Assay Device

Abstract

1. An assay device for use with a sample of biological fluid, comprising an APAP detection unit for detecting and / or measuring N-acetyl-p-aminophenol (APAP) in the sample of biological fluid, and an ALT1 detection unit for detecting and / or measuring alanine aminotransferase 1 (ALT1) in the sample of biological fluid, wherein the APAP detection unit and the ALT1 detection unit act in conjunction as part of an assay process.

Description

[Technical field]

[0001] The present invention relates to assay devices and methods useful for samples of biological fluids, and in particular for diagnosing and / or indicating an overdose of N-acetyl-p-aminophenol. [Background technology]

[0002] N-acetyl-p-aminophenol (APAP), also known as paracetamol or acetaminophen, is a widely available painkiller. Generally safe in low doses, overdose of APAP can cause severe liver damage and can be fatal. Overdose can occur if taken more frequently and / or in larger amounts than prescribed. In some cases, overdose is an intentional act intended to harm oneself.

[0003] APAP overdoses or suspected overdoses are common in hospital emergency departments. The correct treatment depends on the circumstances, including how much APAP was taken and how long it has been since it was taken. Typically, a patient's blood concentration of APAP is determined by taking a blood sample from the patient and analyzing it in a hospital laboratory. However, with current methods, the patient must wait up to four hours in the emergency room before the sample is taken, and then it takes up to another two hours for the sample to be analyzed and the results reported. In addition, the patient must report when and how much APAP was taken. If the blood concentration of APAP and the time and / or amount of intake are known, medical professionals can use the Rumack-Matthew nomogram to provide treatment guidance, particularly in determining whether to administer acetylcysteine ​​treatment. However, the Rumack-Matthew nomogram cannot be used for patients who take APAP repeatedly or for patients whose time and amount of intake are unknown, for example, when the patient is unable or unwilling to tell when and how much APAP was taken. Because APAP is metabolized in the body, blood levels of APAP alone do not identify the time of ingestion. The rate at which APAP is metabolized in the body varies from patient to patient and can be affected by complex factors such as age and other medications taken at the same time. Therefore, it is not possible to know whether a patient previously ingested a large amount or recently ingested a small amount.

[0004] Therefore, there is a need for a simple and efficient test that can diagnose APAP overdose without the need to determine when and how much APAP a patient ingested. In particular, it would be highly beneficial to provide a test that can be used rapidly at the point of care, providing results within minutes of a patient providing a biological sample. Summary of the Invention

[0005] One object of the present invention is to provide an assay device that can be used, inter alia, to diagnose N-acetyl-p-aminophenol (APAP) overdose in a patient. Another object of the invention is to provide such a device that is simple and efficient to use, capable of providing substantially instantaneous results in a clinical setting.

[0006] The present invention provides an assay device and method for the diagnosis of APAP overdose as set out in the accompanying claims. Further features of the invention will become apparent from the dependent claims and the following description.

[0007] According to a first aspect of the present invention there is provided an assay device for use with a sample of a biological fluid comprising: an N-acetyl-p-aminophenol (APAP) detection unit for detecting and / or measuring APAP in a sample of a biological fluid; an ALT1 detection unit for detecting and / or measuring ALT1 in the sample of said biological fluid; wherein the APAP detection unit and the ALT1 detection unit act in concert as part of the assay process.

[0008] According to a second aspect of the present invention, there is provided a method for diagnosing an overdose of N-acetyl-p-aminophenol (APAP), the method comprising the steps of: introducing a sample of biological fluid obtained from a patient into an assay device comprising an APAP detection unit and an alanine aminotransferase 1 (ALT1) detection unit; detecting and / or measuring APAP in the sample of biological fluid; Detecting and / or measuring ALT1 in a sample of a biological fluid; wherein the measurements of APAP and ALT1 indicate the time and amount of APAP (dosage) to be taken.

[0009] The assay device and method of the present invention can be used to diagnose APAP overdose. After ingestion, APAP rises in blood levels rapidly and then slowly drops as it is metabolized. Excess APAP in the body causes liver damage. ALT1 is a marker of liver damage. Thus, ALT1 levels rise gradually during the time that excess APAP damages the liver. By using APAP and ALT1 levels in combination, clinicians can determine if the administration was recent and not severe (low ALT1, low APAP), recent and severe (low ALT1, high APAP), or not recent and severe (high ALT1, low APAP). This assessment helps determine the most appropriate course of treatment, since ALT1 levels indicate that the APAP overdose was recent, even if the time of ingestion is unknown.

[0010] The assay devices and methods of the present invention typically provide results within minutes, which can be very important information in the treatment of an overdose. The assay devices and methods of the present invention can provide rapid results because they operate directly on biological fluids taken directly from a patient (i.e., without the need for prior processing of the biological fluid sample) and / or simultaneously detect and / or measure APAP and ALT1 in the biological fluid, which provides accurate results without the need for further testing. Furthermore, the assay devices and methods of the present invention can be used to repeatedly test a patient and determine the most appropriate course of treatment.

[0011] An advantage of ALT1 is that it is considered to be a more specific marker of liver damage caused by APAP overdose compared with other liver damage markers such as aspartate aminotransferase (AST) and gamma-glutamyltransferase (GGT).

[0012] Throughout this specification, the words "comprising" and "comprises" mean the inclusion of the specified elements but do not exclude the presence of other elements.

[0013] Any features described herein may be used individually or in combination with each other as appropriate, in particular in the combinations described in the appended claims. Any features of each exemplary embodiment of the invention described herein may also be applied to other aspects or exemplary embodiments of the invention as appropriate. In other words, a person skilled in the art who has access to this specification should consider any features of each aspect or embodiment of the invention as interchangeable and combinable between different aspects of the invention.

[0014] A first aspect of the present invention provides an assay device. The term "assay device" is used herein in relation to all aspects of the invention to mean a device for performing an assay, i.e. a device for performing a research analytical procedure to qualitatively assess and / or measure one or more properties of a sample of a biological fluid.

[0015] The assay device according to the first aspect of the invention is for use with a sample of a biological fluid. The term "biological fluid" is used herein in all aspects relating to the invention to mean a fluid obtained from a human or animal, in particular a living human or animal, and includes a fluid obtained directly from a human or animal, and a liquid after modification or treatment. The modification or treatment can be carried out in any suitable manner, for example by filtration and / or dilution or dissolution with a suitable solvent, for example dilution or dissolution with a suitable buffer. Examples of biological fluids include whole blood, plasma, serum, saliva, sputum and nasal samples, which may be taken from the human or animal body or may have been modified or treated as described herein. Preferably, the sample of biological fluid is whole blood, for example whole blood that has been modified or treated as described herein. For example, the sample may comprise whole blood that has been diluted or dissolved with a suitable solvent, for example a suitable buffer.

[0016] Typically, less than 500 pl of sample is required for use with the assay device and method of the present invention. For example, samples of 200-500 pl may be used. The assay device is suitable for use with blood samples obtained, for example, by finger or heel prick, where the blood sample may have been modified or processed as described herein. References to the amount of sample are intended to refer to the amount of sample that is introduced into the assay device (and consequently the amount of sample may represent the amount of sample that includes a biological fluid obtained from the human or animal body and a suitable solvent). Those skilled in the art will appreciate that the amount of sample may be reduced if the sample undergoes a pre-processing step, for example filtration. The ability to perform tests with small amounts of biological sample makes the tests easier to perform and more comfortable for the patient.

[0017] The detection and / or measurement of APAP and / or ALT1 can be achieved by performing optical absorbance measurements at a specific wavelength or range of wavelengths. Those skilled in the art can easily determine the appropriate wavelength depending on the reagent used. For example, such a specific wavelength may be 615 nm, which corresponds to the peak absorption of the indophenol dye produced by coupling 2-methylphenol (o-cresol) with p-aminophenol.

[0018] The APAP detection unit and the ALT1 detection unit act together as part of the assay process. In other words, in the analysis device in the first aspect of the invention, the same sample of biological fluid is analyzed in both the APAP detection unit and the ALT1 detection unit substantially simultaneously. Typically, a part of the sample of said biological fluid enters the APAP detection unit and another part of the sample of said biological fluid enters the ALT1 detection unit. This means that the results obtained in both units are used to diagnose APAP overdose in a patient. This provides a fast and efficient test, which allows a doctor to act quickly to treat the patient if the test result indicates a problem. Analyzing the same sample of biological fluid in both the APAP detection unit and the ALT1 detection unit also ensures that the detection and / or measurement of APAP and the detection and / or measurement of ALT1 correspond to a single point in time for the patient. Thus, the assay device of the invention may improve the accuracy of the diagnosis of APAP overdose.

[0019] The assay device according to the first aspect of the present invention may further comprise a liquid inlet. The assay device may further comprise a filter located at or near the liquid inlet. In other words, the assay device may comprise an integrated filter. If present, the filter acts to filter the sample of biological fluid in a pre-treatment step. After passing through the filter in the pre-treatment step, the sample of biological fluid passes through the APAP detection unit and the ALT1 detection unit.

[0020] As will be appreciated by those skilled in the art, any suitable filter may be used. Examples of suitable filters include plasma filters, such as Fusion 5 filters from GE Healthcare, Vivid filters from Pall Corporation, CytoSep 1667 filters from Ahlstrom-Munksjb, and Cobetter separation membranes. For example, if the sample of biological fluid is whole blood, the filter separates the plasma from the whole blood, which then enters the APAP detection unit and the ALT1 detection unit for analysis. The use of a filter in the assay device means that the sample of said biological fluid does not need to be processed before testing in the assay device. This reduces the time required for output and allows the patient to be diagnosed on-site. Thus, the assay device of the present invention can be used in single-step methods as described herein. The use of an integral filter in the assay device allows the device to function with small sample volumes, such as those obtained by finger prick or heel prick methods. Such sample volumes can be significantly reduced if the sample requires pre-processing in devices or methods external to the assay device.

[0021] The assay device preferably comprises a reservoir located at or near the liquid inlet, the reservoir preferably being located adjacent to the filter if present. In use, the reservoir receives a sufficient amount of a sample of biological fluid to flow into each of the APAP and ALT1 detection units. Preferably, the reservoir is located such that, in use, the sample of biological fluid flows through the filter into the reservoir, and the biological fluid collects in the reservoir in a sufficient amount to flow into each of the APAP and ALT1 detection units. Preferably, the reservoir comprises an adsorbent pad, preferably an absorbent pad. The reservoir and the filter may be integrated, in which case the reservoir also functions as a filter.

[0022] The reservoir may include a mechanical barrier on its surface to divide the reservoir into a first zone and a second zone. In use, the first zone receives a sample of the biological fluid, and then the second zone receives a chase buffer. The chase buffer is a diluent that flows through the first zone and helps to carry the sample of the biological fluid to each of the APAP and ALT1 detection units. The first zone is preferably located between the second zone and the APAP and ALT1 detection units. The mechanical barrier prevents the chase buffer from mixing with the unfiltered sample of the biological fluid.

[0023] Alternatively, the assay device may comprise a chase buffer receiving area that is separate from the reservoir but in fluid communication with the reservoir. In such a configuration, no mechanical barrier is required at the surface of the reservoir. The chase buffer receiving area suitably comprises an adsorbent pad, preferably an absorbent pad. The adsorbent pad suitably comprises a material that allows the chase buffer to flow quickly through the reservoir. The adsorbent pad suitably comprises glass fibre. The chase buffer receiving portion may comprise a Whatman Fusion 5 pad.

[0024] Preferably, the APAP detection unit and / or the ALT1 detection unit are in fluid communication with the liquid introduction. When a sample of biological fluid is added to the liquid introduction, it can preferably pass through the APAP detection unit and / or the ALT1 detection unit without further input from the user or the addition of a chase buffer. Preferably, both the APAP detection unit and the ALT1 detection unit are in fluid communication with the liquid introduction. The liquid introduction, the APAP detection unit and the ALT1 detection unit are preferably arranged such that the sample of biological fluid flows through the assay device by capillary action (i.e. wicking).

[0025] The assay device may include a reference unit or channel that acts as a baseline. The reference unit or channel receives a portion of the sample of the biological fluid before it enters the APAP and / or ALT1 detection unit, for comparison to the sample after the APAP and / or ALT1 assay is performed. For example, if the APAP detection unit performs an assay that performs a colorimetric measurement, the reference unit or channel provides a baseline color for comparison after the assay is performed.

[0026] Preferably, the APAP detection unit and / or the ALT1 detection unit comprises a channel in which a biological assay is performed. Preferably, the APAP detection unit comprises a channel in which an APAP detection assay is performed, and the ALT1 detection unit comprises a channel in which an ALT1 detection assay is performed. The channel is preferably arranged such that a sample of a biological fluid enters at one end of the channel, travels along the channel, and thereby undergoes a biological assay. Preferably, the width of the channel is 1-2.5 mm, such as 1.5-2.3 mm, for example 2 mm. It has been found that a channel width within this range provides an ideal flow rate of the sample along the channel. A channel width of 3 mm or more may be adversely affected by capillary action, resulting in a reduced flow rate of the sample.

[0027] The channel may comprise an adsorbent pad. By adsorbent pad is meant a piece of material that is absorbent or adsorbent towards biological fluids. Suitably, the adsorbent pad allows the biological fluid to migrate through the adsorbent pad, preferably through capillary action (i.e. wicking). The adsorbent pad may comprise a permeable membrane. Suitably, such a device comprises a backing on which the adsorbent pad is arranged. The backing is suitably arranged on the opposite side of the device to the liquid introduction. Suitably, the adsorbent pad is in the form of a strip or sheet.

[0028] Preferably, the channel comprises a plurality of suction pads. Preferably, each suction pad is in strip or sheet form. Preferably, each suction pad is in contact with at least one other suction pad. Preferably, each suction pad overlaps (preferably longitudinally) with at least one other suction pad. This allows a sample of the biological fluid to flow from one pad to another, preferably via capillary action. Each suction pad preferably has the same width as the channel. Preferably, the length of each suction pad is independently at least 6 mm, such as at least 8 mm. Preferably, the length of the overlap between the suction pads is independently at least 2.5 mm, such as at least 3.5 mm, for example 4 mm. It has been found that if the overlap length is less than 2 mm, the flow of sample from one pad to another may be disadvantageously reduced.

[0029] The chase buffer receiving area is preferably in contact with the reservoir. Preferably, the reservoir comprises an adsorbent pad, the chase buffer receiving area comprises an adsorbent pad and the reservoir overlaps the chase buffer receiving area. In the area of ​​overlap, preferably the chase buffer receiving area is proximal to the liquid introduction (and the reservoir is distal to the liquid introduction). In the area of ​​overlap, preferably the chase buffer receiving area is distal to the backing (and the reservoir is proximal to the backing). In the area of ​​overlap, preferably the chase buffer receiving area is not in contact with the backing and the reservoir is in contact with the backing.

[0030] The assay device according to the first aspect of the present invention comprises an APAP detection unit. The APAP detection unit preferably comprises a reagent that changes color in the presence of APAP or a derivative thereof. The reagent is preferably colorless. The reagent preferably comprises an aromatic or heteroaromatic compound.

[0031] Suitably, the APAP detection unit comprises a first reagent which acts to react with APAP to form an APAP derivative, and a second reagent which acts to react with such derivative to form a coloured product.

[0032] The first reagent preferably acts to hydrolyze APAP to produce p-aminophenol. The first reagent preferably comprises an enzyme, such as aryl acylamidase.

[0033] The second reagent preferably acts to react with p-aminophenol to form a colored product, such as an indophenol dye. Preferably, the second reagent comprises an aromatic or heteroaromatic compound substituted with a hydroxyl group. By "aromatic compound" is meant a compound containing one or more aromatic rings in which the ring atoms are carbon atoms. By "heteroaromatic compound" is meant a compound containing one or more aromatic rings, at least one of which contains one or more heteroatoms. By "heteroatom" is meant an atom other than carbon. Suitable heteroatoms include nitrogen, oxygen, and sulfur.

[0034] The hydroxyl-substituted aromatic compound may include hydrocarbyl-substituted phenols, such as alkyl-substituted phenols. The alkyl-substituted phenols may include phenols substituted with one or more methyl groups. The hydroxyl-substituted heteroaromatic compound may include hydroxyl-substituted quinolines. Examples of suitable second reagents include 2-methylphenol (o-cresol), 2,3-dimethylphenol, 2,5-dimethylphenol, 2,6-dimethylphenol, and 8-hydroxyquinoline. Preferably, the second reagent includes 2-methylphenol.

[0035] The second reagent is preferably mobile in the sample of biological fluid. By "mobile" it is meant that the reagent is not immobilized but is carried by the sample of biological fluid which moves through the device.

[0036] The APAP detection unit may include a catalyst that acts to catalyze the reaction between the APAP derivative and the second reagent. The catalyst preferably acts to catalyze the reaction between p-aminophenol and the second reagent. The catalyst preferably includes a metal salt, such as a salt of sodium, potassium, chromium, manganese, iron, cobalt, nickel, or copper. Examples of suitable catalysts include copper sulfate, manganese chloride, and sodium periodate. Copper sulfate is preferred. Copper sulfate may be in the form of a hydrate, such as copper sulfate pentahydrate.

[0037] The APAP detection unit may include a pH adjuster. The pH adjuster may advantageously increase or decrease the pH to a level at which the colored product can be easily detected. For example, the colored product may exhibit strongest single wavelength absorption in a particular pH range. The pH adjuster may also advantageously increase the rate of reaction between the APAP derivative and the second reagent. Suitably, the pH adjuster acts to increase the pH by at least 7, such as at least 8, for example at least 10. The pH adjuster may act to increase the pH to within the range of 7 to 14, such as 8 to 12, for example 10 to 11. The pH adjuster suitably comprises an ammonium salt, such as ammonium chloride, or a carbonate salt, such as sodium carbonate. Ammonium chloride is preferred.

[0038] Suitably, the APAP detection unit comprises a catalyst and a pH adjuster as defined herein. Preferably, the APAP detection unit comprises copper sulfate and ammonium chloride.

[0039] The APAP detection unit may comprise a first reagent area comprising a first reagent. The first reagent area may comprise an adsorbent pad. The first reagent area may be prepared by applying (e.g. spraying) a solution comprising the first reagent to an adsorbent pad and allowing the adsorbent pad to dry. The adsorbent pad is preferably fast drying. The adsorbent pad preferably comprises glass fibre. The first reagent area may comprise a Whatman Standard 17 pad.

[0040] The APAP detection unit may comprise a second reagent area comprising a second reagent. The second reagent area may comprise an adsorbent pad. The second reagent area may be prepared by applying (e.g., spraying) a solution comprising the second reagent to the adsorbent pad and allowing the adsorbent pad to dry. The adsorbent pad preferably comprises a material that allows the sample of the biological fluid to flow quickly through the second reagent area. The second reagent area may comprise a Whatman Grade 1 pad.

[0041] The APAP detection unit may comprise a reaction area comprising a catalyst and / or a pH adjuster. The reaction area may comprise an adsorbent pad. The reaction area may be prepared by applying (e.g., spraying) a solution comprising the catalyst and / or the pH adjuster to the adsorbent pad and allowing the adsorbent pad to dry. The adsorbent pad preferably comprises a material that allows the sample of the biological fluid to flow quickly through the reaction area. The reaction area may comprise a Whatman Grade 1 pad.

[0042] The APAP detection unit may comprise a wicking pad. The wicking pad is an adsorbent pad, for example an absorbent pad. The wicking pad preferably comprises a material that allows the colored product to be clearly visible, preferably a material that allows the colored product to be visible at low concentrations. The wicking pad preferably comprises glass fiber or cellulose fiber. The wicking pad may comprise a Whatman Standard 17 pad or a Millipore Cellulose Fiber Pad C083.

[0043] Preferably, the APAP detection unit comprises a plurality of suction pads. Preferably, each suction pad is in the form of a strip or sheet. Preferably, each suction pad is in contact with at least one other suction pad. Preferably, each suction pad overlaps (preferably in the longitudinal direction) with at least one other suction pad. Preferably, the width of each suction pad is independently 1 to 2.5 mm, such as 1.5 to 2.3 mm, for example 2 mm. Preferably, the width of each suction pad is independently at least s mm, for example at least 8 mm. Preferably, the length of the overlap between the suction pads is independently at least 2.5 mm, for example at least 3.5 mm, for example 4 mm.

[0044] The first reagent area is preferably in contact with the reservoir. Preferably, the reservoir comprises an adsorbent pad and the first reagent area comprises an adsorbent pad and the reservoir overlaps the first reagent area. In the area of ​​overlap, preferably, the first reagent area is distal to the side of the device comprising the liquid introduction portion (and the reservoir is proximal to the side). In the area of ​​overlap, preferably, the first reagent area is proximal to the backing (and the reservoir is distal to the backing). In the area of ​​overlap, preferably, the first reagent area is in contact with the backing and the reservoir is not in contact with the backing.

[0045] The second reagent area is preferably in contact with the first reagent area. Preferably, the first reagent area comprises an absorbent pad and the second reagent area also comprises an absorbent pad, and the first reagent area overlaps the second reagent area. In the area of ​​overlap, the second reagent area is preferably distal to the side of the device comprising the liquid introduction section (and the first reagent area is proximal to the side). In the area of ​​overlap, the second reagent area is preferably proximal to the backing (and the first reagent area is distal to the backing). In the area of ​​overlap, the second reagent area is preferably in contact with the backing and the first reagent area is not in contact with the backing.

[0046] The reaction area is preferably in contact with the second reagent area. Preferably, the second reagent area comprises an absorbent pad, and the reaction area comprises an absorbent pad and the second reagent area overlaps the reaction area. In the area of ​​overlap, preferably the reaction area is distal to the side of the device comprising the liquid inlet (and the second reagent area is proximal to the side). In the area of ​​overlap, preferably the reaction area is proximal to the backing (and the second reagent area is distal to the backing). In the area of ​​overlap, preferably the reaction area is in contact with the backing and the second reagent area is not in contact with the backing.

[0047] The wicking pad is preferably in contact with the reaction area. Preferably, the reaction area comprises an adsorption pad and the reaction area overlaps with the wicking pad. In the area of ​​overlap, preferably the wicking pad is proximal to the side of the device comprising the liquid introduction (and the reaction area is distal to the side). In the area of ​​overlap, preferably the wicking pad is distal to the backing (and the reaction area is proximal to the backing). In the area of ​​overlap, preferably the wicking pad is not in contact with the backing and the reaction area is in contact with the backing.

[0048] The above overlap orientation has been shown to improve sample flow through the APAP detection unit.

[0049] The APAP detection unit may comprise a read area in contact with the reaction area. The read area is preferably located between the reaction area and the wicking pad. The read area may comprise an adsorption pad and overlap the reaction area with the adsorption pad. The read area may comprise an adsorption pad and overlap the reaction area with the adsorption pad and the wicking pad. Alternatively and preferably, the wicking pad may function as the read area. In such an embodiment, a separate read area is not required.

[0050] The APAP detection unit comprises a first reagent area, a second reagent area, and a reaction area, the first reagent area containing a first reagent operative to hydrolyze APAP to produce p-aminophenol, the second reagent area containing a second reagent operative to react with p-aminophenol to form a colored product, and the reaction area containing a catalyst operative to catalyze the reaction of p-aminophenol with the second reagent.

[0051] The APAP detection unit comprises a first reagent area, a second reagent area, and a reaction area, the first reagent area containing a first reagent acting to hydrolyze APAP to produce p-aminophenol, the second reagent area containing a second reagent acting to react with p-aminophenol to form a colored product, and the reaction area containing a pH adjuster and a catalyst acting to catalyze the reaction of p-aminophenol with the second reagent.

[0052] Preferably, the first reagent comprises an enzyme, the second reagent comprises a hydroxyl-substituted aromatic or heteroaromatic compound, and the catalyst comprises a metal salt.

[0053] Preferably, the first reagent comprises an enzyme, the second reagent comprises a hydroxyl-substituted aromatic or heteroaromatic compound, the pH adjuster comprises an ammonium salt, and the catalyst comprises a metal salt.

[0054] Suitably, the first reagent comprises aryl acylamidase, the second reagent comprises o-cresol and the catalyst comprises copper sulfate.

[0055] Preferably, the first reagent comprises aryl acylamidase, the second reagent comprises o-cresol, the pH adjuster comprises ammonium chloride, and the catalyst comprises copper sulfate.

[0056] The assay device according to the first aspect of the present invention comprises an ALT1 detection unit, preferably comprising a conjugate area and a lateral flow test area, wherein the conjugate area comprises an ALT1 binding agent, and the lateral flow test area comprises a test line and a control line.

[0057] The conjugate area preferably comprises an adsorbent pad. The conjugate area may be prepared by applying (e.g., spraying) a solution containing the ALT1 binding agent to an adsorbent pad and allowing the adsorbent pad to dry. The adsorbent pad is preferably fast drying. The adsorbent pad preferably comprises glass fiber. The conjugate area may comprise a Whatman Standard 17 pad.

[0058] The ALT1 binding agent preferably comprises an ALT1 binding moiety conjugated to a detection species. The ALT1 binding moiety preferably comprises an antibody. The antibody is preferably specific for ALT1 and not ALT2. The detection species is preferably colored, fluorescent and / or magnetic. Preferably, the detection species is a colored species (i.e. a chromophore). The ALT1 binding reagent is preferably suitably mobile in a sample of a biological fluid.

[0059] The conjugate region may comprise a secondary detection reagent. The secondary detection reagent preferably comprises a binding moiety conjugated to a detection species. The binding moiety preferably does not bind to ALT1. The detection species is preferably colored, fluorescent and / or magnetic. Preferably, the detection species is a colored species (i.e. a chromophore). The secondary detection reagent is preferably mobile in the sample of the biological fluid.

[0060] The lateral flow test area suitably comprises an absorbent pad. The lateral flow test area may comprise a nitrocellulose membrane, for example Whatman FF120HP.

[0061] The test line preferably comprises a test reagent that binds to a complex formed between ALT1 and the ALT1-binding agent. Preferably, the test reagent specifically binds to the complex formed between ALT1 and the ALT1-binding agent. By specifically binding, the test reagent does not bind to other molecules, such as free ALT1-binding agent or secondary detection reagents, if present. The test reagent preferably comprises an antibody.

[0062] The test reagent is preferably immobilized. By "immobilized" we mean that the reagent is fixed in place and is not carried by the sample of biological fluid flowing through the device. This causes any complex carried by the sample of biological fluid to accumulate at the test line. This allows the complex to be detected, preferably by the appearance of a colored line.

[0063] The control line comprises a control reagent that binds to the ALT1 binding agent and / or secondary detection agent, if present. The control reagent may comprise an antibody. The control reagent is preferably immobilized, such that any free ALT1 binding agent and / or secondary detection agent accumulates at the control line. This allows the free ALT1 binding agent and / or secondary detection agent to be detected, preferably by the appearance of a colored line.

[0064] Where the conjugate region comprises a secondary detection reagent, the control reagent preferably binds to the secondary detection reagent and not to the ALT1 binding agent, which advantageously allows for reliable detection of the control line regardless of the amount of ALT1 contained in the sample of biological fluid.

[0065] Preferably, the test line comprises a test reagent that binds to a complex formed between ALT1 and the ALT1 binding agent, and the control line comprises a control reagent that binds to the ALT1 binding agent. Alternatively, the test line comprises a test reagent that binds to a complex formed between ALT1 and the ALT1 binding agent, and the control line comprises a control reagent that binds to a secondary detection agent. The test and control reagents are preferably immobilized.

[0066] The test line is preferably closer to the conjugate region than the control line, to ensure that any complex formed between ALT1 and the ALT1-binding agent accumulates at the test line and not at the control line.

[0067] The lateral flow test area may be prepared by applying (e.g. spraying) a solution containing the test reagent onto an absorbent pad to form a test line, applying (e.g. spraying) a solution containing a control reagent onto an absorbent pad to form a control line, and drying the absorbent pad.

[0068] The ALT1 detection unit may include a wicking pad. The wicking pad is an adsorbent pad, for example an absorbent pad. The wicking pad may include a Whatman Fusion 5 pad.

[0069] Preferably, the ALT1 detection unit comprises a plurality of suction pads. Preferably, each suction pad is in the form of a strip or sheet. Preferably, each suction pad is in contact with at least one other suction pad. Preferably, each suction pad overlaps with at least one other suction pad. Preferably, the width of each suction pad is independently 3 to 5 mm, such as 3.5 to 4.5 mm, for example 4 mm. Preferably, the length of each suction pad is independently at least 6 mm, for example at least 8 mm. Preferably, the length of the overlap between the suction pads is independently at least 2.5 mm, for example at least 3.5 mm, for example 4 mm.

[0070] The conjugate region is preferably in contact with the reservoir. Preferably, the reservoir comprises an adsorbent pad and the conjugate region comprises an adsorbent pad and the reservoir overlaps the conjugate region. In the overlapping region, preferably the conjugate region is distal to the side of the device comprising the liquid introduction portion (and the reservoir is proximal to the side). In the overlapping region, preferably the conjugate region is proximal to the backing (and the reservoir is distal to the backing). In the overlapping region, preferably the conjugate region is in contact with the backing and the reservoir is not in contact with the backing.

[0071] The lateral flow test area is preferably in contact with the conjugate area. Preferably, the conjugate area comprises an adsorbent pad and the lateral flow test area comprises an adsorbent pad and the conjugate area overlaps the lateral flow test area. In the area of ​​overlap, preferably the lateral flow test area is distal to the side of the device that comprises the liquid introduction portion (and the conjugate area is proximal to the side). In the area of ​​overlap, preferably the lateral flow test area is proximal to the backing (and the conjugate area is distal to the backing). In the area of ​​overlap, preferably the lateral flow test area is in contact with the backing and the conjugate area is not in contact with the backing.

[0072] The wicking pad is preferably in contact with the lateral flow test area. Preferably, the lateral flow test area comprises an absorbent pad and the lateral flow test area overlaps the wicking pad. In the area of ​​overlap, preferably the wicking pad is proximal to the side of the device comprising the liquid introduction portion (and the lateral flow test area is distal to the side). In the area of ​​overlap, preferably the wicking pad is distal to the backing (and the lateral flow test area is proximal to the backing). In the area of ​​overlap, preferably the wicking pad is not in contact with the backing and the lateral flow test area is in contact with the backing.

[0073] The above overlap orientation has been shown to improve sample flow through the ALT1 detection unit.

[0074] The assay device according to the first aspect preferably comprises a housing with one or more openings through which the results from the APAP detection unit and / or the ALT1 detection unit can be viewed. Preferably, the openings are adjacent to the reaction or read area (if present) of the APAP detection unit and the lateral flow test area of ​​the ALT1 detection unit. The liquid introduction portion is preferably in the shape of the opening in the housing.

[0075] The housing is preferably formed of a material that is impermeable to the sample of biological fluid. Such a material is preferably impermeable to water. Such a material may be a polymeric material. Suitable polymeric materials include acrylonitrile butadiene styrene (ABS), polycarbonate, and polyamide.

[0076] The assay device according to the first aspect of the invention is preferably portable.

[0077] A second aspect of the present invention provides a method for diagnosing APAP overdose.

[0078] Preferably, the results of detection and / or measurement of APAP and ALT1 indicate the time and amount of APAP intake.

[0079] The method according to the second aspect of the invention may be carried out by any suitable assay device, such as, but not limited to, the assay device according to the first aspect of the invention.

[0080] The method according to the second aspect is carried out using a sample of a biological fluid taken from a patient. Such a method does not include the step of taking a sample of the biological fluid from the patient. The sample of the biological fluid is not re-administered to the patient.

[0081] The sample of biological fluid is preferably blood or plasma. Preferably, the blood is whole blood. By whole blood, it is meant unprocessed blood from which no components have been removed. Preferably, the assay device comprises an integral filter and the sample of biological fluid is whole blood. This simplifies the method by eliminating the need for pre-treatment of the sample of biological fluid.

[0082] A sample of the biological fluid is preferably introduced into the assay device through a fluid inlet of the device.

[0083] The method according to the second aspect may further comprise the step of introducing a chase buffer into the assay device after introducing the sample of the biological fluid into the assay device, which aids in flow of the sample of the biological fluid through the device. The chase buffer may be introduced into the assay device via a liquid introduction portion of the device.

[0084] APAP in the biological fluid sample is preferably detected by a color change of the APAP detection unit. APAP in the biological fluid sample may be measured by measuring the color change of the APAP detection unit. For example, the change in absorbance at a single wavelength, for example 615 nm, may be measured. The color change is preferably caused by the reaction of APAP or its derivative with a reagent to form a colored product, for example an indophenol dye. The amount of APAP in the biological fluid sample may be measured by measuring the color change of the product. The color change may be measured using an optical measurement device, for example an immunochromatographic reader from Hamamatsu Photonics. The color change is preferably detected and / or measured in a reaction area or a reading area (for example a wicking pad) of the APAP detection unit. Preferably, the APAP detection unit can detect APAP at a concentration of 10 pg / mL or more.

[0085] ALT1 in the biological fluid sample is preferably detected by a color change, a fluorescence change, and / or a magnetic change in the ALT1 detection unit. ALT1 in the biological fluid sample can be measured by measuring a color, a fluorescence change, and / or a magnetic change in the ALT1 detection unit. For example, a color and / or a fluorescence change may be measured by measuring a change in absorbance at a single wavelength. A color, a fluorescence change, and / or a magnetic change can be measured using an optical measurement device, a fluorescence measurement device, and / or a magnetic measurement device. A suitable optical measurement device and a fluorescence measurement device are the immunochromato reader of Hamamatsu Photonics. A color, a fluorescence change, and / or a magnetic change is preferably detected and / or measured at a test line of the ALT1 detection unit. Such a change is usually caused by accumulation of a complex formed from ALT1 and the ALT1 binding agent at the test line, where the ALT1 binding agent is colored or includes a fluorescent and / or magnetic detection species. Preferably, ALT1 in the biological fluid sample is detected by a color change in the ALT1 detection unit. ALT1 in a sample of a biological fluid is preferably measured by measuring a colour change in an ALT1 detection unit.

[0086] Preferably, APAP in the sample of biological fluid is detected by a color change of the APAP detection unit and ALT1 in the sample of biological fluid is detected by a color, fluorescent and / or magnetic change of the ALT1 detection unit. Preferably, APAP in the sample of biological fluid is detected by a color change of the APAP detection unit and ALT1 in the sample of biological fluid is detected by a color change of the ALT1 detection unit.

[0087] According to a third aspect of the present invention there is provided the use of an assay device according to the first aspect of the present invention for diagnosing an overdose of N-acetyl-p-aminophenol (APAP). [Brief description of the drawings]

[0088] For a better understanding of the present invention, and to illustrate how to carry out exemplary embodiments of the same, reference is made, by way of example only, to the accompanying drawings in which: [Figure 1] FIG. 1 shows a schematic diagram of an assay device according to a first embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0089] 1 shows a schematic diagram of an assay device 100 for use with a sample of a biological fluid according to an embodiment of the present invention. The assay device comprises an N-acetyl-p-aminophenol (APAP) detection unit 110 and an alanine aminotransferase 1 (ALT1) detection unit 120.

[0090] The APAP detection unit 110 and the ALT1 detection unit 120 are positioned to receive the same biological fluid sample drawn from the patient, which in this exemplary embodiment is whole blood. The whole blood is added to a hemofilter 101 that filters out red blood cells and channels plasma onto an absorbent support pad 102.

[0091] From the support pad 102, a portion of the plasma is delivered to the APAP detection unit 110. The APAP detection unit 110 is arranged to detect and / or measure APAP in a sample of a biological fluid. The APAP detection unit 110 comprises a first reagent area 111, a second reagent area 112, a reaction area 113, and a wicking pad 114. The first reagent area 111 comprises an adsorbent pad containing aryl acylamidase. The plasma passes through the first reagent area 111, and the aryl acylamidase hydrolyzes all of the APAP in the plasma to produce p-aminophenol. The second reagent area 112 comprises an adsorbent pad containing o-cresol. The plasma passes through the second reagent area 112 and absorbs the o-cresol. The reaction area 113 comprises an adsorbent pad containing ammonium chloride and copper sulfate pentahydrate. The plasma passes through reaction area 113 where copper sulfate catalyzes the reaction between o-cresol and any p-aminophenol present to form an indophenol dye. The wicking pad 114 is formed from an absorbent material. The wicking pad 114 aids in the movement of the plasma through the APAP detection unit 110 and absorbs any excess plasma.

[0092] The formation of the indophenol dye causes a color change, so that the presence of APAP in the plasma can be detected visually. A quantitative measurement of the amount of APAP in the plasma can be made, if desired, by measuring the dye color change, for example, by using an optical measurement device. The wicking pad 114 is advantageously the preferred location for measuring the color change, since it effectively accumulates the indophenol dye.

[0093] From the support pad 102, another portion of the plasma is directed to the ALT1 detection unit 120. The ALT1 detection unit 120 is arranged to detect and / or measure ALT1 in a sample of a biological fluid. The ALT1 detection unit 120 comprises a conjugate region 121, a lateral flow test region 122, and a wicking pad 125. The conjugate region 121 comprises an adsorbent pad and a movably supported conjugate, the conjugate being an ALT1 antibody conjugated to a dye. The plasma passes through the conjugate region 121 and adsorbs the conjugate. Any ALT1 present in the plasma binds to the ALT1 antibody conjugated to a chromophore to form a colored complex. The lateral flow test region 122 comprises an adsorbent pad with blocked nitrocellulose, on which a test line 123 and a control line 124 are present. The test line 123 contains an immobilized ALT1 capture antibody. The control line 124 contains immobilized conjugate-specific capture antibody. As plasma passes through the lateral flow test region 122, any colored complex present binds to the test line 123 and free dye-conjugated ALT1 antibody binds to the control line 124. Accumulation of colored species at the test line 123 and control line 124 visually indicates the presence of ALT1 in the plasma. The wicking pad 125 is formed from an absorbent material. The wicking pad 225 aids in the movement of plasma through the ALT1 detection unit 120 and absorbs any excess plasma.

[0094] The ALT1 antibody used herein is preferably specific for ALT1 and not ALT2. ALT1 antibodies may be obtained from Bio-Rad's synthetic human combinatorial antibody library (HuCAL).

[0095] The blood filter 101, support pad 102, APAP detection unit 110, and ALT1 detection unit 120 are all supported by a backing material (not shown). The support pad 102, first reagent area 111, second reagent area 112, reaction area 113, wicking pad 114, conjugate area 121, lateral flow test area 122, and wicking pad 125 are all formed from individual adsorbent pads. The pads are arranged in an overlapping manner, which allows plasma to wick from one component to the next.

[0096] Attention is directed to all documents and papers filed contemporaneously with or prior to this specification relating to this application, as well as all documents and papers open to public inspection herewith, and the contents of all such documents and papers are hereby incorporated by reference.

[0097] All features disclosed in this specification (including the accompanying claims and drawings), and / or all steps of any method or process disclosed may be combined in any combination, except combinations in which at least some of the features and / or steps are mutually exclusive.

[0098] Each feature disclosed in this specification (including the accompanying claims, abstract, and drawings), unless expressly stated otherwise, may be replaced by alternative features serving the same, equivalent, or similar purpose. Thus, unless otherwise stated, each feature disclosed is only a generic example of a series of equivalent or similar features.

[0099] The invention is not limited to the details of the above embodiments, and extends to any novel one or any novel combination of features disclosed in this specification (including the accompanying claims and drawings), or any novel one or any novel combination of method or process steps disclosed herein.

Claims

1. 1. An assay device for use with a sample of biological fluid, comprising an APAP detection unit for detecting and / or measuring N-acetyl-p-aminophenol (APAP) in the sample of biological fluid, and an ALT1 detection unit for detecting and / or measuring alanine aminotransferase 1 (ALT1) in the sample of biological fluid, the APAP detection unit and the ALT1 detection unit acting in conjunction as part of an assay process.

2. The assay device of claim 1 further comprising a liquid introduction portion.

3. 3. The assay device of claim 2, further comprising a filter located at or near the liquid introduction portion.

4. 4. The assay device of claim 2 or 3, further comprising a reservoir located at or near the liquid introduction portion.

5. The assay device according to any one of claims 2 to 4, wherein the APAP detection unit and / or the ALT1 detection unit are in fluid communication with the liquid inlet.

6. 6. The assay device of claim 1, wherein the APAP detection unit and / or the ALT1 detection unit comprises a channel in which a biological assay is performed.

7. 7. The assay device of claim 6, wherein the channel comprises a plurality of adsorbent pads, each of which preferably overlaps at least one other adsorbent pad.

8. The assay device according to any one of claims 1 to 7, wherein the APAP detection unit comprises a reagent which changes colour in the presence of APAP or a derivative thereof.

9. 9. An assay device according to any one of claims 1 to 8, wherein the APAP detection unit comprises a first reagent area, a second reagent area and a reaction area, the first reagent area containing a first reagent operative to hydrolyse APAP to produce p-aminophenol, the second reagent containing a second reagent operative to react with p-aminophenol to form a coloured product, and the reaction area containing a catalyst operative to catalyse the reaction between p-aminophenol and the second reagent.

10. 10. The assay device of claim 9, wherein the first reagent comprises aryl acylamidase, the second reagent comprises o-cresol, and the catalyst comprises copper sulfate.

11. The assay device of any one of claims 1 to 10, wherein the ALT1 detection unit comprises a conjugate area and a lateral flow test area, the conjugate area comprising an ALT1 binding agent, and the lateral flow test area comprises a test line and a control line.

12. 12. The assay device of claim 11, wherein the ALT1 binding agent comprises an ALT1 binding moiety conjugated to a detection species.

13. The assay device of claim 12 , wherein the ALT1 binding moiety comprises an antibody.

14. An assay device according to any one of claims 11 to 13, wherein the test line comprises a test reagent which binds to a complex formed from ALT1 and the ALT1 binding agent, and the control line comprises a control reagent which binds to the ALT1 binding agent.

15. 15. The assay device of any one of claims 1 to 14, further comprising a housing with one or more openings through which results from the APAP detection unit and / or the ALT1 detection unit can be viewed.

16. The assay device of any one of claims 1 to 15, which is portable.

17. A method for diagnosing an overdose of N-acetyl-p-aminophenol (APAP), comprising the steps of: introducing a sample of a biological fluid obtained from a patient into an assay device comprising an APAP detection unit and an alanine aminotransferase 1 (ALT1) detection unit; detecting and / or measuring APAP in the sample of biological fluid and detecting and / or measuring ALT1 in the sample of biological fluid; A method comprising:

18. The method of claim 17, wherein the assay device is according to any one of claims 1 to 15.

19. 19. The method of claim 17 or 18, wherein the sample of biological fluid is whole blood or plasma.

20. The method according to any one of claims 17 to 19, wherein APAP in the sample of biological fluid is detected by a color change of an APAP detection unit and / or ALT1 in the sample of biological fluid is detected by a color change of an ALT1 detection unit.

Images