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    • 专利摘要:
    DEVICE AND METHOD FOR DETECTING A TARGET MOLECULE, SYSTEM, AND, KIT Devices and methods for detecting antigens are disclosed. Devices and methods for the detection of foodborne pathogens are disclosed.
    公开号:BR112012002308B1
    申请号:R112012002308-0
    申请日:2010-07-30
    公开日:2021-02-02
    发明作者:Nicholas A. Siciliano；Martin Joseph Bouliane
    申请人:Invisible Sentinel, Inc.；
    IPC主号:
    专利说明:

    Cross Reference to Related Order
    [001] The present application claims priority for U.S. application No. 12 / 533,721, filed on July 31, 2009, which is hereby incorporated by reference in its entirety. Field of the Invention
    [002] The present invention is directed, in part, to a device and assay for the detection of one or more antigens and methods for its use. Fundamentals of the Invention
    [003] Antigen detection is important for many areas of scientific research, diagnostic use and therapeutic uses. There are several ways in which antigens can be detected. Various methods are described in US Patent: 5,160,701, US Patent: 5,141,850, PCT Publication WO 91/12336, US Patent: 5,451,504, US Patent: 5,559,041, European Patent Application No .: 0505636A1, Publication PCT No. WO 88/08534, European Patent Application No. 0284 232A1, US Patent Application Publication No. 20070020768 and US Patent No. RE39664, each of which is hereby incorporated by reference in its entirety. The methods and devices available prior to the invention may still require improvements in the sensitivity or speed at which the results can be obtained. These factors can be important where time is of the essence when trying to determine the presence or absence of an antigen.
    [004] One such area is the detection area for foodborne pathogenic contaminants. Approximately seventy-six million people in the United States are afflicted with a foodborne illness. Of that seventy-six million, approximately 325,000 will become violently ill, requiring hospitalization, and approximately 5,000 will die. Most foodborne illnesses are caused by Salmonella, E. coli, Campylobacter and cost approximately $ 35 billion.
    [005] Current measures that guarantee a safe food supply involve a combination of local, state and federal authorities, as well as an elaborate system of inspectors and surveillance networks. Food manufacturers are controlled by certain regulations of the United States Department of Agriculture, United States Food and Drug Administration, and the National Marine Fisheries Service that are imposed by law. The USDA has created a system of health inspectors who are charged with carrying out daily inspections of meat, production and other consumer products manufactured or processed at the manufacturing and processing facilities. These inspections were created to involve a detailed statistical analysis to better guarantee the safety and sterility of food before reaching the consumer. In addition, most of the meat industry has adopted irradiation techniques to further demonstrate the sterility of the products. At a lower level, local and municipal health departments work to ensure that local distributors, restaurants and retailers follow strict guidelines to ensure a secure food supply. However, despite this elaborate network, food-borne infections are still common.
    [006] As soon as an outbreak is strongly suspected, an investigation begins. The research is done for cases among people who may have been exposed. The symptoms and the time of onset and location of possible cases are determined, and a “case definition” is developed which describes these typical cases. The outbreak is systematically described by time, place and person. A graph is drawn of the number of people who became ill on each successive day to show pictorially when it occurred. Calculating the distribution of cases by age and sex shows who is affected.
    [007] Often, the causative microbe is not known, so stool or blood samples must be collected from sick people and sent to the public health laboratory to make a diagnosis. Each collection and sample can cost more than $ 500 per test and often takes 2 to -4 days for analysis (CDC “Foodborne Infections”).
    [008] Before the present invention, to identify the food or other source of the outbreak, the researchers first interviewed some people with the most typical cases about the exposures they may have had in the few days before becoming ill. In this way, certain potential exposures can be excluded, while others that are mentioned several times appear as possibilities of source. Combined with other information, such as probable sources for the specific microbe involved, the hypotheses are then tested in a formal epidemiological investigation. The researchers conduct systematic interviews on a list of possible exposures with sick people, and with a comparable group of people who are not sick. By comparing how often an exposure is reported by sick people and people in good health, researchers can measure the association of exposure with the disease. Using the probability statistics, the probability of no association is directly calculated.
    [009] Since new food-borne problems arise, there is a need for new devices and methods for the detection of food-borne pathogens. The present invention provides a device for the detection of antigens, such as food-borne bacterial antigens, and fulfills the needs of having a device and assay with greater sensitivity and / or speed of detection. The present invention satisfies other needs as will be discussed here. Summary of the Invention
    [010] In some embodiments, the present invention provides devices for the detection of an antigen. In some embodiments, the devices comprise a housing comprising a first housing member and a second housing member, wherein said housing comprises: an entrance opening in the second housing member; a force member connected to the first housing member; a sliding locking member in contact with the first housing member and which comes into contact with the power member; an antigen detection membrane system comprising in the following order: a conjugated cushion; a permeable membrane; a test membrane; and an absorbent member; and a flexible connecting member connected to the locking member and the coupled pad; wherein at least a portion of each of the conjugated pad, permeable membrane, test membrane, and absorbent member is substantially parallel to each other; wherein the conjugate pad is capable of being compressed against the perimeter of the inlet opening in the second housing member; and wherein the force member makes contact with the absorbent member and is capable of applying pressure substantially perpendicular to the antigen detection membrane system.
    [011] In some embodiments of the devices, the devices further comprise a hydrophobic membrane located between the test membrane and the absorbent member. In some embodiments, the first housing member further comprises a slide button that protrudes from the outer surface of the first housing member, where the slide button is attached to the locking member, where the movement of the slide button moves the member locking.
    [012] In some embodiments, the conjugate pad comprises a first antigen-specific antibody.
    [013] In some embodiments, the antigen recognized by the first antigen-specific antibody is a foodborne pathogen antigen.
    [014] In some embodiments, the present invention provides systems comprising a device as described herein and a buffer container or sample collector.
    [015] The present invention also provides methods for detecting an antigen. Brief Description of Drawings
    [016] Figure 1: Represents a perspective view of a representative device according to some embodiments of the present invention.
    [017] Figure 2: Represent some components of a representative device according to some embodiments of the present invention.
    [018] Figure 3: Represent some components of a representative device according to some embodiments of the present invention.
    [019] Figure 4: Represent some components of a representative device according to some embodiments of the present invention.
    [020] Figure 5: Represent some components of a representative device in various positions according to some embodiments of the present invention.
    [021] Figure 6: Represents a side view of some components of a representative device according to some embodiments of the present invention.
    [022] Figure 7: Represents a side view of some components of a representative device according to some embodiments of the present invention.
    [023] Figure 8: Represents a side view of some components of a representative device according to some embodiments of the present invention.
    [024] Figure 9: Represents a flexible connecting member connected to a conjugated cushion.
    [025] Figure 10: Represents the membranes in a representative housing member. Description of the Forms of Realization
    [026] As used herein and unless otherwise indicated, the term “about” is intended to mean ± 5% of the value it modifies. Thus, about 100 means 95 to 105.
    [027] The present invention provides devices and methods for the detection of antigens or other molecules. In some embodiments, the devices use chromatographic assays. In some embodiments, the assays use the specification of the binding assays to indicate the presence or absence of an antigen.
    [028] The term "capture reagent" refers to a reagent, for example, an antibody or antigen binding protein, capable of binding a target molecule or analyte to be detected in a biological sample. A capture reagent can also be, for example, an oligonucleotide or a peptide.
    [029] The term "detect" or "detection" is used in the broadest sense to include qualitative and / or quantitative measurements of a target analyte.
    [030] The terms "linked" or "linked" can include both direct and indirect linking. Two components that are directly linked together are also in physical contact with each other. The two components that are indirectly linked together, are connected through an intermediate component. For example, Component A can be indirectly linked to Component B if Component A is directly linked to Component C and Component C is directly linked to Component B. Therefore, in such an example, Component A would be said to be indirectly linked to Component B.
    [031] The term "isolated" refers to a molecule that is substantially separated from its natural environment. For example, an isolated protein is one that is substantially separated from the cellular or tissue source from which it is derived.
    [032] The term "purified" refers to a molecule that is substantially free of other material that associates with the molecule in its natural environment. For example, a purified protein is substantially free of cellular material or other proteins in the cell or tissue from which it is derived. The term refers to preparations where the isolated protein is sufficiently pure to be analyzed, or at least 70% to 80% (w / w) purity, at least 80% to 90% (w / w) purity , from 90 to 95% purity; and at least 95%, 96%, 97%, 98%, 99%, or 100% (w / w) purity.
    [033] The terms "specific binding", "specifically binding", and so on, mean that two or more molecules form a complex that is measurable under physiological or test conditions and is selective. An antibody or antigen-binding protein or other molecule is said to "specifically bind" to a protein, antigen or epitope if, under properly selected conditions, such binding is not substantially inhibited, while at the same time non-specific binding is inhibited . The specific binding is characterized by a high affinity and is selective for the compound, proteins, epitope or antigen. The non-specific bond generally has a low affinity. Binding in IgG antibodies, for example, is generally characterized by an affinity of at least about 10-7 M or greater, such as at least about 10-8 M or greater, or at least about 10-9 M or higher, or at least about 10-10 M or greater, or at least about 10-11 M or greater, or at least about 10-12 M or greater. The term is also applicable where, for example, an antigen-binding domain is specific to a particular epitope that is not loaded by numerous antigens, in which case the antibody-binding protein or antigen that carries the antigen-binding domain generally it will not bind to other antigens. In some embodiments, the capture reagent has a KD equal to or less than 10-9 M, 10-10 M or 10-11 M with respect to its binding pair (e.g., antigen). In some embodiments, the capture reagent has a Ka greater than or equal to 109M-1 with respect to its binding pair.
    [034] The capture reagent can also refer, for example, to antibodies. Intact antibodies, also known as immunoglobulins, are typically glycosylated tetrameric proteins composed of two light chains (L) of approximately 25 kDa each, and two heavy chains (H) of approximately 50 kDa each. Two types of light chain, called lambda and kappa, exist in antibodies. Depending on the amino acid sequence of the heavy chain constant domain, immunoglobulins are assigned to five main classes: A, D, E, G and M, and several of them can be further divided into subclasses (isotypes), for example, IgG1, IgG2 , IgG3, IgG4, IgA1 and IgA2. Each light chain is composed of a variable domain N-terminal (V) (VL) and constant domain (C) (CL). Each heavy chain is composed of an N-terminal V domain (VH), three or four C domains (CHs), and a hinge region. The CH domain most proximal to the VH is called CH1. The VH and VL domains consist of four regions of relatively conserved sequences called structural regions (FR1, FR2, FR3, and FR4), which form a scaffold for the three regions of hypervariable sequences (complementarity determining regions, CDRs). CDRs contain most of the residues responsible for specific interactions of the antibody or antigen-binding protein with the antigen. CDRs are referred to as CDR1, CDR2 and CDR3. Consequently, the constituents of CDR in the heavy chain are referred to as H1, H2 and H3, although the constituents of CDR in the light chain are referred to as L1, L2 and L3. CDR3 is the largest source of molecular diversity within the binding protein-binding site to the antibody or antigen. H3, for example, can be as short as two amino acid residues or greater than 26 amino acids. Subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known in the art. For a review of the antibody structure, see Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Eds. Harlow et al., 1988. A person skilled in the art will recognize that each subunit structure, for example, a CH, VH, CL, VL, CDR, and / or FR structure, comprises the active fragments. For example, the active fragments may consist of the part of the VH, VL, or CDR subunit that binds to the antigen, that is, the antigen-binding fragment, or the part of the CH subunit that binds and / or activates a receptor and / or complement Fc.
    [035] Non-limiting examples of binding fragments included within the term "antibody specific antigen" used in this document include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) an F (ab ') 2 fragment, a divalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single subdivision of an antibody, (v) a dAb fragment, consisting of a VH domain, and (vi) an isolated CDR. In addition, although the two Fv fragment domains, VL and VH, are encoded by separate genes, they can be recombinantly joined by a synthetic articulator, creating a single protein chain in which the VL and VH domain pair to form monovalent molecules (known as the single Fv chain (scFv)). The most commonly used articulator is a 15-residue peptide (Gly4Ser) 3, but other articulators are also known in the art. Single chain antibodies are also intended to be included by the term "antibody or antigen-binding proteins", or "antigen-binding fragment" of an antibody. The antibody can also be a polyclonal antibody, monoclonal antibody, chimeric antibody, antigen binding fragment, Fc fragment, single chain antibodies, or any derivative thereof.
    [036] These antibodies are obtained using conventional techniques known to those skilled in the art, and fragments are selected for utility in the same way as intact antibodies. The diversity of antibodies is created by multiple germline genes that encode variable domains and a variety of somatic events. Somatic events include the recombination of variable gene segments with genetic segments of diversity (D) and union (J) to produce a complete VH domain, and the recombination of variable and union genetic segments to produce a complete VL domain. The recombination process itself is imprecise, resulting in the loss or addition of amino acids in the V (D) J connections. These mechanisms of diversity occur in the development of the B cell before exposure to the antigen. After antigenic stimulation, the antibody genes expressed in B cells undergo somatic mutation. Based on the estimated number of segments of the germline gene, the random recombination of these segments, and the VH-VL random pairing, up to 1.6 x 107 different antibodies can be produced (Fundamental Immunology, 3rd ed. (1993), ed Paul, Raven Press, New York, NY). When other processes that contribute to the diversity of antibodies (such as somatic mutation) are taken into account, it is thought that over 1 x 1010 different antibodies may be generated (Immunoglobulin Genes, 2nd ed. (1995), eds. Jonio et al., Academic Press, San Diego, Calif.). Because of the many processes involved in generating antibody diversity, it is unlikely that independently derived monoclonal antibodies with the same specificity to the antigen will have identical amino acid sequences.
    [037] Antibody or antigen-binding protein molecules capable of interacting specifically with antigens, epitopes, or other molecules described in this document, can be produced by methods well known to those skilled in the art. For example, monoclonal antibodies can be produced by the generation of hybridomas according to known methods. Hybridomas formed in this way can then be selected using standard methods, such as the enzyme-linked immunosorbent assay (ELISA) and Biacore analysis, to identify one or more hybridomas that produce an antibody that specifically interacts with a molecule or compound of interest.
    [038] As an alternative to the preparation of monoclonal antibody-secreting hybridomas, a monoclonal antibody in a polypeptide of the present invention can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (for example, an antibody presentation library phage) with a polypeptide of the present invention to thereby isolate members of the immunoglobulin library that bind to the polypeptide. The techniques and kits commercially available for the generation and screening of phage presentation libraries are well known to those skilled in the art. In addition, examples of methods and reagents particularly suitable for use in the generation and screening of the research libraries of the antibody or antigen-binding protein can be found in the literature.
    [039] The term "capture reagent" also includes chimeric antibodies, such as humanized antibodies, as well as fully humanized antibodies. In some embodiments, the capture reagent is a Goat anti-E. coli 0157: H7 antibody Cat #: 70-XG13 (Fitzgerald Industries); E. coli 0157: H7 mono Cat #: 10-E13A (Fitzgerald Industries); E. coli 0157: H7 Cat #: 10C-CR1295M3 (Fitzgerald Industries); E. coli 0157: H7 mono Cat #: 10-E12A (Fitzgerald Industries); or Goat antimouse IgG Cat #: ABSE-020 (DCN).
    [040] In some embodiments, the devices of the present invention comprise a housing comprising a first housing member and a second housing member. In some embodiments, the first and second housing members can be constructed as a single unit. The housing may comprise an entrance opening. The entry opening allows the introduction of a sample in the chromatographic assay. In some embodiments, the first housing member comprises the entrance opening. The inlet opening can be of sufficient size to handle an appropriate volume of a solution that is added to the device. In some embodiments, the size of the opening is large enough to handle about 0.1 to 3 ml, about 0.1 to 2.5 ml, about 0.5 to 2.0 ml, about from 0.1 to 1.0 ml, about 0.5 to 1.5 ml, 0.5 to 1.0 ml, and 1.0 to 2.0 ml.
    [041] In some embodiments, the housing comprises a conjugate pad, a permeable membrane, a membrane test, and / or an absorbent member. In some embodiments, the housing includes an antigen detection membrane system. In some embodiments, the antigen detection membrane system comprises a conjugate pad, a permeable membrane, a test membrane, and an absorbent member. In some embodiments, the antigen detection membrane system is free of a permeable membrane. In some embodiments, the antigen detection membrane system comprises in the following order: a conjugated pad, a permeable membrane, a test membrane, and an absorbent member.
    [042] As used herein, the term "conjugate pad" refers to a membrane or other type of material that may comprise a capture reagent. The conjugate pad may be cellulose acetate, cellulose nitrate, polyamide, polycarbonate, fiberglass, membrane, polyethersulfone, regenerated cellulose (RC), polytetrafluoroethylene, (PTFE), polyester (eg, polyethylene terephthalate), polycarbonate (for example, 4,4-hydroxy-diphenyl-2,2'-propane), aluminum oxide, mixed cellulose ester (for example, the mixture of cellulose acetate and cellulose nitrate), Nylon (for example, polyamide, hexamethylene-diamine, and Nylon 66), polypropylene, PVDF, high-density polyethylene (HDPE) + “aluminum dibenzoate” (DBS) nucleating agent (eg 80u 0.024 HDPE DBS (Porex)), and HDPE. Examples of conjugated pillows also include, Cyclopore® (polyethylene terephthalate), Nucleopore® (polyethylene terephthalate), Membra-Fil® (cellulose acetate and nitrate), Whatman® (cellulose acetate and nitrate), Whatman # 12-S (rayon)), Anopore® (aluminum oxide), Anodisc® (aluminum oxide), Sartorius (cellulose acetate, for example, 5 μm) and Whatman Standard 17 (bonded glass).
    [043] In some embodiments, the conjugate pad or test membrane comprises a capture reagent. In some embodiments, the conjugate pad or test membrane is placed in contact with the capture reagent and then allowed to dry. The conjugate pad or test membrane may also comprise other compositions for preserving the capture reagent such that it can be stably stored at room temperature or under refrigeration or freezing temperatures. In some embodiments, the conjugate pad or test membrane is soaked with a buffer before the capture reagent is applied. In some embodiments, the plug is a blocking plug that is used to prevent nonspecific binding. In some embodiments, the buffer comprises borate, BSA, PVP40 and / or Tween-100. In some embodiments, the buffer is 10 mM borate, 3% BSA, 1% PVP40, and 0.25% Tween-100. In some embodiments, the capture reagent is applied to the pad or membrane in a solution comprising trehalose and sucrose. In some embodiments, the capture reagent is applied to the conjugate pad or test membrane in a solution comprising trehalose, sucrose and phosphate and / or BSA. In some embodiments, the capture reagent is applied in a solution that trehalose 5%, sucrose 20%, phosphate 10 mM, and BSA 1%.
    [044] In some embodiments, the pad or membrane (for example, conjugate pad or test membrane) comprises about 0.5 to about 5.0 μg of a capture reagent, about 1 to about 3 μg of a capture reagent, about 1 to about 2 μg of a capture reagent, about 2 to about 3 μg of a capture reagent, around 2.5 μg of a capture reagent, around 1.5 μg of a capture reagent, or around 2.7 μg of a capture reagent.
    [045] In some embodiments, the permeable membrane is connected to or adheres to a test membrane. In some embodiments, the permeable membrane is laminated to the test membrane. The permeable membrane can be a membrane of any material that allows a sample, such as a fluid sample, to flow through the test membrane. Examples of the test membrane include, but are not limited to, nitrocellulose, cellulose, fiberglass, polyester, polypropylene, nylon, and more. In some embodiments, the permeable membrane comprises an opening. The opening may be present to allow visualization or detection of the test membrane. In some embodiments, the opening in the permeable membrane is substantially the same size as the entrance opening in the housing. Examples of permeable membranes include, but are not limited to, Protran BA83, Whatman, and others.
    [046] As used herein, the "test membrane" refers to a membrane where the detection of a binding pair in a capture reagent occurs. The test membranes include, but are not limited to, a nitrocellulose membrane, a nylon membrane, a polyvinylidene fluoride membrane, a polyethersulfone membrane, and more. The test membrane can be any material that can be used by a person of skill in the art to detect the presence of a capture reagent binding pair (for example, antigen or epitope). The test membrane can also comprise a capture reagent. In some embodiments, the test membrane is contacted with a capture reagent and the capture reagent is allowed to dry and adhere to the test membrane. Examples of test membranes include, but are not limited to, Protran BA83, Whatman, Opitran BA-SA83, and 0.22 μm white plain (Millipore Product No. SA3J036107). The test membrane can comprise a plurality of capture reagents. In some embodiments, the test membrane comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 capture reagents. In some embodiments, the test membrane comprises a plurality of areas each with a different capture reagent. In some embodiments, the plurality of areas does not completely overlap or overlap. By using a plurality of capture reagents, multiple binding pairs (for example, epitopes or antigens) can be detected.
    [047] In some embodiments, the housing also comprises an absorbent member. The absorbent member can also be referred to as a “wick pad” or “wick absorption pad”. The absorbent member absorbs the fluid flowing through the device when the sample is applied to the device and provides the wick-absorbing force that aids in the flow of the sample when it is applied to the device.
    [048] The absorbent member can be any material that can facilitate the flow of the sample through the conjugate pad and into the test membrane. Examples of absorbent members include, but are not limited to, cellulose, superabsorbent polymers, fiberglass pads (e.g., C083 (Millipore)), and more. In some embodiments, the housing comprises a plurality (for example, 2 or more) of absorbent members. In some embodiments, the housing comprises 2, 3, 4 or 5 absorbent members. In some embodiments, the absorbent member comprises one or more membranes up to 10 individual membranes, and each membrane can be of the same or a different material.
    [049] In some embodiments, the device comprises a force member. The power member can be used to apply pressure or compress the other components of the antigen detection membrane system against each other. In some embodiments, the power member may comprise an axis and a tip. The power member may have a mushroom-like shape where the tip is larger than the axis. In some embodiments, the tip is narrower than the axis. The force member comprising a tip and an axis can be a single unit or it can be made up of multiple parts that come into contact with each other to form the force member. For example, the tip can be a unit that can be separated from the shaft. After assembly, the tip and shaft are placed in contact with each other to produce the force member. In another example, the tip and shaft are a cohesive unit and are manufactured together and not as separate parts that are later assembled to form the force member. The power member allows the device to work with the vertical flow instead of relying on the lateral flow.
    [050] The devices described in this document can be used in assays to detect the presence of a capture reagent binding pair. For example, an antigen can be detected by an antibody using the devices of the present invention. The devices of the present invention employ vertical flow. The “vertical flow” refers to the direction that the sample flows through the different membranes and members present in the device. The vertical flow refers to a sample flowing through the membrane (for example, from top to bottom) as opposed to the lateral flow, which refers to a sample flowing through (for example, side by side) to a membrane, pad or absorbent member. In a lateral flow device the membranes and pads are horizontally adjacent to each other substantially on the same plane. In a vertical flow device, each membrane or pad is substantially parallel or completely parallel to each other and occupies substantially different spatial planes in the device. The membranes and pads can occupy similar planes when they are compressed or placed under pressure. In some embodiments, at least part of each membrane or pad is layered on top of each other. In some embodiments, at least a part of each layer of the membrane or pad is substantially parallel to one another. In some embodiments, at least part of each layer is on a different spatial plane than each of the other layers.
    [051] To allow vertical flow to occur efficiently, in some embodiments, the conjugate pad, permeable membrane, test membrane and absorbent member are substantially parallel to each other. In some embodiments, the conjugate pad, permeable membrane, test membrane and absorbent member are present in different spatial planes. In some embodiments, the housing also comprises a hydrophobic membrane that can slow or interrupt the vertical flow of the sample. The hydrophobic membrane may be in contact with the test membrane, which would allow the sample to rest or rest on the test membrane. The interruption can take into account the increased sensitivity and detection. The vertical flow is modulated by the pressure that is applied to the membranes. In some embodiments, pressure is applied perpendicular to the test membrane and / or conjugate pad. The pressure can be applied so that the conjugate pad is compressed against the housing. The compression against the housing can be such that the conjugate is in direct contact with the housing, O-ring, or collar, or through an intermediary so that the conjugate pad and the test membrane are compressed against each other.
    [052] The force member can apply pressure that is substantially perpendicular to the test membrane. Pressure facilitates vertical flow. The pressure allows each layer of the membrane stack to be in contact with another layer. The pressure can also be relieved to stop the flow so that the test sample can support or firm on the test membrane, which can take into account greater sensitivity. The pressure can then be reapplied to allow the vertical flow to continue by letting the sample flow into the absorbent members. The force member can apply pressure in such a way that the conjugate pad comes into contact with a part of the housing. In some embodiments, the conjugate pad comes into contact with the housing when it is not under the pressure exerted by the force member, but after the force member exerting pressure on the conjugate pad is pressed against a part of the housing.
    [053] In some embodiments, the conjugate pad comes into contact with the perimeter of the inlet opening. The inlet opening may also comprise a necklace or other similar feature, such as an O-ring. In some embodiments, the conjugate pad comes into contact with the perimeter of a necklace and / or an O-ring. In some embodiments , the conjugate pad is capable of being compressed against the perimeter of the inlet opening, which may include, in some embodiments, a collar and / or an O-ring.
    [054] “Able to be compressed against the perimeter of the inlet opening” refers to a membrane or cushion (eg, conjugated cushion) being compressed directly in contact with the perimeter of the inlet opening or being compressed against another layer or material (for example, membrane) that is in contact with the perimeter of the inlet opening.
    [055] In some embodiments, the conjugated cushion is not in direct physical contact with the housing, but is in fluid contact with the housing. “Fluid contact” means that if a sample is applied to the device through the inlet opening or other opening, the fluid will come in contact with the conjugate pad. In some embodiments, the conjugate pad may be separated from the housing by another membrane, such as a permeable membrane, where the other membrane is in direct physical contact with the housing or in direct physical contact with the collar or O-ring. sample is applied to the device the fluid can come in contact with the other membrane first and then come in contact with the conjugate pad. This is just one example of the conjugated cushion that is in fluid contact with the housing. There are numerous other embodiments where the conjugated cushion is not in direct physical contact with the housing, the collar, or the O-ring, but is in fluid contact with the housing.
    [056] The force member can apply any pressure that is sufficient to facilitate vertical flow through the different layers of the membrane. In some embodiments, the layers of the device (e.g., conjugated pad, permeable membrane, test membrane, and absorbent member) are compressed under a force selected between about 5 lbf (2.27 kgf) to 100 lbf (45 , 36 kgf), about 5 lbf (2.27 kgf) to 50 lbf (22.7 kgf), about 10 lbf (4.5 kgf) to 40 lbf (18.1 kgf), about 15 lbf ( 6.8 kgf) to 40 lbf (18.1 kgf), about 15 lbf (6.8 kgf) to 25 lbf (11.33 kgf), or about 30 lbf (13.6 kgf) to 40 lbf ( 18.1 kgf). The force can also compress a hydrophobic or impermeable membrane even if it is present in the device.
    [057] In some embodiments, the force member comes into contact with a first surface of an absorbent member. In some embodiments, a conjugate pad comes in contact with a test membrane. In some embodiments, the first surface of a test membrane comes into contact with a permeable membrane. In some embodiments, a second surface of the test membrane comes into contact with a second surface of the absorbent pad. In some embodiments, the device comprises a hydrophobic membrane, and, for example, the hydrophobic membrane comes into contact with a second surface of the test membrane. In some embodiments, the hydrophobic membrane comes into contact with a first surface of the absorbent pad.
    [058] In some embodiments, a first surface of the conjugated pad comes into contact with the housing and a second surface of the conjugated pad comes into contact with a first surface of the permeable membrane, where the second surface of the permeable membrane comes into contact with a first surface of the test membrane, in which a second surface of the test membrane comes into contact with a first surface of the absorbent pad, in which a second surface of the absorbent pad comes into contact with the force member. In some embodiments, the first surface of the conjugated pad comes into contact with a perimeter of the inlet opening of said housing. In some embodiments, the first surface of the conjugated pad comes into contact with a perimeter of a necklace or an O-ring.
    [059] The device can also comprise a connecting member. In some embodiments, the connecting member is flexible or made of flexible material. The flexible material can be, for example, an elastic or elastomeric material. A connecting member can, for example, be attached to a conjugated pad and / or a hydrophobic membrane. The connecting member can also be attached to any membrane or member of the device. Examples of binding members include, but are not limited to, elastomer ligation, rubber ligation, spring, and more. In some embodiments, the connecting member can be produced from a shape memory material. The connecting member makes it possible to create a delay between the movement of the locking member and the movement of the conjugated pad or any other type of membrane or pad to which the connecting member is connected. The movement of the pad or membrane does not happen at the same time when the slider or locking member is moved. Not being limited to any particular theory, when the slider or locking member is moved, energy is accumulated in the connecting member and that energy is used to pull a pad or membrane that is connected to the connecting member after the pressure has been applied. released. In some embodiments, the locking member is removed from the force member (i.e., the force member no longer comes into contact with the locking member) before the conjugate pad is moved or removed. The conjugate pad, in some embodiments, is moved as soon as the compression or pressure being exerted by the force member is completely removed.
    [060] The connecting member can also be connected to a sliding button or locking member. The connecting member can be connected by any means, such as adhesives, staples, ties, and the like to other components. In some embodiments, the membrane or pad has notches in the membrane or pad that allow the connecting member to connect to the membrane or pad. A non-limiting example can be seen in Figure 9.
    [061] In some embodiments, the housing comprises a locking member. The locking member can be a sliding locking member that can move within the device. The locking member can be used to lock the force member in a position such that the force created by the force member after the different layers is maintained. The locking member is, for example, locking the force member in place so that the pressure cannot be relieved, unless the locking member is moved to allow the force member to change position (ie , decreased). The locking member can, for example, fit under the tip of the power member, which must keep the power member in the elevated position. The locking member can also be positioned so that it holds the power member in a particular position (for example, raised or lowered). The locking member can be produced from any material including, but not limited to, plastic and others. The locking member can, for example, come into contact with the power member directly or indirectly through another component that prevents the power member from releasing pressure. In some embodiments, the locking member comes into contact with the force member to compress the conjugate pad.
    [062] The locking member can also contact the bonding member in such a way that the movement of the locking member will move the bonding member, any other membrane (eg, conjugated pad, hydrophobic membrane, test membrane , or an absorbent member) or other component that is connected to the connecting member. For example, if the locking member is moved to relieve pressure from the power member thereby allowing the power member to change position (for example, from the raised to a lower position), the movement of the locking member will also deform / accumulate energy in the connecting member so that it can move the membrane or pad since the pressure has been sufficiently reduced. When the conjugate pad is connected to the connecting member and the locking member is moved, this will also move the conjugate pad once the pressure has been sufficiently reduced. In some embodiments, the pressure is completely removed. The conjugate pad can, for example, be moved in such a way that it is removed from the device. In some embodiments, the conjugate pad is moved to reveal the test membrane through the inlet opening. The amount of the test membrane that is developed will depend on the type of detection that is used. For visual detection more of the test membrane may need to be revealed at the inlet opening. For non-visual detection, for example, fluorescent, infrared, radioactive or chemiluminescent, less of the test membrane may need to be developed. In some embodiments, the conjugate pad is moved so that it can no longer be seen or detected through the inlet opening. In some embodiments, the movement of the conjugate pad may create an opening other than the inlet opening for viewing or detecting the test membrane.
    [063] In some embodiments, the connecting member is also connected to the waterproof or hydrophobic membrane. When the connecting member is moved, the movement will also move or remove the waterproof or hydrophobic membrane. As discussed here, the presence of the impermeable or hydrophobic membrane can allow the test sample to rest or firm on the test membrane by delaying or interrupting the vertical flow. When the impermeable or hydrophobic membrane is moved or removed, by its connection to the connecting member or by other means, the vertical flow is no longer impeded or inhibited.
    [064] In some embodiments, the housing comprises a slide button. A slide button can also be referred to as a slide member. The slider can traverse the internal and external surfaces of the housing. In some embodiments, the sliding button or sliding member projects to an external surface of the housing. In some embodiments, the slider is connected directly or indirectly to the locking member. When the slider is connected (directly or indirectly) to the locking member, the movement of the slider also moves the locking member. The connecting member in some embodiments can be connected to the slider. In some embodiments, the connecting member is connected to both the slide button and the locking member. The slider and locking member can also be built as a single unit.
    [065] In some embodiments, the inlet opening comprises an opening selected from a range of about 0.2 to 20 cm2. In some embodiments, the inlet opening is about 1 to about 2 cm in diameter. In some embodiments, the opening of the inlet is about 1 or about 1.5 cm in diameter. In some embodiments, the opening of the entrance is around 1, around 2, around 3, around 4, or around 5 cm in diameter.
    [066] As discussed here, the conjugate pad may comprise an antigen-specific capture reagent. In some embodiments, the conjugate pad comprises a plurality of antigen-specific capture reagents. In some embodiments, the conjugate pad has 1, 2, 3, 4, or 5 antigen-specific capture reagents. The antigen can be any molecule that can be specifically recognized by a capture reagent. Examples of antigens include a polynucleotide molecule (for example, DNA, RNA, siRNA, antisense oligonucleotide), a peptide, a protein, a saccharide, a polysaccharide, a carbohydrate, and more. The antigen can also refer to the different epitopes present on the same protein or polypeptide.
    [067] The capture reagent can also be, for example, protein A, protein G, and more.
    [068] In some embodiments, the protein is a pathogenic protein. A pathogenic protein refers to a protein that is from a pathogen. Examples of pathogens include, but are not limited to, pathogenic prokaryotic and eukaryotic organisms such as unicellular pathogenic organisms and multicellular parasites. Pathogens can also include protozoan pathogens that include a stage in the life cycle where they are intracellular pathogens. As used herein, the term "intracellular pathogen" means to refer to a virus or pathogenic organism that, at least part of its reproductive or life cycle, exists within a host cell and at that point produces or causes the pathogenic proteins.
    [069] Bacterial pathogens include, but are not limited to, such as bacterial pathogenic gram-positive cocci, which include, but are not limited to: pneumococcus; staphylococci and streptococci. Pathogenic gram-negative cocci are: meningococcal and gonococcal. Pathogenic enteric gram-negative bacilli include: enterobacteriaceae; pseudomonas, acinetobacteria and eikenella; melioidosis; salmonella; shigellosis; hemophile; chancroid; brucellosis; tularemia; yersinia (pasteurella); streptobacillus moniliformis and spirilum; listeria monocytogenes, erysipelothrix rhusiopathiae; diphtheria, cholera; anthrax; donovanosis (inguinal granuloma); and Bartonose. Anaerobic pathogenic bacteria include: tetanus; botulism; another clostridia; tuberculosis; leprosy; and other mycobacteria. Pathogenic spirochete diseases include: syphilis; treponematoses; fambroesia; purupuru and endemic syphilis; and leptospirosis. Other infections caused by higher pathogenic bacteria and pathogenic fungi include: actinomycosis; nocardiosis; cryptococcosis; blastomycosis; histoplasmosis and coccidioidomycosis; candidiasis, aspergillosis, and mucormycosis; sporotrichosis; paracoccidiodomycosis, petrielidiosis, torulopsis, mycetoma and chromomycosis, and dermatophytosis. Rickettsioses include rickettsiae and rickettsioses. Examples of mycoplasma and chlamydia infections include: mycoplasma pneumoniae; venereal lymphogranuloma; psittacosis and perinatal chlamydia infections. Infectious helminths and helminths and infectious eukaryotes thus include: amebiasis; malaria; leishmaniasis; trypanosomiasis; toxoplasmosis; pneumocystis carinii; babesiosis; giardiasis; trichinosis; filariasis; schistosomiasis; nematodes; trematodes or worms; and cestoid infections (tapeworms). The bacteria also include E. coli, a Campylobacter, and a Salmonella.
    [070] In some embodiments, E. Coli is E. coli 0157.
    [071] Examples of viruses include, but are not limited to, HIV, Hepatitis A, B and C, FIV, lentivirus, pestivirus, West Nile Virus, measles, smallpox, bovine pox, ebola, coronavirus, and more. Other pathogens are also disclosed in U.S. Patent Application Publication No. 20080139494, which are incorporated by reference.
    [072] In some embodiments, the pathogen is a foodborne pathogen. The antigen may be present in a foodborne pathogen. Foodborne pathogens are pathogens (for example, viral or bacterial) that cause disease after consuming contaminated food. The food itself does not directly cause the disease, but it is more precisely the consumption of the foodborne pathogen that is present in the food that causes the disease. In some embodiments, the foodborne pathogen is E. coli, Campylobacter, or Salmonella. In some embodiments, the antigen is an antigen selected from a foodborne pathogen antigen. For example, the foodborne pathogenic antigen can be, but is not limited to, chosen from an E. coli antigen, a Campylobacter antigen or a Salmonella antigen. In some embodiments, the antigen is the species-specific O antigen. In some embodiments, the O antigen is the E. coli and / or Salmonella O antigen and can be used for the detection of E. coli and Salmonella. In some embodiments, the antigen is a flagellin antigen. In some embodiments, the antigen is the Campylobacter flagellin antigen.
    [073] In some embodiments, the capture reagent comprises a detection reagent. The detection reagent can be any reagent that can be used to detect the presence of the capture reagent if it binds to its specific binding pair. The capture reagent can comprise a detection reagent directly or the capture reagent can comprise a particle that comprises the detection reagent. In some embodiments, the capture reagent and / or particle comprises a color, colloidal gold, radioactive label, fluorescent label, or a chemiluminescent substrate. The particle can be, for example, a viral particle, a latex particle, a lipid particle, or a fluorescent particle. In some embodiments, colloidal gold has a diameter of: about 20 nm, about 30 nm, or about 40 nm or in the range of about 20 to 30 nm, about 20 to 40 nm, about 30 at 40 nm, or about 35 to 40 nm.
    [074] In some embodiments, the test membrane also comprises one or more capture reagents.
    [075] The capture reagents of the present invention may also include an anti-antibody, that is, an antibody that recognizes another antibody, but is not specific to an antigen, such as, but not limited to, the anti-IgG antibody , anti-IgM, or anti-IgE. Where the test membrane comprises an anti-antibody, such as anti-IgG, anti-IgM or anti-IgE antibody, this non-specific antibody can be used as a positive control to detect whether the conjugate has been released from the conjugate pad. When the sample is applied to the device it allows a first capture reagent to be released from the conjugate pad. As the capture reagent is released and flows through the device, attached to the antigen or not, it can come in contact with the anti-antibody, such as the anti-IgG or anti-IgM antibody, which can then be detected. This detection can be used to show that the device is functioning properly.
    [076] In some embodiments, the test membrane comprises a second antigen-specific capture reagent. In some embodiments, the test membrane comprises a first area comprising a first capture reagent that comprises an anti-IgG capture reagent; and a second area comprising a second antigen-specific capture reagent, in which the first and second areas do not completely overlap or coincide with each other. This non-limiting embodiment can be used to demonstrate that the device is functioning properly and be used to detect the presence of the antigen of interest.
    [077] In some embodiments, the conjugate pad comprises a first antigen-specific capture reagent and the test membrane comprises a second antigen-specific capture reagent, where the first and second antigen-specific capture reagents are bind to the non-competitive epitopes present in the antigen. The device can, for example, employ a two-step sandwich test. The first step is the binding of the antigen to the captured reagent present in the conjugate pad. After binding to the first antigen-specific capture reagent, the antigen can flow through or make contact with the test membrane where a second antigen-specific capture reagent is present. After interaction with the test membrane if the test antigen can bind to the second antigen-specific capture reagent, it will be able to be detected by visualization or using another detection device such as, but not limited to to this, a fluorescent reader. The test membrane and the conjugate pad may comprise additional antigen-specific capture reagents that recognize different antigens or different epitopes. In some embodiments, the test membrane or conjugate pad comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 antigen-specific capture reagents. In some embodiments, the test membrane or conjugate pad comprises a plurality of antigen-specific capture reagents. In some embodiments, each antigen-specific capture reagent recognizes a different antigen or a different epitope on the same antigen.
    [078] "Different antigens" can also refer to the same protein, but a protein that is from different strains of the same organism. Different antigens can also refer to antigens from different organisms. For example, there are many strains of E. coli. Not all strains of E. coli cause foodborne illness. The present invention can be used, for example, to detect an antigen from a pathogenic E. coli strain as opposed to detecting an antigen from a non-pathogenic E. coli strain. In some embodiments, the conjugate pad and / or test membrane comprises an antigen-specific first and second capture reagents, wherein the first and said second capture reagents recognize the different antigens. In some embodiments, the test membrane and / or the conjugate pad comprises a plurality of areas comprising a plurality of antigen-specific capture reagents, wherein the plurality of antigen-specific capture reagents recognize the different antigens. In some embodiments, the plurality of areas does not completely overlap or coincide with one another. In some embodiments, the plurality of antigens is each independently selected from an E. coli antigen, a Campylobacter antigen and a Salmonella antigen. In some embodiments of the present invention, the plurality of antigens is 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 antigens.
    [079] The devices can be housed individually, in pairs, or in multiple configurations. The housing can be leakproof to prevent leakage and can be manufactured from a variety of inert materials, such as polymeric materials. The inlet opening, in some embodiments, may be of sufficient volume to contain any required amount of sample and reagents to be used with the invention.
    [080] Since the membranes or pads of the device are preferably chemically inert, they may have to be activated at any reaction site where it is desirable to immobilize a specific binding reagent against solvent transport. Various methods may be required to reproduce the immobilized reagent according to the specific chemical nature of the reagent. Generally, when the medium is nitrocellulose or a mixed nitrocellulose ester, no special chemical bonding is required for the immobilization of the reagents. Various techniques can be used for other materials and reagents that include functionalization with materials such as carbonyldiimidazole, glutaraldehyde or succinic acid, or treatment with materials such as cyanogen bromide. Other suitable reactions include treatment with Schiff bases and borohydride for the reduction of aldehyde, carbonyl and amino groups. DNA, RNA and certain antigens can be immobilized against solvent transport by cooking the chromatographic material. Cooking can be carried out at temperatures ranging from about 60 ° C to about 120 ° C for times ranging from about five minutes to about 12 hours, and in some embodiments, around 80 ° C during about two hours.
    [081] The present invention also provides systems comprising the devices described herein and a buffer container. The buffer container can be any buffer in which the sample being tested can be mixed with the device, and then applied to it. For example, the sample can be taken from a source and the sample can be mixed with the buffer. The buffer can be a lysis buffer that will lyse the cells or a buffer that maintains the pH of the sample so that the analysis can be done properly. The buffer container can be of any shape and can be included outside or inside the housing of the device.
    [082] In some embodiments, the present invention provides a system that comprises a sample collector. The sample collector can be any material that can take a sample from a source and allow the sample to be tested. For example, the sample collector may be a flexible rod, such as a flexible cotton rod. In some embodiments, the sample collector is an inoculator. In some embodiments, the housing comprises the sample collector and a portion of the sample collector is within the housing. In some embodiments, the sample collector is partly outside and partly inside the housing. In some embodiments, the sample collector is completely outside the housing.
    [083] The present invention also provides kits comprising the devices described herein. The kit can include a device as described in this document, a sample collector, a buffer container, an instruction manual, a positive control, a negative control, or any combination of these. With respect to the kit, a positive control is a sample that is known to contain the antigen that can be detected with the device present in the kit. On the contrary, the negative control must not contain an antigen that can be detected by the kit. The negative control when used in conjunction with the anti-antibody would be able to demonstrate that the device is functioning correctly.
    [084] Buffers can also be included in the present invention. Examples of buffers include, but are not limited to, 1X PBS (10 mM phosphate, 137 mM sodium chloride, 2.7 mM potassium chloride), a wash buffer (eg 10 mM sodium phosphate, 150 mM NaCl , 0.5% Tween-20, 0.05% sodium azide), a membrane buffer (eg 10 mM sodium phosphate, 0.1% sucrose, 0.1% BSA, 0.2%, PVP-40 pH 7.21, filtered with 0.2 μm filter), polyclonal conjugated block buffer (for example, 50 mM borate, 10% BSA, pH 8.93); conjugated polyclonal diluent (for example, 50 mM borate, 1% BSA, pH 9.09), or blocking buffers (for example, 10 mM sodium phosphate, 0.1% sucrose, 0.025% Silwet pH 7.42; 10 mM sodium phosphate, 1% sucrose, 1% trehalose, 0.01% BSA, 0.025% Tween-20, 0.05% sodium azide, 0.025% Silwet pH 7.4; 10 mM sodium phosphate, 0, 1% sucrose, 0.1% BSA, 0.2% PVP-40 pH 7.21). The buffer can also be, but is not limited to, a blocking buffer (for example, 10% BSA in deionized water, pH 7.4 or 1% BSA in deionized water, pH 7.4); 10 mM borate, 3% BSA, 1% PVP40, and 0.25% Tween-100; and so on.
    [085] The conjugate pad and the test membrane can be placed in contact with any of the buffers described here in the presence or absence of a capture reagent and, in some embodiments, allowed to dry.
    [086] Examples of buffers that are lysis buffers include, for example, but are not limited to, 2% Tween (v / v) and 0.1% Triton (v / v); 2% Tween (v / v) and 0.1% SDS (w / v); 2% Tween (v / v) and 0.1% BSA (w / v); 2% Tween (v / v) and 1% BSA (w / v), 0.1% SDS (w / v), 1% BSA (w / v), or any combination of these. The lysis buffers can also be, for example, 5% Tween / PBS; 2% Tween / PBS + 0.1% SDS; 2% Tween / PBS + 1% BSA. Other examples of lysis buffers include, but are not limited to, 5% Tween-80 (v / v); 5% Triton X-100 (v / v); 5% NP40 (v / v); 2% Tween-80 (v / v); 2% Triton X-100 (v / v); 2% NP40 (v / v); 1% Tween-80 (v / v); 1% Triton X-100 (v / v); and 1% NP40 (v / v). Detergents and other buffer components can be made with any suitable protein-appropriate buffer, and include, but are not limited to, water and phosphate buffered saline. The lysis buffers can be used to prepare the samples before the samples make contact with the devices described here. In some embodiments, a lysis buffer is not used. A lysis buffer is not used in a sample when a surface protein or surface antigen is desired to be detected. Consequently, in some embodiments, the sample is not subjected to lysis or the conditions that would induce a cell to be subjected to lysis.
    [087] The present invention also provides methods of detecting an antigen which comprises contacting a sample with a device as described herein, in which the sample comes into contact with the conjugate pad and the test membrane, in which a positive reaction with the test membrane it indicates the presence of the antigen, where the conjugate pad comprises a first antigen-specific capture reagent and the test membrane comprises a second antigen-specific capture reagent. The positive reaction is indicated by the capture reagent present on the test membrane that binds to an antigen in the test sample. The capture reagent on the test membrane is applied to the test membrane so that it will indicate a positive reaction when it binds to your specific antigen. The specific capture reagent can be applied in any way such that when detected, it can form a line, a circle, a adding sign, a broken line, an "X" or any other pattern. In some embodiments, the control line that indicates that the device is functioning correctly will cross the antigen-specific line and when the antigen-specific capture reagent binds to the antigen, the detectable label will form a sum signal.
    [088] In some embodiments, a sample comes into contact with the device, which is then followed by a plug being applied to the device after the sample has come in contact with the device. For example, a sample comprising an antigen can be brought into contact with the conjugate pad in such a way that the sample is transferred to the conjugate pad. After contact with the conjugated cushion a separate solution can be applied to the device to facilitate or initiate vertical flow through the devices described here.
    [089] In some embodiments as described herein the capture reagent is an antibody. In some embodiments, the sample that is tested is a solution, but it can also be a mixture of solution or buffer and solid material that can be applied to the device. The solution will then solubilize the antigen and allow the conjugate pad capture reagent to come into contact with the antigens present in the sample. In some embodiments, the sample comprises a cell lysate. In some embodiments, the cell lysate has been clarified by centrifugation or other means to remove non-soluble materials.
    [090] In some embodiments, the methods comprise contacting a test sample with a sample collector and contacting the sample collector with the device. In some embodiments, the methods comprise bringing the sample collector into contact with a solution or buffer, in which the solution or buffer is applied to the device. In some embodiments, samples are placed in contact with the conjugate pad before the sample comes in contact with the test membrane. In some embodiments, the sample is placed in contact with the conjugate pad and the test membrane simultaneously.
    [091] In some embodiments, the method comprises moving the conjugate pad of the devices described herein, in which the movement of the devices exposes the test membrane for detection. In some embodiments, the locking member moves the conjugate pad. In some embodiments, the conjugate pad is connected to the locking member and / or the slide button member. The antigen on which the method can be used to detect can be any antigen. The antigen can be those that are discussed in this document or any other antigen that can be detected using the methods and devices described here. In some embodiments, the method comprises applying the sample to the device and allowing the sample to flow through the device through vertical flow.
    [092] In some embodiments, the detection or indication of the presence or absence of an antigen occurs in less than 60 seconds. In some embodiments, the detection or indication of the presence or absence of an antigen occurs in about 30 to about 60 seconds. In some embodiments, the detection or indication of the presence or absence of an antigen occurs in less than 2 minutes. In some embodiments, the detection or indication of the presence or absence of an antigen occurs in about 30 seconds.
    [093] Referring to the drawings, in some embodiments, Figures 1 to 10, represent the representative devices, the components of a device, and various views of a device. Figure 1 represents a device comprising a first housing member (10), a buffer container (15), a second housing member (20), a slot for the slide button (25), a slide button (30), an inlet opening (35), a collar (40), and a test membrane (45). Figure 1 represents a test membrane (45) comprising two capture reagents. The first (10) and second (20) housing members can also be referred to as the lower and upper housing members, respectively. In Figure 1, the sample must be applied through the inlet opening (35) and can be allowed to flow vertically through the test membrane (45). In Figure 1, the groove (25) allows the slide button to move, which when connected to the locking member moves the locking member and can, in some embodiments, move the coupled pad and change the position of the power member .
    [094] Figure 2 represents a device comprising a first housing member (10), a second housing member (20), a slot for the slide button (25), a slide button (30), an inlet opening (35), a collar (40), a test membrane (45), a conjugate cushion (50), a plurality of absorbent members (for example, cushions) (55), a connecting member (60), a locking member (65), and a force member (70). Figure 2 represents the conjugate pad (50), the test membrane (45) and the absorbent pad (55) arranged substantially in parallel with each other. The force member (70) when in contact with the absorbent member would be the application of pressure which is substantially perpendicular to the conjugate pad. As can be seen in Figure 2, a sample that is placed in contact with the device through the inlet opening (35) would flow vertically through the conjugate pad (50) to the test membrane (45). Not explicitly shown in Figure 2, but in some embodiments, a permeable membrane is also substantially parallel to the conjugate pad (50) and the test membrane (45), with a first surface of the permeable membrane that comes in contact with a surface of the conjugate pad (50) a second surface of the permeable membrane that comes in contact with a surface of the test membrane (45).
    [095] Figure 3 represents a conjugate pad (45), a permeable membrane (75), a test membrane (45), and a plurality of absorbent members (55). Figure 3 represents the components being substantially in parallel with each other. Figure 3 represents the permeable membrane (75) comprising an opening. This opening can be used to allow visualization and detection of the test membrane results.
    [096] Figure 4 represents a device comprising a first housing member (10), a buffer container (15), a second housing member (20), a slide button (30), a test membrane (45), a conjugate pad (50), a permeable membrane (75), a plurality of absorbent members (e.g., pads) (55), a connecting member (60), a locking member (65), and a force member (70). Figure 4 also represents the force member (70) comprising an axis (72) and a point (71) where the point (71) is larger than the axis (72).
    [097] Figure 5 represents a partial view of a device comprising a first housing member (10), a locking member (65), a slide button (30), and a force member (70). Figure 5 represents the locking member (65) in contact with the power member (70) such that the power member (70) is in an elevated method. Figure 5 also represents the movement of the locking member (65) and the slide button (30) away from the power member (70), which allows the power member to change positions. In some embodiments, the change in position is that the strength member is reduced.
    [098] Figure 6 represents a side section out of sight of a device comprising a first housing member (10), a second housing member (20), a slide button (30), a locking member (65) , a collar (40), an O ring (41), a strength member (70), and a support for the strength member (73). The support for the axle can be, for example, part of the first housing member (10) and is protected differently, for example, for exclusive purposes. Figure 6 represents the button (30) in contact with the locking member (65) in such a way that the movement of the button (30) will move the locking member (65). The movement of the locking member (65) will remove the support from the power member (70), which would allow the power member (70) to change positions. Figure 6 also represents the axis (72) and the tip (71) of the power member. The tip (71) creates an edge where the locking member (65) can slide and support the power member (70).
    [099] Figure 7 represents a partial view of a device comprising a first housing member (10), a second housing member (20), an inlet opening (35), a test membrane (45), a conjugated cushion (50), a plurality of absorbent members (55), a connecting member (60), a locking member (65), and a force member (70). Figure 7 represents the connecting member (60) connected to the conjugate pad (50) and the locking member (65). Figure 7 also represents the conjugate pad being compressed against the second housing member (20) and the perimeter of the inlet opening (35). Figure 7 represents the tip of the force member (71) that applies pressure through contact with the plurality of absorbent members (55). In Figure 9, a sample can be applied to the device through the inlet opening (35), so that the sample comes in contact with the conjugate pad (50) and because of the sample pressure through the vertical flow it comes in contact with the test membrane (45).
    [0100] Figure 8 represents a partial view of a device comprising a first housing member (10), a second housing member (20), an inlet opening (35), a test membrane (45), a conjugated cushion (50), a plurality of absorbent members (55), a connecting member (60), a locking member (65), and a force member (70). Figure 8 represents the movement of the locking member (65), which is connected to the connecting member (60). The movement of the connecting member (60), which is connected to the conjugate pad (50) moves the conjugate pad. Figure 8 represents the test force member (70) that changes positions and that decreases or eliminates the pressure and / or compression of the test membrane (45). Figure 9 also represents the movement of the conjugate pad (50) away from the inlet opening (35), revealing the test membrane (45) for visualization and / or detection.
    [0101] Figure 9 represents a connecting member (60) connected to a conjugate pad (50). Figure 9 represents notches (51) in the conjugate pad (50) as locations for the connecting member (60) to attach. The connecting member can also be connected by other means such as adhesives, clips and other forms of connection.
    [0102] Figure 10 represents a partial view of the device comprising a second housing member (20), a plurality of pads or membranes (80), wherein the plurality of pads comprises a test membrane, a permeable membrane and a or more absorbent members, and the retaining members (85) that can retain the plurality of pads or membranes (80). Figure 10 represents the structures that when the conjugate pad is moved, the plurality of pads remain in place. Any means or other structure can be used to hold the plurality of cushions in place.
    [0103] The invention is now described with reference to the following examples. These examples are provided for illustrative purposes only, and the invention should in no way be construed as being limited by these examples, but it must undoubtedly be construed to cover any and all variations that become evident as a result of the teachings provided here. Those of skill in the art will easily recognize a variety of non-critical parameters that can be altered or modified to produce essentially similar results. EXAMPLES
    [0104] The antibody specific for E. coli 0157: H7 conjugated to colloidal gold was cooked and dried on the conjugate pad. A second antibody specific for E. coli 0157: H7 and an anti-antibody was streaked on a test membrane and mounted on an antigen detection device.
    [0105] A sample containing LPS E. coli 0157 was serially diluted in PBS with concentrations of 100 μg / ml, 50 μg / ml, 25 μg / ml, 12.5 μg / ml, 6.25 μg / ml, 3.125 μg / ml, 1.56 μg / ml, and 0.78 μg / ml. The samples were applied to the device to detect the presence of E. Coli 0157 LPS. The experiments were classified based on signal strength and the results are shown below. PBS was used as a negative control. TL refers to the test line (antigen specific) and CL refers to the control line (non-antigen specific). Detection occurred within 30 to 60 seconds of applying the sample to the conjugate pad. The device can detect the presence of a food-borne antigen.

    [0106] The disclosures of each and all patents, patent applications, publications and access numbers cited herein are hereby incorporated by reference in their entirety.
    [0107] Although this invention has been disclosed with reference to specific embodiments, it is evident that other embodiments and variations of this invention can be devised by others skilled in the art without departing from the true spirit and scope of the invention. The attached claims are intended to be interpreted to include all such embodiments and equivalent variations.
    权利要求:
    Claims (34)
    [0001]
    1. Device for detecting a target molecule characterized by the fact that it comprises: a housing comprising an inlet opening in fluid contact with a conjugated pad; a force member; a sliding locking member contacting the power member; an attachment member contacting the power member; a sliding button contacting the attachment member; and a detection membrane system comprising the conjugate pad, a test membrane, and an absorbent member, in which at least a portion of the conjugate pad, test membrane, and absorbent member are parallel to each other, in which the member of force contacts the detection membrane system and is able to apply pressure perpendicular to the detection membrane system, and in which the slide button moves the slide locking member.
    [0002]
    2. Device according to claim 1, characterized by the fact that the attachment member contacts the conjugated cushion.
    [0003]
    Device according to claim 1, characterized by the fact that the detection membrane system still comprises a permeable membrane.
    [0004]
    4. Device according to claim 1, characterized by the fact that the detection membrane system is compressed by the force member.
    [0005]
    5. Device according to claim 1, characterized in that it additionally comprises a hydrophobic membrane located between the test membrane and the absorbent membrane.
    [0006]
    6. Device according to claim 1, characterized by the fact that the attachment member is a flexible attachment member.
    [0007]
    7. Device according to claim 1, characterized by the fact that the conjugate pad comprises a first capture reagent that binds to the target molecule.
    [0008]
    8. Device according to claim 7, characterized by the fact that the first capture reagent is a first antigen-specific antibody.
    [0009]
    9. Device according to claim 7, characterized by the fact that the target molecule is a polynucleotide, a peptide, a protein, a saccharide, or a carbohydrate.
    [0010]
    10. Device according to claim 8, characterized by the fact that the first antigen-specific antibody is a polyclonal antibody, a monoclonal antibody, a chimeric antibody, an Fc fragment or a single chain antibody.
    [0011]
    11. Device according to claim 7, characterized by the fact that the target molecule is a pathogenic protein or a fragment thereof.
    [0012]
    12. Device according to claim 7, characterized by the fact that the target molecule is a target molecule of a food-borne pathogen.
    [0013]
    13. Device according to claim 12, characterized by the fact that the target molecule of the foodborne pathogen is a polynucleotide.
    [0014]
    14. Device according to claim 7, characterized by the fact that the target molecule is a foodborne pathogen antigen.
    [0015]
    15. Device, according to claim 14, characterized by the fact that the foodborne pathogen antigen is an antigen of a species of E. coli, a species of Campylobacter, a species of Listeria or a species of Salmonella.
    [0016]
    16. Device according to claim 7, characterized in that the first capture reagent additionally comprises colloidal gold, a fluorescent molecule, a radioactive label, an infrared molecule, or a chemiluminescent substrate.
    [0017]
    17. Device according to claim 1, characterized in that the conjugate pad comprises a plurality of capture reagents.
    [0018]
    18. Device according to claim 17, characterized by the fact that the plurality of capture reagents is a plurality of antibodies specific to antigen.
    [0019]
    19. Device according to claim 18, characterized by the fact that the plurality of antigen-specific antibodies bind to different antigens.
    [0020]
    20. Device according to claim 1, characterized in that the test membrane comprises a second capture reagent.
    [0021]
    21. Device according to claim 20, characterized by the fact that the second capture reagent is an antigen-specific antibody.
    [0022]
    22. Device according to claim 20, characterized by the fact that the target molecule is a target molecule of E. coli, Campylobacter, Listeria or Salmonella.
    [0023]
    23. Device according to claim 1, characterized in that the test membrane comprises a plurality of capture reagents.
    [0024]
    24. Device according to claim 23, characterized by the fact that the plurality of capture reagents bind to different target molecules.
    [0025]
    25. Device according to claim 24, characterized by the fact that the plurality of capture reagents is a plurality of antigen-specific antibodies.
    [0026]
    26. Device according to claim 25, characterized by the fact that the plurality of antibodies specific to antigen binds to different antigens.
    [0027]
    27. Device according to claim 1, characterized in that the test membrane comprises a plurality of areas comprising a plurality of capture reagents.
    [0028]
    28. Device according to claim 27, characterized by the fact that the plurality of capture reagents bind to different target molecules, in which the different target molecules are each independently chosen from E. coli, a Campylobacteria, Listeria and Salmonella.
    [0029]
    29. Device according to claim 1, characterized by the fact that the housing comprises a first housing member and a Second housing member.
    [0030]
    30. System characterized by the fact that it comprises a device as defined in claim 1 and a buffer container or a sample collector.
    [0031]
    31. Kit, characterized by the fact that it comprises a device as defined in claim 1 and one or more of a positive control, a negative control, an instruction manual, a buffer container, or a sample collector.
    [0032]
    32. Method for detecting a target molecule, characterized by the fact that it comprises: contacting a sample with the conjugate pad of the device as defined in claim 7, in which the attachment member contacts the conjugate pad, in which the sample flows vertically from the pad conjugate to the test membrane; moving the conjugate pad after a portion of the sample has contacted and drained through the conjugate pad, thereby exposing at least a portion of the test membrane for detection; and identifying a positive or negative reaction for the target molecule.
    [0033]
    33. Method according to claim 32, characterized by the fact that the sample is contacted with the conjugated pad prior to compression of the detection membrane system.
    [0034]
    34. Method according to claim 32, characterized in that the conjugate pad is moved by the movement of the sliding locking member.
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    同族专利:
    公开号 | 公开日
    WO2011014763A1|2011-02-03|
    AU2010278857A1|2012-03-15|
    US8476082B2|2013-07-02|
    BR112012002308A2|2016-05-31|
    EP2460009B1|2015-03-04|
    JP5711740B2|2015-05-07|
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    US20170082613A1|2017-03-23|
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    KR20120063470A|2012-06-15|
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    US20120220054A1|2012-08-30|
    JP2013501223A|2013-01-10|
    SI2460009T1|2015-07-31|
    EP2460009A4|2012-12-26|
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    KR101768876B1|2017-08-17|
    PT2460009E|2015-04-29|
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    EP2460009A1|2012-06-06|
    US8183059B2|2012-05-22|
    BR112012002308B8|2021-07-27|
    RU2012107315A|2013-09-10|
    CN102576021A|2012-07-11|
    PL2460009T3|2015-08-31|
    US20110027908A1|2011-02-03|
    US20130288263A1|2013-10-31|
    IL217769A|2016-09-29|
    US9341624B2|2016-05-17|
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    AU2010278857B2|2014-07-17|
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    法律状态:
    2019-07-16| B07A| Technical examination (opinion): publication of technical examination (opinion) [chapter 7.1 patent gazette]|
    2020-11-24| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2021-02-02| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 10 (DEZ) ANOS CONTADOS A PARTIR DE 02/02/2021, OBSERVADAS AS CONDICOES LEGAIS. |
    2021-07-27| B16C| Correction of notification of the grant|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 30/07/2010, OBSERVADAS AS CONDICOES LEGAIS. PATENTE CONCEDIDA CONFORME ADI 5.529/DF, QUE DETERMINA A ALTERACAO DO PRAZO DE CONCESSAO |
    优先权:
    申请号 | 申请日 | 专利标题
    US12/533,721|2009-07-31|
    US12/533,721|US8012770B2|2009-07-31|2009-07-31|Device for detection of antigens and uses thereof|
    PCT/US2010/043889|WO2011014763A1|2009-07-31|2010-07-30|Device for detection of antigens and uses thereof|
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