Lateral flow device and method of use

Abstract

A lateral flow device, method and kit for performing a lateral flow Western blot assay are provided. The lateral flow device comprises a wicking pad composed of a porous material having a region for applying a substrate comprising immobilized analytes, wherein the wicking pad has a first end, a second end, and two side edges; a first reservoir comprising a stack of multiple reagent layers located on the first end of the wicking pad; a second reservoir comprising an absorbent pad located on the second end of the wicking pad.

Description

[0001] This application claims the benefit of U.S. provisional application 62 / 366,496, filed July 25, 2016, which is incorporated by reference herein in its entirety. BACKGROUND

[0002] Methods for detecting immobilized analytes are frequently used in the biological sciences. For example, conventional blots (e.g., Southern, Northern, Western, Far Western, Eastern, Vacuum, Middle Eastern, Eastern-Western, and Far-Eastern blots, etc.) can be used to detect analytes immobilized on a substrate or membrane or in a matrix (e.g., in agarose or acrylamide). Generally, such blotting techniques involve immobilizing the analyte to be detected and contacting the analyte with a binding reagent (e.g., an antibody). Blots also typically include a plurality of wash steps and / or blocking steps between immobilization and final detection. Such wash and blocking steps consume the limited time and / or reagents of the practitioner and can be a source of error and irreproducibility. SUMMARY

[0003] Provided herein are lateral flow devices and methods of use.

[0004] In one embodiment, the device comprises a wicking pad composed of a porous material having a region for applying a substrate comprising an immobilized analyte (e.g., a protein), wherein the wicking pad has a first end, a second end, and two side edges; a first reservoir comprising a stack of a plurality of reagent layers on the first end of the wicking pad; a second reservoir comprising an absorbent pad on the second end of the wicking pad. In some embodiments, each of the plurality of reagent layers comprises a reagent immobilized in the absorbent pad. In some embodiments, each reagent layer comprises a thin barrier layer coated or bound on a portion of the lower surface. In certain embodiments, each of the plurality of reagent layers comprises a reagent in solution. In certain embodiments, each of the plurality of reagent layers comprises a density agent. In some embodiments, the density agent is selected from the group consisting of glycerol, sucrose, trehalose, dextran, and polyethylene glycol. In certain embodiments, each of the plurality of reagent layers has a different reagent therein.

[0005] In some embodiments where each of the plurality of reagent layers comprises a reagent in solution, the first reservoir is a cylinder or a trough having a fluid flow controller at the first end to control release of the solution from the first reservoir. In certain embodiments, the fluid flow controller is a valve. In some embodiments, the fluid flow controller is a slit having a width in the range of about 0.5 mm to about 2 mm. In some embodiments, the width of the slit is about 0.5 mm or about 1 mm.

[0006] In certain embodiments, the reagents in the reagent layers are selected from the group consisting of primary antibodies, secondary antibodies, first wash solution, and second wash solution. In some embodiments, the plurality of reagent layers, starting with the reagent layer in contact with the wicking pad, comprises a first reagent layer with a primary antibody, a second reagent layer with a first wash solution, a third reagent layer with a secondary antibody, and a fourth reagent layer with a second wash solution. In some embodiments, the plurality of reagent layers, starting with the reagent layer at or near the first end of the first reservoir, comprises a first reagent layer with a primary antibody, a second reagent layer with a first wash solution, a third reagent layer with a secondary antibody, and a fourth reagent layer with a second wash solution. In certain embodiments, the fourth reagent layer has a thickness that is at least twice that of the third reagent layer. In some embodiments, the first reservoir has a fifth reagent layer comprising a second wash solution. In certain embodiments, the volume of the second wash solution in the first reservoir is at least twice the volume of the secondary antibody.

[0007] In embodiments in which each of the plurality of reagent layers is formed from an absorbent pad, at least a portion of the first reagent layer is in intimate contact with the wicking pad.

[0008] In some embodiments having absorbent reagent layers, the device is sealed in a plastic housing. In some embodiments, the plastic housing comprises a molded bottom portion and a planar lid sealed to the bottom portion. In some embodiments, the wicking pad and the reservoirs are dry. In some embodiments, at least one of the wicking pad and the first reservoir is wet.

[0009] In some embodiments having absorbent reagent layers, each of the reagent layers is formed from at least one material selected from the group consisting of cotton, glass fiber, cellulose, cellulose fiber derivative, sintered glass, sintered polymer, sintered metal, and synthetic polymer. In certain embodiments, the synthetic polymer is selected from the group consisting of polyacrylamide, nylon, polypropylene, polyethylene, polystyrene, divinylbenzene, polyvinyl, polyvinylidene fluoride, high density polyvinylidene fluoride, (C2-C6) monoolefin polymer, vinyl aromatic polymer, vinyl aminoaromatic polymer, halogen vinyl polymer, (C1-C6) alkyl (meth) acrylate polymer, (meth) acrylamide polymer, vinylpyrrolidone polymer, vinylpyridine polymer, (C1-C6) hydroxyalkyl (meth) acrylate polymer, (meth) acrylic acid polymer, acrylamidomethylpropane sulfonic acid polymer, N-hydroxyl-containing (C1-C6) alkyl (meth) acrylamide polymer, and acrylonitrile.

[0010] In some embodiments, the substrate is selected from the group consisting of a membrane, glass, plastic, silicon, metal, and metal oxide. In certain embodiments, the membrane is formed from at least one material selected from the group consisting of nitrocellulose, polyvinylidene difluoride, nylon, and polysulfone. In some embodiments, the plastic is selected from the group consisting of polyethylene terephthalate, polypropylene, polystyrene, and polycarbonate.

[0011] Methods of performing a lateral flow assay are also provided. In some embodiments in which the first reservoir is comprised of an absorbent reagent layer, the method comprises: providing a lateral flow device as described above or elsewhere herein; applying a running buffer to the wicking pad; applying a substrate comprising a protein (e.g., a Western blot) to the area for applying a substrate comprising an analyte; optionally wetting the first reservoir with the running buffer; and allowing the running buffer to laterally flow from the first reservoir to the second reservoir such that the reagents in the plurality of reagent layers are sequentially transported in the wicking pad and into contact with the protein on the substrate. In some embodiments, the step of allowing lateral flow comprises allowing the reagents to travel along a tortuous path as they flow through the first reservoir and into the wicking pad.

[0012] In some embodiments in which the device is sealed in a plastic housing and the plastic housing comprises a bottom portion and a lid sealed to the bottom portion, the method further comprises removing the lid and applying the running buffer and the substrate to the wicking pad, followed by placing the lid on the bottom portion to allow lateral flow from the first reservoir to the second reservoir.

[0013] In some embodiments in which the first reservoir is comprised of a plurality of reagent- containing solutions stacked, the method comprises: providing a lateral flow device as described above or elsewhere herein; optionally applying a running buffer to the wicking pad; applying a substrate comprising a protein (e.g., a Western blot) to the area for applying a substrate comprising an analyte; allowing the plurality of reagent layers to laterally flow from the first reservoir to the second reservoir such that the reagents in the plurality of reagent layers are sequentially transported in the wicking pad and into contact with the protein on the substrate.

[0014] In some embodiments, the method further comprises allowing the running buffer or solution to laterally flow from the first reservoir to the second reservoir such that a primary antibody from the first reagent layer binds to its target protein, if present on the substrate, followed by allowing a first wash solution from the second reagent layer to remove unbound primary antibody from the substrate. In some embodiments, the method further comprises allowing the running buffer or solution to laterally flow from the first reservoir to the second reservoir such that a secondary antibody or second detection reagent from the third reagent layer can contact the primary antibody bound to its target protein, if present, on the substrate. In some embodiments, the method further comprises allowing the running buffer or solution to laterally flow from the first reservoir to the second reservoir such that a second wash solution from the fourth reagent layer can remove unbound secondary antibody from the substrate.

[0015] In certain embodiments, the method further comprises applying a substantially uniform pressure to the first reservoir. In some embodiments, the method further comprises applying a substantially uniform pressure to the first and second reservoirs.

[0016] In some embodiments, the method further comprises removing the membrane and detecting binding of the primary antibody to the target protein, if present, after the primary antibody binds to the target protein, if present, and optionally, after the secondary antibody or second detection reagent contacts the primary antibody.

[0017] Kits for performing lateral flow are also provided. In some embodiments, the kits comprise a plurality of absorbent pads for forming the first and second reservoirs and the wicking pad, all of which are described herein. In some embodiments, the kits comprise reagents provided as solutions to be applied by the end user to the absorbent reagent layers (e.g., a primary antibody comprising a label or a binding reagent comprising a primary antibody and a secondary antibody, a wash solution, and / or a running buffer). In some embodiments, the kits comprise one or more absorbent reagent layers, each having a reagent reversibly bound therein. In certain embodiments, some or all of the reagents are dried onto the absorbent reagent layers. In some embodiments, some or all of the reagents are dried onto the absorbent reagent layers or portions thereof in the presence of one or more protein aggregation-modifying agents.

[0018] In some embodiments, the kits further comprise a running buffer for performing lateral flow, and optionally, comprise a blocking agent (e.g., bovine serum albumin, skim milk powder, or casein), a surfactant (e.g., Tween 20 or Triton X-100), a protein aggregation-modifying agent described herein, a macromolecular crowding agent (e.g., dextran, polyethylene glycol, and / or Ficoll), and / or a reagent that facilitates uniform flow of reagents and / or facilitates reactions with molecules on the substrate and minimizes background on the substrate. Additional reagents can be provided in the kit in solid or liquid form. In some embodiments, the kits further comprise instructions for performing the methods described herein. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 A side view of a lateral flow device according to one embodiment is depicted. The lateral flow device comprises a first reservoir having a stack of multiple reagent layers. Each reagent layer is an absorbent pad having a reagent immobilized therein.

[0020] Figure 2A and 2B A lateral flow device according to one embodiment is depicted. In Figure 2A the device is shown in a plastic molded bottom portion of a housing. In Figure 2BThe device is shown in a housing having a plastic molded bottom portion with a lid attached. Figure 2B A Western blot membrane on a wicking pad of the device is also shown.

[0021] Figure 3 A first reservoir of a lateral flow device according to one embodiment is depicted, where each reagent layer is a solution contained in the first reservoir (e.g., a cylinder). The first end of the first reservoir has a fluid flow controller (e.g., a valve) to control the release of the solution from the first reservoir.

[0022] Figures 4A-4C A side view, top view, and bottom view of a first reservoir of a lateral flow device according to one embodiment are depicted, where each reagent layer is a solution contained in the first reservoir (e.g., a trough). Figure 4C A first end of the first reservoir is depicted, having a fluid flow controller (e.g., a slit) to control the release of the solution from the first reservoir.

[0023] Figure 5 A side view of a lateral flow device according to one embodiment is depicted. The lateral flow device includes a first reservoir having a stack of multiple reagent layers. Each reagent layer has a barrier layer adhered or coated on a portion of the lower surface. The barrier layer controls the flow of solution through the reagent layer.

[0024] Figures 6A-10 Immunoblot results using Figure 1 a lateral flow device of 2B are shown. DETAILED DESCRIPTION

[0025] Described herein are lateral flow devices and methods of using such devices that allow for efficient lateral flow detection of proteins immobilized on a substrate (e.g., a Western blot membrane) using specific binding reagents (e.g., antibodies). Lateral flow devices and methods of using such devices have been discovered that sequentially and hands-free deliver different reagents (e.g., specific binding reagents, running buffer, wash solution) to a wicking pad in close contact with a substrate having proteins thereon. In some embodiments, the devices described herein can be assembled in a single-use housing, allowing for an economical and simple assay format.

[0026] I. DEFINITIONS

[0027] The term "analyte" refers to a biomolecule, e.g., a protein, a nucleic acid, a polysaccharide, a lipid, an antigen, a growth factor, a hapten, etc., or a portion thereof. The analyte can be irreversibly immobilized on a surface (e.g., a membrane) and detected as described herein.

[0028] The terms "immobilized" or "embedded" interchangeably refer to molecules (e.g., binding reagents or analytes) that are reversibly or irreversibly immobilized. Reversibly immobilized molecules are immobilized in a manner that allows the molecules or portions thereof (e.g., at least 25%, 50%, 60%, 75%, 80%, or more of the molecules) to be removed from their immobilized location without significant denaturation or aggregation. For example, a solution containing the molecules is contacted with an absorbent material, which absorbs the solution and reversibly immobilizes the molecules, thereby reversibly immobilizing the molecules in or on the absorbent material (e.g., an absorbent pad). The reversibly immobilized molecules can then be removed by wicking the solution from the absorbent material, or by wicking the solution from one region of the absorbent material to another region. In some cases, a solution containing the molecules is contacted with an absorbent material, which absorbs the solution, and then the absorbent material containing the solution is dried, thereby reversibly immobilizing the molecules on the absorbent material. The absorbent material can then be contacted with another solution of the same or different composition, which dissolves the reversibly immobilized molecules, and then the solution is wicked from the absorbent material or wicked from one region of the absorbent material to another region, thereby removing the reversibly immobilized molecules.

[0029] Immobilizing irreversibly immobilized molecules (e.g., binding reagents or analytes) such that they are not or substantially not removed from their location under mild conditions (e.g., a pH of about 4-9, a temperature of about 4-65°C). Exemplary irreversibly immobilized molecules include protein analytes bound to nitrocellulose, polyvinylidene fluoride, nylon, or polysulfone membranes by standard blotting techniques (e.g., electroblotting). Other exemplary irreversibly immobilized molecules include protein analytes bound to glass or plastic (e.g., a microarray, a microfluidic chip, a glass histological slide, or a plastic microtiter plate having wells with bound protein analytes).

[0030] The term "binding reagent" refers to an agent that specifically binds to a molecule (e.g., an analyte). Although antibodies are described in many contexts herein, it is understood that other binding reagents can be used instead of antibodies as preferred by the user. A variety of binding reagents are known in the art, including antibodies, aptamers, affimers, lipocalin proteins (e.g., anticalins), thioredoxin A, bilin-binding proteins, or proteins containing ankyrin repeats, the Z domain of staphylococcal protein A, or fibronectin type III domains.

[0031] The term "specifically binds" refers to the affinity of a molecule (e.g., a binding reagent such as an antibody or antibody fragment) for a target that is at least 2-fold greater, e.g., at least 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25-fold, 50-fold, 100-fold, or 1000-fold or more, than the affinity for a non-target compound.

[0032] The term "antibody" refers to a polypeptide or fragment thereof that comprises a framework region from an immunoglobulin gene, which specifically binds and recognizes an antigen, e.g., a particular analyte. Typically, the "variable region" comprises the antigen binding region (or functional equivalent thereof) of an antibody and is critical to specific binding and affinity. See Paul, Fundamental Immunology (2003). Antibodies include, e.g., chimeric, human, humanized antibodies, or single chain antibodies.

[0033] An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer comprises two identical pairs of polypeptide chains, each pair comprising one "light" (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition. The term light chain variable region (V L ) and heavy chain variable region (V H ) refer to these light and heavy chains, respectively.

[0034] An "isotype" is a class of antibody that is determined by the constant region of the heavy chain. The immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant regions. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the classes of antibodies as IgG, IgM, IgA, IgD, and IgE, respectively.

[0035] Antibodies can exist as full immunoglobulins or any of a number of well-characterized fragments that comprise specific antigen-binding activity. Such fragments can be produced by digestion with various peptidases. Pepsin digests the antibody below the disulfide bridges of the hinge region to produce F(ab)'2, a dimer of Fab which itself is a monomer of the light chain and heavy chain connected by disulfides including part of the variable region. Papain digests the antibody at the single chain linkages at the hinge region to form three Fab fragments, each with a portion of the constant region. H -C HThe F(ab')2 fragment also can be reductively cleaved into a F(ab')2 monomer under mild conditions that break disulfide bonds without cleaving the hinge region between the F(ab')2 dimers, thereby converting the F(ab')2 dimer into a Fab' monomer. The Fab' monomer is essentially an Fab with part of the hinge region (see Fundamental Immunology, Paul ed., 3rd ed. 1993). Although various antibody fragments are defined in terms of the digestion of an entire antibody, one of skill will appreciate that such fragments can be synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies (e.g., single-chain Fv) or those identified using phage display libraries (see, e.g., McCafferty et al., Nature 348:552-554 (1990)).

[0036] As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. As used herein, the term "about" means any value that is within 10% of the stated value. Thus, about "5" means any value that is between 4.5 and 5.5, inclusive of 4.5 and 5.5.

[0037] II. Device

[0038] Figure 1 、 2A FIGS. 1A and IB show one embodiment of a lateral flow device 100 for detecting a protein on a substrate. The lateral flow device 100 includes a wicking pad 102 having a first end 104, a second end 106, two side edges 108, and a region 110 for applying a substrate 112 (e.g., a membrane) containing immobilized analytes (e.g., proteins) to be detected. The lateral flow device 100 also includes a first reservoir 114 located on or at the first end 104 of the wicking pad 102. The first reservoir 114 supplies liquid (e.g., buffer and detection reagents) to the wicking pad 102. In some embodiments, the first reservoir 114 is in intimate contact with the wicking pad 102. The lateral flow device 100 also includes a second reservoir 116 located on or near the second end 106 of the wicking pad 102 and in intimate contact with the wicking pad 102. The second reservoir 116 functions as a pump by wicking liquid from the first reservoir 114 to the dry second reservoir 116.

[0039] The wicking pad 102 is a planar absorbent material on which a substrate 112 containing immobilized analytes (e.g., a Western blot) is placed. The wicking pad 102 can include an illustration / indication or other indication of where the user should place the substrate 112. Alternatively, the illustration / indication can be on the device housing. In some embodiments, the substrate 112 is placed on the wicking pad 102 downstream of the first reservoir 114 and upstream of the second reservoir 116 (e.g., between the first reservoir 114 and the second reservoir 116).

[0040] The wicking pad 102 has a width, a length, and a height (e.g., thickness). The wicking pad 102 can be any size and shape. In certain embodiments, the wicking pad 102 is planar, e.g., the wicking pad 102 can be or approach a rectangular planar shape. In some cases, the length and width of the wicking pad 102 are at least about 2, 5, 10, 100, or more times greater than the height (i.e., thickness). In some embodiments, the wicking pad 102 has an impermeable or substantially impermeable backing.

[0041] Exemplary dimensions of the wicking pad include, but are not limited to, a wicking pad that is at least about 0.25 cm, 0.5 cm, 1 cm, 2 cm, 3 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 10 cm, 12 cm, 15 cm, 20 cm, 30 cm, or more in at least one dimension. Exemplary dimensions of a rectangular planar wicking pad include a wicking pad having a length and width of about 1 cm x 1 cm, 7 x 8.5 cm, 8.5 x 13.5 cm, 10 cm x 15 cm, or 25 x 28 cm. Exemplary dimensions also include 8.5 cm x 9 cm, 7 cm x 9 cm, 8 cm x 10.7 cm, 10 cm x 10 cm, 7 cm x 8.5 cm, 8.3 cm x 7.3 cm, 8 cm x 8 cm, 8.3 cm x 13 cm, 10.8 cm x 13.5 cm. In some embodiments, the wicking pad 102 has a length of 18 cm and a width of 10 cm. In some cases, the wicking pad 102 has a length of 18 ± 0.5, 1, 2, or 3 cm and a width of 10 ± 0.5, 1, 2, or 3 cm.

[0042] In some embodiments, the wicking pad 102 is configured to have a high solution capacity and lateral flow rate. In some cases, the high solution capacity and lateral flow rate are provided by the wicking pad 102 having a substantial height (e.g., thickness). In some cases, the wicking pad 102 has a thickness of about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.75, 0.5, or about 0.2 mm. In some cases, the wicking pad 102 has a thickness of between about 0.05 mm and about 0.5 mm.

[0043] The first reservoir 114 includes a stack of a plurality of reagent layers 118, each reagent layer 118 having a reagent (e.g., a primary antibody, a secondary antibody, a running buffer, and / or a wash buffer) immobilized or embedded therein. In some embodiments, each of the plurality of reagent layers has a different reagent immobilized or embedded therein. The first reservoir 114 is in liquid communication with the wicking pad 102 (i.e., liquid can flow from the first reservoir 114 to the wicking pad 102 when present in the first reservoir 114) and is configured to sequentially deliver reagents from the plurality of reagent layers 118 to the wicking pad 102. The embedded reagents are typically embedded and dried into the absorbent pad of the reagent layer such that they are immobilized until contacted with an aqueous fluid front under lateral flow and released in a user-defined event.

[0044] Each reagent layer 118 has a width Wl, a length LI, and a height Hl (e.g., thickness). In certain embodiments, each reagent layer is planar, e.g., each reagent layer can be or approximate a rectangular plane. In some cases, the length LI and width Wl of each reagent layer is at least about 2, 5, 10, 100, or more times greater than the height (i.e., thickness).

[0045] In the embodiments shown in FIGS. 1A and IB, the first reservoir 114 is a rectangular tank having a plurality of reagent layers 118. In some embodiments, the plurality of reagent layers 118 are absorbent pads having reagents immobilized therein. In certain embodiments, each of the plurality of reagent layers 118 has a different reagent immobilized therein. In some embodiments, the plurality of reagent layers 118 are solutions having reagents therein. In some embodiments, each of the plurality of reagent layers 118 has a different reagent therein. Figure 1 2A In the embodiments shown in FIGS. 1A and IB, the first reservoir 114 is a rectangular tank having a plurality of reagent layers 118. In some embodiments, the plurality of reagent layers 118 are absorbent pads having reagents immobilized therein. In certain embodiments, each of the plurality of reagent layers 118 has a different reagent immobilized therein. In some embodiments, the plurality of reagent layers 118 are solutions having reagents therein. In some embodiments, each of the plurality of reagent layers 118 has a different reagent therein. Figure 3 In the embodiments shown in FIGS. 1A and IB, the first reservoir 114 is a rectangular tank having a plurality of reagent layers 118. In some embodiments, the plurality of reagent layers 118 are absorbent pads having reagents immobilized therein. In certain embodiments, each of the plurality of reagent layers 118 has a different reagent immobilized therein. In some embodiments, the plurality of reagent layers 118 are solutions having reagents therein. In some embodiments, each of the plurality of reagent layers 118 has a different reagent therein.

[0046] In some embodiments, the first reservoir 314 is a rectangular tank having a plurality of reagent layers 318. In some embodiments, the plurality of reagent layers 318 are absorbent pads having reagents immobilized therein. In certain embodiments, each of the plurality of reagent layers 318 has a different reagent immobilized therein. In some embodiments, the plurality of reagent layers 318 are solutions having reagents therein. In some embodiments, each of the plurality of reagent layers 318 has a different reagent therein. Figures 4A-4C ​), each reagent layer 318 is a solution having a reagent therein. The first reservoir 314 includes a first end 320 and a second end 324. The first end 320 has a fluid flow controller 322 (e.g., a slit) to control the release of the solution from the first reservoir 314. In embodiments where the fluid flow controller 322 is a slit, the slit can have a width W2 (e.g., the short dimension) ranging from about 0.5 mm to about 2 mm and a length L2 (e.g., the long dimension) ranging from about 8.5 cm to about 20 cm. In some embodiments, the width W2 is 0.5 mm, 1 mm, or 2 mm. In certain embodiments, the length L2 is 8.5 cm, 9.5 cm, 13.5 cm, or 20 cm. The dimensions of the slit and the viscosity of the reagent solution can affect the rate at which the solution exits the slit and can be adjusted according to user preference. For example, as the width W2 decreases, the rate at which the solution exits the slit also decreases, which increases the overall lateral flow processing time of the device 100.

[0047] The fluid flow controller 322 and / or the opening 326 on the second end 324 can be sealed or covered with a removable metal or plastic foil (e.g., tape).

[0048] Referring again to Figure 1 -2B. In some embodiments, each reagent layer is sized to match the width of the wicking pad 102 and has a width that is at least about 3 times, 4 times, 5 times, 6 times, 8 times, 10 times, 13 times, 17 times, 20 times, 27 times, or more greater than the length.

[0049] Exemplary dimensions of each reagent layer include, but are not limited to, at least about 0.25 cm, 0.5 cm, 1 cm, 2 cm, 2.5 cm, 3 cm, 3.5 cm, 4 cm, 5 cm, 6 cm, 7 cm, 8 cm, 8.5 cm, 9.5 cm, 10 cm, 13.5 cm, 20 cm, or more in at least one dimension. Exemplary dimensions of each rectangular planar reagent layer include, but are not limited to, a length LI and a width Wl of about 0.5 cm x 8.5 cm, 1 cm x 1 cm, 2.5 cm x about 8.5 cm, 2 x 13.5 cm, 3 x 13.5 cm, or 3.5 cm x 20 cm. As used herein, "length LI" is based on the direction of flow and is the shortest dimension. In some embodiments, each reagent layer has a length LI of 3 cm and a width Wl of 10 cm. In some cases, each reagent layer has a length LI of 1 ± 0.5, 1, or 2 cm and a width Wl of 14 ± 0.5 cm.

[0050] In some embodiments, each reagent layer 118 is formed of an absorbent porous material and is configured to have a high solution capacity. In some cases, the high solution capacity is provided by the reagent layer having a substantial height (e.g., thickness). In some cases, the thickness of the reagent layer is about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.75, 0.5, or about 0.2 mm. In some cases, the thickness of the reagent layer is between about 0.05 mm and about 0.5 mm.

[0051] Due to the presence of the plurality of pores, each reagent layer 118 typically has a large surface area. The large surface area can increase the reagent layer's loading capacity for one or more reagents or one or more solutions containing reagents. In some embodiments, the reagent layer 118 has a specific surface area of at least about 0.001 m 2 / g, 0.02 m 2 / g, 0.1 m 2 / g, 0.5 m 2 / g, 1 m 2 / g, 10 m 2 / g, or more, as measured by standard techniques.

[0052] In some embodiments, each reagent layer and / or wicking pad 102 can have a particular pore size, a particular average pore size, or a particular pore size range. For example, each reagent layer and / or wicking pad 102 can comprise pores of 0.1 μιη, pores of 0.2 μιη, pores of 0.45 μιη, or pores of 1, 2, 4, 5, 6, 7, 8, 10, 15, 20 μιη, or pores of greater than about 20 μιη. For another example, each reagent layer and / or wicking pad 102 can comprise pores having an average size of 0.1, 0.2, 0.45, 1, 2, 4, 5, 6, 7, 8, 10, 15, or 20 μιη or greater. For yet another example, each reagent layer and / or wicking pad 102 can comprise pores having a size range of about 0.1-8 μιη, 0.2-8 μιη, 0.45-8 μιη, 1-8 μιη, 0.1-4 μιη, 0.1-2 μιη, 0.1-1 μιη, 0.1-0.45 μιη, 0.2-8 μιη, 0.2-4 μιη, 0.2-2 μιη, 0.2-1 μιη, 0.2-0.45 μιη, 0.45-8 μιη, 0.45-4 μιη, 0.45-2 μιη, 0.45-1 μιη. In some cases, each reagent layer and / or wicking pad 102 can comprise pores having a size of less than about 20 μιη. For example, each reagent layer and / or wicking pad 102 can be composed of a material in which at least about 50%, 60%, 70%, 80%, 90% or more of the pores have a size of less than about 20, 15, 10, or 5 μιη. In some cases, the pores in the reagent layer are large enough to accommodate one or more proteins of normal size (e.g., about 1 nm). For example, the pores can have a size of at least 1 nm, at least 5 nm, at least 10, 100, or 500 nm. Alternatively, at least 50%, 60%, 70%, 80%, 90% or more of the pores can have a size of greater than 1, 5, 10, 50, 100, or 500 nm. As used herein, pore size can be measured in terms of half diameter or diameter. In some cases, each reagent layer and / or wicking pad 102 comprises a porous polyethylene, e.g., a porous polyethylene having a pore size of 0.2 to 20 microns, or 1 to 12 microns. Each reagent layer and / or wicking pad 102 can have different pore sizes in different regions of the pad or layer. For example, the wicking pad 102 can have a lateral flow region having a different pore size or pore size range.

[0053] Each reagent layer and / or wicking pad 102 can be treated or functionalized to minimize non-specific reagent binding, increase lateral flow, increase wicking, or reduce protein aggregation. For example, each reagent layer and / or wicking pad 102 or portions thereof can be treated to alter the hydrophilicity or hydrophobicity of the treated area. In some cases, altering the hydrophilicity or hydrophobicity of a reagent layer can increase binding reagent loading, reduce binding reagent aggregation or denaturation, create a masked zone (where binding reagent is excluded or not loaded), or direct binding reagent flow when the reagent layer is wet. In some cases, a reagent layer contains a protein aggregation modifier as described herein.

[0054] Referring to Figure 5 In some embodiments, each reagent layer 418 has a barrier layer 440 coated or adhered to a portion of the lower surface to control the flow of reagent solution through the first reservoir 414 of the lateral flow device 400. In some embodiments, the barrier layer 440 is positioned such that solution flowing sequentially from the reagent layers 418 follows a tortuous path 442 to the wicking pad 402. For example, some alternating reagent layers can have a barrier layer on substantially the entire lower surface except for a first region 444 of the lower surface proximate the first edge 446. Other alternating reagent layers can have a barrier layer 440 on substantially the entire lower surface except for a second region 448 of the lower surface proximate the second edge 450. In certain embodiments, the barrier layer 440 covers the width of the lower surface of each reagent layer. In some embodiments, the first and / or second region 444, 448 of a reagent layer that does not contain a barrier layer covers the width of the reagent layer 418. In some embodiments, the barrier layer 440 can extend beyond the first edge 446 or the second edge 450 of a reagent layer such that solution will flow through a region that does not have a barrier layer rather than down the edge of a reagent layer between which a barrier layer extends. The thin barrier layer 440 can be formed from materials including, but not limited to, plastic foils, metal foils, glass, and / or wax. Plastic from which the barrier layer 440 can be formed includes, but is not limited to, polyvinylidene chloride, low-density polyethylene, polyethylene terephthalate, polypropylene, polystyrene, and / or polycarbonate. Methods by which the barrier layer 440 can be adhered to the reagent layer 418 can include adhesion with adhesive, heat adhesion, or organic solvent adhesion with or without pressure.

[0055] Each reagent layer can be labeled or marked to record the source, composition, or location of the reversibly immobilized binding reagent (e.g., primary antibody). For example, one or more regions containing reversibly immobilized binding reagent can be macroscopically distinguishable, such that one of skill in the art can determine the location of the reversibly immobilized binding reagent. In some cases, the name (e.g., anti-phospho-PIK3), identification (e.g., catalog number), quantity, batch number, etc. of the binding reagent can be printed, embossed, or otherwise indicated on a portion of the reagent layer. In some cases, each reagent layer is labeled or marked such that the proper orientation for the lateral flow blotting device 100 can be discerned.

[0056] In some embodiments, the plurality of reagent layers 118, 218, 318 includes a first reagent layer containing a primary antibody (e.g., a labeled primary antibody) and a second reagent layer containing a first wash solution. In some embodiments, the reagent layers 118, 218, 318 are distinct and include a first reagent layer containing a primary antibody, a second reagent layer containing a first wash solution, a third reagent layer containing a secondary antibody or a second detection reagent, and a fourth reagent layer containing a second wash solution. Figures 1-4A Figures 1-2B The first layer is in close contact with the wicking pad 102, as shown in FIG. 1A. Figure 3 4A The first layer is a layer at or near the first end 220, 320 of the first reservoir 214, 314, as shown in FIGS. 2A and 3A. In some embodiments, the plurality of reagent layers 118, 218, 318 has two or more layers of the same reagent. In such embodiments having two layers of the same reagent, the first reservoir 114, 214, 314 will include twice the volume of the reagent. For example, in some embodiments, the plurality of reagent layers 118, 218, 318 further includes a fifth reagent layer containing a second wash solution, such that the first reservoir 114, 214, 314 includes two layers or twice the volume of the second wash solution. In embodiments where the reagent layers 118 are formed of an absorbent material, one or more of the reagent layers 118 can be at least twice the thickness of the other layers. For example, the fourth reagent layer having the second wash solution can be at least twice the thickness of the third reagent layer having the secondary antibody.

[0057] Each reagent layer 118, 218, 318 can further include a density agent. Having a density agent in each reagent layer 118, 218, 318 of the first reservoir 114, 214, 314 can keep the reagents in discrete areas to minimize mixing and allow the reagents to be delivered to the wicking pad 102 in a timed, sequential manner. Examples of density agents include, but are not limited to, glycerol, sucrose, trehalose, dextran, and polyethylene glycol.

[0058] ​​Each reagent layer 118, 218, 318 can also include one or more dyes or indicators for monitoring the lateral flow of the test agent from the first reservoir 114 and into / through the wicking pad 102 (e.g., to monitor progress).

[0059] The second reservoir 116 acts as a wicking "pump" for the lateral flow system and includes one or more absorbent pads. In some embodiments, the second reservoir 116 can be the wicking pad 102. The second reservoir 116 is in liquid communication with the wicking pad 102 (i.e., when liquid is present in the wicking pad 102, it can flow from the wicking pad 102 to the second reservoir 116).

[0060] The reagent layer 118, the wicking pad 102, and the second reservoir 116 are typically formed from a water-absorbing material and can be made, for example, from natural fibers, synthetic fibers, glass fibers, or mixtures thereof. Non-limiting examples include cotton, glass, and combinations thereof. There are many commercially available materials for diagnostic use from suppliers such as Ahlstrom, GE, PALL, Millipore, Sartorius, S&S, and the like.

[0061] The water-absorbing material can include, but is not limited to, polymer-containing materials. The polymer can be in the form of polymer beads, polymer membranes, or polymer monoliths. In some cases, the polymer is cellulose. Cellulose-containing pads include paper, cloth, woven or nonwoven cellulose substrates. Cloth pads include those containing natural cellulose fibers such as cotton or wool. Paper pads include those containing natural cellulose fibers (e.g., cellulose or regenerated cellulose) as well as those containing cellulose fiber derivatives including, but not limited to, cellulose esters (e.g., nitrocellulose, cellulose acetate, triacetate cellulose, propionate cellulose, acetate-propionate cellulose, acetate-butryate cellulose, and sulfate cellulose), and cellulose ethers (e.g., methyl cellulose, ethyl cellulose, ethyl methyl cellulose, hydroxyethyl cellulose, hydroxyethyl methyl cellulose, hydroxypropyl methyl cellulose, ethyl hydroxyethyl cellulose, and carboxymethyl cellulose). In some cases, the cellulose pad contains rayon. In some cases, the pad is paper, such as various papers.

[0062] The water-absorbing material can also include, but is not limited to, sintered materials. For example, the water-absorbing material can contain sintered glass, sintered polymer, or sintered metal, or combinations thereof. In some cases, the sintered material is formed by sintering one or more powdered glasses, powdered polymers, or powdered metals. In other cases, the sintered material is formed by sintering one or more glass, metal, or polymer fibers. In other cases, the sintered material is formed by sintering one or more glass, polymer, or metal beads.

[0063] The absorbent material may also contain, but is not limited to, one or more non-cellulose polymers, such as synthetic polymers, natural polymers, or semi-synthetic polymers. For example, the material may contain polyesters, such as polyglycolic acid, polylactic acid, polycaprolactone, polyethylene adipate, polyhydroxyalkanoates, polyhydroxybutyrate, poly(3-hydroxybutyrate-co-3-hydroxyvalerate), polyethylene terephthalate, polybutylene terephthalate, 1,3-propylene terephthalate, and polyethylene naphthalate. In some cases, the polymer is spunbond, such as spunbond polyester.

[0064] Other synthetic polymers include, but are not limited to: nylon, polypropylene, polyethylene, polystyrene, divinylbenzene, polyvinyl ether, polydifluoroethylene, high-density polydifluoroethylene, polyacrylamide, (C2-C6) monoolefin polymers, vinyl aromatic polymers, vinyl amino aromatic polymers, halogenated vinyl polymers, (C1-C6) alkyl (meth)acrylate polymers, (meth)acrylamide polymers, vinylpyrrolidone polymers, vinylpyridine polymers, (C1-C6) hydroxyalkyl (meth)acrylate polymers, (meth)acrylic acid polymers, acryloylaminomethylpropanesulfonic acid polymers, N-hydroxyl-containing (C1-C6) alkyl (meth)acrylamide polymers, acrylonitrile, or mixtures of any of the foregoing substances.

[0065] In some embodiments, the reagent layer 118 is formed of a non-absorbent material in which the liquid flows by capillary action. Such materials include, but are not limited to, high-density or ultra-high molecular weight polyethylene sheets manufactured by Porex Technologies Corp.

[0066] In some embodiments, the wick pad 102 may be backed to prevent flow on the bottom surface. This can be achieved, for example, by using an adhesive backing that adheres to the wick pad. The properties of the adhesive may affect test performance (i.e., flow characteristics, reagent stability, etc.), so the adhesive can be optimized for the desired test. In some embodiments, the adhesive may be part of the molded bottom portion of the device 100.

[0067] The substrate 112 is generally planar in shape and can be, for example, a membrane formed from nitrocellulose, polyvinylidene fluoride, nylon, or polysulfone. Other materials that can form the substrate 112 include, but are not limited to, glass, plastic, silicon, metal, and / or metal oxide, either bare or functionalized with a polymer. Plastic materials that can form the substrate 112 include, but are not limited to, polyethylene terephthalate, polypropylene, polystyrene, and / or polycarbonate. Examples of polymers used to functionalize the surface of a substrate formed from a metal or metal oxide include glycidoxypropyltriethoxysilane; poly-L-lysine; polybrene; polyethylene glycol polymers; dextran polymers; aminopropylsilane; caroxysilane; hydrogels and polymer brushes; and / or self-assembled monolayers of, for example, functionalized alkyl mercaptans, dendrimers, or oligonucleotides.

[0068] The wicking assembly can be contained and sealed in a waterproof housing 120. In some embodiments, the housing 120 will be plastic or other inexpensive waterproof material. The housing 120 can be, for example, vacuum molded or injection molded or otherwise constructed. In some embodiments, the housing 120 includes a molded bottom portion and a generally planar lid or plate that mates with (e.g., snap fits with) the bottom portion. Figure 2B One example of such a housing 120 is shown. In this embodiment, the wicking pad, as well as the first and second reservoirs 114, 116, are contained in a hole or portion of the molded bottom portion. In some embodiments, the housing 120 (e.g., the bottom portion and / or the lid) does not contact the wicking pad 102. In certain embodiments, the bottom portion is planar and the first and second reservoirs 114, 116 are enclosed by a molded lid. In some embodiments, the lid is molded such that when the lid is attached to the bottom portion, the lid contacts the first reservoir 114 and exerts an even and downward force on the first reservoir 114. The even and downward force exerted across the first reservoir 114 results in even contact between the reagent layers 118. In certain embodiments, the lid is provided in more than one section. For example, the lid can include a first section and a second section. The first section can be removable and can cover the first reservoir 114 and the substrate area 110 of the device 100. The second section can cover the second reservoir 116 and can be removable or can be welded to the bottom portion.

[0069] A. Exemplary Detection Reagents

[0070] i. Binding reagent

[0071] Binding reagents are described herein for detecting an analyte. In some cases, the binding reagent is an antibody (e.g., a primary or secondary antibody). A primary antibody can be used to bind to an analyte. In some cases, the primary antibody is labeled such that the primary antibody, and thus the analyte, can be detected. In some cases, the primary antibody is detected by binding to a labeled secondary binding reagent, such as a labeled secondary antibody. In some cases, a tertiary binding reagent is used to detect a complex containing the analyte and the primary binding reagent and the secondary binding reagent.

[0072] Binding reagents can be provided on or in one or more reagent layers 118, 218 in the first reservoir 114, 214, or can be supplied separately. In some cases, the reagent layer 118 contains one or more binding reagents dried thereon. The dried binding reagent can be reconstituted by contacting the reagent layer 118 with an aqueous solution. In some cases, the aqueous reconstitution buffer can contain one or more re-wetting reagents, including a salt, a buffer, or a protein aggregation modifier as described herein. Alternatively, the binding reagent can be present in solution, and the binding reagent reversibly immobilized in the reagent layer 118 by immersing the reagent layer in the solution. In some cases, the binding reagent is stored in the reagent layer 118, 218 (e.g., in the first reservoir 114, 214). For example, the binding reagent can be stored dry, substantially dry, or in solution in the reagent layer for at least about 1 day, 3 days, 7-10 days, at least about 1 month, 2 months, 3 months, 6 months, 1 year, or more. In some cases, the binding reagent and the reagent layer are adapted to be stored (e.g., at about 4, 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, 22, 25, 30, 35, or 37 °C or higher) for at least about 1 day, 3 days, 7-10 days, at least about 1 month, 2 months, 3 months, 6 months, 1 year, or more.

[0073] ii. Label

[0074] The analyte can be detected by detecting a label attached to the binding reagent. The label can be attached directly to the binding reagent (e.g., by a covalent or other bond to the primary antibody) or can be attached indirectly (e.g., using a chelator or linker molecule). The terms "label" and "detectable label" are used synonymously herein. In some embodiments, each label (e.g., a first label attached to a first binding reagent, a second label attached to a second binding reagent, etc.) generates a detectable signal and the signals (e.g., a first signal generated by the first label, a second signal generated by the second label, etc.) are distinguishable. In some embodiments, the two or more binding reagent labels comprise the same type of reagent (e.g., a first fluorescent reagent as a first label and a second fluorescent reagent as a second label). In some embodiments, the two or more binding reagent labels (e.g., a first label, a second label, etc.) collectively generate a detectable signal that is not generated in the absence of one or more of the labels.

[0075] Examples of detectable labels include, but are not limited to, biotin / streptavidin labels, nucleic acid (e.g., oligonucleotide) labels, chemically reactive labels, fluorescent labels, enzyme labels, radioactive labels, quantum dots, polymer dots, mass labels, colloidal gold, and combinations thereof. In some embodiments, the labels can include optical reagents such as chromophores, fluorescent reagents, phosphorescent reagents, chemiluminescent reagents, and the like. A variety of reagents (e.g., dyes, probes, or indicators) are known in the art and can be used in the present application. (See, e.g., Invitrogen, The Handbook—A Guide to Fluorescent Probes and Labeling Technologies, 10th Edition (2005)). Chromophores include coenzymes or cofactors that have a detectable absorbance. In some cases, the binding reagent can be detected by detecting the intrinsic absorbance of the peptide bond at, e.g., 220 or 280 nm.

[0076] Fluorescent agents can include various organic and / or inorganic small molecules or various fluorescent proteins and derivatives thereof. For example, fluorescent agents can include, but are not limited to, cyanines, phthalocyanines, porphyrins, indocyanines, rhodamines, phenoxazines, phenylxanthenes, phenothiazines, phenoselenazines, fluoresceins (e.g., FITC, 5-carboxyfluorescein, and 6-carboxyfluorescein), benzoporphyrins, squarilium dyes, dipyrrolyprymidine ketones, naphthacenes, quinolines, pyrazines, corroles, azinones, phenanthridines, rhodamines (e.g., TAMRA, TMR, and rhodamine red), acridines, anthraquinones, chalcogenopyrylium analogs, chlorins, naphthalocyanines, methine dyes, indolium dyes, azo compounds, azulene blue, azulenium blue, triphenylmethane dyes, indoles, benzindoles, indole carbocyanines, benzindole carbocyanines, BODIPY TM and BODIPY TM derivatives, and analogs thereof. In some embodiments, the fluorescent agent is an Alexa Fluor dye. In some embodiments, the fluorescent agent is a polymer dot or a quantum dot. Fluorescent dyes and fluorescent labeling reagents include those commercially available from, for example, Invitrogen / Molecular Probes (Eugene, OR) and Pierce Biotechnology, Inc. (Rockford, IL). In some embodiments, the optical reagent is an intercalating dye. In some embodiments, 2, 3, 4, 5, or more binding reagents are each labeled with an optical reagent such as a fluorescent agent (e.g., a first binding reagent is labeled with a first fluorescent label, a second binding reagent is labeled with a second fluorescent label, etc.), and each binding reagent labeled with an optical reagent is detected by detecting a signal generated by the optical reagent (e.g., a fluorescent signal generated by a fluorescent label). In some embodiments, all of the binding reagents are labeled with an optical reagent, and each binding reagent labeled with an optical reagent is detected by detecting a signal generated by the optical reagent.

[0077] In some embodiments, the label is a radioisotope. Radioisotopes include radionuclides that emit gamma rays, positrons, beta and alpha particles, and X-rays. Suitable radionuclides include, but are not limited to: 225 Ac、 72 As、 211 At、 11 B、 128 Ba、 212 Bi、 75 Br、 77 Br、 14 C、 109 Cd、 62 Cu、 64 Cu、67 Cu, 18 F, 67 Ga, 68 Ga, 3 H, 166 Ho, 123 I, 124 I, 125 I, 130 I, 131 I, 111 In, 177 Lu, 13 N, 15 O, 32 P, 33 P, 212 Pb, 103 Pd, 186 Re, 188 Re, 47 Sc, 153 Sm, 89 Sr, 99m Tc, 88 Y and 90 Y. In some embodiments, 2, 3, 4, 5, or more binding agents are each labeled with a radioisotope (e.g., a first binding agent is labeled with a first radioisotope, a second binding agent is labeled with a second radioisotope, etc.), and each binding agent labeled with a radioisotope is detected by detecting the radiation generated by the radioisotope. For example, one binding agent can be labeled with a gamma emitter, and one binding agent can be labeled with a beta emitter. Alternatively, the binding agents can be labeled with radionuclides that emit the same particle (e.g., alpha, beta, or gamma) at different energies, where the different energies are distinguishable. In some embodiments, all of the binding agents are labeled with a radioisotope, and each labeled binding agent is detected by detecting the radiation generated by the radioisotope.

[0078] In some embodiments, the label is an enzyme, and the binding reagent is detected by detecting a product generated by the enzyme. Examples of suitable enzymes include, but are not limited to, urease, alkaline phosphatase, (horseradish) hydrogen peroxidase (HRP), glucose oxidase, beta-galactosidase, luciferase, alkaline phosphatase, and an esterase that hydrolyzes fluorescein diacetate. For example, a horseradish peroxidase detection system can be used with a chromogenic substrate tetramethylbenzidine (TMB), which produces a soluble product detectable at 450 nm in the presence of hydrogen peroxide, or with a chemiluminescent substrate (e.g., Clarity from Bio-Rad Laboratories), which produces detectable light. An alkaline phosphatase detection system can be used with a chromogenic substrate p-nitrophenyl phosphate, which produces a soluble product that is easily measured at 405 nm. A beta-galactosidase detection system can be used with a chromogenic substrate o-nitrophenyl-beta-D-galactoside (ONPG), which produces a soluble product that is measurable at 410 nm. A urease detection system can be used with a substrate such as urea bromocresol purple (Sigma Immunochemicals, St. Louis, MO). In some embodiments, 2, 3, 4, 5, or more binding reagents are each labeled with an enzyme (e.g., a first binding reagent is labeled with a first enzyme, a second binding reagent is labeled with a second enzyme, etc.), and each binding reagent labeled with an enzyme is detected by detecting a product generated by the enzyme. In some embodiments, all binding reagents are labeled with an enzyme, and each binding reagent labeled with an enzyme is detected by detecting a product generated by the enzyme.

[0079] In some embodiments, the label is an affinity tag. Examples of suitable affinity tags include, but are not limited to, biotin, peptide tags (such as FLAG tag, HA tag, His tag, Myc tag, S tag, SBP tag, Strep tag, eXact tag), and protein tags (such as GST tag, MBP tag, GFP tag).

[0080] In some embodiments, the label is a nucleic acid label. Examples of suitable nucleic acid labels include, but are not limited to, an oligonucleotide sequence, single-stranded DNA, double-stranded DNA, RNA (such as mRNA or miRNA), or a DNA-RNA hybrid. In some embodiments, the nucleic acid label is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 nucleotides in length.

[0081] In some embodiments, the label is a nucleic acid barcode. As used herein, a "barcode" is a short nucleotide sequence (e.g., at least about 4, 6, 8, 10, or 12 nucleotides long) on a uniquely defined label or a second molecule bound to a labeled binding agent. The length of the barcode sequence determines how many unique samples can be distinguished. For example, a 4-nucleotide barcode can distinguish no more than 4 4 i.e., 256 samples; a 6-nucleotide barcode can distinguish no more than 4096 different samples; and an 8-nucleotide barcode can index no more than 65,536 different samples. The use of barcoding technology is well known in the art, see, e.g., Katsuyuki Shiroguchi et al., "Digital RNA sequencing minimizes sequence-dependent bias and amplification noise with optimized single-molecule barcodes," PNAS (Jan. 24, 2012); 109(4): 1347-52; and Smith, AM, et al., "Highly-multiplexed barcode sequencing: an efficient method for parallel analysis of pooled samples," Nucleic Acids Research (July 2010); 38(13): el42.

[0082] In some embodiments, the label is a "click" chemistry moiety. Click chemistry uses simple, robust reactions (such as copper-catalyzed cycloaddition of azides and alkynes) to establish intermolecular linkages. For a review of click chemistry, see Kolb et al., Agnew Chem 40:2004-2021 (2001). In some embodiments, the click chemistry moiety (such as an azide or alkyne moiety) can be detected using another detectable label (such as a fluorescently labeled, biotinylated, or radiolabeled alkyne or azide moiety).

[0083] Techniques for linking detectable labels to binding agents such as proteins (e.g., antibodies) are well known. For example, a review of common protein labeling techniques can be found in Biochemical Techniques: Theory and Practice, John F. Robyt and Bernard J. White, Waveland Press, Inc. (1987). Other labeling techniques are reviewed in, for example, R. Haugland, Excited States of Biopolymers, Steiner, ed., Plenum Press (1983); Fluorogenic Probe Design and Synthesis: A Technical Guide, PE Applied Biosystems (1996); and G. T. Herman, Bioconjugate Techniques, Academic Press (1996).

[0084] In some embodiments, two or more labels (e.g., a first label, a second label, etc.) in combination generate a detectable signal that is not generated in the absence of one or more of the plurality of labels. For example, in some embodiments, each label is an enzyme, and the activity of the enzymes in combination generates a detectable signal to indicate the presence of the label (and thus each labeled protein). Examples of enzymes that generate a detectable signal in combination include pairs of assays, such as the pair of assays using hexokinase and glucose-6-phosphate dehydrogenase; and chemiluminescent assays for NAD(P)H coupled to assays for glucose-6-phosphate dehydrogenase, beta-D-galactosidase, or alkaline phosphatase. See, e.g., Maeda et al., J Biolumin Chemilumin 1989, 4: 140-148.

[0085] B. Protein aggregation modifying agents

[0086] Protein aggregation modifiers are described herein. Protein aggregation modifiers can be employed to reduce or eliminate aggregation or denaturation of a binding agent such as a protein (e.g., an antibody) stored in or delivered from a reagent layer. For example, a protein aggregation modifier can be used to reduce or eliminate aggregation or denaturation of a primary antibody stored in or delivered from a reagent layer 118, 218. In some cases, a protein aggregation modifier can be used to facilitate lateral flow of a binding agent in a lateral flow region 110 of a wicking pad 102.

[0087] In some cases, a protein aggregation modifier that acts to displace a protein from an air-water interface and thereby protect the protein from denaturation and aggregation is particularly effective at reducing aggregation of a binding agent immobilized on a reagent layer. In other cases, a protein aggregation modifier directly affects the stability of a binding agent by binding to and / or stabilizing the binding agent. In other cases, a protein aggregation modifier acts to shift the equilibrium away from a denatured or unfolded state and thereby reduce aggregation. For example, in some cases, the interaction between a protein aggregation modifier and a binding agent is thermodynamically disfavored due to strong repulsion between the amide backbone of the binding agent and the protein aggregation modifier. Thus, the presence of a protein aggregation modifier disfavors unfolding of a binding agent, as unfolding exposes more amide backbone surface to the protein aggregation modifier.

[0088] A protein aggregation modifier can be one or more of a cyclodextrin, a nonionic surfactant, an ionic surfactant, a zwitterionic surfactant, a non-detergent sulfobetaine, a simple sugar, a polysaccharide, a polyol, an organic solvent, an aggregation-modifying protein, a disordered peptide sequence, an amino acid, a redox reagent, a lyoprotectant, a cryoprotectant, or a chaotropic agent.

[0089] Cyclodextrins can be, but are not limited to, a-cyclodextrin, β-cyclodextrin, γ- cyclodextrin, (2,3,6-tri-O-methyl)-β-cyclodextrin, (2,3,6-tri-O-methyl)-β-cyclodextrin, (2- hydroxy)propyl-β-cyclodextrin, (2-hydroxy)propyl-γ-cyclodextrin, random methyl-β- cyclodextrin, random methyl-γ-cyclodextrin, carboxymethyl-β-cyclodextrin, carboxymethyl- γ-cyclodextrin, 6-mono-deoxy-6-monoamino-β-cyclodextrin, sulfobutyl-β-cyclodextrin, 6-amino-6-deoxy-β-cyclodextrin, acetyl β-cyclodextrin, succinyl a-cyclodextrin, succinyl β- cyclodextrin, succinyl γ-cyclodextrin, (2,3,6-tri-O-benzoyl)-β-cyclodextrin, succinyl-(2- hydroxypropyl)-β-cyclodextrin, or succinyl-(2-hydroxypropyl)-γ-cyclodextrin. Cyclodextrins can also be cyclodextrin polymers containing one or more of the foregoing cyclodextrin molecules. Other cyclodextrins are known in the art and include, for example, those described on the World Wide Web at cyclodextrin.com. Exemplary concentrations of cyclodextrins are, but are not limited to, about 1 mM, 2 mM, 2.5 mM, 5 mM, 7.5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 50 mM, 75 mM, or 100 mM.

[0090] Non-ionic surfactants can be polyethylene-sorbitan-fatty acid esters, polyethylene- polypropylene glycols, or polyoxyethylene-stearates. Polyethylene-sorbitan-fatty acid esters can be polyethylene (20)-sorbitan esters (Tween 20 TM ) or polyoxyethylene (20)-sorbitan monooleate (Tween 80 TM ). Polyethylene-polypropylene glycols can be polyoxypropylene-polyoxyethylene block copolymers, such as those sold under the trade name or Poloxamer TM . Polyoxyethylene-stearates can be, for example, those sold under the trade name Myrj TM . Exemplary polyoxyethylene monolauryl ethers include those sold under the trade name Brij TM , such as Brij-35. Exemplary concentrations of non-ionic surfactants are, but are not limited to, about 0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.5%, 0.75%, 1%, 2%, 2.5%, 5%, 7.5%, or about 10% w / w, w / v, or v / v.

[0091] The ionic surfactant can be an anionic surfactant or a cationic surfactant. Anionic surfactants useful in the present application can be, but are not limited to, soaps, including alkali metal soaps, such as sodium, potassium, or ammonium salts of aliphatic carboxylic acids (typically fatty acids), such as sodium stearate. Other anionic surfactants include organic amine soaps, such as organic amine salts of aliphatic carboxylic acids (typically fatty acids), such as triethanolamine stearate. Cationic surfactants useful in the present application include, but are not limited to, ammonium salts, such as octadecyl ammonium chloride or quaternary ammonium compounds, such as benzalkonium chloride. The ionic surfactant can include sodium, potassium, or ammonium salts of alkyl sulfates, such as sodium dodecyl sulfate or sodium octyl sulfate. Exemplary concentrations of ionic surfactants are, but are not limited to, about 0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.5%, 0.75%, 1%, 2%, 2.5%, 5%, 7.5%, or about 10% w / w, w / v, or v / v.

[0092] Zwitterionic surfactants have both a cationic and anionic center attached to the same molecule. For example, the cationic portion is based on a primary, secondary, or tertiary amine or a quaternary ammonium cation. The anionic portion can be a sulfonate, such as in CHAPS (3[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate). Other anionic groups are sulfobetaines, such as cocamidopropyl hydroxysulfobetaine, or betaines, such as cocamidopropyl betaine, cocamidopropyl betaine, or lauramidopropyl betaine. Exemplary concentrations of zwitterionic surfactants are, but are not limited to, about 0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.5%, 0.75%, 1%, 2%, 2.5%, 5%, 7.5%, and about 10% w / w, w / v, or v / v.

[0093] Non-detergent sulfobetaines (NDSB) have a short hydrophobic group that is unable to aggregate to form micelles and a sulfobetaine hydrophilic group, thus NDSB are not considered detergents. Exemplary NDSB include, but are not limited to, NDSB 256, NDSB 221, NDSB 211, NDSB 201, NDSB 195, 3-(4-tert-butyl-l-pyridinium)-l-propanesulfonate, 3-(l-pyridinium)-l-propanesulfonate, 3-(benzyldimethylammonio)propanesulfonate, or dimethylethylpropanesulfonammonium. Exemplary concentrations of NDSB include, but are not limited to, about 0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.5%, 0.75%, 1%, 2%, 2.5%, 5%, 7.5%, and about 10% w / w, w / v, or v / v.

[0094] Polyols are compounds having multiple hydroxyl functional groups. In some cases, polyols can alter the aggregation or denaturation behavior of proteins through a variety of mechanisms. For example, in some cases, polyols can shift the equilibrium toward the folded state by providing thermodynamically unfavorable interactions with the protein backbone. Alternatively, in some cases, polyols can bind to and stabilize the folded state of a protein.

[0095] Polyols can be simple sugars, such as sucrose, mannitol, sorbitol, inositol, xylitol, erythritol, glucose, galactose, raffinose, or trehalose. Polyols can also be polysaccharides, such as dextran, starch, hydroxyethyl starch, or polymers containing one or more simple sugars described herein. Glycerol, ethylene glycol, polyethylene glycol, pentaerythritol propoxylate, and pentaerythritol propoxylate, and combinations thereof are also exemplary polyols.

[0096] Organic solvents can be, but are not limited to, those known to inhibit the denaturation, unfolding, or aggregation of one or more proteins. A variety of suitable organic solvents are known in the art. For example, organic solvents can include ethanol, butanol, propanol, phenol, dimethylformamide, 2-methyl-2,4-pentanediol, 2,3-butanediol, 1,2-propanediol, 1,6-hexanediol, or dimethylsulfoxide.

[0097] Aggregation-modifying proteins can be proteins known in the art to inhibit the denaturation, unfolding, or aggregation of one or more proteins. Exemplary aggregation-modifying proteins include, but are not limited to, albumins, protein chaperones, and heat shock proteins. Albumins are water-soluble, moderately soluble in concentrated salt solutions, and subject to thermal denaturation. Exemplary albumins include serum albumin (e.g., bovine, equine, or human serum albumin) or ovalbumin (e.g., chicken egg white protein). Other exemplary aggregation-modifying proteins include casein, gelatin, ubiquitin, lysozyme, or late embryogenesis abundant (LEA) proteins. LEA proteins include LEA I, LEA II, LEA III, LEA IV, LEA V, or atypical LEA proteins. LEA proteins are known in the art and described, for example, in Goyal K. et al., Biochemical Journal 288 (Part 1), 151-57, (2005).

[0098] Protein aggregation-modifying agents can also be amino acids. In some cases, amino acids can serve an oxidoreduction function to maintain an appropriate oxidation potential for proteins immobilized on substrate 112. Suitable oxidoreduction amino acids include cysteine and cystine. Other amino acids serve to reduce denaturation or aggregation through non-oxidoreduction methods. For example, arginine, glycine, proline, and taurine have been shown to reduce protein aggregation.

[0099] Other redox agents can be used to reduce protein aggregation. Redox agents other than cysteine and cystine can be used to optimize the reduction potential in the substrate 112 to which the protein is immobilized. Exemplary redox agents include mercaptoethanol, dithiothreitol, dithioerythritol, tris(2-carboxyethyl)phosphine, glutathione, glutathione disulfide, and oxidized derivatives thereof, and Cu 2+ .

[0100] The protein aggregation modifier can also include a lyoprotectant, a cryoprotectant, or a chaotropic agent. In some cases, the protein aggregation modifier is a chaotropic agent, such as urea, thiourea, guanidinium salts, cyanate salts, thiocyanate salts, trimethylammonium, tetramethylammonium, cesium, rubidium, nitrate salts, acetate salts, iodide salts, bromide salts, trichloroacetate salts, or perchlorate salts. In certain conditions, such as at low concentrations, chaotropic agents can reduce protein aggregation. Other protein aggregation modifiers include trimethylamine N-oxide.

[0101] The protein aggregation modifier can be a salt. Exemplary salts include, but are not limited to, sodium, potassium, magnesium, or calcium salts of hydrochloric acid, sulfuric acid, or phosphoric acid. The protein aggregation modifier can also be a buffer. Exemplary buffers include, but are not limited to, tris(hydroxymethyl)aminomethane (TRIS), TAPSO, MES, HEPES, PIPES, CAPS, CAPSO, MOPS, MOPSO, or sodium or potassium phosphate, sodium or potassium carbonate, sodium or potassium bicarbonate, sodium or potassium citrate, sodium or potassium acetate, or sodium or potassium borate buffers.

[0102] The protein aggregation modifier can be provided at any suitable concentration. In some cases, the protein is provided in the form of an aqueous solution containing the binding agent and the protein aggregation modifier. In such cases, the solution can be contacted with the reagent layer and optionally dried. Exemplary concentrations of the protein aggregation modifier in the aqueous binding agent solution include, but are not limited to, about 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 4%, 5%, 10%, 20%, or about 25% or more w / v of the solution. Other exemplary concentrations include, but are not limited to, about 1 μΜ, 5 μΜ, 10 μΜ, 25 μΜ, 50 μΜ, 75 μΜ, 100 μΜ, 150 μΜ, 200 μΜ, 300 μΜ, 500 μΜ, 750 μΜ, 1 mM, 5 mM, 10 mM, 25 mM, 50 mM, 100 mM, 150 mM, 200 mM, 300 mM, 500 mM, and 1 M.

[0103] In some cases, a protein aggregation modifier is provided on the reagent layer. Exemplary compositions containing a protein aggregation modifier and a reagent layer contain about 0.001 wt%, 0.005 wt%, 0.01 wt%, 0.05 wt%, 0.1 wt%, 0.5 wt%, 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, or about 10 wt%, 20 wt%, or about 25 wt% of one or more protein aggregation modifiers.

[0104] Protein aggregation modifiers can be provided in any suitable combination. For example, in some cases, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of the aforementioned protein aggregation modifiers can be used to reduce the aggregation of binding reagents reversibly immobilized on the reagent layer. In some cases, the reagent layer contains a protein aggregation modifier before contacting the binding reagent solution, and the binding reagent solution contains the same or different protein aggregation modifiers. In some cases, the reagent layer contains a protein aggregation modifier before contacting the binding reagent solution, and the binding reagent solution does not contain a protein aggregation modifier. In some cases, the binding reagent solution contains a protein aggregation modifier before contacting the reagent layer, while the reagent layer or the area to be contacted does not contain a protein aggregation modifier.

[0105] method

[0106] The use will now be described. Figure 1 The method for performing lateral flow Western blot determination using the lateral flow apparatus depicted in –3 and 5. The method begins by placing substrates 112, 412 face down on (e.g., in close contact with) wicking pad 102, which may be provided pre-wetted or may be pre-wetted by the user with, for example, a running buffer. In some embodiments, substrates 112, 412 are placed on wicking pads 102, 402 downstream of first reservoirs 114, 214, 314, 414 and upstream of second reservoirs 116, 416 (e.g., between the first and second reservoirs).

[0107] The first reservoirs 114, 214, 314, 414 are then formed by stacking one or more reagent layers 118, 218, 318, 418 (e.g., primary antibody, first washing solution, secondary antibody or second detection reagent, second washing solution) on the first ends 104, 404 of the wicking pads 102, 402. In embodiments where the reagent layers are formed of absorbent material ( Figures 1-2B5) Reagent layers 118, 418 are either dry-stacked or pre-wetted with a reagent-containing solution or running buffer (user or supplier operation) (e.g., for reagent layers with dry-embedded reagents). In embodiments where reagent layers 118, 418 are dry-stacked, running buffer can be applied to the first reservoirs 114, 414 after stacking the dry reagent layers 118, 418. In embodiments where reagent layers 218, 318 are solutions ( Figure 3 and 4A The solution is stacked in a container (e.g., in a cylinder or tank). In some embodiments, the stacking of reagent layers 118, 218, 318, 418 begins with the layer in contact with the wick pads 102, 402. Figures 1-2B (or most similar to core suction pads 102 and 402) Figures 3-4A The reagent layers 118, 218, 318, and 418 are stacked in the following order: a first reagent layer with a primary antibody, a second reagent layer with a first wash solution, a third reagent layer with a secondary antibody or a second detection reagent, and a fourth reagent layer with a second wash solution. In some embodiments, the stack of reagent layers includes two or more layers of the same reagent (or twice the volume). In some embodiments, a fifth reagent layer with a second wash solution (e.g., a second layer of the second wash solution) is included, such that the first reservoir has twice the volume of the second wash solution. In some embodiments, the reagent layer with the second wash solution is omitted to allow more time for the secondary antibody or the second detection reagent to bind to the primary antibody.

[0108] In embodiments where reagents are fixed in absorbent layers 118, 418, a running buffer is applied to the absorbent layers 118, 418 in the first reservoirs 114, 414 to initiate sequential flow of reagents from the first reservoirs 114, 414 to the wick pads 102, 402. Alternatively, liquid in the pre-wetted reagent layers is allowed to flow into the wick pads 102, 402. In embodiments where reagent layers 218, 318 are in solution form ( Figure 3 –4C), allowing the solution to flow sequentially from the first reservoirs 214, 314 to the first end 104 of the wick pad 102 or to one or more dry or pre-wetted absorbent pads on the first end 104 of the wick pad 102.

[0109] In embodiments where a valve 222 is provided at the first end 220 of the first reservoir 214, the valve 222 is opened to allow solution to leave the first reservoir 214. Figure 3 In some embodiments, the solution is allowed to flow out from the slit 322 on the first end 320 of the first reservoir 314. Figures 4A-4CIn embodiments where a slit 322 is present at the first end 320 of the first reservoir 314, the slit is first sealed with a removable metal or plastic foil (e.g., a strip), then the first reservoir 314 is filled with a reagent solution, and the foil is then removed to initiate the flow of solution from the first reservoir 314. In embodiments where a reversibly sealed opening 324 is present at the second (top) end 324 of the first reservoir 314, flow of solution is initiated by removing or piercing a seal (e.g., a metal or plastic seal) before or simultaneously with removing the foil covering the slit 322.

[0110] Running buffers or solutions are aspirated from first reservoirs 114, 414 into dry second reservoirs 116, 416, said running buffers or solutions carrying reagents (e.g., primary antibody, first wash solution, and, if necessary, secondary antibody and second wash solution) and these reagents are sequentially contacted by side flow with substrates 112, 412 on which proteins are immobilized. In some embodiments ( Figure 5 As the reagent flows through the first reservoir 414 and into the wick pad 402, the reagent flows along a curved path 442. The primary antibody in the first reagent layer is transferred within the wick pads 102 and 402, contacting proteins on the substrates 112 and 412, and, if present, binding to target proteins on the substrates 112 and 412. In some embodiments, the run buffer / solution flowing from the first reservoirs 114, 214, 314, 414 to the second reservoirs 116 and 416 also allows the first wash solution in the second reagent layer to be transferred within the wick pads 102 and 402, causing unbound primary antibodies to be removed from the substrates 112 and 412. In some embodiments, the run buffer / solution flowing from the first reservoirs 114, 214, 314, 414 to the second reservoirs 116, 416 also allows the secondary antibody or second detection reagent in the third reagent layer to be delivered in the wick pads 102, 402 and contact the primary antibody on the substrates 112, 412 that is bound to its target protein (if present). In some embodiments, the run buffer / solution flowing from the first reservoirs 114, 214, 314, 414 to the second reservoirs 116, 416 also allows the second wash solution in the fourth reagent layer to be delivered in the wick pads 102, 402, causing unbound secondary antibody to be removed from the substrate 112. In some embodiments, the volume of the second wash solution applied to and delivered in the wick pads 102, 402 is twice the volume of the secondary antibody applied to and delivered in the wick pads 102, 402.

[0111] In some embodiments, a substantially uniform pressure is applied to the first reservoir 114, 314, 414, and optionally to the second reservoir 116, 416, after the lateral flow is initiated and / or during the lateral flow to improve the contact of the first reservoir or the first and second reservoirs with the wicking pad 102, 402. For example, a weight can be placed on top of one or both reservoirs to push the one or both reservoirs toward the wicking pad 102, 402.

[0112] In some embodiments, the binding of the primary antibody to the target protein (and optionally the binding of the secondary antibody or second detection reagent to the primary antibody) is tracked during the lateral flow by visual inspection or using a detector. In some embodiments, the substrate 112, 412 is removed from the lateral flow device and the binding of the primary antibody to the target protein, if present, is detected. In some embodiments, the antibody bound to the target protein is visualized and / or detected by using the detectable moieties and / or labels described herein. Suitable labels and / or moieties are detected by spectroscopic, photochemical, biochemical, immunochemical, isotopic, electrical, optical, chemical, or mass spectrometric techniques.

[0113] Typically, if the device is supplied in a housing, once the reagent layer is applied to the wicking pad 102, a lid or cover is placed back on the device to minimize evaporation and to apply uniform pressure to the first and second reservoirs 114, 116. The lid can snap fit onto the base of the housing to apply uniform pressure, or the lid can be loosely placed on top of the base, and then the base with the lid can be placed into a drawer-style container that slides into a box. A sponge can be placed on the first and second reservoirs before the lid is attached or placed to help apply uniform pressure to the reservoirs. The entire process requires minimal user interaction with the consumables.

[0114] Many absorbent bibulous pad materials, wicking pad materials, and antibody application materials are known in the art from which selections can be made to control the volume, to control the flow rate of the system, to ensure uniform flow, and to ensure complete delivery of the antibody / reagent from the first reservoir. Other methods of influencing the delivery time of the reagent / antibody are also possible, such as using a tortuous path in the wicking pad or controlling the contact area of an absorbent pad with immobilized antibody, thereby controlling the rate of antibody removal. In addition, other embodiments of controlling the lateral flow process can be engineered into the plastic housing where the surface can contain sloped regions to retard or accelerate the flow of liquid using gravity.

[0115] Figure 2A and 2BShown is a consumable device that holds a membrane of a single mini-gel size. Typically users perform Western blots using membranes referred to as midblots, which are usually 2x wider than mini-membranes. In other Western blot applications, users can cut mini- and / or mid-membranes into smaller sections that correspond to several lanes of the original gel used for protein electrophoresis and transfer. Thus, in some embodiments, the consumable lateral flow device 100 can be sized to accommodate a mini- or mid-membrane. In other embodiments, separate ridges can be molded into or otherwise present in the base of the consumable that the membrane sections can be placed into. In this latter arrangement, smaller first reservoirs loaded with different antibodies can be placed at the head of each section to facilitate Western blot probing of each section of the membrane with different antibodies in the same device.

[0116] In other embodiments of this Western blot lateral flow device, antibodies can be mixed and loaded into the first reagent layer of the first reservoir to facilitate multiplexed detection of targets in a single sample.

[0117] IV. Kits

[0118] Kits for performing lateral flow Western blot assays according to the methods described herein are provided. Kits comprising devices as described herein are also provided. In some embodiments, the kits include a first reservoir comprising a stack of multiple reagent layers on a first end of a wicking pad and a second reservoir comprising an absorbent pad on a second end of the wicking pad, all of which are described herein. In some embodiments, the kits comprise reagents in liquid form (e.g., primary antibodies comprising a label or binding reagents for primary and secondary antibodies, wash solutions, and / or running buffers). In some embodiments, the kits comprise one or more absorbent reagent layers, each having a reagent reversibly bound therein. In certain embodiments, some or all of the reagents are dried onto the absorbent reagent layers. In some embodiments, some or all of the reagents are dried onto the absorbent reagent layers or portions thereof in the presence of one or more protein aggregation-modifying agents. In some embodiments, the reagents are provided as solutions that are applied onto the absorbent reagent layers by the end user. In some cases, the solutions for each reagent can have different densities to minimize mixing of different reagents. In some embodiments, the float described in US 6641517 can be provided for use with a cylinder or trough to minimize mixing of reagents while stacking solutions with different densities. An exemplary embodiment of the float 326 is shown in FIG. 4.

[0119] In some embodiments, the kit includes a plastic tray having two, four, or more quadrants for the user to apply reagent solutions to the absorbent pads prior to forming the stack of reagent layers 118.

[0120] In some embodiments, the kit contains a running buffer and / or blocking agent (e.g., bovine serum albumin and / or skim milk powder), a surfactant (e.g., Tween 20 or Triton X-100), a protein aggregation modifier as described herein, a crowding agent (e.g., dextran, polyethylene glycol, and / or Ficoll), and / or a reagent that facilitates uniform flow of reagents and / or facilitates reactions with molecules on the substrate and minimizes background on the substrate. Additional reagents can be provided in the kit as a solid (e.g., a powder) or in liquid form (e.g., as a solution). In some embodiments, the kit also includes instructions for performing the methods described herein.

[0121] IV. Examples

[0122] These examples illustrate the use of the lateral flow devices as shown in FIGS. 2A-2B and as described herein to perform Western blot assays. Figure 1

[0123] Example 1 - Detection of tubulin and GAPDH from HEK293 cell lysate using different dilutions of secondary antibody

[0124] ​Freeze-dried HEK293 protein lysate (PrecisionAb Control Lysate VLY001 by Bio-Rad Laboratories) was reconstituted in IX Laemmli sample buffer containing 40 mM DTT and denatured by heating at 100 °C for 5 minutes. A two-fold dilution series of lysate (20 ug to 0.04 ug) was loaded onto 4-20% TGX mini gels (Bio-Rad Laboratories) and run at 250 V for 25 minutes. Individual gels were transferred to PVDF membranes using a Transblot Turbo apparatus and pre-loaded transfer pack using a 1.3 A x 7 min setting. Following transfer, the membranes were briefly rinsed in IX PBS buffer and then placed in a lateral flow buffer containing 1% casein, IX PBS buffer, 0.1% Tween 20 and placed on a rocker for 10 minutes to block. While the membranes were incubating in the lateral flow buffer, a primary antibody was prepared by mixing 5 ul each of rabbit anti-tubulin polyclonal Ab (Cell signaling Technologies #2148) and rabbit anti-GAPDH mAb (Cell Signaling Technologies #5174) antibody into 5 ml of lateral flow buffer. A secondary antibody (goat anti-rabbit IgG-HRP conjugate, Cell Signaling Technologies #7074) was prepared in three different dilutions (1 : 1000, 1 : 5000 and 1 : 10000) in lateral flow buffer. A separate tray with 4 chambers of ~2.5 x 10 cm was used to wet the reagent layers with antibody and wash solutions. The following solutions were pipetted into the chambers: 4.5 ml primary antibody was pipetted into chamber 1, 4.5 ml lateral flow buffer was pipetted into chamber 2 (wash 1), 4.5 ml secondary antibody was pipetted into chamber 3, and 27 ml lateral flow buffer was pipetted into chamber 4 (wash 2) containing a stack of 30 layers of Transblot Turbo pad material. A piece of blotting paper was added to each of chambers 1 to 3 to completely absorb the solutions, thereby reversibly immobilizing the reagents in the paper.

[0125] Blot detection was performed as follows. Paper wicking pads (1 CHR paper by GE Healthcare) were cut to 8.7 cm x 18.7 cm and placed in the bottom of a vacuum moulding tray as Figure 2AThe 9 layers of approximately 4 cm x 9.5 cm blotting paper (Bio-Rad) were placed on one end of the wicking pad to act as a pump. The paper wick was wetted with 3.5 ml of lateral flow buffer and rolled to remove air bubbles. The membrane was removed from the enclosure and inverted (antigen side down) onto the wicking pad with the low molecular weight proteins closest to the pump; air bubbles were removed by rolling. The layers of blotting paper containing the absorbed primary antibody, solution of wash 1, secondary antibody, and solution of wash 2, respectively, were stacked on the end of the wicking pad opposite the pump with the primary antibody layer contacting the wicking pad (i.e., the primary antibody layer was the first (or bottom) layer in the stack of reagent layers). A sponge of similar dimensions to the wicking pad was placed on top of the antibody / buffer reservoir stack and the pump. A plastic lid was placed on top of the sponge and the lid was clipped to the bottom tray using long binder clips. Each of the three different dilutions of secondary antibody was tested using a separate device. The devices were left undisturbed on a horizontal surface at room temperature. After 5 hours, the membranes were removed from the devices and washed in IX PBS for 1 x 5 minutes. Detection was then performed using Clarity chemiluminescent substrate (Bio-Rad Laboratories) according to the instructions. Figure 6A , 6B and 6C are images of the three blot membranes corresponding to 1:1000, 1:5000, and 1:10000 dilutions of the secondary antibody, respectively. Images were acquired from 8 second exposures of the blots using a Chemidoc MP imager from Bio-Rad. The images show that both target antigens were detected at all three dilutions of the secondary antibody.

[0126] Example 2 - Detection of PCNA antigen in HEK293 lysate on PVDF and nitrocellulose membranes

[0127] Conditions were as described in Example 1. The primary antibody was a mouse anti-PCNA mAb (Bio-Rad Laboratories, #VMA00018) diluted 1:1000 in lateral flow buffer. The secondary antibody was a goat anti-mouse IgG-HRP conjugate (Bio-Rad Laboratories, #STAR207P) diluted 1:1000 in lateral flow buffer. Blot images were taken using a Chemidoc MP imager from 0.7 second exposures (see Figure 7A and 7B ). The images show that the target antigen was detected at multiple dilutions on both types of membrane material, with PVDF showing better sensitivity than nitrocellulose.

[0128] Example 3 - Detection of GSK3a / b antigen in HEK293 lysate on PVDF membranes

[0129] Conditions were as described in Example 1. Primary antibody was mouse anti-GSK3a / b mAb (Bio-Rad Laboratories, #VMA00342) diluted 1 : 1000 in lateral flow buffer. Secondary antibody was goat anti-mouse IgG-HRP conjugate (Bio-Rad Laboratories, #STAR207P) diluted 1 : 1000 in lateral flow buffer. Images were taken from the blots using a Chemidoc MP imager from a 2 second exposure as shown in Figure 8 The image shows the target antigen was detected at multiple dilutions.

[0130] Example 4 - Detection of PARP antigen in HEK293 lysate on PVDF membrane

[0131] Conditions were as described in Example 1. Primary antibody was mouse anti-PARP mAb (Bio-Rad Laboratories, #VMA00016) diluted 1 : 1000 in lateral flow buffer. Secondary antibody was goat anti-mouse IgG-HRP conjugate (Bio-Rad Laboratories, #STAR207P) diluted 1 : 1000 in lateral flow buffer. Images were taken from the blots using a Chemidoc MP imager from a 6 second exposure as shown in Figure 9 The image shows the target antigen was detected at multiple dilutions.

[0132] Example 5 - Detection of hRAS antigen in HEK293 lysate on PVDF membrane

[0133] Conditions were as described in Example 1. Primary antibody was mouse anti-hRAS (Bio-Rad Laboratories, #VMA00040) diluted 1 : 1000 in lateral flow buffer. Secondary antibody was goat anti-mouse IgG-HRP conjugate (Bio-Rad Laboratories, #STAR207P) diluted 1 : 1000 in lateral flow buffer. Images were taken from the blots using a Chemidoc MP imager from a 12 second exposure as shown in Figure 10 The image shows the target antigen was detected at multiple dilutions.

[0134] Examples 1-5 illustrate that the lateral flow devices described herein can sequentially deliver Western blot reagents (e.g., specific binding reagents, running buffer, wash solution) and without user intervention to blot on a wicking pad.

[0135] All patents, patent applications, and other published reference materials cited in this specification are fully incorporated by reference herein in their entirety.

Claims

1. A lateral flow device comprising: a wicking pad comprised of a porous material, the wicking pad having a region for applying a substrate comprising an immobilized analyte; wherein the wicking pad has a first end, a second end, and two sides; a first reservoir comprising a stack of a plurality of reagent layers on the first end of the wicking pad, wherein each of the plurality of reagent layers comprises a reagent in solution; and a second reservoir comprising an absorbent pad on the second end of the wicking pad.

2. The apparatus of claim 1, wherein, The reagent solution comprises a density agent.

3. The apparatus of claim 2, wherein, The density agent is selected from the group consisting of glycerol, sucrose, trehalose, dextran, and polyethylene glycol.

4. The apparatus of claim 1, wherein, Each of the plurality of reagent layers has a different reagent therein.

5. The apparatus of claim 1, wherein, The first reservoir is a cylinder or a trough having a fluid flow controller at the first end to control release of the solution from the first reservoir.

6. The apparatus of claim 5, wherein, The fluid flow controller is a valve.

7. The apparatus of claim 5, wherein, The fluid flow controller is a slit.

8. The apparatus of claim 7, wherein, The slit has a width in the range of 0.5 mm to 2 mm.

9. The apparatus of claim 7, wherein, The slit has a width of about 0.5 mm or about 1 mm.

10. The apparatus of claim 1, wherein, The reagent is selected from the group consisting of a primary antibody, a secondary antibody, a first wash solution, and a second wash solution.

11. The apparatus of claim 4, wherein, The plurality of reagent layers, starting with the reagent layer at or near the first end of the first reservoir, comprises a first reagent layer with a primary antibody, a second reagent layer with a first wash solution, a third reagent layer with a secondary antibody, and a fourth reagent layer with a second wash solution.

12. The apparatus of claim 11, wherein, The fourth reagent layer has a thickness that is at least twice that of the third reagent layer.

13. The device of claim 11, further comprising a fifth reagent layer comprising a second wash solution.

14. The apparatus of claim 11, wherein, The second wash solution has a volume that is at least twice the volume of the secondary antibody.

15. The apparatus of claim 1, wherein, The analyte is a protein.

16. The apparatus of claim 1, wherein, The wicking pad is dry.

17. The apparatus of claim 1, wherein, The wicking pad is wet.

18. The apparatus of claim 1, wherein, The substrate is selected from the group consisting of glass, plastic, silicon, metal, and metal oxide.

19. The apparatus of claim 1, wherein, The substrate is a membrane.

20. The apparatus of claim 19, wherein, The membrane is formed from at least one material selected from the group consisting of nitrocellulose, polyvinylidene fluoride, nylon, and polysulfone.

21. A kit for lateral flow, the kit comprising the device of claim 1.

22. A method of performing a lateral flow assay, the method comprising: providing the device of claim 1; applying a substrate comprising a protein to the region for applying a substrate comprising an analyte; and causing lateral flow of the plurality of reagent layers from the first reservoir to the second reservoir such that the plurality of reagents are sequentially transported in the wicking pad and into contact with the protein on the substrate. The method further comprises applying a running buffer to the wicking pad.

23. The method of claim 22, wherein, The step of causing lateral flow comprises causing a primary antibody from the first reagent layer to bind to its target protein, if present, on the substrate, and then causing a first wash solution in the second reagent layer to remove unbound primary antibody from the substrate.

24. The method of claim 22 or 23, wherein, The step of causing lateral flow further comprises causing a secondary antibody or a second detection reagent from the third reagent layer to contact the primary antibody bound to its target protein, if present, on the substrate.

25. The method of claim 24, wherein, The step further comprises causing a second wash solution from the fourth reagent to remove unbound secondary antibody from the substrate.

26. The method of claim 25, wherein, 27. The method of claim 24, further comprising, after the primary antibody binds to the target protein, if present, removing the substrate and detecting the binding of the primary antibody to the target protein, if present. ​ 28. The method of claim 25, further comprising removing the substrate after the secondary antibody or second detection reagent is contacted with the primary antibody and detecting binding of the primary antibody to the target protein, if present.

Images