, at least one microchannel is less than about 5 mm wide.

[0048] In still yet a further aspect of the above system, the top layer, the bottom layer, or the top and bottom layers are generally hydrophilic.

[0049] In yet another aspect of the above system, the overall composition of the top layer, the bottom layer, or both the top and bottom layers, respectively, comprises the same or different materials.

[0050] In still yet further aspects of the above-described systems, the hydrophobic layer, the top layer, the bottom layer, or any combination thereof, has a combined thickness of from about 100 to about 600 microns.

[0051] In still yet further embodiments of the above-described systems, the hydrophobic layer has a thickness of about 50 to about 200 microns.

[0052] In still yet further aspects of the above-described systems, the top layer, the bottom layer, or each of the top and bottom layers has a thickness of about 50 to about 200 microns.

[0053] In still yet a further aspect of the above system, the upper layer includes a sample inlet.

[0054] In still yet a further aspect of the above-described system, the hydrophobic layer and optionally the bottom layer include an opening below the sample inlet.

[0055] In still yet a further aspect of the above system, the sample inlet comprises a separation membrane.

[0056] In still yet a further aspect of the above system, the separation membrane is a plasma separation membrane.

[0057] In still yet a further aspect of the above-described system, the system further comprises a hydrophilic mesh or film located above the separation membrane, below the separation membrane, or both above and below the separation membrane.

[0058] In still further aspects of the above-described systems, the opening in the hydrophobic layer is connected to a first end of at least one microchannel.

[0059] In still further aspects of the above-described systems, the system further comprises a flocculating agent. In some aspects, the flocculating agent is coated or incorporated into one or more of the top layer, hydrophobic layer, bottom layer, upper substrate, separation membrane, hydrophilic mesh or hydrophilic film, or any combination thereof. In some aspects, the flocculating agent comprises lectin, Merquat-100, concanavalin A, DEAE-dextran, poly-L-lysine, polyvinylpyrrolidone, poly(2-(dimethylamino)ethyl methacrylate), or any combination thereof.

[0060] In still other embodiments of the above-described systems, the filter allows passage of particles or molecules smaller than about 0.7 microns, about 0.6 microns, about 0.5 microns, about 0.4 microns, or about 0.3 microns.

[0061] In still further aspects of the above-described systems, a pressure differential between the blood retention chamber and the serum retention chamber allows whole blood to move from the blood retention chamber through the filter to produce serum and / or plasma that is collected in the serum retention chamber.

[0062] In yet another aspect of the system, the reactor includes an opening.

[0063] In yet another aspect of the system, the microsampling device includes a housing, a microneedle, a lancet, a microlancet, a blade, a microblade, a microscrew, or any combination thereof, coupled to the housing, and a receptacle coupled to the housing; a capillary blood sample is collected in the receptacle.

[0064] In yet another aspect of the system, the receptacle is removably coupled to the housing.

[0065] In yet another aspect of the system, the micro-sampling device further includes a cap coupled to the receptacle, the cap sealing the capillary blood sample within the receptacle.

[0066] In yet another aspect of the system, the micro-sampling device further includes an actuator movable relative to the housing.

[0067] In still other aspects, the system has a plasma separation device in fluid communication with the opening at any point along the reactor. In yet further aspects, the plasma separation device is placed in fluid communication with the opening at one end, side, or center of the reactor. In still yet further aspects, the plasma separation device is placed in fluid communication with the opening at an end or side of the reactor at an angle.

[0068] In still another aspect, the system further comprises a transfer tube. In some additional aspects, the transfer tube comprises a cap or stopper.

[0069] In yet another aspect of the system, the plasma separation device includes an inlet for receiving the capillary blood sample from the microsampling device and an outlet for the processed capillary blood sample to exit the plasma separation device. In some aspects, the outlet of the plasma separation device is in fluid communication with an opening in the reactor. In yet another aspect, the outlet of the plasma separation device is in fluid communication with a cap or stopper of the transfer tube.

[0070] In still yet a further aspect of the system, a cap or stopper of the transfer tube is in fluid communication with an opening in the reactor.

[0071] In yet another aspect of the system, the receptacle is squeezed to force the capillary blood sample through the plasma separation device and into a reactor or transfer tube.

[0072] In yet another aspect of the system, the receptacle includes a plunger for forcing the capillary blood sample through the plasma separation device and into a reactor or transfer tube.

[0073] In yet a further aspect of the system, the plasma separation device is integrated within the receptacle.

[0074] In yet another aspect of the system, the receptacle is a reactor.

[0075] In a further aspect of the system, the plasma separation device is integrated within the reactor.

[0076] In yet another aspect of the system, the plasma separation device is integrated into the transfer tube.

[0077] In yet a further aspect of the system, the plasma separation device comprises a filter, a membrane, a synthetic paper, or any combination thereof.

[0078] In still further aspects of the system, the amount of (i) UCH-L1, GFAP, or UCH-L1 and GFAP; or (ii) CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof, is determined (a) within about 25 minutes from the time the sample is collected; (b) within less than about 20 minutes from the time the sample is collected; (c) within about 4 to about 20 minutes from the time the sample is collected; (d) within about 15 to about 18 minutes from the time the sample is collected; or (e) within less than about 18 minutes from the time the sample is collected.

[0079] In yet further embodiments of the system, the amounts of (i) UCH-L1, GFAP, or UCH-L1 and GFAP; or (ii) CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof, are communicated by a device. In further embodiments, the amounts of (i) UCH-L1, GFAP, or UCH-L1 and GFAP; or (ii) CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof, are communicated in a document and / or spreadsheet, on a mobile device, on a computer, on a website, in an email, or any combination thereof. In yet another embodiment, the amounts of (i) UCH-L1, GFAP, or UCH-L1 and GFAP; or (ii) CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof, are communicated by display on a device (e.g., a point-of-care device, a non-point-of-care device, etc.). In yet another aspect, at least portions of the system may be used in a distributed setting.

[0080] In still further aspects of the above-described systems, communicating the determined amount of (i) UCH-L1, GFAP or a combination thereof; or (ii) CK-MB, β-hCG, TSH, homocysteine, free T4 or any combination thereof in the sample comprises communicating the level of (i) UCH-L1, GFAP or a combination thereof; or (ii) CK-MB, β-hCG, TSH, homocysteine, free T4 or any combination thereof in the sample.

[0081] In yet another aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: performing at least one assay for (i) ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof; or (2) CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof, in a blood sample obtained from the subject to determine the amount of (i) UCH-L1, GFAP, or UCH-L1 and GFAP; or (ii) CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof; and 1. A method comprising communicating the amount of (i) UCH-L1, GFAP, or UCH-L1 and GFAP; or (ii) CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof, determined in the sample; The sample is collected by use of a syringe, a standard needle, or a combination thereof; Furthermore, the sample a) a hydrophobic layer having first and second ends and including at least one microchannel defining a path for capillary fluid flow; and an upper layer adjacent to the hydrophobic layer, wherein a surface of the upper layer facing the hydrophobic layer is hydrophilic; or b) a pre-evacuated container or tube having an inlet and an outlet end; a blood holding chamber at the inlet end of the container or tube; a removable serum holding chamber at the outlet end of the container or tube; A filter disposed within the container between the blood holding chamber and the serum holding chamber. The plasma is processed prior to performing the assay using a plasma separation device comprising an apparatus having Regarding the method.

[0082] In the above-described methods, the blood sample is a venous blood sample or a capillary blood sample. In some aspects, the sample is collected in a decentralized or centralized setting.

[0083] In still further aspects of the above-described methods, the assay comprises: (I) (a) an anti-UCH-L1 antibody that binds to UCH-L1 to determine the amount of UCH-L1 in the sample; (b) an anti-GFAP antibody that binds to GFAP to determine the amount of GFAP in the sample; or (c) a combination of (a) and (b); or (II) (a) an anti-CK-MB antibody that binds to CK-MB to determine the amount of CK-MB in the sample; (b) an anti-β-hCG antibody that binds to β-hCG to determine the amount of β-hCG in the sample; (c) an anti-TSH antibody that binds to TSH to determine the amount of TSH in the sample; (d) an anti-homocysteine ​​antibody that binds to homocysteine ​​to determine the amount of homocysteine ​​in the sample; (e) an anti-free T4 antibody that binds to free T4 to determine the amount of free T4 in the sample; or (f) any combination of (a) through (e). The method includes contacting the

[0084] In yet a further aspect of the above method, the blood sample is capillary blood and the assay comprises: (I) (a) if the assay is for GFAP, the GFAP assay comprises a scaling factor of GFAP in the capillary sample compared to GFAP in the venous sample of about 1.0:1.0; (b) if the assay is for UCH-L1, the UCH-L1 assay comprises a scaling factor of UCH-L1 in the capillary sample compared to UCH-L1 in the venous sample of about 2.5:1.0 to about 1.5:1.0; or (c) a combination of (a) and (b); or (II) (a) if the assay is for CK-MB, the CK-MB assay comprises a conversion factor of CK-MB in the capillary sample relative to CK-MB in the venous sample of about 0.5:1.0 to about 1:0:1.2; (b) if the assay is for β-hCG, the β-hCG assay comprises a conversion factor of β-hCG in the capillary sample relative to β-hCG in the venous sample of about 0.8:1.0 to about 1:0:1.4; (c) if the assay is for TSH, the TSH assay comprises a conversion factor of β-hCG in the capillary sample relative to β-hCG in the venous sample of about 0.75:1.0 to about 1.2:1.0. (d) if the assay is for homocysteine, the homocysteine ​​assay includes a conversion factor of homocysteine ​​in the capillary sample compared to homocysteine ​​in the venous sample of from about 1.2:1.0 to about 0.9:1.0; (e) if the assay is for free T4, the free T4 assay includes a conversion factor of free T4 in the capillary sample compared to free T4 in the venous sample of from about 0.8:1.0 to about 1.2:1.0; or (f) any combination of (a)-(e).

[0085] In still further aspects of the above-described methods, if the assay is (a) an assay for GFAP, UCH-L1, or GFAP and UCH-L1, the method is used to aid in the diagnosis and assessment of a subject who has suffered or may have suffered a head injury; (b) if the assay is for CK-MB, the method is used to diagnose myocardial infarction in a subject; (c) if the assay is for β-hCG, the method is used to determine whether the subject is pregnant; or (d) if the assay is for TSH, the method is used to assess thyroid function in a subject, diagnose thyroid disease in a subject, or administer steroids to a subject. (e) if the assay is for homocysteine, the method is used to diagnose hyperhomocysteinemia, homocystinuria, or hyperhomocysteinemia and homocystinuria in a subject, or to treat a subject with hyperhomocysteinemia, homocystinuria, or hyperhomocysteinemia and homocystinuria; or (f) if the assay is for free T4, the method is used to assess thyroid function in a subject, diagnose thyroid disease in a subject, treat thyroid disease in a subject, or any combination thereof.

[0086] In still further aspects of the above-described methods, the amount of (a) UCH-L1, GFAP, and UCH-L1 and UCH-L1 and GFAP; or (b) CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof, is transmitted within (a) about 25 minutes from the time the sample is collected; (b) less than about 20 minutes from the time the sample is collected; (c) about 4 to about 20 minutes from the time the sample is collected; (d) about 15 to about 18 minutes from the time the sample is collected; or (e) less than about 18 minutes from the time the sample is collected.

[0087] In still further aspects of the above-described methods, the amount of UCH-L1, GFAP, CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof, is communicated (a) by a point-of-care device or a non-point-of-care device, in a document and / or spreadsheet, on a mobile device, on a computer, on a website, in an email, or any combination thereof; or (b) by display on a point-of-care device or a non-point-of-care device.

[0088] In still further aspects of the above-described methods, communicating the determined amount of (i) UCH-L1, GFAP or a combination thereof; or (ii) CK-MB, β-hCG, TSH, homocysteine, free T4 or any combination thereof in the sample comprises communicating the level of (i) UCH-L1, GFAP or a combination thereof; or (ii) CK-MB, β-hCG, TSH, homocysteine, free T4 or any combination thereof in the sample.

[0089] In still further aspects of the above-described methods, the assays used in the above-described methods can be analog assays, digital assays, or a combination of analog assays or digital assays. In still further aspects of the above-described methods, the subject is a human.

[0090] In yet a further embodiment of the above method, the device in a) further comprises a bottom layer adjacent to the hydrophobic layer, the surface of the bottom layer facing the hydrophobic layer being hydrophilic.

[0091] In yet a further aspect of the above method, the top layer, the hydrophobic layer, the bottom layer, or any combination thereof, are adhered to one another.

[0092] In still further aspects of the above-described methods, the at least one microchannel extends longitudinally along a portion of the hydrophobic layer to an opening at the second end.

[0093] In still other embodiments of the above method, at least one microchannel is less than about 80 mm in length.

[0094] In still yet further aspects of the above method, at least one microchannel is less than about 5 mm wide.

[0095] In still yet a further aspect of the above method, the top layer, the bottom layer, or the top and bottom layers are generally hydrophilic.

[0096] In still yet further aspects of the above-described methods, the overall composition of the top layer, the bottom layer, or both the top and bottom layers, respectively, comprises the same or different materials.

[0097] In still yet further aspects of the above-described methods, the hydrophobic layer, the top layer, the bottom layer, or any combination thereof, has a combined thickness of from about 100 to about 600 microns.

[0098] In still yet further aspects of the above method, the hydrophobic layer has a thickness of about 50 to about 200 microns.

[0099] In still yet further aspects of the above-described methods, the top layer, the bottom layer, or each of the top and bottom layers has a thickness of from about 50 to about 200 microns.

[0100] In still yet a further aspect of the above method, the top layer includes a sample inlet.

[0101] In still yet further aspects of the above-described methods, the hydrophobic layer and optionally the bottom layer comprise an opening below the sample inlet.

[0102] In still yet a further aspect of the above method, the sample inlet comprises a separation membrane.

[0103] In still yet a further aspect of the above method, the separation membrane is a plasma separation membrane.

[0104] In still yet further aspects of the above-described methods, the separation membrane (e.g., plasma separation membrane) further comprises a hydrophilic mesh or hydrophilic film located above the separation membrane, below the separation membrane, or both above and below the separation membrane.

[0105] In still further aspects of the above-described methods, the opening in the hydrophobic layer is connected to a first end of at least one microchannel.

[0106] In still further aspects of the above methods, the method further comprises a flocculating agent, hi some aspects, the flocculating agent is coated or incorporated into or on one or more of the top layer, hydrophobic layer, bottom layer, upper substrate, separator membrane, hydrophilic mesh or hydrophilic film, or any combination thereof.

[0107] In still further aspects of the above-described methods, the agglutinating agent comprises lectin, Merquat-100, concanavalin A, DEAE-dextran, poly-L-lysine, polyvinylpyrrolidone, poly(2-(dimethylamino)ethyl methacrylate), or any combination thereof.

[0108] In further embodiments of the above methods, the filter allows passage of particles or molecules smaller than about 0.7 microns, about 0.6 microns, about 0.5 microns, about 0.4 microns, or about 0.3 microns.

[0109] In still yet further aspects of the above-described methods, a pressure differential between the blood retention chamber and the serum retention chamber allows whole blood to move from the blood retention chamber through the filter to produce serum and / or plasma that is collected in the serum retention chamber.

[0110] In yet another aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: a plasma separation device for processing a whole blood sample obtained from a subject into serum and / or plasma; a reactor that receives serum and / or plasma from a subject and contains assays for (i) ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof; or (ii) CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof; and and an apparatus for analyzing the reactor to provide the amount of (i) UCH-L1, GFAP, or UCH-L1 and GFAP; or (ii) CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof, in the sample. A system comprising: The plasma separation device a) a hydrophobic layer having first and second ends and including at least one microchannel defining a path for capillary fluid flow; and an upper layer adjacent to the hydrophobic layer, wherein a surface of the upper layer facing the hydrophobic layer is hydrophilic; or b) a pre-evacuated container or tube having an inlet and an outlet end; a blood holding chamber at the inlet end of the container or tube; a removable serum holding chamber at the outlet end of the container or tube; A filter disposed within the container between the blood holding chamber and the serum holding chamber. The present invention relates to a system including an apparatus having:

[0111] In still further aspects of the system, the amount of (i) UCH-L1, GFAP, or UCH-L1 and GFAP; or (ii) CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof, is determined (a) within about 25 minutes from the time the sample is collected; (b) within less than about 20 minutes from the time the sample is collected; (c) within about 4 to about 20 minutes from the time the sample is collected; (d) within about 15 to about 18 minutes from the time the sample is collected; or (e) within less than about 18 minutes from the time the sample is collected.

[0112] In some aspects, in the above-described systems, the blood sample is a venous blood sample or a capillary blood sample.

[0113] In yet another aspect, in the above-described system, the samples are collected in a decentralized or centralized setting.

[0114] In a still further aspect, in the above system, the device in a) further comprises a bottom layer adjacent to the hydrophobic layer, wherein the surface of the bottom layer facing the hydrophobic layer is hydrophilic.

[0115] In still other aspects, in the above-described systems, the top layer, the hydrophobic layer, the bottom layer, or any combination thereof, are adhered to one another.

[0116] In still further aspects, in the above-described system, at least one microchannel extends longitudinally along a portion of the hydrophobic layer to an opening at the second end.

[0117] In still further embodiments, in the above system, at least one microchannel is less than about 80 mm in length.

[0118] In still further embodiments, in the above system, at least one microchannel is less than about 5 mm wide.

[0119] In yet a further aspect, in the above system, the top layer, the bottom layer, or the top and bottom layers are generally hydrophilic.

[0120] In still further aspects, in the above-described systems, the overall composition of the top layer, the bottom layer, or both the top and bottom layers, respectively, comprises the same or different materials.

[0121] In still further embodiments, in the above-described systems, the hydrophobic layer, the top layer, the bottom layer, or any combination thereof, has a combined thickness of about 100 to about 600 microns.

[0122] In yet a further embodiment, in the above system, the hydrophobic layer has a thickness of about 50 to about 200 microns.

[0123] In yet a further aspect, in the above system, the top layer, the bottom layer, or each of the top and bottom layers has a thickness of about 50 to about 200 microns.

[0124] In a still further aspect, in the system described above, the upper layer includes a sample inlet.

[0125] In a still further aspect, in the system described above, the hydrophobic layer and optionally the bottom layer comprise an opening below the sample inlet.

[0126] In a still further aspect, in the system described above, the sample inlet comprises a separation membrane.

[0127] In yet a further aspect, in the above-described system, the separation membrane is a plasma separation membrane. In yet a further aspect, the separation membrane (e.g., plasma separation membrane) further comprises a hydrophilic mesh or film located above the separation membrane, below the separation membrane, or both above and below the separation membrane.

[0128] In still further embodiments, in the system described above, the opening in the hydrophobic layer is connected to a first end of at least one microchannel.

[0129] In still further aspects of the above-described systems, the system further comprises a flocculating agent, hi some aspects, the flocculating agent is coated or incorporated into or on one or more of the top layer, the hydrophobic layer, the bottom layer, the upper substrate, the separator membrane, the hydrophilic mesh or the hydrophilic film, or any combination thereof.

[0130] In still further embodiments of the above-described system, the agglutinating agent comprises lectin, Merquat-100, concanavalin A, DEAE-dextran, poly-L-lysine, polyvinylpyrrolidone, poly(2-(dimethylamino)ethyl methacrylate), or any combination thereof.

[0131] In still further embodiments of the above-described systems, the filter allows passage of particles or molecules smaller than about 0.7 microns, about 0.6 microns, about 0.5 microns, about 0.4 microns, or about 0.3 microns.

[0132] In yet a further aspect of the above-described system, a pressure differential between the blood retention chamber and the serum retention chamber allows whole blood to move from the blood retention chamber through the filter to produce serum and / or plasma that is collected in the serum retention chamber.

[0133] In yet further embodiments of the system, the amounts of (i) UCH-L1, GFAP, or UCH-L1 and GFAP; or (ii) CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof, are communicated by a device. In further embodiments, the amounts of (i) UCH-L1, GFAP, or UCH-L1 and GFAP; or (ii) CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof, are communicated in a document and / or spreadsheet, on a mobile device, on a computer, on a website, in an email, or any combination thereof. In yet another embodiment, the amounts of (i) UCH-L1, GFAP, or UCH-L1 and GFAP; or (ii) CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof, are communicated by display on a device (e.g., a point-of-care device, a non-point-of-care device, etc.). In yet another aspect, at least portions of the system may be used in a distributed setting.

[0134] In still further aspects of the above-described systems, communicating the determined amount of (i) UCH-L1, GFAP or a combination thereof; or (ii) CK-MB, β-hCG, TSH, homocysteine, free T4 or any combination thereof in the sample comprises communicating the level of (i) UCH-L1, GFAP or a combination thereof; or (ii) CK-MB, β-hCG, TSH, homocysteine, free T4 or any combination thereof in the sample. [Brief explanation of the drawings]

[0135] [Figure 1] FIG. 1 illustrates a system for diagnosing and assessing a subject who has sustained or may have sustained a head injury according to one embodiment of the present disclosure. [Figure 2A] 2A-2E show the operational steps of the system of FIG. [Figure 2B] 2A-2E show the operational steps of the system of FIG. [Figure 2C] 2A-2E show the operational steps of the system of FIG. [Figure 2D] 2A-2E show the operational steps of the system of FIG. [Figure 2E] 2A-2E show the operational steps of the system of FIG. [Figure 3] FIG. 3 illustrates an additional and alternative embodiment of the system of FIG. [Figure 4] FIG. 4 illustrates an additional and alternative embodiment of the system of FIG. [Figure 5] FIG. 5 illustrates an additional and alternative embodiment of the system of FIG. [Figure 6A] 6A-6C show additional and alternative embodiments of the system of FIG. [Figure 6B] 6A-6C show additional and alternative embodiments of the system of FIG. [Figure 6C] 6A-6C show additional and alternative embodiments of the system of FIG. [Figure 7] FIG. 7 shows one embodiment of the apparatus of the present disclosure that can be used as a plasma separation device. [Figure 8] FIG. 8 shows another embodiment of the apparatus of the present disclosure that can be used as a plasma separation device. [Figure 9] FIG. 9 shows a further embodiment of the apparatus of the present disclosure used as a plasma separation device as described in Example 1. [Figure 10]10 shows a device including an apparatus of the present disclosure operably connected, removably coupled, or in fluid communication with a sample analysis cartridge, wherein the second end of the microchannel of the apparatus is in fluid communication with a sample application area in the sample analysis cartridge. [Figure 11] Figure 11 shows GFAP (pg / mL) concentration readings in TBI plasma cartridges for donors collected in the study described in Example 3. Included are individual results (left) and mean results with 95% CI (right). Each error bar was generated using the 95% confidence interval of the mean. [Figure 12] Figure 12 shows UCH-L1 concentration readings (pg / mL) in TBI plasma cartridges for donors collected in the study described in Example 3. Individual results (left) and average results (right) are included. [Figure 13] Figure 13 shows GFAP plasma readings (pg / mL) for only venous and capillary plasma samples in TBI plasma cartridges for donors collected in the study described in Example 4. Included are individual results (left) and mean results with 95% CI (right). Each error bar was generated using the 95% confidence interval of the mean. [Figure 14] Figure 14 shows UCH-L1 plasma readings (pg / mL) for only venous and capillary plasma samples in TBI plasma cartridges for donors collected in the study described in Example 4. Included are individual results (left) and mean results with 95% CI (right). Each error bar was generated using the 95% confidence interval of the mean. [Figure 15] Figure 15 shows CK-MB concentration results individually (left) and by mean with 95% confidence intervals (right) as described in Example 5. Each error bar was generated using the 95% confidence interval of the mean. [Figure 16]Figure 16 shows β-hCG concentration results from phases 3.2-3.3 individually (left) and by mean with 95% confidence interval (right), as described in Example 6. Each error bar is generated using the 95% confidence interval of the mean. DETAILED DESCRIPTION OF THE INVENTION

[0136] The present disclosure relates to systems and methods for determining the amount of ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), CK-MB, β-hCG, thyroid-stimulating hormone (TSH), homocysteine, free thyroxine (free T4), or any combination thereof, in a capillary blood sample obtained from a subject (e.g., a human subject). In some aspects, the methods and systems include performing at least one assay for UCH-L1, GFAP, or a combination thereof. In still other aspects, the assay includes contacting the capillary blood sample with: (a) an anti-UCH-L1 antibody that binds to UCH-L1 to determine the amount of UCH-L1 in the sample; (b) an anti-GFAP antibody that binds to GFAP to determine the amount of GFAP in the sample; or (c) a combination of (a) and (b). In other aspects, the methods and systems include performing at least one assay for CK-MB, β-hCG, TSH, homocysteine, free thyroxine (free T4), or any combination thereof. In still other aspects, the assay includes contacting a capillary blood sample with (a) an anti-CK-MB antibody that binds to CK-MB to determine the amount of CK-MB in the sample; (b) an anti-β-hCG antibody that binds to β-hCG to determine the amount of β-hCG in the sample; (c) an anti-TSH antibody that binds to TSH to determine the amount of TSH in the sample; (d) an anti-homocysteine ​​antibody that binds to homocysteine ​​to determine the amount of homocysteine ​​in the sample; (e) an anti-free T4 antibody that binds to free T4 to determine the amount of free T4 in the sample; or (f) any combination of (a)-(e). In still other aspects, the methods include collecting the sample (1) in a decentralized setting; (2) without the use of a syringe, standard needle, or a combination thereof; (3) by a user untrained in collecting blood samples from subjects; (4) by a robot; (5) by a blood collection device applied to oneself or another; or (6) by any combination of (1)-(5). In still further aspects, the amount of UCH-L1, GFAP, CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof determined in the capillary blood sample is transmitted within about 4 to about 40 minutes from the time the sample is collected.In some aspects, the amount of UCH-L1, GFAP, or a combination thereof determined in a capillary blood sample obtained according to the methods described herein can be used to aid in the diagnosis and / or assessment of whether a subject has suffered, may have suffered, or is suspected of having suffered a head injury, such as a traumatic brain injury (TBI).

[0137] In other embodiments, the amount of CK-MB determined in a capillary blood sample obtained according to the methods described herein can be used to diagnose myocardial infarction in a subject. In still other embodiments, the amount of β-hCG determined in a capillary blood sample obtained according to the methods described herein can be used to determine whether a subject is pregnant. In still other embodiments, the amount of TSH determined in a capillary blood sample obtained according to the methods described herein can be used to assess thyroid function in a subject, diagnose thyroid disease in a subject, and / or treat thyroid disease in a subject. In still other embodiments, the amount of homocysteine ​​determined in a capillary blood sample obtained according to the methods described herein can be used to diagnose hyperhomocysteinemia and / or homocystinuria in a subject, or treat a subject with hyperhomocysteinemia and / or homocystinuria. In still other embodiments, the amount of free T4 determined in a capillary blood sample obtained according to the methods described herein can be used to assess thyroid function in a subject, diagnose thyroid disease in a subject, treat thyroid disease in a subject, or any combination thereof.

[0138] In still yet other aspects, the method includes processing the sample prior to performing the assay. In some aspects, the sample is processed using centrifugation. In still other aspects, the sample is processed using a plasma separation device.

[0139] In other embodiments, the system includes a microsampling device for collecting a capillary blood sample from a subject and a reactor for receiving the capillary blood sample. In additional embodiments, the system includes a plasma separation device for producing a processed capillary blood sample from the capillary blood. The system further includes an instrument for analyzing the reactor to determine the amount of UCH-L1, GFAP, CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof, in the sample.

[0140] The section headings used in this section and throughout the disclosure herein are for organizational purposes only and are not intended to be limiting.

[0141] 1.Definition Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, the preferred methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0142] As used herein, the terms "comprise(s)," "include(s)," "having," "having," "can be," and "containing" are open-ended transitional phrases, transitional terms, or transitional words that do not exclude additional acts or structures. The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments that "comprise," "consist of," or "consist essentially of" the embodiments or elements presented herein, whether expressly stated or otherwise.

[0143] In this specification, to recite numerical ranges, each intervening number with the same precision is expressly contemplated. For example, for the range of 6 to 9, the numbers 7 and 8 are also contemplated in addition to 6 and 9, and for the range of 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0144] As used herein, the term "affinity matured antibody" refers to an antibody that has undergone a process to improve the affinity (i.e., K D , k d or k aThe term "affinity matured" is used to refer to an antibody with one or more alterations in one or more CDRs that result in an improvement over a parent antibody that does not have those alterations. Exemplary affinity matured antibodies have nanomolar or even picomolar affinities for the target antigen. Various procedures for generating affinity matured antibodies are known in the art, including screening combinatorial antibody libraries prepared using BioDisplay technology. For example, Marks et al., BioTechnology, 10:779-783 (1992) describe affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and / or framework residues has been described by Barbas et al., Proc. Nat. Acad. Sci. USA, 91:3809-3813 (1994); Schier et al., Gene, 169:147-155 (1995); Yelton et al., J. Immunol., 155:1994-2004 (1995); Jackson et al., J. Immunol., 154(7):3310-3319 (1995), and Hawkins et al., J. Mol. Biol., 226:889-896 (1992). Selective mutagenesis positions and selective mutations at contact or hypermutation positions with activity-enhancing amino acid residues are described in U.S. Patent No. 6,914,128 B1.

[0145] As used herein, "amount" refers to a specified (e.g., more or less) content or number, e.g., the number is a level, such as a position on a real or imaginary scale of amount or content, or a concentration, e.g., the relative amount of a given substance contained within a solution or in a particular volume of space, e.g., the amount of solute per unit volume of solution.

[0146] As used herein, "analog assay" refers to an assay in which the presence and / or amount of an analyte in a test sample is determined by measuring the total signal (e.g., fluorescence, color, etc.) produced by the analyte in the entire reaction mixture (e.g., reactor). In an analog assay, noise is indistinguishable from the signal. An example of an analog assay is an assay in which the presence and / or amount of an analyte is determined by measuring the total signal produced from multiple beads or microparticles contained in a reactor.

[0147] As used herein, "antibody" and "antibodies" include monoclonal antibodies, polyspecific antibodies, human antibodies, humanized antibodies (fully humanized antibodies or partially humanized antibodies), avian (e.g., duck or goose) antibodies, shark antibodies, whale antibodies, and mammalian antibodies, including non-primate (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamster, guinea pig, cat, dog, rat, mouse, etc.) antibodies or non-human primate (e.g., monkey, chimpanzee, etc.) antibodies. "Antibody" refers to, but is not limited to, animal antibodies, recombinant antibodies, chimeric antibodies, single-chain Fvs ("scFvs"), single-chain antibodies, single-domain antibodies, Fab fragments, F(ab') fragments, F(ab') fragments, disulfide-linked Fvs ("sdFvs"), and anti-idiotypic ("anti-Id") antibodies, dual-domain antibodies, dual-variable domain (DVD) or triple-variable domain (TVD) antibodies (dual-variable domain immunoglobulins and methods for producing them are described in Wu, C. et al., Nature Biotechnology, 25(11):1290-1297 (2007) and PCT International Application No. 2001 / 058956, the contents of each of which are incorporated herein by reference), and functionally active, epitope-binding fragments of any of the above. Antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an analyte-binding site. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. For simplicity, antibodies to an analyte will frequently be referred to herein as either "anti-analyte antibodies" or simply "analyte antibodies" (e.g., anti-UCH-L1 or UCH-L1 antibodies, anti-GFAP or GFAP antibodies, anti-CK-MB or CK-MB antibodies, anti-β-hCG or β-hCG antibodies, anti-TSH or TSH antibodies, anti-homocysteine ​​or homocysteine ​​antibodies, or anti-free T4 or free T4 antibodies).

[0148] As used herein, the term "antibody fragment" refers to a portion of an intact antibody that contains the antigen-binding site or variable region. The portion does not contain the heavy chain constant domain of the Fc region of the intact antibody (i.e., CH2, CH3, or CH4, depending on the antibody isotype). Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, Fab'-SH fragments, F(ab')2 fragments, Fd fragments, Fv fragments, diabodies, single-chain Fv (scFv) molecules, single-chain polypeptides containing only one light chain variable domain, single-chain polypeptides containing three CDRs of a light chain variable domain, single-chain polypeptides containing only one heavy chain variable region, and single-chain polypeptides containing three CDRs of a heavy chain variable region.

[0149] As used herein, "aperture" refers to an opening, hole, or gap.

[0150] "Area under the curve" or "AUC" refers to the area under the ROC curve. The AUC under the ROC curve is a measure of accuracy. An AUC of 1 represents a perfect test, while an AUC of 0.5 represents a non-significant test. Preferred AUCs may be at least about 0.700, at least about 0.750, at least about 0.800, at least about 0.850, at least about 0.900, at least about 0.910, at least about 0.920, at least about 0.930, at least about 0.940, at least about 0.950, at least about 0.960, at least about 0.970, at least about 0.980, at least about 0.990, or at least about 0.995.

[0151] "Beta-human chorionic gonadotropin (hCG)" or "β-hCG" refers to the beta (β) subunit of human chorionic gonadotropin (hCG). Human chorionic gonadotropin is a sialoglycoprotein with a molecular weight of approximately 46,000 daltons. HCG is secreted primarily by placental trophoblasts immediately after implantation of the fertilized egg into the uterine wall. The rapid rise in serum hCG levels after conception makes it a useful marker for early confirmation and monitoring of pregnancy. Physiologically, hCG appears to maintain the corpus luteum, thereby allowing the synthesis of progesterone and estrogen to support the endometrium. As an uncomplicated pregnancy progresses, the placenta assumes responsibility for the production of these hormones. Serum hCG levels increase to a peak concentration, then decrease and reach a steady state. HCG circulates as an intact molecule in the serum of normal women during uncomplicated pregnancies. The subunits are rapidly cleaved and eliminated by the kidney. The placental hormone hCG is similar to luteinizing hormone (LH), follicle-stimulating hormone (FSH), and thyroid-stimulating hormone (TSH). All are glycoproteins consisting of two noncovalently linked subunits, designated alpha and beta, each with a carbohydrate side chain attached. The alpha subunits of these glycoproteins are highly similar. In contrast, the beta subunit portion determines the biological and immunochemical specificity. The beta subunits of hCG and LH show considerable homology in amino acid content. The specific amino acid residues in the beta subunit of hCG confer immunochemical specificity. In addition, β-hCG is also used to evaluate other diseases in which this analyte is implicated, such as the diagnosis and management of tumors of ovarian, placental, or testicular origin, gestational trophoblastic disease, or hCG-producing germ cell tumors.

[0152] As used herein, the terms "beads" and "particles" are used interchangeably and refer to a substantially spherical solid support. One example of a bead or particle is a microparticle. Microparticles that may be used herein may be of any type known in the art. For example, beads or particles may be magnetic beads or particles. Magnetic beads / particles may be ferromagnetic, ferrimagnetic, paramagnetic, superparamagnetic, or ferrofluid. Exemplary ferromagnetic materials include Fe, Co, Ni, Gd, Dy, CrO2, MnAs, MnBi, EuO, and NiO / Fe. Examples of ferrimagnetic materials include NiFe2O4, CoFe2O4, and Fe3O4 (or FeO.Fe2O3). Beads may be magnetic and have a solid core portion surrounded by one or more nonmagnetic layers. Alternatively, the magnetic portion may be a layer surrounding the nonmagnetic core. Microparticles can be of any size that works in the methods described herein, for example, from about 0.75 to about 5 nm, or from about 1 to about 5 nm, or from about 1 to about 3 nm.

[0153] As used herein, the term "binding protein" refers to a monomeric or multimeric protein that binds to and forms a complex with a binding partner, such as a polypeptide, antigen, compound, or other molecule, or any type of substrate. A binding protein specifically binds to a binding partner. Binding proteins include antibodies, as well as antigen-binding fragments thereof and various other forms and derivatives thereof known in the art and described herein below, and other molecules containing one or more antigen-binding domains that bind to antigen molecules or specific sites (epitopes) on antigen molecules. Thus, binding proteins include, but are not limited to, tetrameric immunoglobulin antibodies, IgG molecules, IgG1 molecules, monoclonal antibodies, chimeric antibodies, CDR-grafted antibodies, humanized antibodies, affinity-matured antibodies, and fragments of any such antibodies that retain the ability to bind to antigen.

[0154] As used herein, the term "bispecific antibody" is used to refer to full-length antibodies generated by quadroma technology (see Milstein et al., Nature, 305(5934):537-540 (1983)), chemical conjugation of two different monoclonal antibodies (see Staerz et al., Nature, 314(6012):628-631 (1985)), or knob-into-hole (KIH) or similar techniques that introduce mutations within the Fc region (see Holliger et al., Proc. Natl. Acad. Sci. USA, 90(14):6444-6448 (1993)), resulting in multiple different immunoglobulin molecular species, only one of which is the functional bispecific antibody. A bispecific antibody binds to one antigen (or epitope) in one of its two binding arms (one HC / LC pair) and to a different antigen (or epitope) in its second arm (a different HC / LC pair). By this definition, a bispecific antibody has two significantly different antigen-binding arms (both specificity and CDR sequences) and is monovalent for each antigen to which it binds.

[0155] As used herein, a "capillary blood sample" refers to a sample of blood from capillaries obtained (e.g., extracted) through a subject's skin (not a vein) using a syringe, needle, or any other suitable device, or a combination thereof. For example, a whole blood sample may be obtained from the skin of the fingers and / or toes, hands, feet (including heels), earlobes, locations on the arms and / or legs, chest, back, head, or any combination thereof. In some embodiments, the capillary blood sample is whole blood, serum, or plasma. In yet other embodiments, the capillary blood sample contains primarily capillary blood, but may also contain or include a small amount or percentage of interstitial fluid.

[0156] As used herein, "cartridge" refers to a hollow container and / or chip containing one or more substances and / or components (e.g., liquids, reagents (e.g., antibodies and / or antigens), and / or particles (e.g., beads or microparticles)) for insertion into an apparatus (e.g., a point-of-care device). In some embodiments, the cartridge has one or more openings. In some embodiments, the cartridge is a microfluidic cartridge.

[0157] As used herein, "communicating an amount" refers to communicating an amount in terms of presence (i.e., a qualitative measure) or in terms of level(s) (i.e., a quantitative measure), such that "amount" refers to presence or level, as described herein. In some aspects, "communicating an amount" refers to communicating the presence (e.g., a qualitative measure) of a biomarker. In other aspects, "communicating an amount" refers to communicating the level (e.g., a quantitative measure) of a biomarker.

[0158] "Creatine kinase-myoglobin-bound" or "CK-MB" refers to an 84,000 molecular weight enzyme that represents a significant proportion of the creatine kinase present in myocardial tissue. The appearance of CK-MB in a biological sample, such as serum, in the absence of major muscle trauma can indicate cardiac damage and thus myocardial infarction.

[0159] As used herein, "coupled" or "linked" refers to two or more components held together by any suitable means. Thus, in some embodiments, a description of two or more moieties or components being "coupled" is intended to mean that the moieties are linked or operative together, either directly or indirectly, for example, through one or more intermediate moieties or components.

[0160] As used herein, "CDR" refers to a "complementarity-determining region" within an antibody variable sequence. There are three CDRs in each of the heavy and light chain variable regions. Starting from the N-terminus of the heavy or light chain, these regions are designated "CDR1," "CDR2," and "CDR3" for each variable region. As used herein, the term "CDR set" refers to a group of three CDRs occurring in a single variable region that bind to an antigen. Thus, an antigen-binding site may contain six CDRs, including the CDR sets from each of the heavy and light chain variable regions. A polypeptide containing a single CDR (e.g., CDR1, CDR2, or CDR3) may be referred to as a "molecular recognition unit." Crystal structure analysis of antigen-antibody complexes supports the finding that amino acid residues in the CDRs form extensive contacts with the bound antigen, with the most extensive antigen contact occurring with the heavy chain CDR3. Thus, the molecular recognition units may be primarily responsible for the specificity of the antigen-binding site. In general, the CDR residues are directly and most substantially involved in influencing binding to antigen.

[0161] The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any antibody variable region, but also provides the precise residue boundaries that define the three CDRs. These CDRs may be referred to as "Kabat CDRs." Chothia and colleagues (Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987); and Chothia et al., Nature, 342:877-883 (1989)) have described the Kabat CDRs in a numbered manner. It was found that certain subportions within the CDRs adopt nearly identical peptide backbone conformations despite the large diversity at the amino acid sequence level. These subportions were designated "L1," "L2," and "L3," or "H1," "H2," and "H3," with "L" and "H" designating the light chain and heavy chain regions, respectively. These regions are sometimes referred to as "Chothia CDRs," and they have boundaries that overlap with the Kabat CDRs. For other boundaries defining CDRs that overlap with the Kabat CDRs, see Padlan, FASEB, and others. J., 9:133-139 (1995); and MacCallum, J. Mol. Biol., 262(5):732-745 (1996). Still other CDR boundary definitions may not strictly adhere to the system defined herein and may be shorter or longer in light of predicted or experimental findings that a particular residue or group of residues, or even the entire CDR, does not significantly affect antigen binding, yet still overlap with the Kabat CDRs. While the methods used herein may use CDRs defined according to any of these systems, certain embodiments use the Kabat-defined CDRs or the Chothia-defined CDRs.

[0162] A "clinically relevant time frame" refers to a time frame (e.g., seconds, minutes, or hours) that a careful and prudent medical practitioner (e.g., doctor, nurse, paramedic, or other) would reasonably consider the results of one or more biomarker tests in relation to an imaging procedure such as a head CT scan, or in accordance with guidelines established by an oversight entity (e.g., a standard setting body such as the World Health Organization (WHO), a medical review committee, a regulatory approval authority such as the FDA, EMEA, or other).

[0163] A "component," "components," or "at least one component" generally refers to a capture antibody, detection reagent or detection conjugate, calibrator, control, sensitivity panel, container, buffer, diluent, salt, enzyme, cofactor for enzyme, detection reagent, pretreatment reagent / solution, substrate (e.g., in solution), stop solution, etc. that may be included in a kit for assaying a test sample, such as a patient whole blood sample, serum sample, or plasma sample, according to the methods described herein and other methods known in the art. Some components may be in solution or may be lyophilized for reconstitution for use in the assay.

[0164] As used herein, "correlates to" means compared to.

[0165] As used herein, "CT scan" refers to a computed tomography (CT) scan. CT scans combine a series of X-ray images taken from different angles and use computer processing to create cross-sectional images or slices of bones, blood vessels, and soft tissues within the body. CT scans may use X-ray CT, positron emission tomography (PET), single-photon emission computed tomography (SPECT), computed axial tomography (CAT scan), or computer-assisted tomography. CT scans may be conventional CT scans or spiral / helical CT scans. In conventional CT scans, the scan is acquired slice by slice, stopping after each slice and moving to the next slice, for example, from the upper abdomen to the pelvis. Conventional CT scans require the patient to hold their breath to avoid motion artifacts. Spiral / helical CT scans are continuous scans acquired in a spiral, and the scanned images are continuous, making the process much faster.

[0166] A head CT scan is "negative" for TBI if no intracranial lesion(s) are observed in images taken from a subject who has sustained, may have sustained, or is suspected of having sustained a head injury. To further clarify, a subject's head CT scan is "negative" for TBI if no lesions are found or identified; however, in some embodiments, even if the head CT is negative, the subject may still experience symptoms (e.g., of TBI). Given that not all injuries or lesions can be visualized by head CT, most subjects will have a negative head CT for TBI. Consequently, the methods and assays described herein can be used to provide an assessment or determination of subjects with a negative head CT who may still have a TBI.

[0167] As used interchangeably herein, "decentralize," "decentralized," or "decentralized," in the context of testing, refers to the performance of one or more medical tests and / or assays outside of traditional medical settings (e.g., hospitals, doctors' offices, freestanding research sites, etc.) at one or more locations, such as an urgent care clinic, retail clinic, pharmacy, grocery or convenience store, residence (e.g., house, apartment, etc.), workplace, and / or government office (e.g., National Transportation Safety Administration). "Hybrid decentralized" or "hybrid decentralized" refers to a situation in which a subject or patient collects a sample at their residence and / or workplace and ships the sample to a laboratory, avoiding a specialized collection location (e.g., a hospital, doctors' office, or freestanding specimen collection or research site).

[0168] As used herein, the term "determined by an assay" refers to the determination of a reference level by any suitable assay. In some embodiments, the determination of a reference level can be achieved by the same type of assay as that applied to a sample derived from a subject (e.g., by immunoassays, clinical chemistry assays, single molecule detection assays, protein immunoprecipitation, immunoelectrophoresis, chemical analysis, SDS-PAGE and Western blot analysis or protein immunostaining, electrophoretic analysis, protein assays, competitive binding assays, functional protein assays, or chromatographic or spectroscopic methods such as high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC / MS)). In some embodiments, the determination of a reference level can be achieved by the same type of assay as that applied to a sample derived from a subject, under the same assay conditions. As mentioned herein, the present disclosure presents exemplary reference levels (e.g., calculated by comparing reference levels at different time points). It is well within the skill of one of ordinary skill in the art to adapt the present disclosure to other assays and obtain assay-specific reference levels for these other assays based on the descriptions provided by the present disclosure. For example, a set of training samples including samples obtained from human subjects known to have suffered a head injury (more specifically, samples obtained from human subjects known to have suffered (i) a mild TBI; and / or (ii) a moderate, severe, or moderate-to-severe TBI) and samples obtained from human subjects known not to have suffered a head injury can be used to derive assay-specific reference levels. It is understood that a reference level "determined by an assay" and having a recited level of "sensitivity" and / or "specificity" is used herein to refer to a reference level determined to provide the method with the recited sensitivity and / or specificity when employed in the disclosed method. It is well within the skill of one of ordinary skill in the art to determine the sensitivity and specificity associated with a given reference level in the disclosed method, e.g., by repeating statistical analyses of the assay data using multiple different possible reference levels.

[0169] In practice, when distinguishing subjects as having or not having traumatic brain injury, or as having mild versus moderate, severe, or moderate-to-severe traumatic brain injury, one of skill in the art will balance the effect of raising the cutoff on sensitivity and specificity. Raising or lowering the cutoff has well-defined and predictable effects on sensitivity and specificity, as well as other standard statistical measures. It is well known that raising the cutoff improves specificity but likely worsens sensitivity (the proportion of people who have the disease and test positive). In contrast, lowering the cutoff improves sensitivity but worsens specificity (the proportion of people who do not have the disease and test negative). The ramifications of detecting traumatic brain injury or determining mild versus moderate, severe, or moderate-to-severe traumatic brain injury will be readily apparent to one of skill in the art. In distinguishing whether a subject has or does not have traumatic brain injury or mild versus moderate, severe, or moderate-severe traumatic brain injury, a higher cutoff improves specificity as more true negatives (i.e., subjects with no traumatic brain injury, no mild traumatic brain injury, no moderate traumatic brain injury, no severe traumatic brain injury, or no moderate-severe traumatic brain injury) are distinguished from subjects with traumatic brain injury, mild traumatic brain injury, moderate traumatic brain injury, severe traumatic brain injury, or moderate-severe traumatic brain injury. However, at the same time, raising the cutoff necessarily decreases sensitivity, as it reduces the number of cases identified as positive overall, as well as the number of true positives. Conversely, a lower cutoff improves sensitivity as more true positives (i.e., subjects with traumatic brain injury, mild traumatic brain injury, moderate traumatic brain injury, severe traumatic brain injury, or moderate-severe traumatic brain injury) are distinguished from subjects without traumatic brain injury, mild traumatic brain injury, moderate traumatic brain injury, severe traumatic brain injury, or moderate-severe traumatic brain injury. However, at the same time, specificity necessarily decreases, as lowering the cutoff increases the number of cases identified as positive overall, as well as the number of false positives.

[0170] In general, a high sensitivity value will help a person skilled in the art to rule out a disease or condition (such as traumatic brain injury, mild traumatic brain injury, moderate traumatic brain injury, severe traumatic brain injury, or moderate-severe traumatic brain injury), while a high specificity value will help a person skilled in the art to include a disease or condition. Whether a person skilled in the art wants to rule out or include a disease depends on the consequences for the patient for each type of error. Therefore, without fully disclosing the underlying information on how the value was selected, it is not possible to know or predict the exact balancing used to derive the test cutoff. The balancing of sensitivity against specificity and other factors will vary on a case-by-case basis. For this reason, it may be preferable to provide alternative cutoff (e.g., reference) values ​​for a physician or medical professional to choose from.

[0171] As used herein, a "derivative" of an antibody may refer to an antibody that has one or more modifications to its amino acid sequence compared to the original or parent antibody and may exhibit a modified domain structure. A derivative may not only adopt an amino acid sequence capable of specifically binding to a target (antigen), but may also adopt the typical domain configuration found in a natural antibody. Typical examples of antibody derivatives are antibodies coupled to other polypeptides, rearranged antibody domains, or antibody fragments. A derivative may also contain at least one additional compound, e.g., a protein domain, linked by covalent or non-covalent bonds. Linkage may be based on gene fusion according to methods known in the art. Additional domains present in an antibody-containing fusion protein may preferably be linked by a flexible linker, advantageously a peptide linker, comprising multiple hydrophilic, peptide-linked amino acids of sufficient length to span the distance between the C-terminus of the additional protein domain and the N-terminus of the antibody, or vice versa. The antibody may have a conformation suitable for biological activity or may be linked to an effector molecule that selectively binds, for example, a solid support, a biologically active substance (e.g., a cytokine or growth hormone), a chemical agent, a peptide, a protein, or a drug.

[0172] As used herein, "digital assay" refers to an assay in which an analyte is captured and molecules of the analyte are isolated and interrogated (e.g., to detect the presence and / or amount of the analyte in a sample). In a digital assay, noise is separated from signal. In a digital assay, the result is assigned a value of 1 or 0. Examples of digital assays include one or more of the following (which may overlap, but are not mutually exclusive): single molecule detection assays, nanowell assays, single molecule enzyme-linked immunosorbent assays, direct capture counting assays, etc.

[0173] The term "dependence drug" is used herein to refer to one or more addictive substances (e.g., drugs) taken for non-medical reasons (e.g., recreational and / or psychoactive effects). Excessive overdose on, use of, or dependence on such addictive drugs is often referred to as "substance abuse." Examples of addictive drugs include alcohol, barbiturates, benzodiazepines, cannabis, cocaine, hallucinogens (e.g., ketamine, mescaline (peyote), PCP, psilocybin, DMT, and / or LSD), methaqualone, opioids, amphetamines (including methamphetamine), anabolic steroids, inhalants (i.e., substances containing volatile substances containing psychoactive properties, such as, for example, nitrites, spray paints, cleaning fluids, markers, glues, etc.), and combinations thereof.

[0174] As used herein, the term "bispecific antibody" refers to a full-length antibody (see PCT Publication No. 02 / 02773) that can bind to two different antigens (or epitopes) in each of its two binding arms (HC / LC pair). Thus, a bispecific binding protein has two identical antigen-binding arms with identical specificity and identical CDR sequences, and is bivalent for each antigen to which it binds.

[0175] As used herein, the term "dual variable domain" refers to two or more antigen-binding sites on a binding protein, which can be a bivalent binding protein (two antigen-binding sites), a tetravalent binding protein (four antigen-binding sites), or a multivalent binding protein. A DVD can be monospecific, i.e., capable of binding to one antigen (or one specific epitope), or multispecific, i.e., capable of binding to two or more antigens (i.e., two or more epitopes of the same target antigen molecule or two or more epitopes of different target antigens). A preferred DVD-binding protein comprises two heavy chain DVD polypeptides and two light chain DVD polypeptides and is referred to as a "DVD immunoglobulin" or "DVD-Ig." Such DVD-Ig binding proteins are therefore tetrameric, similar to IgG molecules but providing more antigen-binding sites than IgG molecules. Thus, each half of a tetrameric DVD-Ig molecule is similar to half of an IgG molecule, containing a heavy chain DVD polypeptide and a light chain DVD polypeptide, but unlike the heavy and light chain pair of an IgG molecule, which results in a single antigen-binding domain, the heavy and light chain pair of a DVD-Ig results in two or more antigen-binding sites.

[0176] Each antigen-binding site of a DVD-Ig binding protein may be derived from a donor ("parent") monoclonal antibody and may therefore comprise a heavy chain variable domain (VH) and a light chain variable domain (VL) with the CDRs involved in binding to the antigen, for a total of six CDRs per antigen-binding site. Thus, a DVD-Ig binding protein that binds to two different epitopes (i.e., two different epitopes on two different antigen molecules or two different epitopes on the same antigen molecule) comprises an antigen-binding site derived from a first parent monoclonal antibody and an antigen-binding site of a second parent monoclonal antibody.

[0177] The design, expression, and characterization of DVD-Ig binding molecules are described in PCT Publication No. 2007 / 024715, U.S. Patent No. 7,612,181, and Wu et al., Nature Biotech., 25:1290-1297 (2007). A preferred example of such a DVD-Ig molecule comprises a heavy chain comprising the structural formula: VD1-(X1)n-VD2-C-(X2)n, where VD1 is a first heavy chain variable domain, VD2 is a second heavy chain variable domain, C is a heavy chain constant domain, X1 is a linker provided that it is not a CH1, X2 is an Fc region, and n is 0 or 1, preferably 1; and a light chain comprising the structural formula: VD1-(X1)n-VD2-C-(X2)n, where VD1 is a first light chain variable domain, VD2 is a second light chain variable domain, C is a light chain constant domain, X1 is a linker provided that it is not a CH1, X2 does not comprise an Fc region, and n is 0 or 1, preferably 1. Such a DVD-Ig can comprise two such heavy chains and two such light chains, where each chain comprises variable domains linked in tandem with no intervening constant region between the variable domains, and the heavy and light chains can associate to form tandem functional antigen-binding sites, and a pair of heavy and light chains can associate with another pair of heavy and light chains to form a tetrameric binding protein with four functional antigen-binding sites. In another example, a DVD-Ig molecule can comprise heavy and light chains each comprising three variable domains (VD1, VD2, VD3) linked in tandem with no intervening constant region between the variable domains, and the pair of heavy and light chains can associate to form three antigen-binding sites, and a pair of heavy and light chains can associate with another pair of heavy and light chains to form a tetrameric binding protein with six antigen-binding sites.

[0178] In a preferred embodiment, a DVD-Ig binding protein not only binds to the same target molecule as its parent monoclonal antibodies, but also possesses one or more desirable properties of one or more of the parent monoclonal antibodies. Preferably, such additional properties are one or more antibody parameters of the parent monoclonal antibodies. Antibody parameters that can be attributed to a DVD-Ig binding protein derived from one or more of the parent monoclonal antibodies include, but are not limited to, antigen specificity, antigen affinity, potency, biological function, epitope recognition, protein stability, protein solubility, production efficiency, immunogenicity, pharmacokinetics, bioavailability, tissue cross-reactivity, and binding to orthologous antigens.

[0179] DVD-Ig binding proteins bind to at least one epitope of UCH-L1, GFAP, CK-MB, β-hCG, TSH, homocysteine, or free T4. Non-limiting examples of DVD-Ig binding proteins include DVD-Ig binding proteins that bind to one or more epitopes of UCH-L1, GFAP, CK-MB, β-hCG, TSH, homocysteine, or free T4, DVD-Ig binding proteins that bind to an epitope of human UCH-L1, GFAP, CK-MB, β-hCG, TSH, homocysteine, or free T4 and an epitope of UCH-L1, GFAP, CK-MB, β-hCG, TSH, homocysteine, or free T4 of another species (e.g., mouse), and DVD-Ig binding proteins that bind to an epitope of human UCH-L1, GFAP, CK-MB, β-hCG, TSH, homocysteine, or free T4 and an epitope of another target molecule.

[0180] As used herein, "dynamic range" refers to the range over which the readout of an assay is proportional to the amount of target molecule or analyte in the sample being analyzed.

[0181] "Epitope" or "epitopes" or "epitope of interest" refers to a site on any molecule that can be recognized and bind to a complementary site on its specific binding partner. The molecule and specific binding partner are part of a specific binding pair. For example, an epitope can be on a polypeptide, protein, hapten, carbohydrate antigen (such as, but not limited to, a glycolipid, glycoprotein, or lipopolysaccharide), or polysaccharide. The specific binding partner can be, but is not limited to, an antibody.

[0182] As used herein, "Fab (fragment antigen-binding) fragment" or "Fab fragment" refers to an antibody fragment that binds to an antigen and contains one complete light chain and part of one heavy chain, which is one antigen-binding site. Fab is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains. Fab is composed of one constant domain and one variable domain from each heavy and light chain. The variable domain contains a paratope (antigen-binding site) at the amino terminus of the monomer, which includes a set of complementarity-determining regions. Thus, each arm of the Y binds to an epitope on the antigen. Fab fragments can be produced as described in the art, for example, using papain, an enzyme that can be used to cleave an immunoglobulin monomer into two Fab fragments and an Fc fragment, or can be produced by recombinant means.

[0183] As used herein, the term "F(ab')2 fragment" refers to an antibody produced by pepsin digestion of a whole IgG antibody, removing most of the Fc region while leaving a portion of the hinge region intact. The F(ab')2 fragment is a bivalent fragment with a molecular weight of approximately 110 kDa, due to the two antigen-binding F(ab) portions linked together by disulfide bonds. Bivalent antibody fragments (F(ab')2 fragments) are smaller than whole IgG molecules, allowing for better tissue penetration and facilitating better antigen recognition in immunohistochemistry. The use of F(ab')2 fragments also avoids nonspecific binding to Fc receptors or protein A / G on live cells. F(ab')2 fragments can bind to and precipitate antigens.

[0184] As used herein, the term "framework" (FR) or "framework sequence" may refer to the remaining sequence of a variable region, excluding the CDRs. The exact definition of a CDR sequence may be determined by different systems (see, e.g., above), and the meaning of a framework sequence is subject to different interpretations accordingly. The six CDRs (CDR-L1, CDR-L2, and CDR-L3 of the light chain and CDR-H1, CDR-H2, and CDR-H3 of the heavy chain) also divide the framework regions on the light and heavy chains into four subregions (FR1, FR2, FR3, and FR4) on each chain, where CDR1 is located between FR1 and FR2, CDR2 is located between FR2 and FR3, and CDR3 is located between FR3 and FR4. As noted by others, when a specific subregion is not designated as FR1, FR2, FR3, or FR4, the framework region refers to the combination of FRs within the variable region of a single, native immunoglobulin chain. As used herein, FR refers to one of the four subregions, and FR refers to two or more of the four subregions that constitute a framework region. Human heavy and light chain FR sequences are known in the art that can be used as heavy and light chain "acceptor" framework sequences (or simply, "acceptor" sequences) to humanize non-human antibodies using techniques known in the art. In one embodiment, the human heavy and light chain acceptor sequences are selected from framework sequences listed in publicly available databases, such as V-base (hypertext transferprotocol: / / vbase.mrc-cpe.cam.ac.uk / ) or the international ImMunoGeneTics® (IMGT®) information system (hypertext transferprotocol: / / imgt.cines.fr / texts / IMGTrepertoire / LocusGenes / ).

[0185] As used herein, a "functional antigen-binding site" may refer to a site on a binding protein (e.g., an antibody) that is capable of binding to a target antigen. The antigen-binding affinity of an antigen-binding site may not be as strong as that of the parent binding protein, e.g., the parent antibody, from which the antigen-binding site is derived, but the ability to bind to an antigen must be measurable using any one of a variety of known methods for assessing antigen-binding proteins, e.g., antibodies. Furthermore, the antigen-binding affinity of each of the antigen-binding sites of a multivalent protein, e.g., a multivalent antibody herein, does not need to be quantitatively the same.

[0186] "GFAP" is used herein to describe glial fibrillary acidic protein, a protein that is encoded by the GFAP gene in humans and can be produced (e.g., by recombinant means in other species).

[0187] "GFAP status" can refer to the level or amount of GFAP at a point in time (e.g., a point in time involving a single measurement of GFAP), the level or amount of GFAP associated with monitoring (e.g., monitoring involving repeated testing to identify increases or decreases in GFAP amounts in a subject), the level or amount of GFAP associated with treatment for TBI (whether primary and / or secondary brain injury), or a combination thereof.

[0188] As used herein, "Glasgow Coma Scale (GCS)" or "GCS" refers to a 15-point scale (described, for example, in 1974 by Graham Teasdale and Bryan Jennett, Lancet 1974;2:81-4) that provides a practical method for assessing impairment in the level of consciousness in patients who have suffered brain injury. The test measures best motor response, verbal response, and eye opening response with these values: I. best motor response (6: follows two-part request; 5: brings hand to head / neck stimulus above collarbone; 4: bends arm sharply at elbow but features are not primarily abnormal; 3: bends arm at elbow with features clearly primarily abnormal; 2: extends arm from elbow; 1: no arm / leg movement, no interfering factors; NT: paralysis or other limiting factor); II. verbal response (5: gives name, place, and date correctly; 4: disoriented but communicates coherently; 3: understandable words; 2: moans / growls only; 1: no audible response, no interfering factors; NT: factors interfering with communication) and III. eye opening (4: opens before stimulation; 3: after verbal or shouted request; 2: after fingertip stimulation; 1: does not open eyes at any time, no interfering factors; NT: closed due to local factors). The final score is determined by adding the values ​​of I + II + III. If the GCS score is 13-15, the subject is considered to have a mild TBI. If the GCS score is 9-12, the subject is considered to have a moderate TBI. If the GCS score is 8 or less, typically 3-8, the subject is considered to have a severe TBI.

[0189] As used herein, "Glasgow Outcome Scale (GOS)" refers to a global scale for functional outcome frequently used in potential TBI situations that grades a patient's condition into one of five categories: death, vegetative state, severe disability, moderate disability, or good recovery. The "Extended Glasgow Outcome Scale (GOSE)" or "GOSE," used interchangeably herein, provides a more detailed categorization into eight categories by subdividing the severe disability, moderate disability, and good recovery categories into upper and lower categories, as shown in Table 1.

[0190] [Table 1]

[0191] As used interchangeably herein, a "higher throughput assay analyzer" or a "non-point-of-care device" refers to a device that is neither a point-of-care device nor a disposable device. A higher throughput assay analyzer or a non-point-of-care device refers to any device that does not meet either of the limitations of a point-of-care or disposable device defined herein. In some embodiments, a "higher throughput assay analyzer" or a "non-point-of-care device" refers to an instrument that (a) can be relatively large compared to a handheld point-of-care device, e.g., ranging in size from a benchtop instrument (e.g., typically considered to be low or medium throughput) to a large room-sized or multi-room-sized instrument (e.g., typically considered to be high throughput); (b) is not a handheld instrument; (c) is capable of performing assays on two or more clinical samples simultaneously; and (d) is any combination of (a)-(c). A higher throughput assay analyzer can be a clinical chemistry analyzer, an immunoassay analyzer, or a combination thereof. Exemplary higher throughput assay analyzers or non-point-of-care devices include, for example, the ARCHITECT or Alinity platforms produced by Abbott Laboratories.

[0192] "Homocysteine" or "HCY" refers to a thiol-containing amino acid produced by the intracellular demethylation of methionine. Homocysteine ​​is exported into plasma, where it circulates mostly in its oxidized form, bound to plasma proteins as protein-HCY mixed disulfides and albumin. Smaller amounts of reduced homocysteine ​​and disulfide homocystine (HCY-SS-HCY) are present. Total homocysteine ​​(tHCY) represents the sum of all homocysteine ​​species (free plus protein-bound) found in serum or plasma. Homocysteine ​​is metabolized to either cysteine ​​or methionine. In the vitamin B6-dependent transsulfuration pathway, homocysteine ​​is irreversibly catabolized to cysteine. The majority of homocysteine ​​is remethylated to methionine, primarily by the folate- and cobalamin-dependent enzyme methionine synthase. Homocysteine ​​accumulates and is excreted in the blood when these reactions are impaired. Impaired homocysteine ​​metabolism results in hyperhomocysteinemia (increased levels of homocysteine ​​in plasma or serum) or homocystinuria (high plasma levels cause homocysteine ​​to be excreted in the urine). Hyperhomocysteinemia is caused by nutritional and genetic deficiencies. The majority (two-thirds) of cases of elevated homocysteine ​​in the general population are due to deficiencies of folic acid, vitamin B6, and vitamin B12. Severely elevated concentrations of total homocysteine ​​are found in subjects with homocystinuria, a rare genetic disorder of an enzyme involved in homocysteine ​​metabolism. Patients with homocystinuria exhibit mental retardation, premature arteriosclerosis, and arterial and venous thromboembolism. Other less severe genetic defects that result in moderately elevated levels of total homocysteine ​​are also found. Studies have investigated the relationship between elevated homocysteine ​​concentrations and cardiovascular disease (CVD), indicating homocysteine ​​as an important marker for risk assessment. In the presence of known coronary artery disease (CAD), it has been shown to be a strong independent marker of subsequent CAD-related death. In intermediate-risk patients, elevated homocysteine ​​levels are associated with the presence of coronary artery calcification. Elevated homocysteine ​​levels in these patients are independent of coronary heart disease (CHD) risk factors.

[0193] Elevated homocysteine ​​has been suggested to be a modifiable independent risk factor for CAD, stroke, and deep vein thrombosis. Studies have also identified elevated homocysteine ​​as a strong independent risk factor for the development of various forms of dementia, including Alzheimer's disease. Increased tHCY is associated with an increased risk of pregnancy complications (preeclampsia, recurrent early pregnancy loss, premature delivery, low birth weight, and placental abruption or infarction). Maternal hyperhomocysteinemia is associated with birth defects such as neural tube defects, orofacial clefts, clubfoot, and Down syndrome.

[0194] "Human thyroid-stimulating hormone" or "TSH" refers to a glycoprotein with a molecular weight of approximately 28,000 daltons synthesized by basophilic cells (thyrotrope cells) in the anterior pituitary gland. TSH is composed of two noncovalently linked subunits, designated alpha and beta. While the alpha subunit of TSH is common to luteinizing hormone (LH), follicle-stimulating hormone (FSH), and human chorionic gonadotropin (hCG), the beta subunit of these glycoproteins is hormone-specific and confers biological and immunological specificity. Both the alpha and beta subunits are required for biological activity. By interacting with specific receptors on the surface of thyroid cells, TSH stimulates the production and secretion of metabolically active thyroid hormones, thyroxine (T4) and triiodothyronine (T3). T3 and T4 are responsible for regulating diverse biochemical processes throughout the body that are essential for normal development, metabolic activity, and neurological activity. The synthesis and secretion of TSH is stimulated by the hypothalamic tripeptide thyrotropin-releasing hormone (TRH) in response to low levels of circulating thyroid hormones. Elevated levels of T3 and T4 suppress TSH production by a classic negative feedback mechanism. Other evidence also indicates that somatostatin and dopamine exert inhibitory control over TSH release, suggesting that the hypothalamus can provide both inhibitory and stimulatory influences on pituitary TSH production. Dysregulation at any level of the hypothalamic-pituitary-thyroid axis results in either underproduction (hypothyroidism) or overproduction (hyperthyroidism) of T4 and / or T3.

[0195] In cases of primary hypothyroidism, T3 and T4 levels are low, while TSH levels are significantly elevated. In cases of pituitary dysfunction due to either endogenous hypothalamic or pituitary disease; i.e., central hypothyroidism, normal or marginally elevated basal TSH levels are often observed despite significantly decreased T4 and / or T3 levels. These inadequate TSH values ​​are due to the frequently observed decreased TSH bioactivity in such cases. Routine TRH stimulation is recommended to confirm the diagnosis in such cases. Secondary hypothyroidism typically results in an impaired TSH response to TRH, while in tertiary hypothyroidism, the TSH response to TRH may be normal, prolonged, or impaired. Primary hyperthyroidism (e.g., Graves' disease, nodular goiter) is associated with high levels of thyroid hormone and suppressed or undetectable levels of TSH. TRH stimulation testing has been used to diagnose hyperthyroidism. Hyperthyroid patients exhibit subnormal responses to TRH testing. In addition, high doses of glucocorticoids, somatostatin, dopamine, and replacement doses of thyroid hormone reduce or completely blunt the TSH response to TRH.

[0196] As used herein, the term "hydrophilic," such as with reference to a "hydrophilic material" (eg, a membrane, film, etc.), refers to a material that has a water contact angle of less than about 40 degrees.

[0197] As used herein, the term "hydrophobic," such as with reference to a "hydrophobic material" (eg, a membrane, film, etc.), refers to a material that has a water contact angle of greater than about 80 degrees.

[0198] As used herein, the term "humanized antibody" is used to describe antibodies that contain heavy and light chain variable region sequences derived from a non-human species (e.g., mouse), but in which at least a portion of the VH and / or VL sequences have been altered to make them more "human-like," i.e., more similar to human germline variable sequences. A "humanized antibody" is an antibody, or a variant, derivative, analog, or fragment thereof, that immunospecifically binds to an antigen of interest and contains framework (FR) regions that have substantially the amino acid sequence of a human antibody and complementarity-determining regions (CDRs) that have substantially the amino acid sequence of a non-human antibody. As used herein, the term "substantially" in the context of a CDR refers to a CDR that has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of the non-human antibody CDR. A humanized antibody comprises substantially all of at least one, but typically two, variable domains (Fab, Fab', F(ab')2, FabC, Fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. In certain embodiments, the humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically the Fc region of a human immunoglobulin. In some embodiments, the humanized antibody contains at least the variable domain of a heavy chain as well as a light chain. The antibody may also contain the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, the humanized antibody contains only a humanized light chain. In some embodiments, the humanized antibody contains only a humanized heavy chain. In specific embodiments, the humanized antibody contains only humanized variable domains of the light chain and / or humanized heavy chain.

[0199] The humanized antibody may be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including, without limitation, IgG1, IgG2, IgG3, and IgG4. The humanized antibody may comprise sequences from more than one class or isotype, and particular constant domains may be selected to optimize desired effector functions using techniques well known in the art.

[0200] The framework regions and CDRs of a humanized antibody need not exactly correspond to the parental sequences. For example, the donor antibody CDR or consensus framework may be mutagenized by substitution, insertion, and / or deletion of at least one amino acid residue so that the CDR or framework residue at this site does not correspond to that of the donor antibody or consensus framework. However, in preferred embodiments, such mutations are not extensive mutations. Typically, at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% of the humanized antibody residues correspond to those of the parental FR and CDR sequences. As used herein, the term "consensus framework" refers to a framework region within a consensus immunoglobulin sequence. As used herein, the term "consensus immunoglobulin sequence" refers to a sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related immunoglobulin sequences (see, e.g., Winnaker, "From Genes to Clones" (Verlagsgesellschaft, Weinheim, 1987)). Thus, a "consensus immunoglobulin sequence" may comprise a "consensus framework region" and / or a "consensus CDR." Within a family of immunoglobulins, each position within the consensus sequence is occupied by the amino acid that occurs most frequently at that position within the family. If two amino acids occur equally frequently, either may be included within the consensus sequence.

[0201] As used herein, "identical" or "identity" in the context of two or more polypeptide or polynucleotide sequences can mean that the sequences have a specified percentage of residues that are the same over a specified region. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over a specified region, and determining the number of positions in both sequences where identical residues occur to determine the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to determine the percentage of sequence identity. If the two sequences are of different length, or the alignment results in one or more sticky ends, and the specified comparison region includes only a single sequence, the residues of the single sequence are included in the denominator but not the numerator of the calculation.

[0202] "Head injury" or "head injury," used interchangeably herein, refers to any trauma to the scalp, skull, or brain. Such injury can involve only a minor bump on the skull or can be a severe brain injury. Such injury includes primary brain injury and / or secondary brain injury. Primary brain injury occurs during the initial insult and results from displacement of the brain's physical structures. More specifically, primary brain injury is physical damage to the parenchyma (tissue, blood vessels) that occurs during the traumatic event, resulting in shearing and compression of surrounding brain tissue. Secondary brain injury occurs subsequent to the primary injury and can involve a series of cellular processes. More specifically, secondary brain injury refers to changes that develop over a period of time (hours to days) after the primary brain injury. Secondary brain injury includes an entire cascade of cellular, chemical, tissue, or vascular changes within the brain that contribute to further destruction of brain tissue.

[0203] Head injuries can be closed or open (penetrating). A closed head injury is trauma to the scalp, skull, or brain without penetration of the skull by an impacting object. An open head injury is trauma to the scalp, skull, or brain with penetration of the skull by an impacting object. Head injuries can be caused by physical shaking of a person, by external mechanical or other forces resulting in closed or open head injuries (vehicle accidents, such as with a car, plane, train, etc.; blows to the head, such as with a baseball bat or from a firearm), cerebrovascular accidents (e.g., stroke), one or more falls (e.g., during sports or other activities), blunt impact from an explosion or blast (collectively, "blast injury"), and other types of blunt force trauma. Alternatively, head injuries can be caused by ingestion of and / or exposure to fire, chemicals, toxins, or a combination of chemicals and toxins. Examples of such chemicals and / or toxins include mold, asbestos, pesticides and insecticides, organic solvents, paints, adhesives, gases (such as carbon monoxide, hydrogen sulfide, and cyanide), organometallics (such as methylmercury, tetraethyl lead, and organotins), and / or one or more drugs of abuse. Alternatively, head injury may result from a subject suffering from an autoimmune disease, metabolic disorder, brain tumor, hypoxia, viral infection (e.g., SARS-CoV-2), fungal infection, bacterial infection, meningitis, hydrocephalus, or any combination thereof. In some cases, it is not possible to ascertain whether any such event or injury has occurred. For example, there may be no medical history for the patient or subject, the subject may be unable to speak, the subject may be aware of what event they have been exposed to, etc. Such circumstances are described herein as the subject "possibly having suffered a head injury." In certain embodiments herein, closed head injury does not include, and specifically excludes, cerebrovascular accidents such as stroke.

[0204] As used herein, "interstitial fluid" refers to the fluid that surrounds cells and / or fills the spaces between cells. Interstitial fluid can contain or include a mixture of water, ions, and small solutes that are forced out of the blood by the systolic pressure created when the heart pumps.

[0205] As used herein, "intracranial lesion" refers to an area of ​​damage within the brain. Intracranial lesions can be abnormalities seen on a CT scan or brain imaging test, such as magnetic resonance imaging (MRI). On a CT or MRI scan, brain lesions can appear as dark or bright spots that do not resemble normal brain tissue.

[0206] As used herein, an "isolated polynucleotide" can mean a polynucleotide that, by its origin, is not associated with all or part of a polynucleotide with which the "isolated polynucleotide" is found in nature; that is operably linked to a polynucleotide with which it is not linked in nature; or that does not exist in nature as part of a larger sequence (e.g., by genomic, cDNA, or synthetic origin, or some combination thereof).

[0207] As used herein, "label" and "detectable label" refer to a moiety conjugated to an antibody or analyte such that the reaction of the antibody with the analyte is detectable; an antibody or analyte so labeled is said to be "detectably labeled." A label can provide a signal that is detectable by visual or instrumental means. Various labels include signal generators such as chromogens, fluorescent compounds, chemiluminescent compounds, and radioactive compounds. Representative examples of labels include moieties that provide light, e.g., acridinium compounds, and moieties that provide fluorescence, e.g., fluorescein. Other labels are also described herein. In this regard, a moiety itself may not be detectable, but may become detectable upon reaction with yet another moiety. The term "detectably labeled" is intended to encompass such labels.

[0208] Any suitable detectable label known in the art may be used. For example, detectable labels include radioactive labels (such as 3H, 14C, 32P, 33P, 35S, 90Y, 99Tc, 111In, 125I, 131I, 177Lu, 166Ho, and 153Sm), enzyme labels (such as horseradish peroxidase, alkaline peroxidase, glucose 6-phosphate dehydrogenase), chemiluminescent labels (such as acridinium esters, thioesters, or sulfonamides; luminol, isoluminol, phenanthridinium esters), fluorescent labels (such as fluorophores, fluoroisotopes, etc.), and the like. The label may be a fluorescent label (e.g., 5-fluorescein, 6-carboxyfluorescein, 3'6-carboxyfluorescein, 5(6)-carboxyfluorescein, 6-hexachlorofluorescein, 6-tetrachlorofluorescein, fluorescein isothiocyanate, etc.), rhodamine, phycobiliprotein, R-phycoerythrin, quantum dots (e.g., zinc sulfide-capped cadmium selenide), calorimetric labels, or immunopolymerase chain reaction labels. Introductions to labels, labeling procedures, and label detection can be found in Polak and Van Noorden, "Introduction to Immunocytochemistry," 2nd ed., Springer Verlag, NY (1997), and Haugland, "Handbook of Fluorescent Probes and Research Chemicals" (1996), a combination handbook and catalog published by Molecular Probes, Inc., Eugene, Oregon. Fluorescent labels can be used in FPIA (see, e.g., U.S. Patent Nos. 5,593,896, 5,573,904, 5,496,925, 5,359,093, and 5,352,803, which are incorporated by reference herein in their entireties).Acridinium compounds can be used as detectable labels in homogeneous chemiluminescent assays (see, e.g., Adamczyk et al., Bioorg. Med. Chem. Lett., 16:1324-1328 (2006); Adamczyk et al., Bioorg. Med. Chem. Lett., 4:2313-2317 (2004); Adamczyk et al., Bioorg. Med. Chem. Lett., 14:3917-3921 (2004) and Adamczyk et al., Org. Lett., 5:3779-3782 (2003)).

[0209] In one embodiment, the acridinium compound is acridinium-9-carboxamide. Methods for preparing acridinium 9-carboxamide are described in Mattingly, J. Biolumin. Chemilumin., 6:107-114 (1991); Adamczyk et al., J. Org. Chem., 63:5636-5639 (1998); Adamczyk et al., Tetrahedron, 55:10899-10914 (1999); Adamczyk et al., Org. Lett., 1:779-781 (1999); Adamczyk et al., Bioconjugate Chem., 11:714-724 (2000); Mattingly et al., "Luminescence Biotechnology: Instruments and Applications," edited by Dyke, KV, CRC Press: Boca Raton, CA. Raton, 77-105 (2002); Adamczyk et al., Org. Lett., 5:3779-3782 (2003); and U.S. Pat. Nos. 5,468,646, 5,543,524, and 5,783,699 (each of which is incorporated by reference in its entirety for its teachings herein).

[0210] Another example of an acridinium compound is acridinium-9-carboxylate aryl ester. An example of the acridinium-9-carboxylate aryl ester of Formula II is 10-methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate (commercially available from Cayman Chemical, Ann Arbor, MI). Methods for preparing acridinium-9-carboxylate aryl esters are described in McCapra et al., Photochem. Photobiol., 4:1111-21 (1965); Razavi et al., Luminescence, 15:245-249 (2000); Razavi et al., Luminescence, 15:239-244 (2000) and U.S. Pat. No. 5,241,070 (each of which is incorporated herein by reference in its entirety for its teachings). Such acridinium-9-carboxylate aryl esters are effective chemiluminescent indicators for hydrogen peroxide generated in the oxidation of an analyte by at least one oxidase, in terms of signal intensity and / or signal rapidity. The chemiluminescent process of acridinium-9-carboxylate aryl esters is completed rapidly, i.e., in less than one second, while the chemiluminescence of acridinium-9-carboxamides lasts for more than two seconds. However, acridinium-9-carboxylate aryl esters lose their chemiluminescent properties in the presence of proteins. Therefore, their use requires the absence of proteins during signal generation and detection. Methods for separating or removing proteins from a sample are well known to those skilled in the art and include, but are not limited to, ultrafiltration, extraction, precipitation, dialysis, chromatography, and / or digestion (see, e.g., Wells, "High Throughput Bioanalytical Sample Preparation. Methods and Automation Strategies," Elsevier (2003)).The amount of protein removed or separated from the test sample can be about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. Further details regarding acridinium-9-carboxylate aryl esters and their uses are set forth in U.S. patent application Ser. No. 11 / 697,835, filed April 9, 2007. The acridinium-9-carboxylate aryl ester can be dissolved in any suitable solvent, such as degassed anhydrous N,N-dimethylformamide (DMF) or aqueous sodium cholate.

[0211] A "linking sequence" or "linking peptide sequence" refers to a naturally occurring or artificial polypeptide sequence connected to one or more polypeptide sequences of interest (e.g., full-length sequences, sequence fragments, etc.). The term "connected" refers to the joining of the linking sequence to the polypeptide sequence of interest. Preferably, such polypeptide sequences are joined by one or more peptide bonds. A linking sequence can have a length of about 4 to about 50 amino acids. Preferably, the length of a linking sequence is about 6 to about 30 amino acids. A naturally occurring linking sequence can be modified by amino acid substitution, addition, or deletion to create an artificial linking sequence. Linking sequences can be used for many purposes, including use in recombinant Fabs. Exemplary linking sequences include, but are not limited to: (i) a histidine (His) tag, such as a 6xHis tag having the amino acid sequence HHHHHH (SEQ ID NO: 3), is useful as a linking sequence to facilitate isolation and purification of polypeptides and antibodies of interest; and (ii) an enterokinase cleavage site, such as a His tag, is used in the isolation and purification of proteins and antibodies of interest. Enterokinase cleavage sites, in conjunction with His tags, are often used in the isolation and purification of proteins and antibodies of interest. A variety of enterokinase cleavage sites are known in the art. Examples of enterokinase cleavage sites include, but are not limited to, the amino acid sequence DDDDK (SEQ ID NO: 4) and its derivatives (e.g., ADDDDK (SEQ ID NO: 5)). (iii) Other sequences can also be used to link or connect the light chain variable region and / or heavy chain variable region of a single-chain variable region fragment. Examples of other linking sequences can be found in Bird et al., Science, 242:423-426 (1988); Huston et al., PNAS USA, 85:5879-5883 (1988); and McCafferty et al., Nature, 348:552-554 (1990). Linking sequences can also be modified for additional functions, such as drug conjugation or conjugation to a solid support. In the context of the present disclosure, the monoclonal antibody may contain linking sequences such as, for example, a His tag, an enterokinase cleavage site, or both.

[0212] "Magnetic resonance imaging" or "MRI," as used interchangeably herein, refers to a medical imaging technique used in radiology to produce images of the anatomy and physiological processes of the body, both in health and disease (e.g., referred to interchangeably herein as an "MRI," an "MRI procedure," or an "MRI exam." MRI is a form of medical imaging that measures the response of atomic nuclei in body tissues to radio frequency radio waves when placed in a strong magnetic field, producing images of internal organs. MRI scanners are based on the science of nuclear magnetic resonance (NMR) and use strong magnetic fields, radio waves, and field gradients to produce images of the inside of the body.

[0213] As used herein, the term "microchannel" refers to a channel having cross-sectional dimensions (i.e., height and width) that are less than about 200 μm. In some embodiments, the channel has a cross-sectional dimension of less than about 150 μm. In still other embodiments, the channel has a cross-sectional dimension of less than about 100 μm.

[0214] As used herein, the term "microsampling device" refers to any device known in the art suitable for extracting capillary blood through the skin. While a sample obtained through the skin using a microsampling device primarily contains capillary blood, it is understood that the sample may also contain a small amount or percentage of interstitial fluid. In some embodiments, the microsampling device can contain between about 0.1 mL and about 4 mL of capillary blood. In some other embodiments, the device contains a plurality of microneedles, lancets or microlancets, blades or microblades, microscrews, or any combination thereof. In some embodiments, the plurality of microneedles, lancets or microlancets, blades or microblades, microscrews, or any combination thereof can be rotatable. In still other embodiments, the plurality of microneedles, lancets or microlancets, blades or microblades, microscrews, or any combination thereof is non-rotatable. In some embodiments, the microsampling device generates and / or uses a stored vacuum to draw the skin into the device and / or activates multiple microneedles, lancets or microlancets, blades or microblades, microscrews, or any combination thereof to cut the skin. Exemplary microsampling devices that may be used in the methods described herein include the TAP device available from YourBio Health, Inc. (Cambridge, MA), as well as the devices described in U.S. Patent No. 9,113,836, the contents of which are incorporated herein by reference; the Tasso+, Tasso-M20, and Tasso-ST devices available from Tasso, Inc. (Seattle, WA); the One Draw device available from Draw Bridge Health (San Diego, CA); the PBS-1000 from PreciHealth (Neuchatel, Switzerland); or the Loop blood collection device available from Loop Medical (Lausanne, Switzerland).In other embodiments, examples of microsampling devices include fingerstick devices. In some embodiments, the microsampling device may comprise a band-aid, dressing, or other suitable material that can be applied or dispensed onto an area of ​​skin once the sample has been obtained and / or once the device has been removed and / or detached from the skin.

[0215] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible natural mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen. Furthermore, in contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Monoclonal antibodies herein include, inter alia, "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological properties.

[0216] As used herein, the term "multivalent binding protein" is used to refer to a binding protein that comprises two or more antigen-binding sites (also referred to herein as "antigen-binding domains"). Multivalent binding proteins are preferably engineered to have three or more antigen-binding sites and are generally not naturally occurring antibodies. The term "multispecific binding protein" refers to binding proteins that can bind to two or more targets, related or unrelated, including binding proteins capable of binding to two or more different epitopes of the same target molecule.

[0217] "Negative predictive value" or "NPV," used interchangeably herein, refers to the probability that a subject will have a negative outcome given that they have a negative test result.

[0218] As used herein, "normalize" or "normalizing" refers to adjusting the amount of an analyte (e.g., UCH-L1, GFAP, CK-MB, β-hCG, TSH, homocysteine, and / or free T4) determined in a capillary blood sample obtained from a subject based on the amount of the same analyte in venous blood. In some embodiments, for example, normalizing can include multiplying a factor (e.g., a correlation or conversion factor) by the amount of the analyte in the capillary blood sample. As described herein, a "conversion factor" is generally in the form of a conversion factor described for (i) the same analyte in a capillary sample (i.e., a capillary whole blood sample) compared to the analyte in a venous sample (i.e., a venous whole blood sample); or (ii) the same analyte in a venous sample (i.e., a venous whole blood sample) compared to the analyte in a capillary sample (i.e., a capillary whole blood sample). Theoretically, the value of the conversion factor should be comparable to the conversion factor obtained for a capillary plasma sample versus a venous plasma sample (e.g., depending on the efficiency of plasma separation and other factors apparent to one skilled in the art).

[0219] As used herein, "operably coupled" or "operably connected" means that several elements or assemblies that are movable between a first position and a second position or configuration, respectively, are coupled such that movement of the first element from one position / configuration to another causes the second element to similarly move between positions / configurations. It is noted that a first element may be "operably coupled" to another element, but the opposite is not true. When movement is possible between a first element and another element, and vice versa, the elements are said to be "reciprocally operably coupled."

[0220] A "point-of-care device" refers to a device used to provide medical diagnostic testing at or near the point of care (i.e., typically outside a laboratory), at the time and place of patient care (such as a hospital, clinic, urgent care facility or other medical care facility, the patient's home, a nursing home and / or long-term care facility and / or hospice facility, etc.). Examples of point-of-care devices include point-of-care devices made by Abbott Laboratories (Abbott Park, IL) (e.g., i-STAT and i-STAT Alinity, Universal Biosensors (Rowville, Australia)) (see U.S. Patent Application Publication No. 2006 / 0134713), Axis-Shield PoC AS (Oslo, Norway), and Clinical Lab Products (Los Angeles, USA).

[0221] "Positive predictive value" or "PPV," used interchangeably herein, refers to the probability that a subject will have a positive outcome given that they have a positive test result.

[0222] "Quality control reagents" in the context of the immunoassays and kits described herein include, but are not limited to, calibrators, controls, and sensitivity panels. "Calibrators" or "standards" (e.g., multiple, one or more) are typically used to establish a calibration curve for interpolating the amount of an analyte, such as an antibody or analyte. Alternatively, a single calibrator that is near a reference or control level (e.g., a "low," "medium," or "high" level) can be used. Multiple calibrators (i.e., more than one calibrator or varying amounts of calibrators) can be used to comprise a "sensitivity panel."

[0223] As used herein, "reactor" refers to a holder or receptacle, e.g., a vessel, receptacle, tube, and / or cartridge, in or on which an assay is performed. In some embodiments, a reactor can have one or more openings.

[0224] A "receiver operating characteristic" (ROC) curve or "ROC" curve refers to a graphical plot illustrating the performance of a binary classifier system as its discrimination threshold is varied. For example, an ROC curve can be a plot of the true positive rate against the false positive rate for different possible cutoff points of a diagnostic test. An ROC curve is created by plotting the proportion of true positives among positives (TPR = true positive rate) against the proportion of false positives among negatives (FPR = false positive rate) at various threshold situations. TPR is also known as sensitivity, and FPR is [1 - specificity or true negative rate]. ROC curves demonstrate a trade-off between sensitivity and specificity (any increase in sensitivity is accompanied by a decrease in specificity); the closer the curve is to the left and then upper boundaries of ROC space, the more accurate the test; the closer the curve is to the 45-degree diagonal of ROC space, the less accurate the test; the slope of the tangent at the cutoff point gives the likelihood ratio (LR) for this value of the test, and the area under the curve is a measure of the accuracy of the test.

[0225] The terms "recombinant antibody" and "recombinant antibodies" refer to antibodies prepared by one or more steps, including recombinantly cloning a nucleic acid sequence encoding all or part of one or more monoclonal antibodies into a suitable expression vector, and then expressing the antibody in a suitable host cell. The terms include, but are not limited to, recombinantly produced monoclonal antibodies, chimeric antibodies, humanized antibodies (fully or partially humanized), multispecific or multivalent structures formed from antibody fragments, bifunctional antibodies, heteroconjugate Abs, DVD-Igs®, and other antibodies described in (i) herein (dual variable domain immunoglobulins and methods for producing them are described in Wu, C. et al., Nature Biotechnology, 25:1290-1297 (2007)). As used herein, the term "bifunctional antibody" refers to an antibody that contains a first arm that has specificity for one antigenic site and a second arm that has specificity for a different antigenic site, i.e., a bifunctional antibody has dual specificities.

[0226] As used herein, a "reference level" refers to an assay cutoff value used to evaluate diagnostic, prognostic, or therapeutic efficacy, and is herein linked or associated with various clinical parameters (e.g., the presence of disease, the stage of disease, the severity of disease, disease progression, non-progression, or improvement, etc.). As used herein, an "absolute amount" refers to the absolute value of the change or difference between at least two assay results taken or sampled at different time points, which, like a reference level, is herein related or associated with various clinical parameters (e.g., the presence of disease, the stage of disease, the severity of disease, disease progression, non-progression, or improvement, etc.). As used herein, an "absolute value" refers to the magnitude of the actual number (e.g., the difference between two compared levels (e.g., a level taken at a first time point and a level taken at a second time point)), regardless of its sign, i.e., whether it is positive or negative. The UCH-L1 and / or GFAP or CK-MB, β-hCG, TSH, homocysteine ​​and / or free T4 reference levels referred to herein are derived from venous blood.

[0227] The present disclosure provides exemplary reference levels and absolute amounts (e.g., calculated by comparing reference levels at different time points). However, it is well known that reference levels and absolute amounts may vary depending on the properties of the immunoassay (e.g., the antibody used, the reaction conditions, the purity of the sample, etc.), and assays can be compared and standardized. Furthermore, it is well within the skill of one of ordinary skill in the art to adapt the present disclosure herein to other immunoassays and obtain immunoassay-specific reference levels and absolute amounts for these other immunoassays based on the descriptions provided by this disclosure. While the exact values ​​of reference levels and absolute amounts may vary between assays, the findings described herein are generally applicable and can be extrapolated to other assays.

[0228] As used herein, "removably coupled" or "removably connected" means that one component is coupled to another component in an essentially temporary manner. That is, the two components are coupled in a manner that allows for easy connection or separation of the components without damaging the components. Thus, "removably coupled" components can be readily uncoupled and recoupled without damaging the components.

[0229] As used herein, a "result" refers to an item of information obtained by performing an assay. In one aspect, the result is the amount of a biomarker (e.g., UCH-L1, GFAP, CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof) in a test sample (e.g., a capillary blood sample). In another aspect, the result is identifying the presence of a biomarker (e.g., UCH-L1, GFAP, CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof) in the sample. The result can be displayed visually (e.g., as a readout).

[0230] As used herein, "risk assessment," "risk classification," "risk identification," or "risk stratification" of a subject (e.g., a patient) refers to the assessment of factors, including biomarkers, to predict the risk of developing a future event, including disease onset or disease progression, so that treatment decisions for the subject can be made on a more informed, condition-based basis.

[0231] As used herein, the term "plasma separation device" refers to an apparatus or device that can be used to separate components of whole blood (e.g., red blood cells and white blood cells) from serum, plasma, or serum and plasma using a separation system such as, for example, at least one membrane, filter, synthetic paper (e.g., microprismatic scaffold), or any combination thereof. For example, membranes and / or filters that can be used in plasma separation devices can comprise at least one of polycarbonate, polysulfone, polyester, polyethylene, polyurethane, and polypropylene. In some embodiments, the membranes and / or filters are pretreated (e.g., with one or more polycations, zwitterions, one or more non-covalent surface treatments (e.g., PEGMA, HEMA, BSA, O2 plasma, etc.)). In other embodiments, the membranes and / or filters are not pretreated. In still further embodiments, the filters that can be used are gravity-assisted separation systems. Examples of plasma separation devices that can be used in the methods described herein include the devices described in U.S. Patent Application Publication No. 2020 / 0124508, the contents of which are incorporated herein by reference. In some aspects, the plasma separation device does not include a lateral flow device.

[0232] As used herein, the "sensitivity" of an assay refers to the proportion of subjects with a positive outcome who are correctly identified as positive (e.g., correctly identifying a subject who has the disease or medical condition for which the subject is being tested). For example, this can include correctly identifying a subject as having a TBI separately from subjects who do not have a TBI, correctly identifying a subject with a moderate, severe, or moderate-severe TBI separately from subjects who have a mild TBI, correctly identifying a subject as having a mild TBI separately from subjects who have a moderate, severe, or moderate-severe TBI, correctly identifying a subject as having a moderate, severe, or moderate-severe TBI separately from subjects who do not have a TBI, or correctly identifying a subject as having a mild TBI separately from subjects who do not have a TBI, etc.

[0233] As used herein, the "specificity" of an assay refers to the proportion of subjects whose outcome is negative who are correctly identified as negative (e.g., the proportion of subjects who correctly identify themselves as not having the disease or medical condition for which they are being tested). For example, this can include correctly identifying subjects who do not have a TBI separately from those who have a TBI; correctly identifying subjects who do not have a moderate, severe, or moderate-severe TBI separately from those who have a mild TBI; correctly identifying subjects as not having a mild TBI separately from those who have a moderate, severe, or moderate-severe TBI, etc.

[0234] A "series of calibration compositions" refers to a plurality of compositions containing known amounts of UCH-L1, GFAP, UCH-L1, CK-MB, β-hCG, TSH, homocysteine, or free T4, each of which differs from the other compositions in the series by the amount of UCH-L1, GFAP, UCH-L1, CK-MB, β-hCG, TSH, homocysteine, or free T4.

[0235] As used interchangeably herein, "solid phase" or "solid support" refers to any material to which (1) one or more capture agents or specific binding partners for capture or (2) one or more detection agents or specific binding partners for detection can be attached and / or attract and immobilize. The solid phase can be selected for its intrinsic ability to attract and immobilize capture agents. Alternatively, the solid phase has attached a linking agent capable of attracting and immobilizing (1) the capture agent or specific binding partner for capture or (2) the detection agent or specific binding partner for detection. For example, the linking agent can comprise a charged substance that is oppositely charged relative to the capture agent (e.g., the specific binding partner for capture) or detection agent (e.g., the specific binding partner for detection) itself or a charged substance conjugated to (1) the capture agent or specific binding partner for capture or (2) the detection agent or specific binding partner for detection. Generally, the linking agent can be any binding partner (preferably a specific binding partner) that can be immobilized on (attached to) a solid phase and immobilize (1) a capture agent or a specific binding partner for capture, or (2) a detection agent or a specific binding partner for detection, via a binding reaction. The linking agent allows for indirect binding of the capture agent to the solid phase material before or during the performance of the assay. For example, the solid phase can be plastic, derivatized plastic, magnetic or non-magnetic metal, glass, or silicon, including, for example, test tubes, microtiter wells, sheets, beads, microparticles, chips, and other configurations known to those skilled in the art.

[0236] As used herein, "specific binding" or "specifically binding to" can refer to the interaction of an antibody, protein, or peptide with a second chemical species, where the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure, rather than proteins in general. If an antibody is specific for epitope "A," then the presence of a molecule containing epitope A (or free A, unlabeled A) in a reaction containing labeled "A" and the antibody will reduce the amount of labeled A bound to the antibody.

[0237] A "specific binding partner" is a member of a specific binding pair. A specific binding pair comprises two distinct molecules that specifically bind to one another through chemical or physical means. Thus, in addition to the specific binding pair of antigen and antibody of a typical immunoassay, other specific binding pairs can include biotin and avidin (or streptavidin), carbohydrates and lectins, complementary nucleotide sequences, effector and receptor molecules, cofactors and enzymes, enzymes and enzyme inhibitors, and the like. Furthermore, specific binding pairs can include members that are analogs of the original specific binding member, e.g., analyte-analogs. Immunoreactive specific binding members include antigens, antigen fragments, and monoclonal and polyclonal antibodies, as well as antibodies, including complexes and fragments thereof, whether isolated or recombinantly produced.

[0238] As used herein, "statistically significant" refers to the likelihood that a relationship between two or more variables is caused by something other than random chance. Statistical hypothesis testing is used to determine whether the results of a data set are statistically significant. In statistical hypothesis testing, a statistically significant result is reached whenever the observed p-value for the test statistic is less than the significance level defined for the study. The p-value is the probability of obtaining a result at least as extreme as the result observed if the null hypothesis were true. Examples of statistical hypothesis analysis include the Wilcoxon matched-rank test, t-test, chi-square test, or Fisher's exact test. As used herein, "significant" refers to a change that has not been determined to be statistically significant (e.g., may not have been subjected to statistical hypothesis testing).

[0239] As used interchangeably herein, "subject" and "patient" refer to any vertebrate, including, but not limited to, mammals (e.g., cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats and mice, non-human primates (e.g., monkeys such as cynomolgus or rhesus monkeys, chimpanzees), and humans). In some embodiments, the subject can be human or non-human. In some embodiments, the subject is human. The subject or patient may be undergoing other forms of treatment.

[0240] Thyroxine or T4 circulates in the blood as an equilibrium mixture of free and serum protein-bound hormone. Thyroxine-binding globulin (TBG), albumin, and prealbumin bind approximately 75%, 10%, and 15% of total circulating T4, respectively. T4 binding by these proteins is such that less than 0.03% exists in the circulation as unbound free T4. This small percentage of total T4 represents the biologically active, physiologically available hormone. As free T4 is absorbed by target cells, equilibrium reestablishes circulating free T4 levels. If changes occur in either the concentration or affinity of serum-binding proteins, equilibrium results in the maintenance of a constant level of free T4. Thus, in various normal (pregnancy) and abnormal (familial dysalbuminergic hyperthyroxinemia, FDH) conditions, or as a result of the administration of certain drugs (e.g., furosemide and fenclofenac), target tissues are guaranteed to receive the required amount of hormone. Because free T4 is less sensitive to changes in serum binding proteins, free T4 levels often provided the best indicator of thyroid dysfunction.

[0241] As used herein, "treating," "treating," or "treatment" are each used interchangeably to describe preventing, alleviating, or inhibiting the progression of a disease and / or injury, or one or more symptoms of such a disease to which such term applies. Depending on the condition of the subject, the term also refers to preventing a disease, including preventing the onset of a disease or preventing symptoms associated with a disease. Treatment can be performed acutely or chronically. The term also refers to reducing the severity of a disease or symptoms associated with such a disease before the onset of the disease. Such prevention or reduction of the severity of a disease before onset refers to the administration of a pharmaceutical composition to a subject who is not affected by the disease at the time of administration. "Preventing" also refers to preventing the recurrence of a disease or one or more symptoms associated with such a disease. "Treatment" and "therapeutically" refer to the act of treating, where "treating" is as defined above.

[0242] As used herein, "transfer tube" refers to a container or receptacle used to transfer a fluid (e.g., a capillary blood sample) from one location to a second location (e.g., to a reactor or from a plasma separation device).

[0243] "Traumatic brain injury" or "TBI," used interchangeably herein, refers to a complex injury with a wide range of symptoms and disability. TBI is most often an acute event similar to other injuries. TBI can be classified as "mild," "moderate," or "severe." Causes of TBI are diverse and include, for example, being physically shaken by a person, a motor vehicle accident, a weapon injury, a cerebrovascular accident (e.g., stroke), a fall, an explosion or blast, and other types of blunt force trauma. Other causes of TBI include ingestion of and / or exposure to one or more fires, chemicals or toxins (e.g., mold, asbestos, pesticides and insecticides, organic solvents, paints, glues, gases (e.g., carbon monoxide, hydrogen sulfide, and cyanides), organometallics (e.g., methylmercury, tetraethyl lead, and organotins), one or more drugs of abuse, or a combination thereof). Alternatively, TBI may occur in subjects suffering from an autoimmune disease, metabolic disorder, brain tumor, hypoxia, viral infection (e.g., SARS-CoV-2, meningitis, etc.), fungal infection (e.g., meningitis), bacterial infection (e.g., meningitis), or any combination thereof. Young adults and the elderly are the age groups at highest risk for TBI. In certain embodiments herein, traumatic brain injury or TBI does not include, and specifically excludes, cerebrovascular accidents such as stroke.

[0244] As used herein, "mild TBI" refers to a head injury in which a subject may or may not experience loss of consciousness. In subjects who experience loss of consciousness, the loss of consciousness is typically brief, usually lasting only a few seconds or minutes. Mild TBI is also referred to as concussion, mild head trauma, mild TBI, mild brain injury, and mild head injury. While MRI and CT scans are often normal, individuals with mild TBI may have cognitive problems such as headache, difficulty thinking, memory problems, attention deficits, mood swings, and frustration.

[0245] Mild TBI is the most common type of TBI and is often overlooked at the time of initial injury. Typically, subjects have a GCS score between 13 and 15 (e.g., 13-15 or 14-15). Of people with mild TBI, 15 percent (15%) have symptoms that persist for three months or more. Common symptoms of mild TBI include fatigue, headache, visual disturbances, memory loss, decreased attention / concentration, sleep disturbances, vertigo / loss of balance, irritability (emotional disturbance), depression, and seizures. Other symptoms associated with mild TBI include nausea, loss of smell, sensitivity to light and sound, mood changes, confusion or confusion, and / or slowed thinking.

[0246] As used herein, "moderate TBI" refers to a brain injury in which loss of consciousness and / or confusion and disorientation occurs for 1 to 24 hours, and the subject has a Glasgow Coma Scale (GCS) score of 9 to 13 (e.g., 9 to 12 or 9 to 13). Individuals with moderate TBI may have abnormal brain imaging results. As used herein, "severe TBI" refers to a brain injury in which loss of consciousness occurs for more than 24 hours, memory loss after injury or penetrating skull injury occurs for more than 24 hours, and the subject has a Glasgow Coma Scale (GCS) score of 3 to 8. Deficits range from high-level cognitive impairment to coma. Survivors may have limited arm or leg function, speech or language abnormalities, loss of thinking skills, or emotional problems. Individuals with severe injury may remain in a prolonged state of incapacity. For many people with severe TBI, prolonged rehabilitation is often required to maximize function and independence.

[0247] As used herein, "moderate-severe" TBI refers to a range of brain injury (e.g., over time) that includes changes from moderate to severe TBI over time, and thus encompasses moderate TBI alone, severe TBI alone, and combined moderate-severe TBI. For example, in some clinical situations, a subject may initially be diagnosed with a moderate TBI, but over time (minutes, hours, or days), the subject develops a severe TBI (e.g., in the setting of a cerebral hemorrhage). Alternatively, in some clinical situations, a subject may initially be diagnosed with a severe TBI, but over time (minutes, hours, or days), the subject develops a moderate TBI. Such subjects are considered examples of patients that can be classified as "moderate-severe." Common symptoms of moderate to severe TBI include difficulties with attention, concentration, distractibility, memory, processing speed, confusion, perseveration, impulsivity, language processing and / or "executive function", inability to understand spoken words (receptive aphasia), difficulty speaking and being understood (expressive aphasia), slurred speech, very fast or very slow speech, problems reading, problems writing, difficulty interpreting touch, temperature, movement, limb position and fine discrimination, difficulty integrating or patterning sensory impressions into psychologically meaningful data, partial or total loss of vision, weakness and double vision (diplopia), blurred vision, difficulty judging distances. Symptoms of TBI include problems reading, involuntary eye movements (nystagmus), light intolerance (photophobia), hearing problems such as reduced or lost hearing, ringing in the ears (tinnitus), hypersensitivity to sound, loss or diminished sense of smell (anosmia), loss or diminished sense of taste, seizures associated with epilepsy, which can be of several types and may involve problems with consciousness, sensory perception or movement, bowel and bladder control, sleep disturbances, loss of energy, changes in appetite, regulation of internal temperature, menstrual difficulties, addictive behaviors, problems with emotional capacity or stability, lack of motivation, irritability, aggression, depression, disinhibition, or cognitive deficits including denial / lack of consciousness. Subjects with moderate to severe TBI can have a Glasgow Coma Scale (GCS) score of 3 to 12 (including a range of 9 to 12 for moderate TBI and 3 to 8 for severe TBI).

[0248] "Ubiquitin carboxy-terminal hydrolase L1" or "UCH-L1," as used interchangeably herein, refers to the deubiquitinating enzyme encoded by the UCH-L1 gene in humans. UCH-L1, also known as ubiquitin carboxyl-terminal esterase L1 and ubiquitin thiolesterase, is a member of a gene family whose products hydrolyze small C-terminal appendages of ubiquitin to generate ubiquitin monomers.

[0249] "UCH-L1 status" can refer to the level or amount of UCH-L1 at a point in time (such as a point in time involving a single measurement of UCH-L1), the level or amount of UCH-L1 associated with monitoring (such as monitoring involving repeated testing to identify increases or decreases in UCH-L1 amounts in a subject), the level or amount of UCH-L1 associated with treatment for traumatic brain injury (whether primary and / or secondary brain injury), or a combination thereof.

[0250] As used herein, the term "variant" is used to describe a peptide or polypeptide that differs in amino acid sequence by amino acid insertion, deletion, or conservative substitution but retains at least one biological activity. Representative examples of "biological activity" include the ability to be bound by a specific antibody or the ability to stimulate an immune response. As used herein, the term "variant" is also used to describe a protein with an amino acid sequence substantially identical to a reference protein, with the amino acid sequence retaining at least one biological activity. Conservative amino acid substitutions, i.e., replacing an amino acid with a different amino acid with similar properties (e.g., hydrophilicity, degree of charge, and distribution of charged regions), are recognized in the art as typically involving minor changes. As understood in the art, these minor changes can be identified, in part, by examining the hydrophobicity index of an amino acid (Kyte et al., J. Mol. Biol. 157:105-132 (1982)). The hydrophobicity index of an amino acid is based on its hydrophobicity and charge. It is known in the art that amino acids with similar hydrophilicity indices may be substituted and still retain protein function. In one embodiment, amino acids with hydrophilicity indices of ±2 are substituted. The hydrophilicity of amino acids can also be used to identify substitutions that result in proteins that retain biological function. Consideration of the hydrophilicity of amino acids in the context of a peptide allows for calculation of the maximum local average hydrophilicity of the peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity (U.S. Pat. No. 4,554,101, incorporated herein by reference). Substitution with amino acids with similar hydrophilicity values ​​can result in peptides that retain biological activity, such as immunogenicity as understood in the art. Substitutions can be made with amino acids with hydrophilicity values ​​within ±2 of each other. Both the hydrophobicity index and hydrophilicity value of an amino acid are influenced by the specific side chain of the amino acid.Consistent with this observation, it is understood that amino acid substitutions that are compatible with biological function depend on the relative similarity of the amino acids, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties, particularly the side chains of those amino acids. "Variant" can also be used to refer to antigenically reactive fragments of anti-UCH-L1 antibodies that differ in amino acid sequence from the corresponding anti-UCH-L1 antibody fragment but are still antigenically reactive and can compete with the corresponding anti-UCH-L1 antibody fragment for binding to UCH-L1. "Variant" can also be used to describe polypeptides or fragments thereof that have been processed differently, such as by proteolysis, phosphorylation, or other post-translational modification, but that retain their antigenic reactivity.

[0251] As used herein, the term "vector" is used to describe a nucleic acid molecule capable of carrying another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, into which additional DNA segments can be ligated. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and mammalian episomal vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. Since plasmids are the most commonly used form of vector, the terms "plasmid" and "vector" can be used interchangeably. However, other forms of expression vectors, such as viral vectors (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions, can also be used. In this regard, RNA forms of vectors (including viral RNA vectors) can also be used in the context of the present disclosure.

[0252] As used herein, "venous blood" refers to a blood sample obtained from a subject's vein using a syringe, needle, or a combination thereof, or any suitable device. In some embodiments, the venous blood sample is obtained by a trained medical clinician, such as a physician, phlebotomist, nurse, laboratory technician, or a combination thereof. In some embodiments, the venous blood sample is whole blood, serum, or plasma.

[0253] Unless otherwise specified, technical and scientific terms used herein shall have the same meaning as commonly understood by those skilled in the art. For example, the terminology used in connection with, and techniques relating to, cell and tissue culture methods, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well-known terminology and techniques commonly used in the art. The meaning and scope of terms shall be clear, but in the unlikely event of any potential ambiguity, the definitions provided herein shall take precedence over any dictionary or external definitions. Furthermore, unless otherwise required by context, singular terms shall include the plural, and plural terms shall include the singular.

[0254] 2. Methods for determining the amount or presence of UCH-L1, GFAP, or a combination thereof in a capillary blood sample obtained from a subject In one embodiment, the present disclosure relates to an improved method for determining the amount (e.g., a quantitative measure) or presence (e.g., a qualitative measure) of UCH-L1, GFAP, or UCH-L1 and GFAP in a capillary blood sample obtained from a subject (e.g., a human subject). The amount or presence of UCH-L1, GFAP, or UCH-L1 and GFAP determined according to the methods described herein can be used to assess whether a subject (e.g., a human subject) has suffered or may have suffered a head injury and / or to aid in the diagnosis and assessment thereof. The methods and devices described herein can aid in determining the extent of traumatic brain injury in a subject (e.g., a human subject) with actual or suspected head injury, for example, determining whether the subject (e.g., a human subject) has mild traumatic brain injury, moderate traumatic brain injury, severe traumatic brain injury, or moderate-severe traumatic brain injury. As used herein, "determining whether a subject (e.g., a human subject) has mild traumatic brain injury, moderate traumatic brain injury, severe traumatic brain injury, or moderate-severe traumatic brain injury" refers to the fact that the above-described methods, when used, for example, in conjunction with other information (e.g., clinical assessment data), can determine that a subject is more likely than not to have mild traumatic brain injury, moderate traumatic brain injury, severe traumatic brain injury, or moderate-severe traumatic brain injury. The method can include performing an assay on a sample obtained from a subject (e.g., a human subject) within about 24 hours after an actual or suspected head injury to measure or detect a level of a biomarker for traumatic brain injury, such as ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof, in the sample, and determining whether the subject (e.g., a human subject) has suffered a mild, moderate, severe, or moderate-to-severe traumatic brain injury (TBI). In some embodiments, if the amount of the biomarker in the sample is higher than a reference level for the biomarker (e.g., UCH-L1, GFAP, or a combination of UCH-L1 and GFAP), the subject is determined to have a mild, moderate, severe, or moderate-to-severe TBI.

[0255] In some aspects, the sample obtained from a subject (e.g., a human subject) is a capillary blood sample, such as whole blood, serum, or plasma. In some aspects, the capillary blood sample is whole blood. In other aspects, the capillary blood sample is serum. In still other aspects, the capillary blood sample is plasma.

[0256] A capillary blood sample can be obtained by extracting a sample through a subject's skin (e.g., fingers and / or toes, hand, foot (including heel), earlobe, location on arm and / or leg, chest, back, head, or any combination thereof, etc.). In some embodiments, a capillary blood sample is extracted from a finger or toe. In other embodiments, an entire capillary blood sample is extracted from an arm or leg. In still other embodiments, a capillary blood sample is obtained from a hand or foot. In still other embodiments, a capillary blood sample is obtained from the chest or back. In still other embodiments, a capillary blood sample is obtained from an earlobe. In still other embodiments, a capillary blood sample is obtained from the head.

[0257] In some embodiments, the capillary blood sample obtained from the subject is obtained without using a syringe, needle (e.g., a 21-gauge needle, a butterfly needle, etc.), or any other suitable device, or any combination thereof, typically used to collect blood (e.g., venous blood). Instead, the capillary blood sample is obtained using a blood collection device that is administered to the subject or another person. Examples of blood collection devices that are administered to the subject or another person include microsampling devices. Examples of microsampling devices that may be used herein include the TAP device available from YourBio Health, Inc. (Cambridge, MA), as well as the devices described in U.S. Pat. No. 9,113,836, the contents of which are incorporated herein by reference; the Tasso+, Tasso-M20, and Tasso-ST devices available from Tasso, Inc. (Seattle, WA); the One Draw device available from Draw Bridge Health (San Diego, CA); the PBS-1000 from PreciHealth (Neuchatel, Switzerland); or the Loop blood collection device available from Loop Medical (Lausanne, Switzerland).

[0258] In other embodiments, capillary blood samples are obtained or collected from subjects in a decentralized setting, for example, capillary blood samples can be obtained or collected from urgent care clinics, pharmacies, grocery stores or other convenience stores, residences, workplaces, and / or government offices.

[0259] Additionally or alternatively, in still further embodiments, the capillary blood sample is obtained from the subject by a user who is not trained in blood collection (e.g., other than a trained phlebotomist, nurse, medical assistant, and / or physician). For example, the capillary blood sample can be obtained by the subject themselves, a relative, a friend, a colleague, a teacher, a pharmacist, and / or any other individual. In still further embodiments, the capillary blood sample is obtained from the subject by a robot.

[0260] In some embodiments, the capillary blood sample obtained from the subject is less than about 4 mL in volume. In some embodiments, the capillary blood sample obtained from the subject is less than about 3 mL. In some embodiments, the capillary blood sample obtained from the subject is less than about 2 mL. In some embodiments, the volume of a capillary blood sample obtained from a subject is less than about 3.9 mL, about 3.8 mL, about 3.7 mL, about 3.6 mL, about 3.5 mL, about 3.4 mL, about 3.3 mL, about 3.2 mL, about 3.1 mL, about 3.0 mL, about 2.9 mL, about 2.8 mL, about 2.7 mL, about 2.6 mL, about 2.5 mL, about 2.4 mL, about 2.3 mL, about 2.2 mL, about 2.1 mL, about 2.0 mL, about 1.9 mL, about 1.8 mL, about 1.7 mL, about 1.6 mL, about 1.5 mL, about 1.4 mL, about 1.3 mL, about 1.2 mL, about 1.1 mL, about 1.0 mL, about 0.9 mL, about 0.8 mL, about 0.7 mL, about 0.6 mL, or about 0.5 mL. In some embodiments, when the collected sample is whole blood, a larger volume of capillary blood can be obtained.

[0261] In some embodiments, the capillary blood sample obtained from the subject is whole blood that has been subjected to further or additional processing before determining the amount of UCH-L1, GFAP, or UCH-L1 and GFAP in the sample. In some embodiments, the sample is processed using centrifugation. In other embodiments, the sample is processed using a plasma separation device that can include at least one filter, membrane, and / or synthetic paper. For example, in some embodiments, the plasma separation device that can be used is the device described in Section 4. The plasma separation device can separate whole blood into serum and / or plasma, which can then be used in the methods described herein.

[0262] In other embodiments, the plasma separation device may be in fluid communication with, or operably linked, coupled, and / or removably coupled to, the microsampling device as part of a system, as described in more detail in Section 5. In other embodiments, the plasma separation device may be integrated into the microsampling device, as described in more detail in Section 5.

[0263] In still other embodiments, the plasma separation device may be in fluid communication with, or operably linked, coupled, and / or removably coupled to, an opening in the reactor, as described in more detail in Section 5. For example, in some embodiments, the reactor is a cartridge, such as a cartridge used in a point-of-care device.

[0264] In still other embodiments, the plasma separation device may be in fluid communication with, or operably linked, coupled, and / or removably coupled to, a transfer tube, as described in further detail in Section 5. In these embodiments, the transfer may be in fluid communication with, or operably linked, coupled, and / or removably coupled to, a reactor, as described in further detail in Section 5. In still further embodiments, the plasma separation device may be integrated into the transfer tube. In still other embodiments, the transfer tube includes a cap or stopper.

[0265] In some aspects, the method can include obtaining a sample within about 24 hours of actual or suspected injury to the subject and contacting the sample with an antibody to a biomarker for TBI, such as UCH-L1, GFAP, or a combination thereof, to form an antibody-biomarker complex. More specifically, the sample can be contacted with (a) an anti-UCH-L1 antibody; (b) an anti-GFAP antibody; or (c) an anti-UCH-L1 antibody and an anti-UCH-L1 antibody. The method also includes detecting the resulting antibody-biomarker complex.

[0266] In some embodiments, the sample is taken from a subject (eg, a human subject) within about 24 hours of an actual or suspected injury to the head. For example, a sample can be collected from a subject (e.g., a human subject) within about 0 minutes, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 60 minutes, about 90 minutes, within about 2 hours, within about 3 hours, within about 4 hours, within about 5 hours, within about 6 hours, within about 7 hours, within about 8 hours, within about 9 hours, within about 10 hours, within about 11 hours, within about 12 hours, within about 13 hours, within about 14 hours, within about 15 hours, within about 16 hours, within about 17 hours, within about 18 hours, within about 19 hours, within about 20 hours, within about 21 hours, within about 22 hours, within about 23 hours, or within about 24 hours after an actual or suspected injury to the head.

[0267] The amount of UCH-L1, GFAP, or UCH-L1 and GFAP in a sample obtained from a subject can be determined using an analog assay, a digital assay, or a combination of analog and digital assays. In some embodiments, the amount of UCH-L1, GFAP, or UCH-L1 and GFAP is determined using an analog assay. In other embodiments, the amount of UCH-L1, GFAP, or UCH-L1 and GFAP can be determined using a digital assay. In some embodiments, a digital assay is used when the ratio between the capillary and venous blood volumes of UCH-L1, GFAP, or UCH-L1 and GFAP is equal to or less than 1, or is expected to be so. In still other embodiments, an analog assay is used when the ratio between the capillary and venous blood volumes of UCH-L1, GFAP, or UCH-L1 and GFAP is greater than 1, or is expected to be so.

[0268] Examples of assays that can be used to determine the amount of UCH-L1, GFAP, or UCH-L1 and GFAP include immunoassays, such as enzyme immunoassays (EIA), enzyme-linked immunosorbent assays (ELISA), fluorescent immunoassays, chemiluminescence immunoassays (CLIA), radioimmunoassays (RIA), microparticle enzyme immunoassays (MEIA), and turbidimetric immunoassays. In yet other embodiments, the assay can be a clinical chemistry assay, such as, for example, a photometric, spectrophotometric, absorbance, fluorescent, turbidimetric, nephelometric, potentiometric, and / or electrophoretic assay. In still further embodiments, the assay can be a combination of an immunoassay and a clinical chemistry assay. In yet other embodiments, the assay can be a single molecule detection assay.

[0269] At least one assay for GFAP and at least one assay for UCH-L1 may be performed simultaneously. Alternatively, an assay for GFAP and an assay for UCH-L1 may be performed sequentially. The assays may be performed sequentially in any order. For example, an assay for GFAP may be performed first, followed by an assay for UCH-L1. As another example, an assay for UCH-L1 may be performed first, followed by an assay for GFAP.

[0270] In some embodiments, at least one assay for GFAP and / or at least one assay for UCH-L1 is or can be performed in less than about 30 minutes. In some other embodiments, at least one assay for GFAP and / or at least one assay for UCH-L1 is or can be performed in less than about 25 minutes. In still further embodiments, at least one assay for GFAP and / or at least one assay for UCH-L1 is or can be performed in less than about 20 minutes. In still other embodiments, at least one assay for GFAP and / or at least one assay for UCH-L1 is or can be performed in less than about 18 minutes. In still other embodiments, at least one assay for GFAP and / or at least one assay for UCH-L1 is or can be performed in about 4 to about 20 minutes. In still further embodiments, the at least one assay for GFAP and / or the at least one assay for UCH-L1 are each performed or capable of being performed in about 15 to about 18 minutes.

[0271] In some embodiments, at least one assay for GFAP and / or at least one assay for UCH-L1 is or can be performed in about 4 minutes. In some embodiments, at least one assay for GFAP and at least one assay for UCH-L1 is or can be performed in about 5 minutes. In some embodiments, at least one assay for GFAP and / or at least one assay for UCH-L1 is or can be performed in about 6 minutes. In some embodiments, at least one assay for GFAP and / or at least one assay for UCH-L1 is or can be performed in about 7 minutes. In some embodiments, at least one assay for GFAP and / or at least one assay for UCH-L1 is or can be performed in about 8 minutes. In some embodiments, at least one assay for GFAP and / or at least one assay for UCH-L1 is or can be performed in about 9 minutes. In some embodiments, at least one assay for GFAP and / or at least one assay for UCH-L1 is or can be performed in about 10 minutes. In some embodiments, at least one assay for GFAP and / or at least one assay for UCH-L1 is or can be performed in about 11 minutes. In some embodiments, at least one assay for GFAP and / or at least one assay for UCH-L1 is or can be performed in about 12 minutes. In some embodiments, at least one assay for GFAP and / or at least one assay for UCH-L1 is or can be performed in about 13 minutes. In some embodiments, at least one assay for GFAP and / or at least one assay for UCH-L1 is or can be performed in about 14 minutes.In some embodiments, at least one assay for GFAP and / or at least one assay for UCH-L1 is or can be performed in about 15 minutes. In some embodiments, at least one assay for GFAP and / or at least one assay for UCH-L1 is or can be performed in about 16 minutes. In some embodiments, at least one assay for GFAP and / or at least one assay for UCH-L1 is or can be performed in about 17 minutes. In some embodiments, at least one assay for GFAP and / or at least one assay for UCH-L1 is or can be performed in about 18 minutes. In some embodiments, at least one assay for GFAP and / or at least one assay for UCH-L1 is or can be performed in about 19 minutes. In some embodiments, at least one assay for GFAP and / or at least one assay for UCH-L1 is or can be performed in about 20 minutes.

[0272] In some embodiments, the subject receives a Glasgow Coma Scale (GCS) score before or after the assay is performed. In some embodiments, the subject (e.g., a human subject) is suspected of having moderate, severe, or moderate-severe traumatic brain injury based on the GCS (Glasgow Coma Scale) score. In some embodiments, a baseline level of a biomarker, such as UCH-L1, GFAP, or a combination thereof, correlates with the subject having moderate, severe, or moderate-severe traumatic brain injury. In some embodiments, a baseline level of a biomarker, such as UCH-L1, GFAP, or a combination thereof, correlates with a GCS (Glasgow Coma Scale) score of 9 to 13 (moderate TBI). In some embodiments, a baseline level of a biomarker, such as UCH-L1, GFAP, or a combination thereof, correlates with a GCS (Glasgow Coma Scale) score of 3 to 8 (severe TBI). In some embodiments, baseline levels of biomarkers such as UCH-L1, GFAP, or a combination thereof correlate with a GCS (Glasgow Coma Scale) score of 3 to 12 (moderate, severe, or moderate-severe TBI). In some embodiments, a subject is suspected of having mild traumatic brain injury based on the GCS (Glasgow Coma Scale) score. In some embodiments, baseline levels of biomarkers such as UCH-L1, GFAP, or a combination thereof correlate with a subject having mild traumatic brain injury. In some embodiments, baseline levels of biomarkers such as UCH-L1, GFAP, or a combination thereof correlate with a GCS (Glasgow Coma Scale) score of 13 to 15 (mild TBI).

[0273] Generally, a reference level of a biomarker, such as UCH-L1, GFAP, or a combination thereof, can also be used as a benchmark against which to assess results obtained when assaying a test sample for a biomarker, such as UCH-L1, GFAP, or a combination thereof. Generally, in making such a comparison, the reference level of a biomarker, such as UCH-L1, GFAP, or a combination thereof, is obtained by performing or executing a particular assay a sufficient number of times and under appropriate conditions so as to link or correlate the presence, amount, or concentration of the analyte with a particular stage or endpoint of TBI or with a particular symptom. Typically, the reference level of a biomarker, such as UCH-L1, GFAP, or a combination thereof, is obtained using an assay of a reference subject (or population of subjects). The measured biomarker, such as UCH-L1, GFAP, or a combination thereof, can include fragments thereof, degradation products thereof, and / or enzymatic cleavage products thereof.

[0274] In certain embodiments, the baseline level may be correlated to a control subject (eg, a human subject) who has not suffered a head injury.

[0275] In some embodiments, the baseline level of UCH-L1 is about 320 to about 400 pg / mL. In other embodiments, the baseline level of UCH-L1 is about 360 pg / mL. In yet further embodiments, the baseline level of UCH-L1 is about 400 pg / mL.

[0276] In some embodiments, the baseline level of UCH-L1 is about 320 to about 400 pg / mL and the sample is obtained from the subject within about 24 hours or less. In other embodiments, the baseline level of UCH-L1 is about 360 pg / mL and the sample is obtained from the subject within about 24 hours or less. In still other embodiments, the baseline level of UCH-L1 is about 400 pg / mL and the sample is obtained from the subject within about 24 hours or less.

[0277] In some embodiments, the baseline level of GFAP is about 25 to about 40 pg / mL. In other embodiments, the baseline level of GFAP is about 30 pg / mL. In yet further embodiments, the baseline level of GFAP is about 35 pg / mL.

[0278] In some embodiments, the baseline level of GFAP is about 25 to about 40 pg / mL and the sample is obtained from the subject within about 24 hours or less. In other embodiments, the baseline level of GFAP is about 30 pg / mL and the sample is obtained from the subject within about 24 hours or less. In still other embodiments, the baseline level of GFAP is about 35 pg / mL and the sample is obtained from the subject within about 24 hours or less.

[0279] In some embodiments, the baseline level of UCH-L1 is about 360 pg / mL and the baseline level of GFAP is about 30 pg / mL. In still other embodiments, the baseline level of UCH-L1 is about 400 pg / mL and the baseline level of GFAP is about 35 pg / mL. In still further embodiments, the baseline level of UCH-L1 is about 360 pg / mL and the baseline level of GFAP is about 30 pg / mL, and the sample is obtained from the subject within about 24 hours or less. In still other embodiments, the baseline level of UCH-L1 is about 400 pg / mL and the baseline level of GFAP is about 35 pg / mL, and the sample is obtained from the subject within about 24 hours or less.

[0280] In some embodiments, the methods include performing at least one assay for UCH-L1 and at least one assay for GFAP in at least one sample obtained from the subject, and determining whether the amounts (e.g., levels) of GFAP and UCH-L1 are elevated in the subject based on the results of the assays. In some embodiments, the methods include determining that the amounts of GFAP and UCH-L1 are elevated in the subject. In some embodiments, the methods include determining that the amounts of GFAP and UCH-L1 are elevated in the subject if the amount of GFAP in the sample is equal to or greater than 30 pg / mL and the level of UCH-L1 is below about 360 pg / mL, cannot be determined by an assay for UCH-L1, or is not reported by an assay for UCH-L1. In some embodiments, the methods include determining that the amounts of GFAP and UCH-L1 are elevated in the sample obtained from the subject if the level of GFAP is equal to or greater than about 30 pg / mL and the level of UCH-L1 is equal to or greater than about 360 pg / mL. In some aspects, the methods include determining that the amount of GFAP and UCH-L1 in the subject is elevated if the level of GFAP cannot be determined by an assay for GFAP or is not reported by an assay for GFAP and the level of UCH-L1 is equal to or greater than about 360 pg / mL.

[0281] In some embodiments, the method includes determining that the amount of GFAP and UCH-L1 in the subject is not elevated. In some embodiments, the method includes determining that the amount of GFAP and UCH-L1 in the subject is not elevated if the level of GFAP in the sample is below about 30 pg / mL and the level of UCH-L1 in the sample is below about 360 pg / mL.

[0282] In some embodiments, the method includes determining that assays for UCH-L1 and GFAP should be repeated. In some embodiments, the method includes determining that assays for UCH-L1 and GFAP should be repeated if the amount of GFAP is below about 30 pg / mL and the level of UCH-L1 cannot be determined by or is not reported by an assay for UCH-L1. In some embodiments, the method includes determining that assays for UCH-L1 and GFAP should be repeated if the amount of GFAP cannot be determined by or is not reported by an assay for GFAP and the level of UCH-L1 is below about 360 pg / mL. In some embodiments, the method includes determining that assays for UCH-L1 and GFAP should be repeated if the amount of GFAP cannot be determined by or is not reported by an assay for GFAP and the level of UCH-L1 cannot be determined by or is not reported by an assay for UCH-L1.

[0283] In some embodiments, the methods include performing at least one assay for UCH-L1 and at least one assay for GFAP on at least one sample obtained from the subject, and determining whether the amounts of GFAP and UCH-L1 are elevated in the subject based on the results of the assays. In some embodiments, the methods include determining that the amounts of GFAP and UCH-L1 are elevated in the subject. In some embodiments, the methods include determining that the amounts of GFAP and UCH-L1 are elevated in the subject if the amount of GFAP in the sample is greater than or equal to 35 pg / mL and the amount of UCH-L1 is below about 400 pg / mL, cannot be determined by an assay for UCH-L1, or is not reported by an assay for UCH-L1. In some embodiments, the methods include determining that the amounts of GFAP and UCH-L1 are elevated in the subject if the amount of GFAP is greater than or equal to about 35 pg / mL and the level of UCH-L1 is greater than or equal to about 400 pg / mL. In some aspects, the methods include determining that the amount of GFAP and UCH-L1 in the subject is elevated if the amount of GFAP cannot be determined by an assay for GFAP or is not reported by an assay for GFAP and the amount of UCH-L1 is equal to or greater than about 400 pg / mL.

[0284] In some embodiments, the method includes determining that the amount of GFAP and UCH-L1 in the subject is not elevated. In some embodiments, the method includes determining that the amount of GFAP and UCH-L1 in the subject is not elevated if the amount of GFAP in the sample is below about 35 pg / mL and the amount of UCH-L1 in the sample is below about 400 pg / mL.

[0285] In some embodiments, the method includes determining that assays for UCH-L1 and GFAP should be repeated. In some embodiments, the method includes determining that assays for UCH-L1 and GFAP should be repeated if the amount of GFAP is below about 35 pg / mL and the amount of UCH-L1 cannot be determined by or is not reported by an assay for UCH-L1. In some embodiments, the method includes determining that assays for UCH-L1 and GFAP should be repeated if the amount of GFAP cannot be determined by or is not reported by an assay for GFAP and the amount of UCH-L1 is below about 400 pg / mL. In some embodiments, the method includes determining that assays for UCH-L1 and GFAP should be repeated if the amount of GFAP cannot be determined by or is not reported by an assay for GFAP and the amount of UCH-L1 cannot be determined by or is not reported by an assay for UCH-L1.

[0286] In some embodiments, the methods further include performing a head computed tomography (CT) scan, a magnetic resonance imaging (MRI) procedure, or both a CT scan or an MRI procedure on the subject if the subject's levels of GFAP and UCH-L1 are elevated. For example, in some embodiments, the methods further include performing a head CT scan on the subject if the subject's levels of GFAP and UCH-L1 are elevated. As another example, in some embodiments, the methods further include performing an MRI procedure on the subject if the subject's levels of GFAP and UCH-L1 are elevated. In some embodiments, the methods further include performing a head CT scan and an MRI procedure on the subject if the subject's levels of GFAP and UCH-L1 are elevated.

[0287] In still further embodiments, results are obtained after the amount of UCH-L1 and / or GFAP in a capillary blood sample is determined using the methods described herein. In some further embodiments, the results may be further processed. Specifically, this further processing includes selecting a conversion factor for comparing the amount of UCH-L1 and / or GFAP in a capillary whole blood or plasma sample with the amount of UCH-L1 and / or GFAP in venous whole blood or plasma.

[0288] In some embodiments, when the assay is for UCH-L1, the conversion factor can be about 2.5:1.0 (capillary whole blood or plasma vs. venous whole blood or plasma) to about 1.5:1.0 ((capillary whole blood or plasma vs. venous whole blood or plasma)). Without wishing to be bound by any theory, it is believed that the higher levels of UCH-L1 demonstrated in capillary blood samples may be the result of noise levels rather than, for example, due to subjects suffering from traumatic brain injury.

[0289] In other embodiments, when the assay is an assay for GFAP, the conversion factor can be about 1.0:1.0 (capillary whole blood or plasma to venous whole blood or plasma).

[0290] Once the conversion factor is selected, processing can further include normalizing the amount of UCH-L1 and / or GFAP in the capillary whole blood or plasma sample by the amount of UCH-L1 and / or GFAP from the venous whole blood or plasma by applying the conversion factor to the amount of UCH-L1 and / or GFAP in the sample. For example, the amount of UCH-L1 and / or GFAP in the capillary whole blood or plasma sample can be multiplied by the conversion factor to obtain a normalized amount of UCH-L1 and / or GFAP in the sample.

[0291] In some aspects, the processing of the amount of UCH-L1 and / or GFAP (e.g., the results) may be performed by a processing system including a computer processor and non-transitory computer memory including one or more computer programs configured to select a conversion factor for comparing the amount of UCH-L1 and / or GFAP in the capillary whole blood or plasma sample with the amount of UCH-L1 and / or GFAP in the venous whole blood or plasma, and apply the selected conversion factor to the amount of UCH-L1 and / or GFAP in the sample, thereby normalizing the amount of UCH-L1 and / or GFAP in the capillary whole blood or plasma sample with the amount of UCH-L1 and / or GFAP from the venous whole blood or plasma.

[0292] The results or processed results may be communicated (e.g., reported) for further analysis, interpretation, processing, and / or display. The results may be communicated (e.g., reported) by computer, in a document and / or spreadsheet, on a mobile device (e.g., a smartphone), on a website, in an email, or any combination thereof. In some embodiments, the results may be displayed not as numbers, but as visual signals (e.g., lines or bars) that can be read / interpreted by a reading device or the naked eye.

[0293] In some embodiments, the results of UCH-L1, GFAP, or UCH-L1 and GFAP determined in the sample are communicated, such as by display on the device. In further embodiments, the results are displayed as an indication that the amount of UCH-L1 and / or GFAP in the subject is elevated, not elevated, or that the assay(s) for UCH-L1 and / or GFAP should be repeated.

[0294] Suitable devices for use in the methods described herein include higher throughput assay analyzers (e.g., the ARCHITECT platform sold by Abbott Laboratories) or point-of-care devices (e.g., the i-STAT and i-STAT Alinity devices sold by Abbott Laboratories) that can contain a user interface that can display the determination. Other devices for use in the methods described herein include lateral flow devices (e.g., lateral flow devices equipped with a reader).

[0295] In some embodiments, the UCH-L1, GFAP, or UCH-L1 and GFAP results are or can be communicated within about 4 minutes to about 40 minutes from the time the sample is collected (e.g., from the time of injury or suspected injury). In other embodiments, the results are or can be communicated within about 4 minutes to about 30 minutes from the time the sample is collected (e.g., from the time of injury or suspected injury). In still other embodiments, the results are or can be communicated within about 4 minutes to about 20 minutes from the time the sample is collected (e.g., from the time of injury or suspected injury). In some embodiments, results are obtained within about 40 minutes or less, about 39 minutes or less, about 38 minutes or less, about 37 minutes or less, about 36 minutes or less, about 35 minutes or less, about 34 minutes or less, about 33 minutes or less, about 32 minutes or less, about 31 minutes or less, about 30 minutes or less, about 29 minutes or less, about 28 minutes or less from the time the sample was collected (e.g., from the time of injury or suspected injury). The delivery may be or be capable of being delivered in about 27 minutes or less, about 26 minutes or less, about 25 minutes or less, about 24 minutes or less, about 23 minutes or less, about 22 minutes or less, about 21 minutes or less, about 20 minutes, about 19 minutes, about 18 minutes, about 17 minutes, about 16 minutes, about 15 minutes, about 14 minutes, about 13 minutes, about 12 minutes, about 11 minutes, about 10 minutes, about 9 minutes, about 8 minutes, about 7 minutes, about 6 minutes, about 5 minutes, or about 4 minutes.

[0296] In some embodiments of the above-described methods, the time from sample collection (from the subject) to the time the results for GFAP, UCH-L1, or GFAP and UCH-L1 are communicated is less than about 30 minutes. In other embodiments of the above-described methods, the time from sample collection to the time the results for GFAP, UCH-L1, or GFAP and UCH-L1 are communicated is less than about 25 minutes. In some other embodiments of the above-described methods, the time from sample collection to the time the results for GFAP, UCH-L1, or GFAP and UCH-L1 are communicated is less than about 20 minutes. In still further embodiments of the above-described methods, the time from sample collection to the time the results for GFAP, UCH-L1, or GFAP and UCH-L1 are communicated is less than about 18 minutes. In some embodiments of the above-described methods, the time from sample collection to the time the results for GFAP, UCH-L1, or GFAP and UCH-L1 are communicated is less than about 15 minutes. In some embodiments of the above-described methods, the time from sample collection to the time results for GFAP, UCH-L1, or GFAP and UCH-L1 are communicated is about 4 to about 20 minutes. In some embodiments of the above-described methods, the time from sample collection to the time results for GFAP, UCH-L1, or GFAP and UCH-L1 are communicated is about 15 to about 20 minutes.

[0297] In some embodiments, the device contains software for performing one or more tasks, including implementing the methods and algorithms described herein. In some embodiments, the device contains software for automatically determining the appropriate next step in the methods and algorithms as described herein. For example, the device can contain software that determines the amount or presence of an analyte of interest. The software can display this determination, such as in a graphical user interface.

[0298] In some aspects, the device stores software that instructs the processor to perform given tasks. In some aspects, the software stores machine-readable instructions that instruct the processor to perform given tasks. The machine-readable instructions may be one or more executable programs or portion(s) of an executable program for execution by a computer. The program may be embodied in software stored on a non-transitory computer-readable storage medium, such as a CD-ROM, floppy disk, hard drive, DVD, Blu-ray disk, or memory associated with the processor. Alternatively, the entire program and / or portions thereof may instead be executed by a device other than a processor and / or may be embodied in firmware or dedicated hardware. Additionally or alternatively, the process may be implemented by one or more hardware circuits (e.g., discrete and / or integrated analog and / or digital circuits, FPGAs, ASICs, comparators, operational amplifiers (op-amps), logic circuits, etc.) structured to perform corresponding operations without executing software or firmware.

[0299] Machine-readable instructions may be stored in one or more of a compressed format, an encrypted format, a fragmented format, a compiled format, an executable format, a packaged format, etc. The machine-readable instructions described herein may be stored as data (e.g., portions of instructions, code, representations of code, etc.) that can be used to create, manufacture, and / or produce machine-executable instructions. For example, machine-readable instructions may be fragmented and stored on one or more storage devices and / or computing devices (e.g., servers). The machine-readable instructions may require one or more of installing, modifying, adapting, updating, combining, adding, configuring, decrypting, decompressing, unpacking, distributing, rearranging, editing, etc. to make the instructions directly readable, interpretable, and / or executable by computing devices and / or other machines. For example, machine-readable instructions may be stored in multiple portions that are individually compressed, encrypted, and stored on separate computing devices, and the portions, when decrypted, decompressed, and combined, form a set of executable instructions that execute a program such as those described herein.

[0300] In another example, machine-readable instructions may be stored in a state that can be read by a computer, but require the addition of a library (e.g., a dynamic link library (DLL)), a software development kit (SDK), an application programming interface (API), etc., to execute the instructions on a particular computing device or other device. In another example, the machine-readable instructions may need to be configured (e.g., stored settings, data entries, recorded network addresses, etc.) before the machine-readable instructions and / or corresponding program(s) can be executed, in whole or in part. Thus, disclosed machine-readable instructions and / or corresponding program(s) are intended to encompass such machine-readable instructions and / or program(s) regardless of the particular format or state of the machine-readable instructions and / or program(s) when stored or otherwise being stored or transferred.

[0301] The machine-readable instructions described herein may be expressed in any past, present, or future command language, scripting language, programming language, etc. For example, the machine-readable instructions may be expressed using any of the following languages: C, C++, Java, C#, Perl, Python, JavaScript, HyperText Markup Language (HTML), Structured Query Language (SQL), Swift, etc.

[0302] The machine-readable instructions may be stored on a non-transitory computer and / or machine-readable medium, such as a hard disk drive, flash memory, read-only memory, compact disk, digital versatile disk, cache, random access memory, and / or any other storage device or storage disk on which information is stored for any period of time (e.g., long term, permanently, for a short period of time, for temporary buffering, and / or for caching of information). As used herein, the term non-transitory computer-readable medium is expressly defined to include any type of computer-readable storage device and / or storage disk, but to exclude propagated signals and to exclude transmission media.

[0303] 3. A method for determining the amount or presence of CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof in a capillary blood sample obtained from a subject In one embodiment, the present disclosure relates to an improved method for determining the amount (e.g., a quantitative measure) or presence (e.g., a qualitative measure) of CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof in a capillary blood sample obtained from a subject (e.g., a human subject). In some aspects, the amount or presence of CK-MB determined in a capillary blood sample obtained according to the methods described herein can be used to diagnose myocardial infarction. In some aspects, if the amount of CK-MB in the sample is greater than a reference level of a biomarker (e.g., CK-MB), the subject is determined to have myocardial infarction.

[0304] In still other embodiments, the amount or presence of β-hCG determined in a capillary blood sample obtained according to the methods described herein can be used to determine whether a subject is pregnant. In some embodiments, if the amount of β-hCG in the sample is greater than a reference level for a biomarker (e.g., β-hCG), the subject (e.g., a female subject) is determined to be likely pregnant.

[0305] In still other embodiments, the amount or presence of TSH determined in a capillary blood sample obtained according to the methods described herein can be used to diagnose thyroid disease in a subject, treat thyroid disease in a subject, or any combination thereof. In some embodiments, if the amount of TSH in the sample is greater than a reference level of a biomarker (e.g., TSH), the subject is determined to have thyroid disease.

[0306] In still other aspects, the amount or presence of homocysteine ​​determined in a capillary blood sample obtained according to the methods described herein can be used to diagnose hyperhomocysteinemia, homocystinuria, or hyperhomocysteinemia and homocystinuria in a subject, or to treat a subject with hyperhomocysteinemia, homocystinuria, or hyperhomocysteinemia and homocystinuria, or any combination thereof. In some aspects, if the amount of homocysteine ​​in the sample is greater than the reference level of a biomarker (e.g., homocysteine), the subject is determined to have hyperhomocysteinemia, homocystinuria, or hyperhomocysteinemia and homocystinuria.

[0307] In still other embodiments, the amount or presence of free T4 determined in a capillary blood sample obtained according to the methods described herein can be used to diagnose a thyroid disease in a subject, treat a thyroid disease in a subject, or any combination thereof. In some embodiments, if the amount of free T4 in the sample is greater than a reference level of a biomarker (e.g., free T4), the subject is determined to have a thyroid disease.

[0308] In some aspects, the sample obtained from a subject (e.g., a human subject) is a capillary blood sample, such as whole blood, serum, or plasma. In some aspects, the capillary blood sample is whole blood. In other aspects, the capillary blood sample is serum. In still other aspects, the capillary blood sample is plasma.

[0309] A capillary blood sample can be obtained by extracting a sample through a subject's skin (e.g., fingers and / or toes, hand, foot (including heel), earlobe, location on arm and / or leg, chest, back, head, or any combination thereof, etc.). In some embodiments, a capillary blood sample is extracted from a finger or toe. In other embodiments, an entire capillary blood sample is extracted from an arm or leg. In still other embodiments, a capillary blood sample is obtained from a hand or foot. In still other embodiments, a capillary blood sample is obtained from the chest or back. In still other embodiments, a capillary blood sample is obtained from an earlobe. In still other embodiments, a capillary blood sample is obtained from the head.

[0310] In some embodiments, the capillary blood sample obtained from the subject is obtained without using a syringe, needle (e.g., a 21-gauge needle, a butterfly needle, etc.), or any other suitable device, or any combination thereof, typically used to collect blood (e.g., venous blood). Instead, the capillary blood sample is obtained using a blood collection device that is administered to the subject or another person. Examples of blood collection devices that are administered to the subject or another person include microsampling devices. Examples of microsampling devices that may be used herein include the TAP device available from YourBio Health, Inc. (Cambridge, MA), as well as the devices described in U.S. Pat. No. 9,113,836, the contents of which are incorporated herein by reference; the Tasso+, Tasso-M20, and Tasso-ST devices available from Tasso, Inc. (Seattle, WA); the One Draw device available from Draw Bridge Health (San Diego, CA); the PBS-1000 from PreciHealth (Neuchatel, Switzerland); or the Loop blood collection device available from Loop Medical (Lausanne, Switzerland).

[0311] In other embodiments, capillary blood samples are obtained or collected from subjects in a decentralized setting, for example, capillary blood samples can be obtained or collected from urgent care clinics, pharmacies, grocery stores or other convenience stores, residences, workplaces, and / or government offices.

[0312] Additionally or alternatively, in still further embodiments, the capillary blood sample is obtained from the subject by a user who is not trained in blood collection (e.g., other than a trained phlebotomist, nurse, medical assistant, and / or physician). For example, the capillary blood sample can be obtained by the subject themselves, a relative, a friend, a colleague, a teacher, a pharmacist, and / or any other individual. In still further embodiments, the capillary blood sample is obtained from the subject by a robot.

[0313] In some embodiments, the capillary blood sample obtained from the subject is less than about 4 mL in volume. In some embodiments, the capillary blood sample obtained from the subject is less than about 3 mL. In some embodiments, the capillary blood sample obtained from the subject is less than about 2 mL. In some embodiments, the volume of a capillary blood sample obtained from a subject is less than about 3.9 mL, about 3.8 mL, about 3.7 mL, about 3.6 mL, about 3.5 mL, about 3.4 mL, about 3.3 mL, about 3.2 mL, about 3.1 mL, about 3.0 mL, about 2.9 mL, about 2.8 mL, about 2.7 mL, about 2.6 mL, about 2.5 mL, about 2.4 mL, about 2.3 mL, about 2.2 mL, about 2.1 mL, about 2.0 mL, about 1.9 mL, about 1.8 mL, about 1.7 mL, about 1.6 mL, about 1.5 mL, about 1.4 mL, about 1.3 mL, about 1.2 mL, about 1.1 mL, about 1.0 mL, about 0.9 mL, about 0.8 mL, about 0.7 mL, about 0.6 mL, or about 0.5 mL. In some embodiments, when the collected sample is whole blood, a larger volume of capillary blood can be obtained.

[0314] In some embodiments, the capillary blood sample obtained from the subject is whole blood that has been subjected to further or additional processing before determining the amount of CK-MB, β-hCG, or CK-MB and β-hCG in the sample. In some embodiments, the sample is processed using centrifugation. In other embodiments, the sample is processed using a plasma separation device that can include at least one filter, membrane, and / or synthetic paper. For example, in some embodiments, a plasma separation device that can be used is the device described in Section 4. The plasma separation device can separate whole blood into serum and / or plasma, which can then be used in the methods described herein.

[0315] In other embodiments, the plasma separation device may be in fluid communication with, or operably linked, coupled, and / or removably coupled to, the microsampling device as part of a system, as described in more detail in Section 5. In other embodiments, the plasma separation device may be integrated into the microsampling device, as described in more detail in Section 5.

[0316] In still other embodiments, the plasma separation device may be in fluid communication with, or operably linked, coupled, and / or removably coupled to, an opening in the reactor, as described in more detail in Section 5. For example, in some embodiments, the reactor is a cartridge, such as a cartridge used in a point-of-care device.

[0317] In still other embodiments, the plasma separation device may be in fluid communication with, or operably linked, coupled, and / or removably coupled to, a transfer tube, as described in further detail in Section 5. In these embodiments, the transfer may be in fluid communication with, or operably linked, coupled, and / or removably coupled to, a reactor, as described in further detail in Section 5. In still further embodiments, the plasma separation device may be integrated into the transfer tube. In still other embodiments, the transfer tube includes a cap or stopper.

[0318] In some embodiments, with respect to the biomarker CK-MB, the method can include obtaining a sample within about 24 hours of an actual or suspected myocardial infarction and contacting the sample with an antibody to CK-MB to allow formation of an antibody-biomarker complex. More specifically, the sample can be contacted with an anti-CK-MB antibody. The method also includes detecting the resulting antibody-biomarker complex.

[0319] In some embodiments, the sample is taken from the subject (eg, a human subject) within about 24 hours of an actual or suspected myocardial infarction injury. For example, a sample can be collected from a subject (e.g., a human subject) within about 0 minutes, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 60 minutes, about 90 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours after an actual or suspected myocardial infarction.

[0320] In other aspects, with respect to the biomarker β-hCG, the method can include obtaining a sample from a subject, e.g., a subject suspected of being pregnant, and contacting the sample with an antibody to β-hCG to allow for the formation of an antibody-biomarker complex. More specifically, the sample can be contacted with an anti-β-hCG antibody. The method also includes detecting the resulting antibody-biomarker complex.

[0321] In yet other embodiments, with respect to the biomarkers TSH and / or T4, the method can include obtaining a sample from a subject suspected of or suffering from a thyroid dysfunction or thyroid disease, or being treated for a thyroid disease, or any combination thereof.

[0322] In still other aspects, with respect to the biomarker homocysteine, the method can include obtaining a sample from a subject suspected of, suffering from, or diagnosed as having hyperhomocysteinemia, homocystinuria, or hyperhomocysteinemia and homocystinuria, or being treated for hyperhomocysteinemia, homocystinuria, or hyperhomocysteinemia and homocystinuria.

[0323] The amount of CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof in a sample obtained from a subject can be determined using an analog assay, a digital assay, or a combination of analog and digital assays. In some embodiments, the amount of CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof is determined using an analog assay. In other embodiments, the amount of CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof can be determined using a digital assay. In some embodiments, a digital assay is used when the ratio of CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof between capillary whole blood or plasma and venous whole blood or plasma is equal to or less than 1, or is expected to be so. In still other embodiments, an analog assay is used when the ratio of CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof between capillary blood and venous blood is greater than 1, or is expected to be so.

[0324] Examples of assays that can be used to determine the amount of CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof include immunoassays, such as enzyme immunoassays (EIAs), enzyme-linked immunosorbent assays (ELISAs), fluorescent immunoassays, chemiluminescence immunoassays (CLIAs), radioimmunoassays (RIAs), microparticle enzyme immunoassays (MEIAs), turbidimetric immunoassays, etc. In yet other embodiments, the assay can be a clinical chemistry assay, such as, for example, a photometric, spectrophotometric, absorbance, fluorescent, turbidimetric, nephelometric, potentiometric, and / or electrophoretic assay. In still further embodiments, the assay can be a combination of an immunoassay and a clinical chemistry assay. In yet other embodiments, the assay can be a single molecule detection assay.

[0325] At least one of the assays for CK-MB, β-hCG, TSH, homocysteine, and / or free T4 may be performed simultaneously. Alternatively, the assays for β-hCG, TSH, homocysteine, and / or free T4, hCG may be performed sequentially. The assays may be performed sequentially in any order.

[0326] In some embodiments, at least one assay for CK-MB, at least one assay for β-hCG, an assay for TSH, an assay for homocysteine, and / or an assay for free T4 is or can be performed in less than about 30 minutes. In some other embodiments, at least one assay for CK-MB, at least one assay for β-hCG, an assay for TSH, an assay for homocysteine, and / or an assay for free T4 is or can be performed in less than about 25 minutes. In some embodiments, at least one assay for CK-MB, at least one assay for β-hCG, an assay for TSH, an assay for homocysteine, and / or an assay for free T4 is or can be performed in less than about 20 minutes. In some embodiments, at least one assay for CK-MB, at least one assay for β-hCG, an assay for TSH, an assay for homocysteine, and / or an assay for free T4 is or can be performed in less than about 18 minutes. In still other embodiments, at least one assay for CK-MB, at least one assay for β-hCG, an assay for TSH, an assay for homocysteine, and / or an assay for free T4 is or can be performed in about 4 to about 20 minutes, hi some embodiments, at least one assay for CK-MB, at least one assay for β-hCG, an assay for TSH, an assay for homocysteine, and / or an assay for free T4 is or can be performed in about 15 to about 18 minutes, respectively.

[0327] In some embodiments, at least one assay for CK-MB, at least one assay for β-hCG, an assay for TSH, an assay for homocysteine, and / or an assay for free T4 is or can be performed in about 4 minutes. In some embodiments, at least one assay for CK-MB, at least one assay for β-hCG, an assay for TSH, an assay for homocysteine, and / or an assay for free T4 is or can be performed in about 5 minutes. In some embodiments, at least one assay for CK-MB, at least one assay for β-hCG, an assay for TSH, an assay for homocysteine, and / or an assay for free T4 is or can be performed in about 6 minutes. In some embodiments, at least one assay for CK-MB, at least one assay for β-hCG, an assay for TSH, an assay for homocysteine, and / or an assay for free T4 is or can be performed in about 7 minutes. In some embodiments, at least one assay for CK-MB, at least one assay for β-hCG, an assay for TSH, an assay for homocysteine, and / or an assay for free T4 is or can be performed in about 8 minutes. In some embodiments, at least one assay for CK-MB, at least one assay for β-hCG, an assay for TSH, an assay for homocysteine, and / or an assay for free T4 is or can be performed in about 9 minutes. In some embodiments, at least one assay for CK-MB, at least one assay for β-hCG, an assay for TSH, an assay for homocysteine, and / or an assay for free T4 is or can be performed in about 10 minutes.In some embodiments, at least one assay for CK-MB, at least one assay for β-hCG, an assay for TSH, an assay for homocysteine, and / or an assay for free T4 is or can be performed in about 11 minutes. In some embodiments, at least one assay for CK-MB, at least one assay for β-hCG, an assay for TSH, an assay for homocysteine, and / or an assay for free T4 is or can be performed in about 12 minutes. In some embodiments, at least one assay for CK-MB, at least one assay for β-hCG, an assay for TSH, an assay for homocysteine, and / or an assay for free T4, an assay for TSH, an assay for homocysteine, and / or an assay for free T4 is or can be performed in about 13 minutes. In some embodiments, at least one assay for CK-MB, at least one assay for β-hCG, an assay for TSH, an assay for homocysteine, and / or an assay for free T4 is or can be performed in about 14 minutes. In some embodiments, at least one assay for CK-MB, at least one assay for β-hCG, an assay for TSH, an assay for homocysteine, and / or an assay for free T4 is or can be performed in about 15 minutes. In some embodiments, at least one assay for CK-MB, at least one assay for β-hCG, an assay for TSH, an assay for homocysteine, and / or an assay for free T4 is or can be performed in about 16 minutes. In some embodiments, at least one assay for CK-MB, at least one assay for β-hCG, an assay for TSH, an assay for homocysteine, and / or an assay for free T4 is or can be performed in about 17 minutes.In some embodiments, at least one assay for CK-MB, at least one assay for β-hCG, an assay for TSH, an assay for homocysteine, and / or an assay for free T4 is or can be performed in about 18 minutes. At least one assay for CK-MB, at least one assay for β-hCG, an assay for TSH, an assay for homocysteine, and / or an assay for free T4 is or can be performed in about 19 minutes. In some embodiments, at least one assay for CK-MB, at least one assay for β-hCG, an assay for TSH, an assay for homocysteine, and / or an assay for free T4 is or can be performed in about 20 minutes.

[0328] Generally, a reference level of a biomarker, such as CK-MB, β-hCG, TSH, homocysteine, or free T4, or any combination thereof, can also be used as a benchmark for evaluating results obtained after assaying a test sample for a biomarker, such as CK-MB, β-hCG, TSH, homocysteine, or free T4, or any combination thereof. Generally, when making such a comparison, the reference level of a biomarker, such as CK-MB, β-hCG, TSH, homocysteine, or free T4, or any combination thereof, is obtained by performing or executing a particular assay a sufficient number of times and under appropriate conditions such that a correlation or association between analyte presence, amount, or concentration and a particular stage or endpoint or with a particular symptom of a disease, disorder, and / or condition can be obtained. Typically, the reference level of a biomarker, such as CK-MB, β-hCG, TSH, homocysteine, or free T4, or any combination thereof, is obtained by assaying a reference subject (or population of subjects). The biomarkers measured, such as CK-MB, β-hCG, TSH, homocysteine ​​or free T4 or any combination thereof, can include fragments thereof, degradation products thereof and / or enzymatic cleavage products thereof.

[0329] In certain embodiments, the reference level may be correlated to a control subject (e.g., a human subject) who has never had a myocardial infarction (e.g., in the case of CK-MB) or is not pregnant (e.g., in the case of β-hCG), does not suffer from a thyroid dysfunction or thyroid disease (e.g., in the case of TSH and / or free T4), or does not suffer from hyperhomocysteinemia, homocystinuria, or hyperhomocysteinemia and homocystinuria (e.g., in the case of homocysteine).

[0330] In still further embodiments, once the amount of CK-MB, β-hCG, TSH, homocysteine, or free T4, or any combination thereof, is determined using the methods described herein, a result is obtained. The result can be further processed. Specifically, the further processing includes selecting a conversion factor for comparing the amount of CK-MB, β-hCG, TSH, homocysteine, and / or free T4 in a capillary whole blood or plasma sample with the amount of the same analyte in venous whole blood or plasma.

[0331] In some embodiments, when the assay is for CK-MB, the conversion factor can be about 0.5:1.0 (capillary whole blood or plasma vs. venous whole blood or plasma) to about 1:0:1.2 (capillary whole blood or plasma vs. venous whole blood or plasma). In other embodiments, the conversion factor is about 0.8:1.0 (capillary whole blood or plasma vs. venous whole blood or plasma). In still further embodiments, when the assay is for CK-MB, the conversion factor can be about 1.2:1.0 (venous whole blood or plasma vs. capillary whole blood or plasma) to about 1:0.5 (venous whole blood or plasma vs. capillary whole blood or plasma). In other embodiments, the conversion factor is about 1:0.8 (venous whole blood or plasma vs. capillary whole blood or plasma).

[0332] In other embodiments, when the assay is for β-hCG, the conversion factor can be from 0.8:1.0 (capillary whole blood or plasma to venous whole blood or plasma) to about 1.0:1.4 (capillary whole blood or plasma to venous whole blood or plasma). In other embodiments, the conversion factor is about 1:1 (capillary whole blood or plasma to venous whole blood or plasma). In yet other embodiments, when the analyte to be detected is β-hCG, the conversion factor can be from 1.4:1.0 (venous whole blood or plasma to capillary whole blood or plasma) to about 1:0.8 (venous whole blood or plasma to capillary whole blood or plasma). In other embodiments, the conversion factor is about 1:1 (venous whole blood or plasma to capillary whole blood or plasma).

[0333] In other embodiments, when the assay is for TSH, the conversion factor can be from about 0.60:1.0 (capillary whole blood or plasma to venous whole blood or plasma) to about 1.5:1.0 (capillary whole blood or plasma to venous whole blood or plasma), hi other embodiments, the conversion factor is from about 0.75:1.0 (capillary whole blood or plasma to venous whole blood or plasma) to about 1.2:1.0 (capillary whole blood or plasma to venous whole blood or plasma).

[0334] In still other embodiments, when the assay is for homocysteine, the conversion factor can be from 1.5:1.0 (capillary whole blood or plasma to venous whole blood or plasma) to about 0.60:1.0 (capillary whole blood or plasma to venous whole blood or plasma). In other embodiments, the conversion factor is from about 1.2:1.0 (capillary whole blood or plasma to venous whole blood or plasma) to about 0.9:1.0 (capillary whole blood or plasma to venous whole blood or plasma).

[0335] In still other embodiments, when the assay is for free T4, the conversion factor can be from 0.60:1.0 (capillary whole blood or plasma to venous whole blood or plasma) to about 1.5:1.0 (capillary whole blood or plasma to venous whole blood or plasma). In other embodiments, the conversion factor is from about 0.8:1.0 (capillary whole blood or plasma to venous whole blood or plasma) to about 1.2:1.0 (capillary whole blood or plasma to venous whole blood or plasma).

[0336] Once the conversion factor is selected, processing further includes normalizing the amount of CK-MB, β-hCG, TSH, homocysteine, and / or free T4 in the capillary whole blood or plasma sample by the amount of the same analyte from venous whole blood or plasma by applying the conversion factor to the amount of the analyte (e.g., CK-MB, β-hCG, TSH, homocysteine, and / or free T4) in the sample. For example, the amount of the analyte in the capillary whole blood or plasma sample can be multiplied by the conversion factor to obtain the normalized amount of the analyte in the sample.

[0337] In some embodiments, the processing of the amounts (e.g., results) of CK-MB, β-hCG, TSH, homocysteine, and / or free T4 may be performed by a processing system including a computer processor and non-transitory computer memory containing one or more computer programs configured to select a conversion factor for comparing the amounts of CK-MB, β-hCG, TSH, homocysteine, and / or free T4 in the capillary whole blood or plasma sample with the amounts of the same analytes in venous whole blood or plasma, and to apply the selected conversion factor to the amounts of CK-MB, β-hCG, TSH, homocysteine, and / or free T4 in the sample, thereby normalizing the amounts of CK-MB, β-hCG, TSH, homocysteine, and / or free T4 in the sample by the amounts of the same analytes from venous whole blood or plasma.

[0338] The results or processed results may be communicated (e.g., reported) for further analysis, interpretation, processing, and / or display. The results may be communicated (e.g., reported) by computer, in a document and / or spreadsheet, on a mobile device (e.g., a smartphone), on a website, in an email, or any combination thereof. In some embodiments, the results may be displayed not as numbers, but as visual signals (e.g., lines or bars) that can be read / interpreted by a reading device or the naked eye.

[0339] In some embodiments, the results of CK-MB, β-hCG, TSH, homocysteine, or free T4, or any combination thereof, determined in the sample are communicated, such as by display on the device. In further embodiments, the results are displayed as an indication that the amount of CK-MB, β-hCG, TSH, homocysteine, or free T4, or any combination thereof, in the subject is elevated, not elevated, or that the assay(s) for CK-MB, β-hCG, TSH, homocysteine, or free T4, or any combination thereof, should be repeated.

[0340] Suitable instruments for use in the methods described herein include higher throughput assay analyzers (e.g., the ARCHITECT platform sold by Abbott Laboratories) or point-of-care devices (e.g., the i-STAT and i-STAT Alinity devices sold by Abbott Laboratories) that can contain a user interface capable of displaying determinations.

[0341] In some embodiments, the results for CK-MB, β-hCG, TSH, homocysteine, or free T4, or any combination thereof, are or can be communicated within about 4 minutes to about 40 minutes from the time the sample is collected. In other embodiments, the results are or can be communicated within about 4 minutes to about 30 minutes from the time the sample is collected. In still other embodiments, the results are or can be communicated within about 4 minutes to about 20 minutes from the time the sample is collected. In some embodiments, the results are or can be communicated within about 40 minutes or less, about 39 minutes or less, about 38 minutes or less, about 37 minutes or less, about 36 minutes or less, about 35 minutes or less, about 34 minutes or less, about 33 minutes or less, about 32 minutes or less, about 31 minutes or less, about 30 minutes or less, about 29 minutes or less, about 28 minutes or less from the time the sample is collected. The delivery is in less than, about 27 minutes or less, about 26 minutes or less, about 25 minutes or less, about 24 minutes or less, about 23 minutes or less, about 22 minutes or less, about 21 minutes or less, about 20 minutes, about 19 minutes, about 18 minutes, about 17 minutes, about 16 minutes, about 15 minutes, about 14 minutes, about 13 minutes, about 12 minutes, about 11 minutes, about 10 minutes, about 9 minutes, about 8 minutes, about 7 minutes, about 6 minutes, about 5 minutes, or about 4 minutes.

[0342] In some embodiments of the above-described methods, the time from sample collection (from a subject) to the time a result for CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof is communicated is less than about 30 minutes. In other embodiments of the above-described methods, the time from sample collection to the time a result for CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof is communicated is less than about 25 minutes. In still other embodiments of the above-described methods, the time from sample collection to the time a result for CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof is communicated is less than about 20 minutes. In still further embodiments of the above-described methods, the time from sample collection to the time a result for CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof is communicated is less than about 18 minutes. In some embodiments of the above-described methods, the time from sample collection to the time results for CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof are communicated is less than about 15 minutes. In some embodiments of the above-described methods, the time from sample collection to the time results for CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof are communicated is about 4 to about 20 minutes. In some embodiments of the above-described methods, the time from sample collection to the time results for CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof are communicated is about 15 to about 20 minutes.

[0343] In some embodiments, the device contains software for performing one or more tasks, including implementing the methods and algorithms described herein. In some embodiments, the device contains software for automatically determining the appropriate next step in the methods and algorithms as described herein. For example, the device can contain software that determines the amount or presence of an analyte of interest. The software can display this determination, such as in a graphical user interface.

[0344] In some aspects, the device stores software that instructs the processor to perform given tasks. In some aspects, the software stores machine-readable instructions that instruct the processor to perform given tasks. The machine-readable instructions may be one or more executable programs or portions of executable programs for execution by a computer. The programs may be embodied in software stored on a non-transitory computer-readable storage medium, such as a CD-ROM, floppy disk, hard drive, DVD, Blu-ray disk, or memory associated with the processor. Alternatively, the entire program and / or portions thereof may instead be executed by a device other than the processor and / or may be embodied in firmware or dedicated hardware. Additionally or alternatively, the processing may be performed by one or more hardware circuits (e.g., discrete and / or integrated analog and / or digital circuits, FPGAs, ASICs, comparators, operational amplifiers (op-amps), logic circuits, etc.) structured to perform corresponding operations without executing software or firmware.

[0345] The machine-readable instructions may be stored in one or more of a compressed format, an encrypted format, a fragmented format, a compiled format, an executable format, a packaged format, etc. The machine-readable instructions described herein may be stored as data (e.g., portions of instructions, code, representations of code, etc.) that can be used to generate, manufacture, and / or create machine-executable instructions. For example, the machine-readable instructions may be fragmented and stored on one or more storage devices and / or computing devices (e.g., servers). The machine-readable instructions may require one or more of installing, modifying, adapting, updating, combining, supplementing, configuring, decrypting, decompressing, unpacking, distributing, reallocating, compiling, etc. to make them directly readable, interpretable, and / or executable by the computing device and / or other machines. For example, the machine-readable instructions may be stored in multiple portions that are individually compressed, encrypted, and stored on separate computing devices, and that, when decrypted, decompressed, and combined, form a set of executable instructions that execute a program such as those described herein.

[0346] In another example, machine-readable instructions may be stored in a state that can be read by a computer, but require the addition of a library (e.g., a dynamic link library (DLL)), a software development kit (SDK), an application programming interface (API), etc., to execute the instructions on a particular computing device or other device. In another example, the machine-readable instructions may need to be configured (e.g., stored settings, data entries, recorded network addresses, etc.) before the machine-readable instructions and / or corresponding program(s) can be executed, in whole or in part. Thus, disclosed machine-readable instructions and / or corresponding program(s) are intended to encompass such machine-readable instructions and / or program(s) regardless of the particular format or state of the machine-readable instructions and / or program(s) when stored or otherwise being stored or transferred.

[0347] The machine-readable instructions described herein may be expressed in any past, present, or future command language, scripting language, programming language, etc. For example, the machine-readable instructions may be expressed using any of the following languages: C, C++, Java, C#, Perl, Python, JavaScript, HyperText Markup Language (HTML), Structured Query Language (SQL), Swift, etc.

[0348] The machine-readable instructions may be stored in a non-transitory computer and / or machine-readable medium, such as a hard disk drive, flash memory, read-only memory, compact disk, digital versatile disk, cache, random access memory, and / or any other storage device or storage disk, on which information is stored for any duration (e.g., for an extended period of time, permanently, for short instances, for temporary buffering and / or caching of information). As used herein, the term non-transitory computer-readable medium is expressly defined to include any type of computer-readable storage device and / or storage disk, and to exclude propagating signals and to exclude transmission media.

[0349] 4.Plasma separation device In another embodiment, the present disclosure relates to a device that can be used as a plasma separation device. In one aspect, the device includes a hydrophobic layer containing at least one microchannel and an upper layer adjacent to, on, or above the hydrophobic layer. The hydrophobic layer can include or be constructed from at least one hydrophobic material. The hydrophobic material can be a membrane, film, fabric, fiber, filter, microfilm, screen, mesh, or any combination thereof. In one aspect, the hydrophobic layer is a membrane or film. Hydrophobic membranes or films that can be used are those known in the art. Specifically, membranes or films can be used, such as those available from Adhesive Research (Glen Rock, PA), 3M (Minneapolis, Minnesota), and / or Tesa SE (Norderstadt, Germany).

[0350] The hydrophobic layer includes at least one microchannel having first and second ends that define a path for capillary fluid flow of the processed blood sample or blood product (e.g., plasma). In some embodiments, the at least one microchannel extends longitudinally along a portion of the hydrophobic layer from the first end of the microchannel to an opening at the second (e.g., opposite) end. In other embodiments, the at least one microchannel extends along the width of the portion of the hydrophobic layer from the first end of the microchannel to an opening at the second (e.g., opposite) end. In some embodiments, the microchannel contains a first opening connected to the first end of the microchannel. In these embodiments, the processed blood or blood product (e.g., plasma) can flow from the first opening at the first end of the microchannel to the second opening at the second end of the microchannel.

[0351] Openings at the second ends of the microchannels allow the processed blood or blood products (e.g., plasma) to flow out of the device. For example, if the fluid being processed is blood, the plasma can flow by capillary fluid flow from the first end toward a second opening at the second (e.g., opposite) end. The plasma can be collected at the second (e.g., opposite) end using a collection or other device, or, if the device is operably associated with, removably coupled to, or in fluid communication with another device (such as a sample analysis cartridge (e.g., a microfluidic cartridge)), the plasma can continue to flow directly onto or through the device for further processing and / or analysis.

[0352] The microchannels can be of any length. In some embodiments, the microchannels are less than about 80 mm in length. In other embodiments, at least one microchannel is about 70 mm in length, and at least one microchannel is about 60 mm in length, about 55 mm in length, about 50 mm in length, about 45 mm in length, about 40 mm in length, about 35 mm in length, about 30 mm in length, about 25 mm in length, about 20 mm in length, or about 15 mm in length. In other embodiments, at least one microchannel is less than about 5 mm in width, less than about 4.5 mm in width, less than about 4 mm in width, less than about 3 mm in width, less than about 2.5 mm in width, or less than about 2.0 mm in width.

[0353] Moreover, the at least one microchannel can be located anywhere in the hydrophobic layer. For example, the at least one microchannel can be centered in the hydrophobic layer, slightly off-center in the hydrophobic layer, or on or near the side or edge of the hydrophobic layer. In yet other embodiments, the microchannel does not contain a filter or any type of filtering device (e.g., the microchannel does not contain a filter or filtering device at any point between its first and second ends that define a path for capillary fluid flow of the processed blood sample or blood product). Alternatively, in some embodiments, the microchannel contains one or more filters or filtering-type devices.

[0354] In additional embodiments, the hydrophobic layer can have a thickness of about 50 to about 200 microns. In other embodiments, the hydrophobic layer can have a thickness of about 100 to about 200 microns. In some embodiments, the hydrophobic layer can have a thickness of about 100 to about 150 microns.

[0355] The device also includes a top layer adjacent to, or located on, or above the hydrophobic layer. In some embodiments, the top layer is adhered to the hydrophobic layer. In yet further embodiments, the surface of the top layer facing the hydrophobic layer comprises a material or is coated with a hydrophilic material. The hydrophilic material can be an adhesive, a membrane, a film, a fabric, a fiber, a filter, a microfilm, a screen, a mesh, or any combination thereof. In other embodiments, the entire top layer is made or constructed of a hydrophilic material, such as a membrane, a film, a fabric, a fiber, a filter, a microfilm, a screen, a mesh, or any combination thereof. For example, in one embodiment, the top layer is a membrane or a film. In some embodiments, the membrane or film is not constructed from a hydrophilic material, but is coated with a hydrophilic material. The portion of the top layer coated with the hydrophilic material faces the hydrophobic layer. In another embodiment, the entire membrane or film is made or constructed from a hydrophilic material. Hydrophilic membranes or films that can be used are those known in the art. Examples of hydrophilic films that may be used include 9984 diagnostic microfluidic surfactant-free hydrophilic film available from 3M (Minneapolis, MN), Kemafoil H, a hydrophilic coated polyester film available from Coveme (S. Lazzaro di Savena, Italy), and Tesa 62580, a hydrophilic coated polyester film available from Tesa SE (Norderstadt, Germany). In additional embodiments, the top layer can have a thickness of about 50 to about 200 microns. In other embodiments, the top layer can have a thickness of about 100 to about 200 microns. In some embodiments, the top layer can have a thickness of about 100 to about 150 microns.

[0356] When a blood sample or blood product is placed on the upper layer of the device, the blood sample or product flows through the upper layer. As it flows, cellular components of the blood (e.g., red blood cells, white blood cells, platelets, and combinations thereof) are trapped in the pores and / or fibers of the hydrophilic material, allowing the plasma to continue flowing through the hydrophilic material, into the hydrophobic layer, and into the microchannel. Once in the microchannel, the plasma flows by capillary fluid flow from a first end of the channel to an opening at a second (e.g., opposite) end. The plasma can be collected using a collection or other device, or, if the device is operably associated with, removably coupled to, or in fluid communication with another device, such as a sample analysis cartridge (e.g., a microfluidic cartridge), it can continue to flow directly onto or through the device for further processing and analysis.

[0357] In other embodiments, the device optionally includes a bottom layer. The bottom layer is adjacent to or located below or beneath the hydrophobic layer. In these embodiments, the device includes at least three layers—a top layer, a hydrophobic layer, and a bottom layer. In some embodiments, the bottom layer is adhered to the hydrophobic layer. In additional embodiments, the top layer and the bottom layer are each adhered to a hydrophobic layer.

[0358] In yet a further aspect, the surface of the bottom layer facing the hydrophobic layer comprises a material or is coated with a hydrophilic material. The hydrophilic material can be an adhesive, a membrane, a film, a fabric, a fiber, a filter, a microfilm, a screen, a mesh, or any combination thereof. In other aspects, the entire bottom layer is made or constructed from a hydrophilic material, such as a membrane, a film, a fabric, a fiber, a filter, a microfilm, a screen, a mesh, or any combination thereof. For example, in one aspect, the bottom layer is a membrane or a film. In some aspects, the membrane or film is not constructed from a hydrophilic material, but is coated with a hydrophilic material. The portion of the bottom layer coated with the hydrophilic material faces the hydrophobic layer. In another aspect, the entire membrane or film is made or constructed from a hydrophilic material. Hydrophilic membranes or films that can be used are hydrophilic membranes or films known in the art. Examples of hydrophilic films that may be used are 9984 diagnostic microfluidic surfactant-free hydrophilic film available from 3M (Minneapolis, MN), Kemafoil H, a hydrophilic coated polyester film obtained from Coveme (S. Lazzaro di Savena, Italy), and Tesa 62580, a hydrophilic coated polyester film obtained from Tesa SE (Norderstadt, Germany).

[0359] In additional embodiments, the bottom layer can have a thickness of about 50 to about 200 microns. In other embodiments, the bottom layer can have a thickness of about 100 to about 200 microns. In some embodiments, the bottom layer can have a thickness of about 100 to about 150 microns.

[0360] In still further embodiments, the device can contain a protective film adjacent to or located below or beneath the bottom layer. In some embodiments, the protective film is adhered to the bottom layer. The protective film protects the device from moisture and / or other contamination. The protective film can be a plastic film, such as a plastic film with adhesive, cardboard with an adhesive material, a plastic sheet with an adhesive material, or a combination thereof.

[0361] In yet another embodiment, when the device includes a top layer, a hydrophobic layer, and a bottom layer, the combined thickness of the three layers is between about 100 and about 600 microns. In another embodiment, the combined thickness of the three layers is between about 150 and about 600 microns. In another embodiment, the combined thickness of the three layers is between about 200 and about 600 microns. In yet another embodiment, the combined thickness of the three layers is between about 150 and about 500 microns. In yet a further embodiment, the combined thickness of the three layers is between about 200 and about 500 microns.

[0362] Furthermore, in some aspects, when a device includes a top layer, a hydrophobic layer, and a bottom layer, the top layer and the bottom layer can be made of the same material. Alternatively, the top layer and the bottom layer can be made of different materials. For example, the top layer and the bottom layer can be made of or constructed from the same hydrophilic material or different hydrophilic materials. Alternatively, the top and bottom layers can be made of or constructed from a material that is not hydrophilic, but the surface of the layer facing the hydrophobic layer can be coated with a hydrophilic material. Still further, either the top or the bottom layer can be made of or constructed from a material that is not hydrophilic, and the surface facing the hydrophobic layer can be coated with a hydrophilic material, while the other layer can be made entirely from a hydrophilic material.

[0363] In some embodiments, the top layer includes a sample inlet where a blood sample or blood product is placed to begin sample processing through the device. The sample inlet can have any shape. For example, the sample inlet can be circular, oval, rectangular, square, triangular, or any combination thereof. In some embodiments, a hydrophobic transfer material (e.g., transfer tape, etc.) can surround the sample inlet. The hydrophobic transfer material helps prevent the blood sample or blood product from wicking or migrating away from the sample inlet area.

[0364] When the top layer includes a sample inlet, the hydrophobic layer and, optionally, the bottom layer include one or more openings. The openings can have any shape. For example, the openings can be circular, oval, rectangular, square, triangular, or any combination thereof. The openings can have the same shape as the sample inlet, or they can have different shapes. In some embodiments, when a device contains two layers (e.g., a top layer with a sample inlet and a hydrophobic layer), one or more openings can be made in the hydrophobic layer. In yet other embodiments, when a device contains three layers, one or more openings can be made in the hydrophobic layer but not in the bottom layer. In other embodiments, when a device contains three layers, one or more openings can be made in each of the hydrophobic layer and the bottom layer. Each opening in one or more layers can be directly below the sample inlet.

[0365] In some embodiments, the devices described herein are used to separate plasma from whole blood in a horizontal orientation. In other embodiments, the devices are used to separate plasma from whole blood in a vertical orientation. When the device is used vertically, the microchannel can be bent 90 degrees during loading (e.g., keeping the membrane horizontal and the channel vertical), or alternatively, a closed inlet with a funnel-like structure can be used.

[0366] For example, Figure 7 illustrates one embodiment of a device of the present disclosure used in a horizontal orientation, containing a hydrophilic top layer and a hydrophobic layer containing a microchannel having first and second ends. The top layer is adhered to the hydrophobic layer. The hydrophilic top layer includes a sample inlet, and the hydrophobic layer contains a first opening directly below the sample inlet. The first opening in the hydrophobic layer directly below the sample inlet is connected to the first end of the microchannel. When plasma reaches the first opening in the hydrophobic layer, the plasma flows from the opening to the first end of the microchannel and continues to a second opening at the second end (e.g., the opposite end) where it can be collected or allowed to flow directly onto or into a sample analysis cartridge.

[0367] FIG. 8 illustrates another embodiment of a device of the present disclosure used in a horizontal orientation. In this embodiment, the device includes a hydrophilic top layer, a hydrophobic layer, and a hydrophilic bottom layer. The hydrophilic top layer is adhered to the hydrophobic layer, and the bottom layer is adhered to the hydrophobic layer. The hydrophilic top layer includes a hydrophobic transfer tape and a sample inlet. The hydrophobic layer contains a first opening directly below the sample inlet. The first opening in the hydrophobic layer directly below the sample inlet is connected to a first end of a microchannel. When the plasma reaches the first opening in the hydrophobic layer, the plasma flows to the first end of the microchannel and continues to a second opening at a second (e.g., opposite) end, where it can be collected or allowed to flow directly onto or into a sample analysis cartridge.

[0368] In still further embodiments, the sample inlet can further comprise a separation membrane (such as a plasma separation membrane). In some embodiments, the separation membrane is a glass fiber material, such as a membrane, a film, a fabric, a fiber, a filter, a microfilm, a screen, a mesh, or any combination thereof. Moreover, the separation membrane can be any shape. For example, the sample inlet can be circular, oval, rectangular, square, triangular, or any combination thereof. In some embodiments, the separation membrane can have the same shape as the sample inlet. In other embodiments, the separation membrane can have a different shape from the sample inlet.

[0369] The separation membrane can be made from any material known in the art to be useful for separating a blood sample or blood product into its components, e.g., plasma. When a blood sample or blood product is placed on the separation membrane, the blood sample or product flows through the membrane or material. As it flows through the membrane or material, cellular components of the blood (e.g., red blood cells, white blood cells, platelets, and combinations thereof) are trapped in the pores and / or fibers of the membrane or material, thereby allowing the plasma to continue flowing through the sample inlet to a first opening in the hydrophobic layer. The first opening in the hydrophobic layer is connected to the microchannel such that when the plasma reaches the first opening in the hydrophobic layer, it flows to a first end of the microchannel and continues flowing to a second opening at a second (e.g., opposite) end of the microchannel. When the plasma reaches a second opening at the second end of the microchannel, the plasma can be collected using a collection or other device, or, if the apparatus is operably associated with, removably coupled to, or in fluid communication with another device, such as a sample analysis cartridge (e.g., a microfluidic cartridge), the plasma can continue to flow directly onto or through this device for further processing and analysis.

[0370] In some embodiments, once the plasma reaches the first opening, the second opening, or both the first and second openings of the microchannel, a pump, such as an air pump, a foam ring pump, a foil blister pump, a film blister pump, or any combination thereof, can be used to direct and / or distribute the plasma in the microchannel. In some embodiments, the pump can be used to direct and / or distribute the plasma from the first end of the microchannel toward the second end of the microchannel. In still other embodiments, the pump can be used to direct and / or distribute the plasma from the second end of the microchannel toward and / or into another device, such as a sample analysis cartridge (e.g., a microfluidic cartridge). The pump can be connected at any point along the microfluidic channel, for example, at the first opening, along the side, near the second end, or any combination thereof.

[0371] In some embodiments, the separation membrane can be located or placed above, below, or within the sample inlet. For example, in some embodiments, the separation membrane can be cut to be larger than the size of the sample inlet and simply placed on top of the sample inlet. Alternatively, the separation membrane can be cut to be the same size as or slightly smaller than the size of the sample inlet and placed at the inlet. Still further, the separation membrane can be placed below the sample inlet and attached or adhered by any means known in the art, such as an adhesive, glue, etc.

[0372] When the hydrophobic layer and / or bottom layer contains one or more openings, the hydrophobic layer and / or bottom layer can further contain one or more separation membranes located above, below, or within the openings in the layer. The separation membrane can be constructed from the same material as that used for the sample inlet, or from a different material. In still further embodiments, the separation membrane can be located above, below, or within one or more openings. For example, in some embodiments, the separation membrane can be cut to be larger than the size of the opening and simply placed on top of the opening. Alternatively, the separation membrane can be cut to be the same size as or slightly smaller than the size of the opening and placed at the inlet. Still further, the separation membrane can be placed below the opening and attached or adhered by any means known in the art, such as an adhesive, glue, etc.

[0373] In still further embodiments, one or more hydrophilic meshes or films can be adjacent to the separation membrane at the sample inlet and / or one or more openings. For example, the hydrophilic mesh or film can be located above or on top of the separation membrane to facilitate spreading of the blood sample or blood product into the device. Alternatively, the hydrophilic mesh or film can be located below or beneath the separation membrane to help facilitate continued movement of the blood sample or blood product as it is processed to the hydrophobic layer and the first opening connected to the first end of the microchannel.

[0374] In some aspects, the device further comprises an upper substrate material comprising a sample inlet. The upper substrate material is adjacent to or located on or above the top layer. In some aspects, the top layer is adhered to the upper substrate. The upper substrate material can be a membrane, film, fabric, fiber, filter, microfilm, screen, mesh, or any combination thereof. In some aspects, the upper substrate material is made of a hydrophilic material. In other aspects, the upper substrate material is made of a hydrophobic material.

[0375] The upper substrate material includes a sample inlet where a blood sample or blood product is placed to begin sample processing. The sample inlet can have any shape. For example, the sample inlet can be circular, oval, rectangular, square, triangular, or any combination thereof. In some embodiments, a hydrophobic transfer material (e.g., transfer tape, etc.) can surround the sample inlet. The hydrophobic transfer material helps prevent the blood sample or blood product from wicking or migrating away from the sample inlet area.

[0376] In still further aspects, the sample inlet can include a separation membrane. In some aspects, the separation membrane comprises a glass fiber material, such as a membrane, film, fabric, fiber, filter, microfilm, screen, mesh, or any combination thereof. The separation membrane can be made from any material known in the art to be useful for separating a blood sample or blood product into its components, such as plasma. The separation membrane in the upper substrate functions in the same manner as the separation membrane used in the sample inlet of the upper layer.

[0377] In some embodiments, the separation membrane can be located or placed above, below, or within the sample inlet. For example, in some embodiments, the separation membrane can be cut to be larger than the size of the sample inlet and simply placed on top of the sample inlet. Alternatively, the separation membrane can be cut to be the same size as or slightly smaller than the size of the sample inlet and placed at the inlet. Still further, the separation membrane can be placed below the sample inlet and attached or adhered by any means known in the art, such as an adhesive, glue, etc.

[0378] If an upper substrate and a sample inlet are present, one or more openings are created in each of the top layer and the hydrophobic layer, or in each of the top layer, the hydrophobic layer, and optionally the bottom layer. The openings can have any shape. For example, the openings can be circular, oval, rectangular, square, triangular, or any combination thereof. In some embodiments, the openings are the same shape as the sample inlet. In other embodiments, the openings are a different shape from the sample inlet. In some embodiments, if the device contains an upper substrate, one or more openings can be created in the top layer and the hydrophobic layer but not in the bottom layer. In other embodiments, one or more openings can be created in each of the top layer, the hydrophobic layer, and the bottom layer. Each opening in one or more layers can be directly below the sample inlet. One or more openings in each of the top layer, the hydrophobic layer, and / or the bottom layer can also contain one or more separation membranes located above, below, or within the openings in the layers. The separation membranes can be constructed from the same material as that used for the sample inlet, or can be constructed from a different material. Additionally, one or more hydrophilic meshes or films can be adjacent to the separation membrane at the sample inlet and / or one or more openings, e.g., the hydrophilic mesh or film can be located above or below the separation membrane, as previously described herein.

[0379] In yet another aspect, the top layer, hydrophobic layer, bottom layer, hydrophilic mesh or film, separation membrane, or any combination thereof, is either ubiquitous for any analyte or specific to an analyte or class of analytes.

[0380] It was found that the volume of plasma produced by the device depends on the hematocrit (%PCV) of the whole blood sample. Specifically, due to the short vertical separation distance in the device, at higher hematocrits, the volume of plasma produced can be lower than the volume of the microchannel. Plasma flows into the microchannel by capillary action, but if the volume of plasma is not sufficient to fill the entire channel, red blood cells held back by the separation membrane fill the remainder. Thus, as a result, in some embodiments, the whole blood sample used with the device has a hematocrit of less than about 40, less than about 39, less than about 38, less than about 37, less than about 36, less than about 35, less than about 34, less than about 33, less than about 32, less than about 31, less than about 30, less than about 29, less than about 28, less than about 27, less than about 26, less than about 25, less than about 24, less than about 23, less than about 22, less than about 21, less than about 20, less than about 19, less than about 18, less than about 17, less than about 16, or less than about 15. In some embodiments, the whole blood sample used with the device has a hematocrit of between about 15 and about 40. In other embodiments, the whole blood sample used with the device has a hematocrit of between about 20 and about 35. In still other embodiments, the whole blood sample used with the device has a hematocrit of between about 25 and about 35.

[0381] In yet another embodiment, the devices described herein, such as those shown in FIGS. 7-9, can be used in conjunction with a point-of-care device, such as that shown in FIG. 10. The device may be optimized for use with a non-point-of-care device, such as a high-throughput analyzer (e.g., a non-point-of-care device), using routine techniques known in the art. Such optimization can include increasing one or more of the length, width, and / or thickness of the device. For example, in some embodiments, the device can have a thickness of about 600 microns to about 10,000 microns. Moreover, in other embodiments, the length and / or width of the microchannel can be increased. For example, the length of the microchannel can be about 80 mm to about 200 mm, and / or the width of the microchannel can be about 5 mm to about 15 mm. In some embodiments, the device includes one (e.g., a single) microchannel. In other embodiments, the device can contain two or more microchannels. For example, the device can contain at least two microchannels, at least three microchannels, at least four microchannels, or at least five microchannels.

[0382] The devices described herein, such as those shown in Figures 7-9, are passive membrane-based separators. As described herein, whole blood can be applied to the top of the membrane. The hydrophilic fabric allows for more uniform wetting of the top of the membrane and directs the blood to move vertically through the separation medium. Cellular components are retained in the membrane matrix, thereby creating a forward-moving plasma front. The uniform, approximately columnar movement of blood through the separation medium maximizes the volume of plasma generated at the bottom of the membrane. Once the separation membrane is saturated, plasma from the bottom of the membrane is directed into the microchannels by capillary action.

[0383] In yet another embodiment, the device further comprises at least one agglutinating agent for agglutinating red blood cells to form red blood cell clumps for improved separation and cleaner plasma production. In some embodiments, the agglutinating agent is coated or incorporated into one or more of the top layer, hydrophobic layer, bottom layer, upper substrate, separation membrane, hydrophilic mesh, or hydrophilic film, or any combination thereof. Examples of agglutinating agents that may be used include lectin (e.g., soybean lectin), Merquat-100, concanavalin A, DEAE-dextran, poly-L-lysine, polyvinylpyrrolidone, poly(2-(dimethylamino)ethyl methacrylate), or any combination thereof.

[0384] In addition, the device can further include one or more salts that can be used to achieve minimal partial outward protrusion of red blood cells in a whole blood sample. Salts are ionic compounds that dissociate in water. Salts that can be used in the device include both organic and inorganic salts. Examples of salts that can be used include, for example, calcium chloride, potassium chloride, sodium chloride, manganese chloride, magnesium chloride, potassium sulfate, guanidine hydrochloride, and combinations thereof. In yet another embodiment, the top layer, hydrophobic layer, bottom layer, upper substrate, separation membrane, hydrophilic mesh, or hydrophilic film, or any combination thereof, is coated with a coating, such as a surfactant, a hydrophilic coating, or any combination thereof, to improve or increase the speed or rate of separation of plasma and / or serum from whole blood as it passes through any of the layers, substrates, membranes, meshes, files, or any combination thereof.

[0385] The plasma produced using the devices disclosed herein may not be pure plasma, but rather plasma that has been depleted of one or more components of blood (e.g., red blood cells, white blood cells, platelets, and combinations thereof). In some embodiments, the plasma contains about 5% or less red blood cells, white blood cells, and / or platelets by volume. In other embodiments, the plasma contains about 5% or less red blood cells by volume. In other embodiments, the plasma contains about 4% or less red blood cells by volume. In still other embodiments, the plasma contains about 3% or less red blood cells by volume. In still other embodiments, the plasma contains about 2% or less red blood cells by volume. In still yet further embodiments, the plasma contains about 1% or less red blood cells by volume. In other words, in some embodiments, the product produced by the devices described herein is a product with a reduced number of blood cells (e.g., the product is reduced in red blood cells) compared to a whole blood sample or blood product that has not been contacted with a device described herein.

[0386] In another embodiment, the present disclosure relates to a device. In one aspect, the device includes the apparatus previously described herein and at least one sample analysis cartridge (e.g., a microfluidic cartridge) having a sample application area where a test sample is applied. In one aspect, the apparatus is configured with the sample analysis cartridge such that the microchannel is operably linked, removably coupled, or in fluid communication with the sample application area. Specifically, as shown in FIG. 10 , the second end of the microchannel can be operably linked, removably coupled, or in fluid communication with the sample analysis cartridge at the sample application area. In another aspect, the apparatus is included or incorporated as part of a clip, such as a Moby clip, an extended Moby clip, or the like, configured, coupled, or integrated with the sample analysis cartridge such that the microchannel is operably linked, removably coupled, or in fluid communication with the sample application area.

[0387] In another embodiment, the plasma separation device includes a pre-evacuated container or tube having closed inlet and outlet ends, respectively. A pressure differential exists between the inlet and outlet ends of the container or tube. The inlet end of the container or tube includes a cap or septum that can be pierced by a needle or blood collection needle assembly used to obtain a whole blood sample from a subject. The outlet end includes a serum retention chamber (e.g., a filtrate container) that receives serum and / or plasma (e.g., filtrate) produced by the device.

[0388] The inlet end of the container or tube, containing a cap or septum adapted to be penetrated by a needle or a standard blood collection needle assembly, defines a first end of a blood holding chamber that is free to receive a whole blood sample from the needle or blood collection needle assembly for filtration.

[0389] In some embodiments, a filter assembly is proximate the second end of the blood retention chamber. The filter assembly captures cellular components of the blood (e.g., red blood cells) and allows passage of serum and / or plasma components through the assembly to the serum retention chamber at the outlet end. In some embodiments, the filter assembly covers the entire cross-sectional area of ​​the container or tubing. In some embodiments, the filter assembly allows passage of particles or molecules smaller than about 0.7 microns, about 0.6 microns, about 0.5 microns, about 0.4 microns, or about 0.3 microns, functioning similarly to size exclusion chromatography (SEC, also known as gel filtration), whereby smaller sized particles and molecules (e.g., serum and / or plasma) pass through the filter assembly more quickly than larger sized molecules (e.g., red blood cells). The filter assembly can be made of any material or combination of materials that can be used to separate blood components based on size and allow passage of serum and / or plasma components through the assembly to the serum retention chamber. For example, in some embodiments, the filter assembly includes one or more microfiber membranes and / or glass fiber filter materials (e.g., low-density fiber filter materials, etc.). For example, Micro-Strand glass microfibers obtained from Johns Manville (Fruita, CO) may be used. In some embodiments, highly hydrophilic, highly porous materials, such as those available from Porex Filtration Group (South Chesterfield, VA), such as POR 410 or POR 4711, may be used in the filter assembly. In still further embodiments, the filter assembly terminates near the center of the container or tube, and is held and supported by a screen member or other perforated material.

[0390] Optionally, in some aspects, a flow regulator can be placed adjacent to the cap or septum and can be used to regulate the rate of blood flow into the filter assembly, hi other aspects, the flow regulator can be included as part of the blood collection needle assembly.

[0391] Because the container or tube has previously been evacuated, a pressure differential exists in the area surrounding the filter assembly. The pressure at the top of the container or tube in the blood retention chamber is higher than the pressure at the bottom of the container in the serum retention chamber. This pressure differential causes the whole blood sample in the blood retention chamber to move toward and through the filter assembly, where larger red blood cells become trapped and entrained in the filter assembly, and serum and / or plasma (e.g., filtrate) moves at a much faster rate through the filter assembly into the serum retention chamber, where it is collected.

[0392] The serum holding chamber has a hollow space sized to hold serum and / or plasma resulting from the filter assembly. The serum holding chamber is removably attached (e.g., detachable) to a container or tube and can be removed from the container or tube to allow further processing and / or analysis of the serum or plasma. For example, the serum holding chamber can be removed from the container or tube by twisting or sliding the serum holding chamber in a clockwise or counterclockwise motion to break the vacuum. Once the serum holding chamber is removed from the container or tube, the serum or plasma sample can be used for further processing and / or analysis in another device and / or apparatus. For example, the serum or plasma sample in the serum holding chamber can be pipetted into another container or device (e.g., a cartridge) for further processing and / or analysis.

[0393] In other embodiments, plasma separation can occur in a container or tube that has not previously been evacuated. In such embodiments, plasma separation can be performed using any suitable means, including, for example, using size-exclusion chromatography to separate molecules or components in blood by size differences as they pass through a material (e.g., a filter assembly) contained within the container or tube. For example, any material that serves to preferentially slow the movement of red blood cells relative to plasma may be used (e.g., glass or porous beads, membranes, one or more filters, glass or other fibrous materials, or any combination thereof) so that plasma can be collected. Such methods include the use of other than a pressure differential; for example, gravity-fed separation can be used without the use of any pressure differential.

[0394] Examples of pre-evacuated containers or tubes having the above-described components that can be used as plasma separation devices in the methods described in this disclosure include those described in U.S. Pat. No. 9,427,707, the contents of which are incorporated herein by reference.

[0395] In yet other aspects, the plasma separation device described above may be used in connection with capillary blood samples collected (1) in a decentralized setting; (2) without the use of a syringe, standard needle, or a combination thereof; (3) by a user untrained in collecting blood samples from subjects; (4) by a robot; (5) by a blood collection device applied to oneself or another; or (6) any combination thereof.

[0396] In still further aspects, the plasma separation devices described above can be used in connection with blood samples obtained from subjects collected by use of a syringe, a standard needle, or a combination thereof. Such samples can be collected in a decentralized or centralized setting (e.g., in a traditional medical setting (hospital, doctor's office, stand-alone research center, etc.)), by a user trained in blood collection, or a combination thereof. In some aspects, the blood sample obtained from a subject collected by use of a syringe, a standard needle, or a combination thereof is a venous blood sample. In other aspects, the blood sample obtained from a subject collected by use of a syringe, a standard needle, or a combination thereof is a capillary blood sample. For example, in some embodiments, a blood sample (e.g., a venous blood sample and / or a capillary blood sample) can be obtained from a subject using a syringe, a standard needle, or a combination thereof (and optionally, in a centralized setting and / or by a user trained in collecting blood samples from subjects) and processed prior to performing assays for UCH-L1, GFAP, CK-MB, β-hCG, TSH, homocysteine, free T4, and / or any combination thereof using the plasma separation device described above.

[0397] 5. A system for determining the amount of UCH-L1, GFAP, CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof using a capillary blood sample Referring to FIG. 1 , a system 10 is provided for determining the amount of UCH-L1, GFAP, CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof. The amount of UCH-L1, GFAP, CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof can be used to aid in the diagnosis and / or assessment of a subject who has or may have suffered a head injury. In one embodiment, system 10 includes a microsampling device 14, a reactor 22, and an instrument, such as instrument 26 (e.g., a point-of-care device). In other embodiments, the system can use instrument 81 (e.g., a higher-throughput analyzer, as shown in FIG. 6A ) instead of instrument 26. Note that while the shape of reactor 22 shown in FIG. 1 is rectangular, the shape is not critical. For example, in some embodiments, reactor 22 can be tubular. In further embodiments, reactor 22 can be a microfluidic cartridge.

[0398] In some additional embodiments, the system may further include a plasma separation device, 18 (FIG. 1). When plasma separation device 18 is present, microsampling device 14 collects a capillary blood sample from the subject, and plasma separation device 18 produces a processed capillary blood sample (e.g., serum or plasma) from the capillary blood sample.

[0399] In yet a further additional embodiment, the system can further include a transfer tube, 79 (FIG. 6C). The transfer tube 79 can include a cap or stopper 80. The transfer tube 79 also has an opening 83. The opening 83 allows the reaction tube to receive a capillary blood sample or a processed capillary blood sample.

[0400] In some embodiments, the reactor 22 or transfer tube 79 receives the processed capillary blood sample from the plasma separation device 18 .

[0401] Instrument 26 or 81 analyzes reactor 22 to provide a determination of the amount of UCH-L1, GFAP, CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof, in the subject. In some embodiments, the amount determination is communicated as a result. The result may be communicated for further analysis, interpretation, processing, and / or display. The amount may be communicated by computer, in a document and / or spreadsheet, on a mobile device (e.g., a smartphone), on a website, in an email, or any combination thereof.

[0402] In some embodiments, the communicated amount of UCH-L1, GFAP, CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof, may be displayed on a device, such as device 26 or 81. For example, the amount of UCH-L1, GFAP, CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof may be displayed as elevated, not elevated, or that the test (e.g., assay) should be repeated. Various operational steps by system 10 are described with respect to Figures 2A-2E, and Figures 3A-3C are described in further detail herein.

[0403] 2A , micro-sampling device 14 is coupled to subject 30 and a capillary blood sample is collected from subject 30. In the illustrated embodiment, micro-sampling device 14 includes a housing 34 and a receptacle 38 coupled to housing 34. In the illustrated embodiment, the capillary blood sample is collected in receptacle 38. In other aspects, receptacle 38 is removably coupled to housing 34. For example, receptacle 38 can be separated from housing 34 once the capillary blood sample has been collected. In still other aspects, receptacle 38 is reactor 22. In these aspects, when the receptacle is a reactor, it can be removed from micro-sampling device 14 and inserted directly into instrument 26 or 81.

[0404] The microsampling device 14 further includes a microneedle, a lancet, a microlancet, a blade, a microblade, a microscrew, or any combination thereof, coupled to the housing. In some embodiments, the microsampling device 14 includes a plurality of microneedles. In some embodiments, the microsampling device 14 further includes an actuator movable relative to the housing 34. The actuator can actuate the microneedle or similar component into the subject's skin to initiate collection of a capillary blood sample from the subject 30.

[0405] 2B , system 10 can further include a cap 42 coupled to receptacle 38. In some embodiments, cap 42 is attached to receptacle 38 after the receptacle is removed from housing 34. In aspects, cap 42 seals the capillary blood sample within container 38. For example, a threaded configuration can couple cap 42 to receptacle 38. In some embodiments, cap 42 is part of the interface between receptacle 38 and plasma separation device 18.

[0406] 2C, the plasma separation device 18 includes an inlet 46 for receiving the capillary blood sample from the microsampling device 14 and an outlet 50 through which the processed capillary blood sample exits the plasma separation device 18. In the illustrated embodiment, the inlet 46 receives the capillary blood sample from the receptacle 38. In some aspects, the plasma separation device 18 is integrated within the receptacle 38 (e.g., the plasma separation device 18 is not intended to be removed from the receptacle 38 by a user).

[0407] In some other embodiments, the plasma separation device 18 is formed separately from the reactor 22 or the transfer tube 79 (i.e., the plasma separation device 18 is movable and can be separated from the reactor 22 or the transfer tube 79). In other embodiments, the plasma separation device 18 is integrated with the reactor 22 or the transfer tube 79. For example, in some embodiments, the plasma separation device 18 is integrated within the housing 54 of the reactor 22 (i.e., the plasma separation device 18 is not intended to be removed from the reactor 22 or the transfer tube 79 by the user).

[0408] The plasma separation device 18 comprises a filter, a membrane, a synthetic paper, or any combination thereof. In one aspect, the plasma separation device 18 is removably coupled to the reactor 22 or the transfer tube 79 by a detachable coupling 58. In some aspects, the detachable coupling 58 comprises a threaded configuration. In other embodiments, the detachable coupling 58 comprises a retaining member located on the reactor 22 or the transfer tube 79 for holding the plasma separation device 18 in place when the plasma separation device 18 is installed in the reactor 22 or the transfer tube 79. The outlet 50 of the plasma separation device 18 is placed in fluid communication with the opening 62 in the reactor 22 or the opening 83 in the transfer tube 79. In other words, the processed capillary blood sample flows from the outlet 50 of the plasma separation device 18 to the opening 62 in the reactor 22 or the opening 83 in the transfer tube 79.

[0409] In still further embodiments, receptacle 38 is removably coupled to plasma separation device 18 by removable coupling 66 (FIG. 2C). In some embodiments, receptacle 38 is coupled to plasma separation device 18 after plasma separation device 18 is coupled to reactor 22 or transfer tubing 79. In still further embodiments, cap 42 is removed prior to coupling receptacle 38 to plasma separation device 18. In some embodiments, cap 42 remains in place when receptacle 38 is coupled to plasma separation device 18. In some embodiments, cap 42 is pierced when receptacle 38 is coupled to plasma separation device 18, allowing the capillary blood sample to flow into plasma separation device 18.

[0410] In still further aspects, the plasma separation device 18 is removably coupled to the reactor 22 or transfer tube 79 and the receptacle 38. Referring to FIG. 2D , the plasma separation device 18 is positioned between the receptacle 38 and the reactor 22. In other aspects, the plasma separation device 18 is positioned between the receptacle 38 and the transfer tube 79. In some aspects, the receptacle 38 is squeezed by a user or operator to force the capillary blood sample through the plasma separation device 18 and into the reactor 22 or transfer tube 79. In other aspects, the receptacle 38 includes a plunger for pushing the capillary blood sample through the plasma separation device 18 and into the reactor 22 or transfer tube 79. In other embodiments, the capillary blood sample is gravity fed through the plasma separation device 18 into the reactor 22 or transfer tube 79.

[0411] 2D, reactor 22, in some embodiments, is a microfluidic cartridge. The processed capillary blood sample flows from plasma separation device 18 to reactor 22 where an assay is performed. In some embodiments, the assay is for UCH-L1, GFAP, CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof.

[0412] 3, the plasma separation device 18 is placed in fluid communication with the opening 62 at any point along the reactor 22 (e.g., a microfluidic cartridge). In some embodiments, the plasma separation device 18 is placed in fluid communication with the opening 62 at one end, side, or center of the reactor 22 (e.g., a microfluidic cartridge). In some embodiments, as shown in FIG. 4, the plasma separation device 18 is placed in fluid communication with the opening 62 at an angle, such as about 5 degrees, about 10 degrees, about 15 degrees, about 20 degrees, about 25 degrees, about 30 degrees, about 35 degrees, about 40 degrees, about 45 degrees, about 50 degrees, about 55 degrees, about 60 degrees, about 65 degrees, about 70 degrees, about 75 degrees, about 80 degrees, about 85 degrees, or about 90 degrees (e.g., forming an L- or J-shape, forming a right angle), at an end or side of the reactor 22 (e.g., a microfluidic cartridge). In another embodiment, as shown in FIG. 5, the plasma separation device 18 is placed in fluid communication with at least one opening 62 at one end or side of the reactor 22 (eg, a microfluidic cartridge).

[0413] 2E , once the processed capillary blood sample is loaded into the reactor 22, the reactor 22 is inserted into the instrument 26. In the illustrated embodiment, the reactor 22 is inserted into the bottom portion 70 of the handle 74. The instrument 26 includes a display 78 configured to communicate the results of UCH-L1, GFAP, CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof, determined in the sample. For example, the display 78 on the instrument 26 can display the results as indicating that the amount of UCH-L1, GFAP, CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof in the subject is elevated, not elevated, or that the test should be repeated. In some aspects, the results are provided by visual, audio, or tactile feedback.

[0414] 6B , the processed capillary blood sample in transfer tube 79 is loaded into instrument 81. Once loaded, a portion of the processed capillary blood sample is removed from transfer tube 79 and placed into a reactor (e.g., a tube) already contained in instrument 81 to perform an assay for UCH-L1, GFAP, CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof. The portion of the processed capillary blood sample can be removed from transfer tube 79 to the reactor using a mechanical pipetting system or other techniques known in the art. Instrument 81 includes a display 82 configured to communicate the results of the UCH-L1, GFAP, CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof determined in the sample. For example, display 82 on device 81 can display the results as an indication that the amount of UCH-L1, GFAP, CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof in the subject is elevated, not elevated, or that the test should be repeated. In some embodiments, the results are provided by visual, audio, or tactile feedback.

[0415] In some embodiments, results are communicated within about 4 minutes from the time the sample is collected (e.g., from the time of injury or suspected injury). In some embodiments, results are communicated within about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, or about 40 minutes from the time the sample is collected (e.g., from the time of injury or suspected injury). In some embodiments, results are communicated within about 4 minutes to about 40 minutes from the time the sample is collected (e.g., from the time of injury or suspected injury). In some embodiments, results are communicated within about 4 minutes to about 30 minutes from the time the sample is collected (e.g., from the time of injury or suspected injury). In some embodiments, results are communicated within about 4 minutes to about 20 minutes from the time the sample is collected (e.g., from the time of injury or suspected injury).

[0416] Advantageously, portions of system 10 can be used in a decentralized setting. In other words, system 10 is portable. Advantageously, system 10, or portions thereof, can be reused for multiple samples and / or subjects. In some embodiments, microsampling device 14, plasma separation device 18, and reactor 22 or transfer tube 79 are disposable components (i.e., usable for a single test on a single subject); however, instruments 26 and 81 are reusable for the analysis of multiple reactors 22.

[0417] 6. Treatment and monitoring of subjects with head injuries In the above methods, the identified subject can be treated or monitored. In some embodiments, the method further includes treating the subject, such as a human subject, with a traumatic brain injury treatment, such as any treatment known in the art. For example, traumatic brain injury treatment can take various forms depending on the severity of the head injury. For example, in a subject suffering from a mild TBI, treatment can include one or more of rest, refraining from physical activity such as sports, avoiding light or wearing sunglasses when outside in bright light, medication to relieve headaches or migraines, antiemetic medication, etc. Treatment for patients suffering from moderate, severe, or moderate-to-severe TBI can include administration of one or more appropriate medications (e.g., diuretics, anticonvulsant medications, medications to sedate and put the individual into a drug-induced coma, or other pharmaceutical or biopharmaceutical medications (known or later developed for the treatment of TBI)), one or more surgical procedures (e.g., hematoma removal, skull fracture repair, decompressive craniectomy, etc.), airway protection, and one or more therapies (e.g., one or more of rehabilitation, cognitive behavioral therapy, anger management, counseling psychology, etc.). In some embodiments, the method further includes monitoring the subject, such as a human subject. In some embodiments, the subject can be monitored by CT scan or MRI procedure.

[0418] 7. Methods for Measuring UCH-L1 Levels In the methods described above, UCH-L1 levels can be measured by any means, including immunoassays, protein immunoprecipitation, immunoelectrophoresis, chemical analysis, antibody-dependent methods such as SDS-PAGE and Western blot analysis, protein immunostaining, electrophoretic analysis, protein assays, competitive binding assays, functional protein assays, or chromatographic methods such as high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC / MS), or spectroscopic methods. The assays can also be used in clinical chemistry formats that would be known to those skilled in the art.

[0419] In some embodiments, measuring the level of UCH-L1 comprises contacting the sample with a first specific binding member and a second specific binding member. In some embodiments, the first specific binding member is a capture antibody and the second specific binding member is a detection antibody. In some embodiments, measuring the level of UCH-L1 comprises contacting the sample with, simultaneously or sequentially, in any order, (1) a capture antibody (e.g., a UCH-L1 capture antibody) that binds to an epitope on UCH-L1 or a UCH-L1 fragment to form a capture antibody-UCH-L1 antigen complex (e.g., a UCH-L1 capture antibody-UCH-L1 antigen complex), and (2) a detectable label that binds to an epitope on UCH-L1 not bound by the capture antibody to form a UCH-L1 antigen complex. - contacting the sample with a detection antibody (e.g., a UCH-L1 detection antibody) that forms a detection antibody complex (e.g., a UCH-L1 antigen-UCH-L1 detection antibody complex) to form a capture antibody-UCH-L1 antigen-detection antibody complex (e.g., a UCH-L1 capture antibody-UCH-L1 antigen-UCH-L1 detection antibody complex), and measuring the amount or concentration of UCH-L1 in the sample based on a signal generated by the detectable label in the capture antibody-UCH-L1 antigen-detection antibody complex.

[0420] In some embodiments, the first specific binding member is immobilized on a solid support. In some embodiments, the second specific binding member is immobilized on a solid support. In some embodiments, the first specific binding member is a UCH-L1 antibody described below.

[0421] In some embodiments, the sample is diluted or neat. The sample may be about 1 to about 25 microliters, about 1 to about 24 microliters, about 1 to about 23 microliters, about 1 to about 22 microliters, about 1 to about 21 microliters, about 1 to about 20 microliters, about 1 to about 18 microliters, about 1 to about 17 microliters, about 1 to about 16 microliters, about 15 microliters, or about 1 microliter, about 2 microliters, about 3 microliters, about 4 microliters, about 5 microliters, about 6 microliters, about 7 microliters, about 8 microliters, about 9 microliters, about 10 microliters, about 11 microliters, about 12 microliters, about 13 microliters, about 14 microliters, about 15 microliters, about 16 microliters, about 17 microliters, about 18 microliters, about 19 microliters, about 20 microliters, about 21 microliters, about 22 microliters, about 23 microliters, about 24 microliters, or about 25 microliters. In some embodiments, the sample is about 1 to about 150 microliters or less, or about 1 to about 25 microliters or less.

[0422] Some meters other than point-of-care devices (e.g., the ARCHITECT® meter manufactured by Abbott Laboratories and other core testing meters) may be capable of measuring UCH-L1 levels in samples of 25,000 pg / mL or greater.

[0423] Other detection methods may include or be adapted for use in nanopore or nanowell devices. Examples of nanopore devices are described in International Patent Publication No. WO 2016 / 161402, which is incorporated herein by reference in its entirety. Examples of nanowell devices are described in International Patent Publication No. WO 2016 / 161400, which is incorporated herein by reference in its entirety.

[0424] 8.UCH-L1 antibody The methods described herein may use isolated antibodies that specifically bind to ubiquitin carboxy-terminal hydrolase L1 ("UCH-L1") (or fragments thereof), referred to as "UCH-L1 antibodies." UCH-L1 antibodies may be used to assess UCH-L1 status as a measure of traumatic brain injury, or to detect the presence of UCH-L1 in a sample, to quantify the amount of UCH-L1 present in a sample, or to detect the presence of UCH-L1 in a sample and quantify the amount of UCH-L1 in a sample.

[0425] a. Ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) Ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) ("UCH-L1"), also known as "ubiquitin C-terminal hydrolase," is a deubiquitinating enzyme. UCH-L1 is a member of a gene family whose product hydrolyzes the small C-terminal adduct of ubiquitin to generate ubiquitin monomers. UCH-L1 expression is highly specific to neurons and cells of the diffuse neuroendocrine system and their tumors. UCH-L1 is abundant in all neurons (accounting for 1-2% of total brain protein) and is particularly expressed in neurons and testis / ovary. The catalytic triad of UCH-L1 contains a cysteine ​​at position 90, an aspartic acid at position 176, and a histidine at position 161, which contribute to its hydrolase activity.

[0426] Human UCH-L1 has the following amino acid sequence:

[0427] [ka] may have.

[0428] Human UCH-L1 can be a fragment or variant of SEQ ID NO: 1. Fragments of UCH-L1 can be between 5 and 225 amino acids, between 10 and 225 amino acids, between 50 and 225 amino acids, between 60 and 225 amino acids, between 65 and 225 amino acids, between 100 and 225 amino acids, between 150 and 225 amino acids, between 100 and 225 amino acids, or between 175 and 225 amino acids in length. Fragments can include consecutive amino acids derived from SEQ ID NO: 1.

[0429] b.UCH-L1 recognition antibody The antibody is an antibody that binds to UCH-L1, a fragment thereof, an epitope of UCH-L1, or a variant thereof. The antibody may be a fragment of an anti-UCH-L1 antibody, or a variant or derivative thereof. The antibody may be a polyclonal or monoclonal antibody. The antibody may be a chimeric antibody, a single-chain antibody, an affinity-matured antibody, a human antibody, a humanized antibody, a fully human antibody, or an antibody fragment such as a Fab fragment, or a mixture thereof. The antibody fragment or derivative may include a F(ab')2 fragment, an Fv fragment, or an scFv fragment. The antibody derivative may be produced by peptidomimetic. Furthermore, techniques described for producing single-chain antibodies can be adapted to produce single-chain antibodies.

[0430] The anti-UCH-L1 antibody may be a chimeric or humanized anti-UCH-L1 antibody. In one embodiment, both the humanized and chimeric antibodies are monovalent. In one embodiment, both the humanized and chimeric antibodies comprise a single Fab region linked to an Fc region.

[0431] Human antibodies can be derived using phage display technology or transgenic mice expressing human immunoglobulin genes. Human antibodies can be generated and isolated as a result of a human in vivo immune response. See, for example, Funaro et al., BMC Biotechnology, 2008(8):85. Thus, the antibody can be a product of a human and may not be part of the animal repertoire. Because the antibody is of human origin, the risk of reactivity against self-antigens can be minimized. Alternatively, standard yeast display libraries and yeast display technology can be used to select and isolate human anti-UCH-L1 antibodies. For example, a library of naive human single-chain variable fragments (scFvs) can be used to select human anti-UCH-L1 antibodies. Transgenic animals can be used to express human antibodies.

[0432] A humanized antibody may be an antibody molecule derived from a non-human species antibody that binds to a desired antigen, and that has one or more complementarity-determining regions (CDRs) derived from the non-human species and a framework region derived from a human immunoglobulin molecule.

[0433] The antibody is distinguishable from known antibodies in that it possesses a different biological function than antibodies known in the art.

[0434] (1) Epitope The antibody can immunospecifically bind to UCH-L1 (SEQ ID NO: 1), a fragment thereof, or a variant thereof. The antibody can immunospecifically recognize and bind to at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acids within the epitope region. The antibody can immunospecifically recognize and bind to an epitope having at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 consecutive amino acids within the epitope region.

[0435] c. Antibody preparation / production Antibodies can be prepared by any of a variety of techniques, including techniques well known to those of skill in the art. Generally, antibodies can be produced by conventional techniques or by cell culture techniques, including the generation of monoclonal antibodies by transfecting antibody genes, heavy chains, and / or light chains into a suitable bacterial or mammalian cell host, which allows for the production of antibodies, which may be recombinant. The term "transfection" in its various forms is intended to encompass a wide variety of techniques commonly used for the introduction of foreign DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and others. While it is possible to express antibodies in either prokaryotic or eukaryotic host cells, expression of antibodies in eukaryotic cells is preferred, and expression of antibodies in mammalian host cells is most preferred, since such eukaryotic cells (especially mammalian cells) are more likely than prokaryotic cells to assemble and secrete properly folded and immunologically active antibodies.

[0436] Exemplary mammalian host cells for expressing recombinant antibodies include Chinese hamster ovary (CHO) cells (including the dhfr-CHO cells described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216-4220 (1980)) used with the DHFR selectable marker, e.g., as described in Kaufman and Sharp, J. Mol. Biol., 159:601-621 (1982)), NS0 myeloma cells, COS cells, and SP2 cells. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow expression of the antibody within the host cell or, more preferably, secretion of the antibody into the culture medium in which the host cell is grown. The antibody may be recovered from the culture medium using standard protein purification methods.

[0437] Host cells can also be used to produce functional antibody fragments, such as Fab fragments or scFv molecules. It is understood that variations on the above procedures can be implemented. For example, it may be desirable to transfect host cells with DNA encoding functional fragments of the antibody light and / or heavy chains. Recombinant DNA technology can also be used to remove some or all of the DNA encoding one or both of the light and heavy chains that is not necessary for binding to the antigen of interest. Molecules expressed from such truncated DNA molecules are also encompassed by antibodies. Additionally, bifunctional antibodies, in which one heavy chain and one light chain are antibody (i.e., bind to human UCH-L1) and the other heavy and light chains are specific for an antigen other than human UCH-L1, can also be produced by crosslinking an antibody to a second antibody via standard chemical crosslinking methods.

[0438] In a preferred system for recombinantly expressing an antibody or antigen-binding portion thereof, a recombinant expression vector encoding both the antibody heavy and light chains is introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the antibody heavy and light chain genes are each operably linked to a CMV enhancer / AdMLP promoter regulatory element to drive high levels of gene transcription. The recombinant expression vector also carries a DHFR gene, allowing for selection of CHO cells transfected with the vector using methotrexate selection / amplification. Selected transformant host cells are cultured to allow expression of the antibody heavy and light chains, and intact antibody is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells, and recover the antibody from the culture medium. Furthermore, a method for synthesizing a recombinant antibody is by culturing the host cells in an appropriate culture medium until the recombinant antibody is synthesized. The method may further comprise isolating the recombinant antibody from the culture medium.

[0439] Methods for preparing monoclonal antibodies involve the preparation of immortal cell lines capable of producing antibodies with the desired specificity. Such cell lines can be generated from spleen cells obtained from immunized animals. Animals can be immunized with UCH-L1 or fragments and / or variants thereof. The peptide used to immunize the animal can include amino acids encoding human Fc, e.g., the Fc (fragment crystallizable) region or tail region of a human antibody. The spleen cells are then immortalized, for example, by fusion with a myeloma cell fusion partner. Various fusion methods can be used. For example, spleen cells and myeloma cells are combined with a non-ionic detergent for several minutes and then plated at low density on a selective medium that supports the growth of hybrid cells but not myeloma cells. One such technique uses hypoxanthine, aminopterin, and thymidine (HAT) selection. Another technique involves electrofusion. After sufficient time, usually about 1 to 2 weeks, hybrid colonies can be observed. Single colonies are selected and their culture supernatants are tested for binding activity to the polypeptide. Hybridomas with high reactivity and specificity can be used.

[0440] Monoclonal antibodies can be isolated from the supernatant of growing hybridoma colonies. In addition, various techniques can be used to enhance the yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse. The monoclonal antibody can then be collected from the ascites fluid or blood. Contaminants can be removed from the antibody by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction. Affinity chromatography is an example of a method that can be used in the process of purifying the antibody.

[0441] The proteolytic enzyme papain preferentially cleaves IgG molecules into several fragments, two of which (F(ab) fragments) comprise covalent heterodimers, each of which contains an intact antigen-binding site. The enzyme pepsin can cleave IgG molecules into several fragments, including the F(ab')2 fragment, which contains both antigen-binding sites.

[0442] Fv fragments can be generated by preferential proteolytic cleavage of IgM, and in rare cases, IgG or IgA immunoglobulin molecules. Fv fragments can be derived using recombinant methods. Fv fragments comprise a noncovalent VH::VL heterodimer containing an antigen-binding site that retains much of the antigen recognition and binding capabilities of a native antibody molecule.

[0443] Each antibody, antibody fragment, or derivative may comprise a set of heavy and light chain complementarity determining...

Claims

1. Using a point-of-care device or a non-point-of-care device, (A) performing at least one assay for (i) ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof; or (ii) CK-MB, β-hCG, thyroid stimulating hormone (TSH), homocysteine, free thyroxine (free T4), or any combination thereof, in a capillary blood sample obtained from the subject to determine the amount of (i) UCH-L1, GFAP, or a combination thereof; or (ii) CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof; and (B) communicating the amount of (i) UCH-L1, GFAP, or a combination thereof; or (ii) CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof, determined in the sample. A method comprising: the sample is collected from a location on the subject other than a finger, and (1) in a decentralized setting; (2) without the use of a syringe, standard needle, or a combination thereof; (3) by a user who is not trained in collecting blood samples from subjects; (4) by a robot; (5) by a blood collection device that is applied to oneself or another; or (6) by any combination of (1)-(5); Further, (i) the assay is capable of being performed in less than about 30 minutes; (ii) the amount of (a) UCH-L1, GFAP or a combination thereof; or (B) CK-MB, β-hCG, TSH, homocysteine, free T4 or any combination thereof determined in the sample is capable of being transmitted in less than about 30 minutes from the time the sample is collected; or (iii) a combination of (i) and (ii).

2. The method of claim 1 , wherein the sample is collected using a microsampling device.

3. 3. The method of claim 1 or claim 2, wherein the sample is processed prior to performing the assay, and further wherein the sample is whole blood and is processed into serum or plasma.

4. The method of claim 3 , wherein the sample is processed using a plasma separation device.

5. The method of any of claims 2 to 4, wherein the microsampling device (a) comprises a plasma separation device; or (b) is operably linked to a plasma separation device.

6. The plasma separation device comprises: a hydrophobic layer including at least one microchannel having first and second ends and defining a path for capillary fluid flow; and a top layer adjacent to the hydrophobic layer, the surface of the top layer facing the hydrophobic layer being hydrophilic; and the sample comprises blood or a blood product.

7. The method of claim 6 , wherein the device further comprises a bottom layer adjacent to the hydrophobic layer, the surface of the bottom layer facing the hydrophobic layer being hydrophilic.

8. 8. The method of claim 6 or claim 7, wherein the at least one microchannel (a) extends longitudinally along a portion of the hydrophobic layer to an opening at a second end; (b) is less than about 80 mm in length; (c) is less than about 5 mm in width; or (d) any combination of (a)-(c).

9. (a) the top layer, bottom layer, or top and bottom layers are generally hydrophilic; (b) the overall composition of the top layer, bottom layer, or both the top and bottom layers, respectively, comprises the same or different materials; (c) the hydrophobic layer, top layer, bottom layer, or any combination thereof, has a combined thickness of about 100 to about 600 microns; (d) the hydrophobic layer has a thickness of about 50 to about 200 microns; (e) each of the top layer, bottom layer, or top and bottom layers has a thickness of about 50 to about 200 microns; (f) the top layer includes a sample inlet; (g) the hydrophobic layer and optionally the bottom layer comprise an opening below the sample inlet; (h) the sample inlet comprises a separation membrane; (i) the opening in the hydrophobic layer is connected to the first end of the at least one microchannel; (j) the plasma separation device further comprises a flocculating agent; or (k) any combination of (a) to (j); The method according to any one of claims 6 to 8.

10. The plasma separation device comprises: a pre-evacuated container or tube having an inlet and an outlet end; a blood holding chamber at the inlet end of the container or tube; a removable serum holding chamber at the outlet end of the container or tube; A filter disposed within the vessel between the blood-holding chamber and the serum-holding chamber. The method of claim 4, wherein the apparatus comprises:

11. 11. The method of claim 10, wherein the filter allows the passage of particles or molecules smaller than about 0.7 microns, about 0.6 microns, about 0.5 microns, about 0.4 microns, or about 0.3 microns.

12. 12. The method of claim 10 or claim 11, wherein a pressure differential between the blood holding chamber and the serum holding chamber allows whole blood to move from the blood holding chamber through the filter to produce serum and / or plasma that is collected in the serum holding chamber.

13. performing at least one assay for (i) ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof; or (2) CK-MB, β-hCG, thyroid stimulating hormone (TSH), homocysteine, free thyroxine (free T4), or any combination thereof, in a blood sample obtained from the subject to determine the amount of (i) UCH-L1, GFAP, or a combination thereof; or (ii) CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof; and communicating the amount of (i) UCH-L1, GFAP, or a combination thereof; or (ii) CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof, determined in said sample. A method comprising: The sample is collected by use of a syringe, a standard needle, or a combination thereof; Furthermore, the sample a) a hydrophobic layer including at least one microchannel having first and second ends and defining a path for capillary fluid flow; and an upper layer adjacent to the hydrophobic layer, wherein a surface of the upper layer facing the hydrophobic layer is hydrophilic; or b) a pre-evacuated container or tube having an inlet and an outlet end; a blood holding chamber at the inlet end of the vessel or tube; a removable serum holding chamber at the outlet end of the container or tube; A filter disposed within the vessel between the blood-holding chamber and the serum-holding chamber. The method of claim 1, wherein the plasma is treated prior to performing the assay using a plasma separation device comprising an apparatus having the steps of:

14. The method of claim 13, wherein the blood sample is a venous blood sample or a capillary blood sample.

15. 15. The method of claim 13 or claim 14, wherein the samples are collected in a decentralized or centralized setting.

16. 16. The method of any of claims 13-15, wherein the at least one microchannel (a) extends longitudinally along a portion of the hydrophobic layer to an opening at a second end; (b) is less than about 80 mm in length; (c) is less than about 5 mm in width; or (d) any combination of (a)-(c).

17. (a) the top layer, bottom layer, or top and bottom layers are generally hydrophilic; (b) the overall composition of the top layer, bottom layer, or both the top and bottom layers, respectively, comprises the same or different materials; (c) the hydrophobic layer, top layer, bottom layer, or any combination thereof, has a combined thickness of about 100 to about 600 microns; (d) the hydrophobic layer has a thickness of about 50 to about 200 microns; (e) the top layer, bottom layer, or each of the top and bottom layers has a thickness of about 50 to about 200 microns; (f) the top layer includes a sample inlet; (g) the hydrophobic layer and optionally the bottom layer comprise an opening below the sample inlet; (h) the sample inlet comprises a separation membrane; (i) the opening in the hydrophobic layer is connected to the first end of the at least one microchannel; (j) the plasma separation device further comprises a flocculating agent; (k) the filter allows the passage of particles or molecules smaller than about 0.7 microns, about 0.6 microns, about 0.5 microns, about 0.4 microns, or about 0.3 microns; (l) a pressure differential between the blood holding chamber and the serum holding chamber permits whole blood to move from the blood holding chamber through the filter to produce serum and / or plasma that is collected in the serum holding chamber; or (m) any combination of (a) to (l); The method according to any one of claims 13 to 16.

18. The method of claim 14, wherein the sample is a capillary blood sample.

19. The assay further comprises: (I) (a) an anti-UCH-L1 antibody that binds to UCH-L1 to determine the amount of UCH-L1 in the sample; (b) an anti-GFAP antibody that binds to GFAP to determine the amount of GFAP in the sample; or (c) a combination of (a) and (b); or (II) (a) an anti-CK-MB antibody that binds to CK-MB to determine the amount of CK-MB in the sample; (b) an anti-β-hCG antibody that binds to β-hCG to determine the amount of β-hCG in the sample; (c) an anti-TSH antibody that binds to TSH to determine the amount of TSH in the sample; (d) an anti-homocysteine ​​antibody that binds to homocysteine ​​to determine the amount of homocysteine ​​in the sample; (e) an anti-free T4 antibody that binds to free T4 to determine the amount of free T4 in the sample; or (f) any combination of (a)-(e). The method of any one of claims 1 to 18, comprising contacting the

20. The assay comprising: (I) (a) if an assay for GFAP, said GFAP assay comprises a scaling factor of GFAP in a capillary sample compared to GFAP in a venous sample of about 1.0:1.0; (b) if an assay for UCH-L1, said UCH-L1 assay comprises a scaling factor of UCH-L1 in a capillary sample compared to UCH-L1 in a venous sample of about 2.5:1.0 to about 1.5:1.0; or (c) a combination of (a) and (b); or (II) (a) when it is an assay for CK-MB, said CK-MB assay comprises a conversion factor of CK-MB in a capillary blood sample compared to CK-MB in a venous sample of about 0.5:1.0 to about 1:0:1.2; (b) when it is an assay for β-hCG, said β-hCG assay comprises a conversion factor of β-hCG in a capillary blood sample compared to β-hCG in a venous sample of about 0.8:1.0 to about 1:0:1.4; (c) when it is an assay for TSH, said TSH assay comprises a conversion factor of β-hCG in a capillary blood sample compared to TSH in a venous sample of about 0.75:1.0 to about 1.2:1.

0.

20. The method of any of claims 1-12 or 18-19, wherein the homocysteine ​​assay comprises a conversion factor of homocysteine ​​in a capillary sample compared to homocysteine ​​in a venous sample of from about 1.2:1.0 to about 0.9:1.0; (c) if the homocysteine ​​assay is for homocysteine, the homocysteine ​​assay comprises a conversion factor of homocysteine ​​in a capillary sample compared to homocysteine ​​in a venous sample of from about 0.8:1.0 to about 1.2:1.0; (e) if the free T4 assay is for free T4, the free T4 assay comprises a conversion factor of free T4 in a capillary sample compared to free T4 in a venous sample of from about 0.8:1.0 to about 1.2:1.0; or (f) any combination of (a)-(e).

21. Where the assay is (a) an assay for GFAP, UCH-L1, or GFAP and UCH-L1, the method is used to aid in the diagnosis and assessment of a subject who has suffered or may have suffered a head injury; (b) an assay for CK-MB, the method is used to diagnose myocardial infarction in a subject; (c) an assay for β-hCG, the method is used to determine whether a subject is pregnant; (d) an assay for TSH, the method is used to assess thyroid function in a subject, diagnose thyroid disease in a subject, treat or prevent thyroid disease in a subject.

21. The method of any of claims 1 to 20, wherein (e) if the method is an assay for homocysteine, the method is used to diagnose hyperhomocysteinemia, homocystinuria, or hyperhomocysteinemia and homocystinuria in a subject, or to treat a subject having hyperhomocysteinemia, homocystinuria, or hyperhomocysteinemia and homocystinuria; or (f) if the method is an assay for free T4, the method is used to assess thyroid function in a subject, diagnose a thyroid disease in a subject, treat a thyroid disease in a subject, or any combination thereof.

22. 22. The method of any of claims 1-21, wherein the amount of: (a) UCH-L1, GFAP, and UCH-L1 and UCH-L1 and GFAP; or (b) CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof, is delivered within: (a) about 25 minutes from the time the sample is collected; (b) less than about 20 minutes from the time the sample is collected; (c) about 4 to about 20 minutes from the time the sample is collected; (d) about 15 to about 18 minutes from the time the sample is collected; or (e) less than about 18 minutes from the time the sample is collected.

23. The method of any of claims 1 to 12, wherein (a) the point-of-care device comprises a cartridge; or (b) the non-point-of-care device is a higher throughput assay analyzer.

24. 24. The method of any of claims 1-23, wherein the amount of UCH-L1, GFAP, CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof is communicated (a) by a point-of-care device or a non-point-of-care device, in a document and / or spreadsheet, on a mobile device, on a computer, on a website, in an email, or any combination thereof; or (b) by display on a point-of-care device or a non-point-of-care device.

25. The method of any one of claims 1 to 24, wherein the assay is an analogue assay, a digital assay, or a combination of analogue or digital assays.

26. The method of any one of claims 1 to 25, wherein the subject is a human.

27. a microsampling device for collecting a capillary blood sample from a subject; a reactor for receiving the capillary blood sample and comprising: (i) ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof; or (ii) an assay for CK-MB, β-hCG, thyroid stimulating hormone (TSH), homocysteine, free thyroxine (free T4), or any combination thereof; and and analyzing said reactor to provide the amount of (i) UCH-L1, GFAP, or UCH-L1 and GFAP; or (ii) CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof, in said sample. A system including:

28. 30. The system of claim 27, further comprising a plasma separation device for producing a processed capillary blood sample.

29. 29. The system of claim 27 or claim 28, wherein the reactor comprises an opening.

30. 30. The system of claim 28 or claim 29, wherein the plasma separation device is in fluid communication with the opening (a) at any point along the reactor; or (b) at one end, a side, or the center of the reactor; or (c) at an angle at an end or side of the reactor.

31. The system of any of claims 27 to 30, wherein the plasma separation device includes an inlet for receiving the capillary blood sample from the microsampling device and an outlet for the treated capillary blood sample to exit the plasma separation device.

32. 32. The system of claim 31 , wherein the outlet of the plasma separation device is in fluid communication with the opening of the reactor.

33. A system according to any one of claims 27 to 32, wherein at least parts of the system are operable in a distributed setting.

34. The plasma separation device comprises: a) a hydrophobic layer including at least one microchannel having first and second ends and defining a path for capillary fluid flow; and an upper layer adjacent to the hydrophobic layer, wherein a surface of the upper layer facing the hydrophobic layer is hydrophilic; or b) a pre-evacuated container or tube having an inlet and an outlet end; a blood-retaining chamber at the inlet end of the container or tube; a removable serum-retaining chamber at the outlet end of the container or tube; and a filter disposed within the container between the blood-retaining chamber and the serum-retaining chamber. The system according to any one of claims 28 to 33, comprising an apparatus having:

35. 35. The system of claim 34, wherein the device further comprises: a) a bottom layer adjacent to the hydrophobic layer, the surface of the bottom layer facing the hydrophobic layer being hydrophilic.

36. 36. The system of claim 34 or claim 35, wherein the at least one microchannel (a) extends longitudinally along a portion of the hydrophobic layer to an opening at a second end; (b) is less than about 80 mm in length; (c) is less than about 5 mm in width; or (d) any combination of (a)-(c).

37. (a) the top layer, bottom layer, or top and bottom layers are generally hydrophilic; (b) the overall composition of the top layer, bottom layer, or both the top and bottom layers, respectively, comprises the same or different materials; (c) the hydrophobic layer, top layer, bottom layer, or any combination thereof, has a combined thickness of about 100 to about 600 microns; (d) the hydrophobic layer has a thickness of about 50 to about 200 microns; (e) the top layer, bottom layer, or each of the top and bottom layers has a thickness of about 50 to about 200 microns; (f) the top layer includes a sample inlet; (g) the hydrophobic layer and optionally the bottom layer comprise an opening below the sample inlet; (h) the sample inlet comprises a separation membrane; (i) the opening in the hydrophobic layer is connected to the first end of the at least one microchannel; (j) the plasma separation device further comprises a flocculating agent; (k) the filter allows the passage of particles or molecules smaller than about 0.7 microns, about 0.6 microns, about 0.5 microns, about 0.4 microns, or about 0.3 microns; (l) a pressure differential between the blood holding chamber and the serum holding chamber permits whole blood to move from the blood holding chamber through the filter to produce serum and / or plasma that is collected in the serum holding chamber; or (m) any combination of (a) to (l); A system according to any one of claims 34 to 36.

38. a plasma separation device for processing a whole blood sample obtained from a subject into serum and / or plasma; a reactor that receives the serum and / or plasma from the subject and that contains assays for (i) ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillary acidic protein (GFAP), or a combination thereof; or (ii) CK-MB, β-hCG, thyroid stimulating hormone (TSH), homocysteine, free thyroxine (free T4), or any combination thereof; and and analyzing said reactor to provide the amount of (i) UCH-L1, GFAP, or UCH-L1 and GFAP; or (ii) CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof, in said sample. A system comprising: The plasma separation device comprises: a) a hydrophobic layer including at least one microchannel having first and second ends and defining a path for capillary fluid flow; and an upper layer adjacent to the hydrophobic layer, wherein a surface of the upper layer facing the hydrophobic layer is hydrophilic; or b) a pre-evacuated container or tube having an inlet and an outlet end; a blood holding chamber at the inlet end of the vessel or tube; a removable serum holding chamber at the outlet end of the container or tube; A filter disposed within the vessel between the blood-holding chamber and the serum-holding chamber.

23. A system comprising an apparatus having:

39. 39. The system of claim 38, wherein the blood sample is a venous blood sample or a capillary blood sample.

40. 40. The system of claim 38 or claim 39, wherein the samples are collected in a decentralized or centralized setting.

41. The system of any of claims 38 to 40, wherein the device in a) further comprises a bottom layer adjacent to the hydrophobic layer, the surface of the bottom layer facing the hydrophobic layer being hydrophilic.

42. 42. The system of any of claims 38-41, wherein the at least one microchannel (a) extends longitudinally along a portion of the hydrophobic layer to an opening at a second end; (b) is less than about 80 mm in length; (c) is less than about 5 mm in width; or (d) any combination of (a)-(c).

43. (a) the top layer, bottom layer, or top and bottom layers are generally hydrophilic; (b) the overall composition of the top layer, bottom layer, or both the top and bottom layers, respectively, comprises the same or different materials; (c) the hydrophobic layer, top layer, bottom layer, or any combination thereof, has a combined thickness of about 100 to about 600 microns; (d) the hydrophobic layer has a thickness of about 50 to about 200 microns; (e) the top layer, bottom layer, or each of the top and bottom layers has a thickness of about 50 to about 200 microns; (f) the top layer includes a sample inlet; (g) the hydrophobic layer and optionally the bottom layer comprise an opening below the sample inlet; (h) the sample inlet comprises a separation membrane; (i) the opening in the hydrophobic layer is connected to the first end of the at least one microchannel; (j) the plasma separation device further comprises a flocculating agent; (k) the filter allows the passage of particles or molecules smaller than about 0.7 microns, about 0.6 microns, about 0.5 microns, about 0.4 microns, or about 0.3 microns; (l) a pressure differential between the blood holding chamber and the serum holding chamber permits whole blood to move from the blood holding chamber through the filter to produce serum and / or plasma that is collected in the serum holding chamber; or (m) any combination of (a) to (l); A system according to any one of claims 38 to 42.

44. 38. The system of claims 27-37, wherein the micro-sampling device comprises a housing, a microneedle, a lancet, a microlancet, a blade, a microblade, a microscrew or any combination thereof coupled to the housing, and a receptacle coupled to the housing; and the capillary blood sample is collected in the receptacle.

45. 45. The system of claims 27-44, wherein the amount of (i) UCH-L1, GFAP, or UCH-L1 and GFAP; or (ii) CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof, is determined within: (a) about 25 minutes from the time the sample is collected; (b) less than about 20 minutes from the time the sample is collected; (c) about 4 to about 20 minutes from the time the sample is collected; (d) about 15 to about 18 minutes from the time the sample is collected; or (e) less than about 18 minutes from the time the sample is collected.

46. 46. ​​The system of claims 27-45, wherein the amount of (i) UCH-L1, GFAP, or UCH-L1 and GFAP; or (ii) CK-MB, β-hCG, TSH, homocysteine, free T4, or any combination thereof, is communicated (a) by the device; (b) in a document and / or spreadsheet, on a mobile device, on a computer, on a website, in an email, or any combination thereof; or (c) by displaying on the device.

47. 27. The method of any of claims 1 to 26, wherein said communicating the determined amount of (i) UCH-L1, GFAP or a combination thereof; or (ii) CK-MB, β-hCG, TSH, homocysteine, free T4 or any combination thereof in said sample comprises communicating the level of (i) UCH-L1, GFAP or a combination thereof; or (ii) CK-MB, β-hCG, TSH, homocysteine, free T4 or any combination thereof in said sample.

48. 47. The system of any of claims 27 to 46, wherein the communicating the determined amount of (i) UCH-L1, GFAP or a combination thereof; or (ii) CK-MB, β-hCG, TSH, homocysteine, free T4 or any combination thereof in the sample comprises communicating the level of (i) UCH-L1, GFAP or a combination thereof; or (ii) CK-MB, β-hCG, TSH, homocysteine, free T4 or any combination thereof in the sample.

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