Capture reagent for lateral flow immunoassay

Abstract

A lateral flow immunoassay device and associated reagents for confirming the presence of one or more analytes in a liquid sample. The detection zone of the membrane supporting the lateral flow contains an immobilized, dried capture reagent comprising a first antibody conjugated to a first binding partner conjugated to a second binding partner conjugated to a particle. This disclosure generally relates to a lateral flow immunoassay device and associated reagents for confirming the presence of one or more analytes in a liquid sample.

Description

[Technical Field]

[0001] (Field) This disclosure generally relates to lateral flow immunoassay devices and associated reagents for confirming the presence of one or more analytes in a liquid sample. [Background technology]

[0002] (background) Lateral flow devices have been used for the detection of analytes in biological, industrial, and environmental samples. However, immunoassays performed with such devices often suffer from a lack of sensitivity desired for many analytes and applications. A lateral flow immunoassay device that provides increased assay sensitivity while maintaining user convenience is disclosed herein. [Overview of the project] [Means for solving the problem]

[0003] (overview) In one embodiment, the disclosure relates to an immunoassay device for determining an antigen in a sample. The device comprises (a) a membrane supporting a lateral flow of liquid, including a detection zone; and (b) a dry capture reagent in the detection zone, comprising a first antibody that specifically binds to the antigen, wherein the first antibody is conjugated to a second binding partner bound to a particle immobilized in the detection zone. In one embodiment of the disclosure, the first binding partner is biotin, and the second binding partner is streptavidin. Furthermore, the device may include a labeled second antibody that specifically binds to the antigen, wherein the second antibody is diffusively bound in the reagent zone of the immunoassay device. In various embodiments, the labeling may be an enzyme.

[0004] In exemplary embodiments of the present disclosure, the immunoassay device may include a housing that supports its membrane. The housing may include a container for a liquid washing reagent, and the housing may also include a container for a liquid substrate reagent for its labeling substrate.

[0005] In another aspect, the Disclosure relates to a kit comprising an immunoassay device of the Disclosure and a reagent comprising a labeled second antibody that specifically binds to the antigen thereof. The labeling may be an enzyme. The kit may comprise a housing that supports the membrane, the housing may comprise a container for a liquid washing reagent and a container for a liquid substrate reagent comprising a substrate for the enzyme.

[0006] In a further embodiment, the disclosure relates to a method for determining the presence or amount of an antigen in a liquid sample. The method includes adding the sample to the sample application zone of an immunoassay device described herein, and allowing the sample to move by lateral flow to the detection zone. The presence or amount of the label in the detection zone may be measured, and the presence or amount of the label in the detection provides a signal relating to the presence or amount of antigen bound in the detection zone.

[0007] Furthermore, in another embodiment, the Disclosure relates to a method for determining the presence or amount of an antigen in a liquid sample. The method includes the steps of: contacting the sample with a reagent containing a labeled antibody that specifically binds to the antigen to form a mixture; adding the mixture to the sample application zone of an immunoassay device described herein; allowing the mixture to move by lateral flow to the detection zone; and detecting the presence or amount of the label in the detection zone, the presence or amount of the label in the detection providing a signal relating to the presence or amount of the antigen bound in the detection zone.

[0008] (Brief explanation of the drawing) The accompanying drawings (which are included to provide a further understanding of the Disclosure) are incorporated herein and constitute part of this specification, illustrating embodiments of the Disclosure and serving together with the detailed description to explain the principles of the Disclosure. No attempt is made to provide structural details of the Disclosure in more detail than may be necessary for a basic understanding of the Disclosure and the various forms in which it may be implemented. [Brief explanation of the drawing]

[0009] [Figure 1] Panels A and B in Figure 1 show the selectable formats for the lateral flow test strips of this disclosure.

[0010] [Figure 2] Figure 2 shows grayscale images of the detection zones from the lateral flow membrane relating to this disclosure for a series of clinical samples known to be positive for whipworm antigens, which may also include hookworm and roundworm antigens. The signal minus background (SB) was measured by densitometry and is shown for each detection zone (some positive sample spots may not be visible in the figure as a result of the image conversion to grayscale). A legend for the locations of immobilized capture antibodies in the detection zones is also shown. The antigen concentrations in the clinical samples were determined by ELISA. The optical density (ELISA OD) for each ELISA sample is shown.

[0011] [Figure 3]Figure 3 shows grayscale images of the detection zones from the lateral flow membrane according to this disclosure, used in the analysis of a series of recombinant hookworm antigens, recombinant roundworm antigens, and recombinant whipworm antigens at known concentrations. The signal minus background (SB) was measured by densitometry and is shown for each detection zone. Arrows point to sample spots that may not be visible in the figure as a result of the image conversion to grayscale (and other faint sample spots that may be present but not clearly visible in the figure as a result of replication). A legend for the locations of the immobilized capture antibodies in the detection zones is also shown.

[0012] [Figure 4] Figure 4 shows a summary of the results from Figure 3, which illustrates the immunoassays in a lateral flow membrane according to this disclosure for recombinant hookworm antigen, recombinant roundworm antigen, and recombinant whipworm antigen at known concentrations.

[0013] [Figure 5] Figure 5 shows a grayscale image of the detection zone from the lateral flow membrane according to this disclosure, used in the analysis of samples known to be negative for roundworm antigens, hookworm antigens, and whipworm antigens. No positive signals were detected. [Modes for carrying out the invention]

[0014] (explanation) This disclosure relates to an immunoassay device, including reagents, for use in determining analytes in liquid samples. This disclosure provides a lateral flow membrane with a non-diffusively bound capture reagent containing an antibody that specifically binds to an antigen in the sample, the antibody (or other binding partner of the analyte) being biotinylated and bound to streptavidin-coated particles immobilized on the membrane.

[0015] Before describing the present invention in detail, many terms are defined. As used herein, when the singular forms "a", "an", and "the" are used, they include plural referents unless the context clearly indicates otherwise.

[0016] As used herein, the term "analyte" generally refers to a substance or series of substances in a sample that are detected and / or measured. In some embodiments, the analyte is an antigen.

[0017] As used herein, the term "antibody" generally refers to a glycoprotein produced by B lymphocyte cells in response to exposure to an antigen and that specifically binds to that antigen. The term "antibody" is used in its broadest sense and specifically encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments as long as they exhibit the desired biological activity. Antibodies can be produced naturally or can be engineered by methods well known to those skilled in the art.

[0018] As used herein, the term "antibody fragment" refers to a portion of a full-length antibody, generally its antigen-binding domain or variable domain. For example, antibody fragments can include Fab fragments, Fab' fragments, F(ab')2 fragments, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies derived from antibody fragments.

[0019] As used herein, the term "antigen" generally refers to a substance that is capable of reacting with an antibody specific to that antigen under appropriate conditions.

[0020] As used herein, the term "sample" generally refers to a sample of tissue, excreted product, or fluid derived from a human or animal, and includes, but is not limited to, whole blood, plasma, serum, cerebrospinal fluid, lymphatic fluid, abdominal fluid (ascites), external sections of skin, external sections of airways, external sections of intestinal tract, and external sections of urogenital tract, tears, saliva, urine, blood cells, tumors, organs, tissues, feces, and in vitro cell culture components. Samples can also include industrial samples or environmental samples that require analysis. A sample may require mechanical or chemical processing (e.g., separation, filtration, centrifugation, chemical modification of sample components) prior to analysis. As used herein, a sample includes both raw samples and / or processed samples.

[0021] As used herein, the term "immunoassay" generally refers to a test that uses a complex of an antibody and an antigen to generate a measurable response. "Antibody:antigen complex" can be used interchangeably with the term "immune complex". Immunoassays generally include non-competitive immunoassays, competitive immunoassays, homogeneous immunoassays, and heterogeneous immunoassays. In a "competitive immunoassay", an unlabeled analyte (or antigen) in a test sample is measured by its ability to compete with a labeled antigen in the immunoassay. The unlabeled antigen blocks the ability of the labeled antigen to bind because the binding site on the antibody is already occupied. In a "competitive immunoassay", the amount of antigen present in a test sample is inversely related to the amount of signal generated from the label. Conversely, in a "non-competitive immunoassay" (also known as a "sandwich" immunoassay), an analyte is bound between two highly specific antibodies to form a complex, and the amount of antigen is directly proportional to the amount of signal associated with the complex. Immunoassays that require separation of the bound antibody:antigen complex are generally called "heterogeneous immunoassays", and immunoassays that do not require separation of the antibody:antigen complex are generally called "homogeneous immunoassays". One of ordinary skill in the art can readily understand these various immunoassay formats.

[0022] As used herein, the term “label” means a detectable compound that can be conjugated directly or indirectly (e.g., by covalent or non-covalent means, either alone or in an encapsulated state) to an antibody or analog of the Disclosure. The label may be detectable on its own (e.g., chemiluminescent dye, fluorescent label) or, in the case of an enzyme label, may catalyze a chemical change in a substrate compound or substrate composition that is detectable (e.g., an enzyme such as horseradish peroxidase or alkaline phosphatase). Labels used herein may, but are not limited to, alkaline phosphatases; glucose-6-phosphate dehydrogenase ("G6PDH"); horseradish peroxidase (HRP); chemiluminescents (e.g., isoluminol); fluorescents (e.g., fluorescein compounds and rhodamine compounds); ribozymes; and dyes. The label generates a signal that can be detected and, if necessary, measured by means such as detection or direct visualization of electromagnetic radiation.

[0023] As used herein, the terms “membrane” or “matrix” refer to a non-aqueous matrix to which components of an immunoassay (including the reagents of this disclosure) are diffusively or non-diffusively bound. Examples of membranes and matrices include supports partially or entirely formed of glass (e.g., controlled-pore glass), synthetic and natural polymers, polysaccharides (e.g., agarose), polyacrylamide, polystyrene, polyvinyl alcohol, and silicone, which are formed into chromatography, thereby allowing the bound reagents to be washed or separated from the unbound material. In some embodiments, the membrane or matrix may be porous.

[0024] The terms “particle” or “particles” as used in this disclosure include, for example, latex particles, polystyrene particles, or particles of other support materials (e.g., silica, agarose, ceramics, glass, polyacrylamide, polymethyl methacrylate, carboxylic acid-modified latex, melamine, and Sepharose). These particles vary in size from about 0.1 microns to about 100 microns, for example, about 0.1 microns, about 0.5 microns, about 1.0 micron, about 5 microns, about 10 microns, about 20 microns, about 30 microns, about 40 microns, about 50 microns, about 60 microns, about 70 microns, about 80 microns, about 90 microns, or about 100 microns. Particularly useful commercially available materials include carboxylic acid-modified latex, cyanide-activated Sepharose beads, fused silica particles, isothiocyanate glass, polystyrene, and carboxylic acid monodisperse microspheres.

[0025] Particles suitable for use in this disclosure are capable of binding to other substances (e.g., derivatives, linker molecules, or proteins). The ability of the particles to bind to other substances may arise from the particle material and from any surface modifications or functional groups added to the particles. The particles may be functionalized, or can be functionalized, to allow covalent or noncovalent attachment of proteins, linker molecules, or derivatives as described herein. Suitable functional groups include, for example, amines, biotin, streptavidin, avidin, protein A, sulfhydryl, hydroxyl, and carboxyl.

[0026] The term "multiple" refers to an immunoassay that can detect more than one antigen in a single sample from a single experiment.

[0027] The term "capture reagent" refers to a complex containing a binding partner for an analyte (e.g., an antibody that binds to an antigen), which is immobilized on a particle through the interaction of a separate set of binding partners (e.g., biotin and streptavidin). For example, an antibody conjugated to biotin may bind to a particle functionalized with streptavidin.

[0028] All patent references identified herein are incorporated by reference in their entirety.

[0029] Turning to various aspects of the present disclosure, an immunoassay device for determining one or more analytes in a sample is described herein. For example, the device comprises a membrane supporting a lateral flow of liquid, and the device comprises one or more immobilized capture reagents that bind to analytes in the sample.

[0030] In the exemplary embodiments of this disclosure, the membrane supports the lateral flow of a sample and, optionally, other liquid reagents applied to the membrane. In some embodiments, the membrane includes a sample application zone and a detection zone. A sample applied to the membrane, located in the sample application zone, moves through the membrane to the detection zone, where the sample encounters an immobilized capture reagent as described herein. In one embodiment shown in panel A of Figure 1, the sample is mixed with a detection reagent containing a labeled binding partner of the analyte in the sample before the sample is applied to the membrane. In another embodiment shown in panel B of Figure 1, the detection reagent is dried in the reagent zone of the membrane so that the sample solution solubilizes the detection reagent and transports it to the detection zone along with the analyte in the sample (if present).

[0031] The membranes of this disclosure may be continuous or non-continuous, as described herein, insofar as there is fluid communication between the portion of the membrane having the sample application zone and the portion of the membrane having the detection zone. In an alternative embodiment, the reagent zone itself may be located in another membrane that the liquid sample encounters before the portion of the membrane having the detection zone comes into contact with the sample.

[0032] In various embodiments of this disclosure, the capture reagent comprises an analyte binding partner (e.g., an anti-antigen antibody) that binds to an analyte (e.g., an antigen) in the sample, the analyte binding partner being bound to a particle immobilized in the detection zone. In one embodiment, the antibody is bound to the particle through the interaction of another pair of binding partners, one of which is bound to the analyte binding partner and the other to the particle. Examples of the separate set of binding partners include (a) biotin and avidin (streptavidin), (b) digoxigenin and anti-digoxigenin, (c) a hybridizing nucleic acid, and (d) an anti-species antibody (e.g., anti-mouse) and a species antibody (IgG) as the first antibody. One of the binding partners of the separate set of binding partners may be linked to the analyte binding partner by techniques known to those skilled in the art, and the other may be bound to the particle. In one exemplary embodiment, the binding partner of the analyte on the capture reagent is a biotinylated antianalyte antibody, which is bound to streptavidin-coated particles via a biotin-streptavidin interaction. The capture reagent may be deposited in liquid in the detection zone, or it may be dried, thereby immobilizing the capture reagent on a membrane in the detection zone.

[0033] The detection reagent contains a labeled binding partner of the analyte. In various embodiments where the analyte binding partner of the detection reagent and the analyte binding partner of the capture reagent are antibodies, the detection antibody and the capture antibody may be the same or different, depending on the available epitopes on the target antigen in the sample. The label present in the detection zone can be detected. This may be done, for example, visually, or by spectrophotometric or densitometry. For example, the label may be an enzyme that provides a visible signal when in contact with a substrate. An exemplary enzyme is horseradish peroxidase (HRP), for which the substrate is known. In this embodiment, the detection reagent of the present disclosure may be, for example, an anti-antigen antibody conjugated to HRP. The reagent may be added to the sample or dried in the reagent zone of the membrane as described herein.

[0034] Antigen-specific binding partners other than antibodies are known. Examples include nucleic acids (DNA / DNA, DNA / RNA, etc.), proteins, and other peptides that are known to specifically bind to various molecules in biological, industrial, and environmental samples.

[0035] The membranes used in lateral flow devices are made of materials that support the lateral flow of liquid. Suitable materials include fibrous mats composed of synthetic or natural fibers (e.g., glass or cellulose-based materials or thermoplastic polymers, e.g., polyethylene, polypropylene, or polyester); sintered structures composed of particulate materials (e.g., glass or various thermoplastic polymers); or cast membrane films composed of nitrocellulose, nylon, polysulfone, etc. (generally, in practice, synthetic materials). The matrix may also consist of sintered polyethylene microparticles (commonly known as porous polyethylene) (e.g., sintered polyethylene beads). Such materials may have (a) a density between about 0.30 grams and about 0.60 grams per cubic centimeter, for example, between 0.35 grams and 0.55 grams per cubic centimeter; (b) a pore size between about 1 micron and about 50 microns, or between about 5 microns and about 40 microns; and (c) a void volume between about 25 percent and about 75 percent, or between about 40 percent and about 60 percent. For example, the material density may be 0.30 grams, 0.35 grams, 0.40 grams, 0.45 grams, 0.50 grams, 0.55 grams, or 0.60 grams per cubic centimeter. The pore size may be 1 micron, 2 microns, 3 microns, 4 microns, 5 microns, 10 microns, 15 microns, 20 microns, 25 microns, 30 microns, 35 microns, 40 microns, 45 microns, or 50 microns. The void volume may be approximately 25 percent, approximately 30 percent, approximately 35 percent, approximately 40 percent, approximately 45 percent, approximately 50 percent, approximately 55 percent, approximately 60 percent, approximately 65 percent, or approximately 70 percent. Particulate polyethylene composed of cross-linked polyethylene or particulate polyethylene composed of ultra-high molecular weight polyethylene can also be used. An example matrix is ​​FUSION 5, available from Whatman, Inc. (商標)Examples include matrices and various sintered plastic materials from Porex Corporation. The matrices may be selected considering the sample to which they are applied, and other parameters known to those skilled in the art.

[0036] In embodiments of this disclosure, the lateral flow membrane of the device is supported within a housing. In addition to supporting the membrane, the housing may include a container for holding a liquid washing reagent. Furthermore, if the labeling is an enzyme, the housing may include a container for holding a liquid containing a substrate for labeling ("substrate reagent"). Embodiments of housing incorporating the washing reagent and substrate reagent are shown, for example, in U.S. Patent No. 5,627,010, published by IDEXX Laboratories, Inc. (登録商標) It is sold under a brand name. Briefly, when a sample is added to such a device, the sample moves to the detection zone, where the antigen (if present) binds to the capture reagent. The sample liquid contains a labeled second antibody, either added to the sample or present in the reagent zone. If the antigen is present in the sample, the label of the detection reagent is bound to the detection zone through interaction between the capture reagent, the sample antigen, and the second antibody (i.e., an immunoassay sandwich). The operator can activate the device to release the washing reagent and substrate reagent onto the lateral flow membrane, resulting in the removal of unbound reactants from the detection zone by the movement of the washing reagent, and the substrate generating a signal by reacting with the enzyme label bound to the detection reagent. This signal can be read by the operator, for example, by visible means or spectrophotometric means. In some embodiments, the washing reagent and substrate reagent are the same reagent. If the label does not require a substrate for detection (e.g., fluorescent label), the device does not require a substrate reagent.

[0037] In a relevant embodiment, the device may be used in a competitive assay configuration. Thus, the capture reagent is bound in the detection zone, and the analyte-labeled conjugate may be mixed with the sample or present in the reagent zone of the device as described herein. Once a sample is added to the device, the labeled conjugate competes with the analyte in the sample for binding to the capture reagent in the detection zone. The absence of a signal from the label in the detection zone indicates the presence of the analyte in the sample.

[0038] Another aspect of this disclosure relates to a multiplexing device having the ability to determine more than one analyte in a sample. In this aspect, one or more detection reagents may bind to each of the one or more analytes. The labels on the one or more detection reagents bound to each of the one or more analytes may be the same or different, insofar as the labels can be distinguished from one another. For example, if the capture reagents for different analytes in the sample are located in separate regions of the detection zone, the same label may be used on each detection reagent and may be associated with the correct antigen due to the position of the label in the detection zone. If all of those capture reagents are located in regions of the detection zone where the positions of the capture reagents cannot be distinguished from one another, their labels may be distinguished by the wavelengths of light they emit. For example, if the labels are fluorescent or chemiluminescent, each label may emit light at a different wavelength, which is typically detected by a spectrophotometer.

[0039] In another embodiment, the Disclosure relates to a method for determining the presence or amount of one or more antigens in a sample. The method includes adding a sample suspected to contain an analyte(s) to a sample application zone of a device relating to the Disclosure, and enabling the sample to move by lateral flow to a detection zone, where the sample encounters an immobilized capture reagent(s). The detection reagent(s) may be added to the sample or dried in a reagent zone of the membrane as described herein. An operator may detect the presence or amount of the label in the detection zone, the presence or amount of the label providing a signal relating to the presence or amount of an analyte in the sample. As described above, the device may include a housing supporting the membrane, and the device may include containers for washing reagents and substrate reagents such that when the device is activated, the washing reagents wash the detection zone to remove unbound reactants. The substrate for labeling may provide a detectable signal if the sample contains an antigen.

[0040] Some embodiments of this disclosure relate to the use of one or more reagents of this disclosure to detect fecal antigens (including hookworm antigens, roundworm antigens, and whipworm antigens) in a stool sample processed for application to the device in liquid form. In exemplary embodiments, the detection reagent is a detection antibody against one of the fecal antigens (described in the examples below) conjugated to an enzyme (e.g., HRP). One or more capture reagents (including anti-hookworm antigen antibodies, and anti-roundworm antigen antibodies and / or anti-whipworm antigen antibodies) are immobilized in the detection zone of the device as described herein. [Examples]

[0041] (Examples) (Explanation of the assay) As a model system, a sintered matrix immunoassay device format (SNAP) that uses reversible flow on the matrix is ​​used. (登録商標)The sensitivity and specificity of an immunoassay for fecal antigens were determined using a device (IDEXX Laboratories, Inc.) (see, for example, U.S. Patent No. 5726010A) with biotinylated antibodies immobilized on streptavidin-coated particles as the capture reagent. The matrix used was a porous polyethylene pad (Porex Corporation) produced by sintering polyethylene particles. The results show that the assay sensitivity is as high as that of a standard ELISA assay and that there is no nonspecific binding. The results also show that the assay can be multiplexed for the simultaneous detection of multiple antigens.

[0042] (material and method) The following describes the procedure for preparing the capture reagent (streptavidin-coated particles containing biotinylated antibody) and the sample mixed with the HRP-conjugated antibody in liquid or dry form, all of which are SNAP. (登録商標) Used in reversible flow assay devices.

[0043] (Streptavidin-biotinylated antibody particle (capture reagent) preparation protocol)

[0044] 1.5 mL of streptavidin-coated particles (nominal size 0.4 μm to 0.69 μm at 1% w / v) (SVP-05-10 Spherotech) were centrifuged at 17,000 rpm for 10 minutes and resuspended in 750 μL of carbonate buffer (10 mM, pH 10.5). This process was repeated twice, and the pellet was finally resuspended in 750 μL of carbonate buffer (10 mM, pH 10.5) to prepare a 2% w / v particle-containing solution. This solution was sonicated until no aggregation was observed under a microscope. Next, anti-antigen antibodies (anti-whipworm antigen, anti-roundworm antigen, and anti-hookworm antigen) (antibodies prepared in tissue) were biotinylated by conjugating these antibodies to sulfo-NHS-LC-biotin. These antibody conjugates were mixed with streptavidin-coated particles (Spherotech, Inc.) and sonicated until no aggregation was observed. The mixture was incubated at room temperature in end-to-end rotation for 40 minutes, followed by centrifugation at 17,000 rpm for 10 minutes. The supernatant was removed, and the streptavidin-biotinized antibody particles were resuspended in an appropriate volume of solution to achieve the desired %w / v concentration. The solid particle percentage concentration was measured, taking into account the original stock dilution of the streptavidin particles.

[0045] The particle solution is SNAP in the "detection zone" at the desired % concentration in the spotting buffer (10 mM carbonic acid-bicarbonate buffer, pH 10, 2.5% sucrose, 0.05% Tween®-20). (登録商標) The samples were directly spotted / deposited onto a sintered matrix. The volume of each spot was 1 μL. The spotted fragments were dried at 30°C for 10 minutes and stored in a foil bag with a desiccant. The detection zone can be prepared using capture reagents for one or more antigens.

[0046] (Preparation of samples and secondary antibodies (detection reagents) conjugated to HRP)

[0047] Liquid format: A 350 μL sample was prepared by mixing the sample with extraction buffer (pH 10.5) and fecal mass, followed by centrifugation. Secondary antibodies (detection antibodies) that bind to different epitopes on the antigen were conjugated to horseradish peroxidase (HRP) using SMCC chemistry. To conjugate the antibody with HRP, a mixture of conjugate buffer (10 mM PBS buffer / 2 mM EDTA), 0.5 M EDTA, and 5 mg / mL antibody was prepared in a tube. 5 mM DTT was added to the mixture to reduce the antibody, which was then incubated at 18°C–27°C for 30 minutes and desalted using a G-25 column. The reduced antibody was then combined with HRP-SMCC in a molar ratio of 1:1–1:4 and incubated at 18°C–27°C for 2 hours. Any unreacted sulfhydryl groups were capped using 0.5 mM L-Cys. Finally, the conjugated antibody-HRP was characterized by SEC before use in SNAP.

[0048] The antibody-HRP conjugate was added directly to the sample and mixed with it (see panel A in Figure 1). The sample mixture (350 μL) was then subjected to SNAP with the matrix described above. (登録商標) The sample was loaded into the device's sample cup.

[0049] Drying method: In another embodiment (drying method), the secondary antibody conjugated to HRP can be dried into strips by first mixing its conjugate with a solution containing 20% ​​to 25% sucrose / trehalose, and then striping the mixture on a matrix (see panel B in Figure 1). The matrix is ​​then dried at 37°C. The dried matrix can be stored in a foil bag with a desiccant.

[0050] The sample can be prepared as described above, except that it is not mixed with the antibody-HRP conjugate, and can be loaded directly into the sample cup. When the sample passes through the dried conjugate on the strip, the sample contacts the conjugate and solubilizes the conjugate.

[0051] (SNAP (登録商標) (Sample analysis in)

[0052] The strip spotted as described above can be incorporated into a SNAP (登録商標) device that includes an absorbent block, a wash reservoir, and a substrate reservoir. An appropriate sample (depending on liquid form / dry form) can be loaded into the sample cup. Once the sample flow reaches the activation circle (allowing sufficient time for the antigen to bind to the antibody at that spot), the device is activated to sequentially release a wash solution and a substrate solution (e.g., tetramethylbenzidine (TMB)), reversing the direction of the liquid flow on the strip. After about 5 minutes, blue color is visible in the detection zone (as a result of the oxidation of the TMB substrate). Although not required, the addition of sodium azide can stop this color development, but this step is not necessary. The color development indicates that the sample is positive for the antigen.

[0053] (Assay spot density measurement)

[0054] For quantitative measurements to determine assay sensitivity, a densitometer was used to measure the optical density of the assay spots as well as the background density of the membrane. The background density was subtracted from the assay spot density to calculate the sample minus background (S - B).

[0055] (Example 1: Detection of fecal antigen) As shown in Figure 2, the above capture reagent (1 μL of 1.3% w / v streptavidin particles) spotted SNAP (登録商標)The assay (particle-streptavidin-biotin-antibody) in the device was able to detect whipworm antigens in a series of clinical fecal samples identified as positive by a standard ELISA assay with an OD > 0.1. The detection zone shown in Figure 2 also included capture reagents for roundworm antigens and hookworm antigens. As described above, before adding the sample to the device, a secondary antibody-HRP conjugate (anti-hookworm antibody = 2 μg / mL, anti-roundworm antibody (Ab) = 4.5 μg / mL, anti-whipworm antibody (Ab) = 3.0 μg / mL) was added to the sample. The sample solution was added to the device without incubation.

[0056] This assay demonstrated high sensitivity for whipworm antigen-positive clinical samples (which may also have contained hookworm and / or roundworm antigens) at a low concentration of approximately 0.5 ng / mL, comparable to the ELISA sensitivity of 0.1 ng / mL to 0.8 ng / mL. Furthermore, as shown in Figure 2, whipworm antigen was detected without nonspecific binding and without cross-reactivity with samples that were also positive for hookworm and roundworm antigens. The absence of cross-reactivity becomes even clearer when the sample is positive only for whipworm, as neither roundworm nor hookworm indicators were detected.

[0057] Figure 3 shows similar results for the detection zones at known concentrations (0 ng / mL, 0.5 ng / mL, 1 ng / mL, and 5 ng / mL) of the sample antigen. These results are summarized in Figure 4.

[0058] Figure 5 shows the detection zone (corresponding to ELISA OD < 0.1) from negative samples for these three antigens.

[0059] (Example 2: Variation of assay parameters) To evaluate the reaction conditions, two types of fecal masses, 0.1 g / mL and 0.3 g / mL, were tested with samples and antibody (Ab)-HRP under various incubation conditions (N=9-14 for positive samples and N=26-30 for negative samples). As shown in Table 2, incubation of small amounts of fecal mass (0.1 g / mL) and fecal samples with antibody (Ab)-HRP yielded results that were more consistent with / less erroneous than no incubation. For large amounts of fecal mass (0.3 g / mL), incubation at room temperature (RT) was more accurate than incubation at 50°C. [Table 2]

[0060] While the present invention has been described in detail by reference to specific embodiments of the present invention, it is evident that modifications and alterations are possible without departing from the scope of the invention as defined in the appended claims. More specifically, while certain aspects of the present invention are identified herein as particularly advantageous, it is intended that the present invention is not necessarily limited to these specific aspects.

Claims

1. An immunoassay device for determining antigens in a sample, A membrane supporting the lateral flow of the liquid, including the detection zone; A dried capture reagent in the detection zone, comprising a first antibody that specifically binds to the antigen, An immunoassay device comprising, wherein the first antibody is conjugated to a first binding partner which is bound to a second binding partner which is bound to a particle immobilized in the detection zone.

2. The device according to claim 1, wherein the first binding partner is biotin and the second binding partner is streptavidin.

3. A labeled second antibody that specifically binds to the aforementioned antigen. The immunoassay device according to any one of claims 1, further comprising the second antibody being diffused and bound within the reagent zone of the immunoassay device.

4. The immunoassay device according to claim 3, wherein the label is an enzyme.

5. Housing supporting the aforementioned membrane The immunoassay device according to claim 1, further comprising:

6. The immunoassay device according to claim 5, wherein the housing includes a container containing a liquid cleaning reagent.

7. The immunoassay device according to claim 6, wherein the housing further comprises a container containing a liquid substrate reagent containing the substrate for labeling.

8. The immunoassay device according to claim 1, A reagent comprising a labeled second antibody that specifically binds to the aforementioned antigen, A kit that includes this.

9. The kit according to claim 8, wherein the immunoassay device further comprises a housing that supports the membrane.

10. The kit according to claim 9, wherein the housing includes a container containing a liquid cleaning reagent.

11. The kit according to claim 9, wherein the label is an enzyme.

12. The kit according to claim 11, wherein the housing further comprises a container containing a liquid substrate reagent containing a substrate for the enzyme.

13. A method for determining the presence or amount of an antigen in a liquid sample, A step of adding the sample to the sample application zone of the immunoassay device according to claim 3, A step that enables the sample to move to the detection zone by lateral flow; and A step of detecting the presence or amount of the label in the detection zone. A method comprising the presence or amount of the label in the detection providing a signal related to the presence or amount of the antigen bound in the detection zone.

14. The immunoassay device further includes a housing that includes a container for a washing reagent and supports the membrane, and the method is The step of washing the detection zone with the washing reagent. The method according to claim 13, including the method described in claim 13.

15. The immunoassay device further includes a housing that supports the membrane, comprising a container for a washing reagent and a container for the label substrate, and the method is (i) a step of washing the reaction zone by adding the washing reagent to the membrane, and (2) Step of adding the substrate to the reaction zone. The method according to claim 14, including the method described in claim 14.

16. A method for determining the presence or amount of an antigen in a liquid sample, A step of forming a mixture by contacting the sample with a reagent containing a labeled antibody that specifically binds to the antigen; A step of adding the mixture to the sample application zone of the immunoassay device according to claim 1, A step that allows the mixture to move to the detection zone by lateral flow; and A step of detecting the presence or amount of the label in the detection zone. A method comprising the presence or amount of the label in the detection providing a signal related to the presence or amount of the antigen bound in the detection zone.

17. The immunoassay device further includes a housing that supports the membrane, which includes a container for a washing reagent and a container for a substrate for labeling, and the method (i) a step of washing the reaction zone by adding the washing reagent to the membrane, and (2) Step of adding the substrate to the reaction zone. The method according to claim 16, including the method described in claim 16.

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