WO2019081361A1

WO2019081361A1 - Universal lateral flow immunoassay

Info

Publication number: WO2019081361A1
Application number: PCT/EP2018/078678
Authority: WO
Inventors: Nicolas Steven Gee; Isabella PUPITA; Anna SERENI
Original assignee: Expedeon Ltd
Priority date: 2017-10-21
Filing date: 2018-10-19
Publication date: 2019-05-02

Abstract

Disclosed herein are devices and methods for performing lateral flow chromatography. Lateral flow strips of this disclosure include a solid porous material that supports lateral flow of liquid containing a sample and, on the strip, a test area having immobilized thereto an affinity agent for a first tag, and a control area having immobilized thereto an affinity agent for binding of visualization agent, either directly or through a second tag attached to the visualization agent. Lateral flow chromatography is performed on a sample comprising the visualization agent, optionally attached to the second tag and a complex comprising an analyte bound by a first conjugate and a second conjugate. The first conjugate comprises an analyte capture agent attached to the first tag. The second conjugate comprises an analyte detection agent attached to a visualization agent. The complex is captured through the first tag at the capture area and can be detected there through the visualization agent. Visualization agent, optionally attached to a second tag, is captured and detected at the control area.

Description

UNIVERSAL LATERAL FLOW IMMUNOASSAY

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0001] None.

REFERENCE TO RELATED APPLICATIONS

[0002] This application is related to U.S. provisional application 62/575,425, filed October 21 , 2017.

THE NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT [0003] None.

SEQUENCE LISTING

[0004] None.

BACKGROUND

[0005] The lateral flow assay (LFA) or Immunochromatographic test system is widely used for the detection and quantification of analytes in complex mixtures. The technology was first commercialized by Unipath in the 1980s for the detection of pregnancy hormone (hCG) in urine, and the same general approach is now widely used to detect a variety of biomolecules including, antigens, antibodies, viruses, drugs, and DNA, among others.

[0006] The principle behind LFA technology is simple; a complex mixture of analytes, usually in an aqueous medium, is drawn by capillary forces up a nitrocellulose strip. Dried at certain points along the strip in capture zones are antibodies, or other binding agents, whose purpose is to capture analytes or antibody conjugates from the liquid flow. Bound analyte is visualized using a detector molecule, which most commonly comprises an antibody or other binding agent attached to gold or latex particles or to some other detectable substance. The assay is set up either in a sandwich or competitive format, depending on the size of the analyte. For larger molecules a sandwich format is typically used, whereas for molecules that are too small to accommodate the binding of both capture and detector reagents, a competitive format is necessary. [0007] LFA tests are rapid (typically 5-15 minutes) and, as no special equipment or operator skills are needed, the test has become very popular for measuring substances in point-of-care settings and in remote locations. Test samples commonly are blood, saliva or urine but, in principle, analytes in any aqueous fluid can be measured in a lateral flow test.

[0008] The analyte specificity of an LFA test is determined by the antibodies that are dried onto the test strips; for each analyte to be measured a new LFA strip with the appropriate antibody must be developed. In sandwich assays, a matched conjugate for visualization of bound analyte is also required, which often comprises a colored particle attached to an antibody.

[0009] Additional components of an LFA test strip may include a sample pad at one end, onto which the sample is deposited, which may overlap with a conjugate pad impregnated with a dried detection reagent, and an absorbent pad at the opposite end to wick excess liquid and to prevent back flow. The strip is often housed in a plastic cassette with a viewing window and port to apply sample.

[00010] Generally, the sample is applied in a fixed volume to the sample pad, and the total volume of liquid that can be applied to the test strip is dependent on the capacity of the wicking pad.

[00011] While the act of carrying out an LFA test is not demanding, the production of LFA strips requires expensive equipment to dispense capture reagents onto nitrocellulose membranes, a process called 'striping' or 'spraying', followed by assembly with other components and precise cutting into test strips. Expertise is also required in antibody conjugation technologies and methods of drying biomolecules; and some knowledge of the interactions between biological reagents and the various materials in the test strip. Thus, there are many barriers that hinder non-specialists from creating LFA tests.

[00012] Even with expert knowledge and the necessary specialist dispensing and cutting equipment, the process of developing an LFA tests is labor-intensive; weeks or months of effort yield a product that can measure just one, or at best, a few substances, depending on the number of capture zones on the test strip. Each LFA test has a specific purpose and once fabricated LFA strips cannot be reconfigured to measure other analytes of interest. [00013] There is thus a clear need for a more efficient approach of developing LFA tests, which allows one type of test strip to be used to measure different analytes, and easier ways of creating the necessary capture and detection reagents.

[00014] A partial solution to these problems is provided by the gRAD system, which is currently commercialized by BioPorto Diagnostics A/S. This system features a lateral flow strip with a capture zone (the test line, or T line) comprising an immobilized 'biotin binding protein', which in principle can capture any biotinylated antibody. In turn, this biotinylated antibody may capture an analyte from a complex mixture. The production of biotin conjugates requires knowledge of chemistry and certain technical skills e.g. in chromatographic separations. Gold colloid is also provided with the gRAD system to allow the operator to make a detection reagent, but the production of gold conjugates is technically difficult for non-experts, and tedious for those with greater experience. Typically, the production of a gold conjugate requires exploration of varying antibody concentrations, buffer type and pH to optimize conjugate stability and performance. This process is time-consuming and there is no set of conditions that works with all antibodies.

[00015] In a second capture zone (the control line, or C line) there is a mixture of anti-species (mouse, rabbit, and goat) antibodies which allows some of the gold detection conjugate flowing along the strips to be captured. The detection antibody used with the gRAD strip must of course be from a species that can be recognized by the antibodies deposited at the C line.

[00016] At the end of the test, the C line must be visible for the T line result to be valid. This is especially important if a negative result at the T line is obtained, because a C line signal proves that the detector particles have migrated past the T line. The gRAD approach to visualizing the C line is imperfect because deposition of reagents at the T line causes variation in C line intensity.

[00017] In addition to the technical challenges of using the gRAD system, a further weakness is that a biotinylated antibody must be captured on the T line from a complex tissue extract or other fluid, which commonly contain competing biotin ligand. Moreover, for samples whose biotin content is unknown a false negative signal at the T line could easily be obtained and yet a strong signal at the C line would still be obtained. [00018] It is clear from the cost and technical issues noted above that there is a need for far simpler methodologies to create lateral flow reagents and more robust LFA tests.

BRIEF DESCRIPTION OF THE DRAWINGS

[00019] The accompanying drawings, which are incorporated herein and form a part of the specification, illustrate exemplary embodiments and, together with the description, further serve to enable a person skilled in the pertinent art to make and use these embodiments and others that will be apparent to those skilled in the art. The invention will be more particularly described in conjunction with the following drawings wherein:

[00020] FIG. 1 shows an exemplary configuration of an assay of this disclosure. A lateral flow test strip comprises, from right to left, a sample application area, a test line ("T"), a control line ("C") and a wicking pad. The test line comprises an immobilized anti-tag binding agent, such an antibody. The control line comprises a binding agent ("B") that binds a second tag, in this case, a small molecule (such as biotin) coupled to a Visualization agent ("V2"). Liquid containing the analyte in complex flows laterally from right to left. The complex comprises a sandwich including Conjugate 1 (Analyte capture agent bound to Tag), the analyte ("A") and Conjugate 2 (Analyte detection agent bound to Visualization agent ("V1 ").

[00021] FIG. 2 shows the measured assay signal in in millivolts (mV) for the T Line (full) and C line (dotted) for concentration of C-reactive protein ("CRP"). A scorecard provides a score from 0 to 8 for intensity of signal.

[00022] FIG. 3 shows an exemplary implementation of a universal LFA test. A lateral flow test strip comprises, from right to left, a sample application area, a test line ("T"), a control line ("C") and an absorbent pad. The test line comprises an immobilized antibody specific for a tag, digoxigenin. The control line comprises immobilized streptavidin. Liquid containing the analyte in complex flows laterally from right to left. A complex comprises a sandwich including a digoxigenin-linked anti-CRP antibody conjugate, the analyte, CRP, and gold-linked anti-CRP conjugate. This complex is bound to the T line through the digoxigenin tag. At the C line, a gold-biotin conjugate is bound through streptavidin. SUMMARY

[00023] Provided herein is an analyte detection method featuring lateral flow test strips with two capture areas; a T line with an immobilized anti-tag binding agent ("capture area"), and a C line with an immobilized agent ("control area"); plus an analyte capture agent which also bears a tag, and an analyte detection agent capable of binding to the analyte bound to the capture agent; plus, a colored substance capable of binding to the C line; and, in some embodiments, buffers and other accessory reagents.

[00024] An advantage of the present invention is its adaptability to any pair of capture and detection antibodies, which permits the detection of almost any type of analyte, without the need to stripe or spray the capture antibody onto LFA strips; and the ease with which the necessary capture and detection reagents can be made. The test strip also can have a simpler construction than a traditional LFA strip, as, in some embodiments, it lacks a conjugate pad, and the T line signal is not affected by biotin in samples.

[00025] The invention also provides a universal lateral flow kit, comprising test strips and conjugation kits for attaching a tag to one antibody, and stabilized gold particles to another, and a C line detector reagent; and, in some embodiments, buffers, protocol and a scoring card.

DETAILED DESCRIPTION

I. Definitions

[00026] As used herein, the term "label" refers to a chemical, biochemical, or particulate moiety attached to a molecule, such as to a polypeptide. Labels include, for example, tags and visualization agents.

[00027] As used herein, the term "tag" refers to a label that can be captured or immobilized. Tags include, without limitation, biotin, avidin, streptavidin, a nucleic acid comprising a particular nucleotide sequence, and a hapten recognized by an antibody. The tag can be a member of a binding pair, such as biotin/streptavidin or hapten/antibody. In certain embodiments, tags function as indirect visualization agents.

[00028] As used herein, the term "conjugate" refers to a molecule that is bound to a label, either covalently, through a linker or a chemical bond, or noncovalently, through ionic, van der Waals, electrostatic, or hydrogen bonds, such that the molecule can be captured through the label or detected through a label.

[00029] As used herein, the term "affinity agent" refers to agents that specifically bind a target molecule. The term embraces, without limitation, "binding agents", "analyte capture agents" and "analyte detection agents". Affinity agents include, without limitation, antibodies, non-antibody proteins, protein receptors, aptamers and nucleic acids.

[00030] An affinity agent specifically binds a target molecule if the affinity agent binds the target molecule with at least 2-fold greater affinity it does non-target molecules, e.g., with at least any of 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25-fold, 50-fold, or 100- fold greater affinity. For example, a monoclonal antibody raised against a first peptide epitope will specifically bind the first peptide epitope but will not specifically bind a second, different peptide epitope, even if the amino acid sequence of the two epitopes partially overlap.

[00031] As used herein, the term "antibody" means an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigen recognition site or antigen-binding site within the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses intact (i.e., whole, four chain) polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single chain Fv (scFv) mutants, multispecific antibodies such as bispecific antibodies generated from at least two intact antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen recognition site of an antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site so long as the antibodies exhibit the desired biological activity.

[00032] As used herein, the term "visualization agent" refers to an agent detectable by visual, spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. Examples of visualization agents include, without limitation, colorimetric, fluorescent, chemiluminescent, enzymatic, and radioactive labels. Certain visualization agents produce a signal that is directly detectable. Examples include colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, fluorescent labels (e.g., phycoerythrin, fluorescein isothiocyanate, texas red, rhodamine, a green fluorescent protein, a red fluorescent protein, a yellow fluorescent protein), and radioactive labels (e.g., ³H, I, S, C, or P). Other visualization agents produce a detectable signal indirectly. This can be, for example, through the binding of another agent that, itself, comprises a directly detectable visualization agent, such as a labeled antibody. In another example, the visualization agent may be an enzyme (e.g. luciferase), horse radish peroxidase or alkaline phosphatase), which produces a signal by the conversion of a substrate to produce either light or a colored product, such as those generated by the action of horseradish peroxidase on 3, 3', 5,5'- tetramethylbenzidine (TMB), 3,3'-diaminobenzidine (DAB) or 2,2'-azino-bis(3- ethylbenzothiazoline-6-sulphonic acid (ABTS). An indirect label is a label that is detected (primarily or secondarily) by another moiety comprising a direct label. Examples of indirect labels are extraction moieties, such as antibodies, biotin or streptavidin, that bind other molecules which themselves bear a direct label.

[00033] Visualization agents can be detected or measured as follows. Gold or latex particles: visually. Fluorescence: A fluorescent molecule (fluorophore), such as a dye or a protein, are excited with light of specific wavelength. The fluorophore then emits light of a specific wavelength, which can be measured using a detector, such as a photomultiplier tube, CMOS, etc. Luminescence: Chemical reactions can produce light. One example is enzyme Luciferase that oxidizes luciferin and emits photons. This light can be measured using a detector, such as a photomultiplier tube, CMOS, etc.

II. Lateral Flow Strip

[00034] The general configuration of the assay is illustrated in Figure 1.

[00035] The lateral flow strips disclosed herein (also referred to as "test strips") typically comprise a solid support to which is attached to a solid porous material that supports lateral flow and, optionally, an absorbent material functioning as a wicking agent and a sample application area optionally in the form of a sample application pad. The solid support, also referred to as a "backing card", can be made, for example, of a plastic. The solid support material typically comprises nitrocellulose. In certain embodiments it can comprise, e.g., cellulose acetate. A wicking pad, if present, can comprise a bibulous material, such as a glass fiber or cellulose filter. The solid porous material and the wicking pad are arranged in lateral flow contact with each other, that is, fluid can move through the solid porous material into the wicking pad. The sample application area, e.g., the sample application pad, is positioned upstream of the capture area and control area. The wicking material is positioned downstream of the capture area and control area. "Upstream" refers to a direction opposite the direction of liquid flow, while "downstream" refers to a direction that is in the direction of flow.

[00036] The lateral flow strip comprises an anti-tag binding agent deposited and dried at the T line. This agent may be an antibody, as depicted in Figure 1 as a Ύ' shape, though any molecule capable of selectively binding the tag is acceptable.

III. Affinity Agent Complexes

[00037] The complex capture agent immobilized at the capture area captures an affinity agent complex. The affinity agent complex comprises a first conjugate ("conjugate 1"), an analyte and a second conjugate ("conjugate 2"). The first conjugate comprises an analyte capture agent attached to a first tag. The second conjugate comprises an analyte detection agent attached to a visualization agent. Each of the analyte capture agent and the analyte detection agent can specifically bind the analyte.

[00038] The analyte capture agent (ACA) determines the specificity of the test and bears a tag that may be captured by the anti-tag binding agent. The combination of ACA linked to a tag is referred to as conjugate 1. In certain embodiments the analyte capture agent comprises an antibody that specifically binds the target analyte.

[00039] The analyte detection agent (ADA) is conjugated to a visualization agent, V1 , and this hybrid molecule is referred to as conjugate 2. In certain embodiments the analyte detection agent comprises an antibody that specifically binds the target analyte. In certain embodiments, the analyte detection agent binds the analyte at a different epitope than the analyte capture agent or at a second copy of the same epitope.

[00040] When the ACA is anchored at the T line any bound analyte, A, is detected through binding of conjugate 2, which causes a line to appear on the test strip when a directly detectable visualization agent is used.

[00041] The production of the ACA and ADA can be accomplished with minimal effort using methods of the present invention.

[00042] The complex capture agent at the capture area and the detector binding agent at the control area are immobilized at their positions, that is, they do not flow out of their areas during lateral flow chromatography. Antibodies typically are immobilized on nitrocellulose by absorption followed by drying. Other immobilization methods also can be used, for example, covalent attachment.

A. Test Visualization Agent

[00043] The test visualization agent ("V1") may comprise a colored particle, a fluorescent dye or fluorescent protein, or any other substance capable of providing a readout, either directly or indirectly.

[00044] In a particularly preferred embodiment the visualization agent V1 is a colored or fluorescent particle.

[00045] The ADA may be attached to V1 by passive or covalent means.

[00046] In a preferred embodiment V1 is covalently attached to the ADA.

[00047] Preferably V1 is a gold particle with a diameter less than 200 nm, more preferably less than 100nm and even more preferably between about 20nm and 80nm.

[00048] In a particularly preferred embodiment V1 is a coated gold particle, e.g. Innovacoat GOLD™ (available from Expedeon Ltd.).

[00049] In another embodiment, V1 is a fluorescent particle which may afford a lower limit of detection, or permit higher dilutions of sample, or require a lesser amount of sample per test.

[00050] In a preferred embodiment, the fluorescent particle is impregnated with europium (Eu3+) ions, which are visualized under UV light or by using a reader with suitable

excitation/emission characteristics.

[00051] In a preferred embodiment of the invention the tag on the ACA is preferably not present in samples to be evaluated, or found extremely rarely in such samples.

[00052] The tag, if not indigenous, is introduced into the ACA using a reactive tag derivative. Derivatives include, but are not limited to, NHS esters or maleimides, which may be reacted with amines (e.g. lysines) or thiols, respectively, on the ACA using methods well known in the art.

[00053] At the end of the reaction, any unbound tag may be removed from the ACA by desalting or dialysis, or some other separation technique. B. First Tag

[00054] In a particularly preferred embodiment of the invention, the tag is digoxigenin, which is advantageous because it is absent from (almost) all biological materials; it is found only in the plants Digitalis purpurea, Digitalis orientalis and Digitalis lanata (foxgloves).

[00055] Digoxigenin tag may be introduced into the ACA with digoxigenin NHS ester using methods well known in the art, in which case there is one digoxigenin tag per site of modification on the ACA.

[00056] In a particularly preferred embodiment, digoxigenin is attached via a scaffold using a thiol:maleimide strategy (see Examples section below). Advantageously this approach to conjugation does not require a post-conjugation separation step to remove excess tag.

[00057] A molecular scaffold allows the incorporation of several digoxigenin molecules into the ACA without chemical modification of multiple sites on the ACA, which could impair its function. Moreover, avidity of binding with the anti-tag binding agent is enhanced by the plurality of digoxigenin tags on the molecular scaffold.

[00058] The molecular scaffold is a polymer, for example, a sugar polymer, preferably between 10kDa and 1000kDa in size, and more preferably 500KDa or less.

[00059] In a preferred embodiment the scaffold is dextran, which is functionalized with carboxyl groups, of which at least some are converted to amines or other tag-reactive groups. A variety of methods to introduce carboxyl groups and amines into biomolecules are well known in the art.

[00060] In a particularly preferred embodiment the carboxylated dextran is reacted with a diamine or polyamine to furnish amine groups on the scaffold. Preferably the diamine is ethylenediamine.

[00061] In another preferred embodiment a freeze-dried mixture comprising a reactive digoxigenin scaffold derivative is used to make digoxigenin-ACA reagent, which configuration is not currently known in the art (see Examples).

[00062] The interaction of digoxigenin with the anti-tag binding agent at the T line, advantageously, is not affected by biotin in test samples. [00063] In a particularly preferred embodiment the anti-tag binding agent is an anti- digoxigenin antibody.

[00064] The anti-tag antibody preferably is an IgG, though antibody fragments and non- antibody binding agents may also be used. The species of anti-tag antibody is not limiting.

[00065] Many biological samples contained peptidases. Such peptidases would tend to degrade polypeptide-based tags, rendering them un-capturable at the test line. Accordingly, in certain embodiments the first tag is resistant to proteolysis, e.g., resistant to cleavage by proteinases or peptidases. Proteases to which the tag can be resisted include, for example, serine proteases, cysteine proteases, threonine proteases, aspartic proteases, glutamic proteases, metalloproteases and asparagine peptide lyases. For example, the first tag may be one that does not comprise a peptide bond (i.e. that formed by the condensation of amino acids). The tag can be a small organic molecule, e.g., having a mass of no more than 1500 Da or 500 Da.

[00066] In certain embodiments the first tag comprises a moiety that is either not found or that is found in a very small amounts in the type of sample to be tested for the presence of the analyte. That is, the first tag preferably is chosen such that the sample is substantially free of the first tag. A sample is "substantially free" of a tag if the tag is present in the sample at a concentration no more than 1 milligram per ml, no more than 100 pg per ml, no more than 10 pg per ml, no more than 1 pg per ml, no more than 100 ng per ml, no more than 10 ng per ml, or no more than 1 ng per ml. Similarly, a compound is "not substantially found" in a sample if the compound is normally present in the sample in amounts of no more than 1 milligram per ml, no more than 100 pg per ml, no more than 10 pg per ml, no more than 1 pg per ml, no more than 100 ng per ml, no more than 10 ng per ml, or no more than 1 ng per ml. For example, if the intended sample type is blood, the first tag can be a molecule of which blood is essentially free. In certain embodiments, the first tag is not biotin.

C. Analyte

[00067] The analyte, A, is any entity that can bind simultaneously to the ACA and ADA, one of which (ACA) is anchored by the anti-tag binding agent on the T line. [00068] In a preferred embodiment the analyte is a peptide, protein or polynucleotide, e.g., having a specific nucleotide sequence, though a wide range of substances may be detected, limited only by the availability of suitable binding agents.

[00069] Preferably the interactions on the T and C lines involve completely independent binding interactions, i.e., agents containing V1 and V2 can bind only at the T line and C line, respectively.

[00070] The sample comprising the analyte can be, for example, a biological sample. A biological sample is a sample comprising material produced by a living organism. This includes biological fluids (fluids produced by organisms), cells and cell products. Biological samples include, for example, blood, serum, plasma, saliva, throat swab, nasopharyngeal swab, sputum, pleural effusion, bronchial lavage or aspirates, urine, breast milk, colostrum, tears, peritoneal fluid, cerebrospinal fluid, seminal fluid, amniotic fluid, vaginal samples, stool, skin, plant extracts, cell culture supernatant and/or a biopsy (e.g., tissue biopsy or liquid biopsy). As used herein, the term "raw biological sample" refers to a biological sample which has been subject to no more than crude fractionation, homogenization or preservation, such as removal of particulates, filtration, sedimentation, centrifugation, clot removal, addition of protease or nuclease inhibitors and the like. Raw biological samples typically comprise proteases which may degrade peptides used as first tags in the methods described herein. While such action could in theory be neutralized by the addition of protease inhibitors to the raw sample, the number of

proteases/peptidases in a sample and the inhibitor profiles of each enzyme typically are not known, and the risk of degradation of peptide tags remains.

IV. Control Visualization Agent

[00071] The control visualization agent ("V2") may be captured directly on the control area or through an attached tag. A conjugate comprising a control visualization agent and a tag can be referred to as a "detector reagent". Accordingly, the detector binding agent immobilized at the control area can either be directed to the control visualization agent or to a tag that is attached to the control visualization agent in the detector reagent. The control visualization agent can be the same or different than the test visualization agent.

[00072] V2 may be a naturally occurring colored molecule for which a binding agent, B, is known, or it may be a colored substance V2 with an attached small molecule, analogous to the tag used on the ACA, which can be captured by B immobilized at the C line. As used herein, the term "small molecule" refers to a molecule having a molecular mass of less than 1500 Da, e.g., less than 500 Da. The small molecule may be pre-attached to a biomolecule e.g., a protein, before attachment to V2.

[00073] In a preferred embodiment of the present invention B is streptavidin and V2 is a biotinylated gold particle.

[00074] Biotin may be attached to gold by methods well known in the art, e.g., via a thiolated biotin derivative. Such derivatives are not particularly stable as molecules anchored by a single thiol readily dissociate from gold surfaces.

[00075] More preferably biotin is covalently attached to coated gold via an avidly bound surface coat which affords an extremely stable construct for marking the C line.

[00076] While the C line signal in the present invention is not affected by common test samples such as serum, which contain biotin, (see Examples) there are far less serious consequences of a negative C line, compared with a false negative T line, which is possible with a test that uses biotin as the tag at the T line (e.g., the commercial product from BioPorto) and could lead to misdiagnosis.

[00077] Should a sample contain biotin at levels that interfere in the present invention the sample is further diluted and/or the amount of competing multivalent biotin-gold conjugate is increased to obtain a clear C line signal and confirmation that the strip has run correctly.

[00078] In many conventional lateral flow assays, the binding agent at the control line (e.g., an anti-lgG antibody) binds to an antibody attached to the visualization agent (i.e., a conjugate comprising an anti-analyte antibody attached to a visualization agent). For example, the binding agent at the control line can be selected to capture the same conjugate used for detection of the analyte at the test line. In such implementations, the binding agent at the control line must be selected in reference to the analyte detection agent in the conjugate (typically an antibody).

[00079] In the present devices, the test strip can be universal by virtue of a design to capture a visualization agent/conjugate at the control line independently of the capture of a visualization agent/conjugate at the test line. Accordingly, the detector binding agent immobilized at the control area can be selected to bind a visualization agent directly or through a tag, but to not bind the second conjugate, that is, not to bind the conjugate comprising a visualization agent attached to an analyte detection agent. In particular, the binding agent can be selected to not bind the analyte capture agent (e.g., an antibody), used in the second conjugate. Several embodiments are contemplated. In one embodiment, the binding agent can bind something other than an antibody. For example, the binding agent does not bind an immunoglobulin of a specific species type, e.g., is not an anti-species (anti-rabbit, anti-goat, anti-rat) antibody. In another embodiment, the binding agent can bind a tag which is not comprised in the second conjugate. For example, the tag could be biotin, which is not part of the second conjugate, or, if the tag in the control conjugate is an antibody, the binding agent may bind it, but not an antibody that is part of the second conjugate. In another embodiment, binding agent does not bind the visualization agent directly (e.g., if the control visualization agent is the same as the test visualization agent). In this way, the second conjugate will not be captured at the control line. Typically, the detector binding agent can be selected to bind a second tag that is attached to the control visualization agent, which second tag (e.g., biotin) can be different than the first tag used in the first conjugate (e.g., digoxigenin). This prevents visualization agent from being directly captured at the test line. In another embodiment, when the control visualization agent is not linked to an antibody or, if linked to an antibody, the visualization reagent interacts with the C line in a non-antibody-dependent fashion, such an arrangement performs a control without the need for a detector binding agent specific for the analyte detection agent (e.g., an anti-analyte antibody). In these ways, the test strip can be "universal", usable with any analyte and without reference to antibodies used to detect the analyte at the T line. Moreover, the C line maintains constant intensity regardless of whether any detection reagents are deposited at the T line.

V. Methods

[00080] Preferably the sample containing analyte is contacted with the tagged ACA

(conjugate 1 ) and applied to the lateral flow test strip in the sample application area. After the sample has been drawn into the membrane the ACAA 1 construct (conjugate 2) is applied to the test strip.

[00081] More preferably the sample is contacted with conjugate 1 and conjugate 2 prior to application of the mixture to the test strip. [00082] The mixture may be contacted with the test strip in different ways. For example, if the test strip is housed in a plastic casing, the sample might be pipetted into a port hole directly over the sample pad.

[00083] Alternatively, the test strips may be dipped into a small aliquot of sample placed in a tube or other small receptacle e.g. the well of a 96-well plate.

[00084] Preferably, a range of dilutions of sample is tested to ensure that samples with unknown analyte fall within the dynamic range of the assay. This range is established using samples with known amounts of analyte.

[00085] Increasing the concentration of the detection reagents in the present invention may increase the intensity of the T and C-lines, but possibly also the background. After the fluid has passed along the test strip a further application of buffer, optionally, may be made to clear any background signal.

[00086] After complexes and control visualization agents are captured at the test and control lines, respectively, and, optionally, the strip has been washed by application of extra running buffer, the test strips are considered to be developed. Accordingly, provided herein are articles comprising test strips comprising a solid porous material that supports lateral flow. The solid porous material comprises a capture area and a control area. The capture area comprises a complex capture agent to which is bound a complex comprising an analyte bound by a first conjugate comprising an analyte capture agent bound to a first tag, and a second conjugate comprising an analyte detection agent bound to a first visualization agent. The complex is bound to the complex capture agent through the first tag. The control area comprises a detector binding agent bound to a detector reagent comprising a second visualization agent optionally coupled to a second tag. The capture area and control area can both be configured as lines oriented substantially perpendicular to the direction of lateral flow on the test strip.

[00087] LFA strips can be examined by eye, imaged, or read on a lateral flow reader, of which several are commercially available, or estimated using a scoring card.

[00088] The sample or buffers may be supplemented with other agents to facilitate flow of conjugates along the test strip and to reduce background binding. Such materials include but are not limited to proteins e.g. BSA, detergents e.g. Tween 20, and salts, and are well known in the art. VI. Kits

[00089] As used herein, the term "kit" refers to a collection of items intended for use together. The items in the kit may or may not be in operative connection with each other. A kit can comprise, e.g., test strips, reagents, buffers, enzymes, antibodies and other compositions specific for the purpose. A kit can also include instructions for use and software for data analysis and interpretation. A kit can further comprise samples that serve as normative standards. Typically, items in a kit are contained in primary containers, such as vials, tubes, bottles, boxes or bags. Separate items can be contained in their own, separate containers or in the same container. Items in a kit, or primary containers of a kit, can be assembled into a secondary container, for example a box or a bag, optionally adapted for commercial sale, e.g., for shelving, or for transport by a common carrier, such as mail or delivery service.

[00090] Kits provided herein can comprise elements for conjugating tags and/or visualization agents to affinity agents, such as antibodies, visualization agent conjugates, such as biotin- tagged gold particles, and test strips comprising a capture area having an affinity agent for the first tag and a control area having an affinity agent for the second tag.

[00091] In one embodiment, a kit of this disclosure comprises one or a plurality of test strips of this disclosure, having a capture line with immobilized capture agents, e.g., antibodies, to a first tag and a control line with immobilized capture reagents to a second tag. The kit can further include a container containing the first tag, optionally with reagents for conjugating the first tag to an affinity agent. The kit can further include a container containing a first

visualization agent, optionally including reagents for coupling the visualization agent to an affinity reagent. The kit can also include a complex comprising a second visualization agent (which can be the same as the first visualization reagent) coupled to the second tag (which is different than the first tag). The kit also can include a container containing chromatography buffer. The kit also can include one or a plurality of multiwell plates, e.g., 96-well plates. The kit also can include a scoring card for scoring results of immunoassays. Scoring cards can show different intensities of signal, e.g., color, produced by visualization agents and assign a score to each different intensity or intensity range. VII. Systems

[00092] Also provided herein are systems comprising kits as disclosed herein or developed test strips as disclosed herein and a reader for detecting visualization agent. For example, the reader can be a fluorescence detection reader.

[00093] This new approach to lateral flow described in the present invention is particularly powerful when universal LFA strips are combined with freeze dried conjugation kits described herein, providing for the first time a remarkably simple and rapid approach for developing lateral flow tests, requiring no expensive equipment and no specialist skills.

EXEMPLARY EMBODIMENTS

[00094] Exemplary embodiments of the invention include the following:

[00095] 1. A kit comprising: a) one or more test strips, each test strip comprising a solid porous material that conducts lateral flow of a liquid, wherein the solid porous material comprises: (i) a capture area comprising an immobilized complex capture agent that specifically binds a first tag, and (ii) a control area comprising an immobilized detector binding agent that specifically binds a control visualization agent, either directly or through a second tag attached to the control visualization agent; b) a container containing the first tag; c) a container containing the control visualization agent optionally attached to a second tag; and d) a container containing a test visualization agent.

[00096] 2. The kit of embodiment 1 , wherein the control visualization agent is attached to a second tag, and wherein the detector binding agent binds the second tag.

[00097] 3. The kit of embodiment 2, wherein the second tag comprises biotin.

[00098] 4. The kit of embodiment 3, wherein the detector binding agent comprises streptavidin.

[00099] 5. The kit of embodiment 1 , wherein the second tag, if present, is not an antibody.

[000100] 6. The kit of embodiment 1 , wherein the first tag comprises digoxigenin.

[000101] 7. The kit of embodiment 1 , wherein the first tag is not biotin. [000102] 8. The kit of embodiment 1 , wherein the first tag is protease resistant.

[000103] 9. The kit of embodiment 1 , wherein the detector binding agent does not specifically bind an antibody.

[000104] 10. The kit of embodiment 1 , further comprising: e) one, two or three elements selected from: i) chromatography running buffer, ii) one or more multi-well plates, and iii) one or more scoring cards.

[000105] 1 1. The kit of embodiment 1 , further comprising: e) conjugation reagents for conjugating the first tag to an analyte capture agent and/or for conjugating the test visualization agent to an analyte detection agent.

[000106] 12. The kit of embodiment 11 , further comprising a quenching agent.

[000107] 13. The kit of embodiment 1 , wherein the solid porous material comprises nitrocellulose or cellulose acetate.

[000108] 14. The kit of embodiment 1 , wherein the capture area and/or the control area each are in the form of a line.

[000109] 15. The kit of embodiment 1 , wherein the test strip comprises a sample application area positioned downstream of the capture area, and the control area is positioned upstream of the capture area.

[000110] 16. The kit of embodiment 1 , wherein the test strip further comprises a solid support to which the solid porous material is attached.

[000111] 17. The kit of embodiment 1 , wherein the test strip further comprises: (1 ) an absorbent material for sample application located upstream of the capture and control areas and (2) an absorbent material for wicking located downstream of the capture and control areas, wherein each is in lateral flow contact with the solid porous material.

[000112] 18. The kit of embodiment 1 , wherein the test visualization agent and/or the control visualization agent each comprises a colored particle or a fluorescent material. [000113] 19. The kit of embodiment 18, wherein the test visualization agent and/or the control visualization agent each comprises a gold particle, e.g., with a diameter less than about 200 nm e.g. less than about 100 nm or between about 20 nm and 80 nm.

[000114] 20. The kit of embodiment 18, wherein the test visualization agent and/or the control visualization agent each comprises a latex particle.

[000115] 21. The kit of embodiment 18, wherein where in test visualization agent and/or the control visualization agent each comprises a fluorescent material comprising a fluorescent dye or a fluorescent protein.

[000116] 22. The kit of embodiment 18, wherein where in the control visualization agent comprises a gold-biotin conjugate.

[000117] 23. The kit of embodiment 1 , wherein each test strip further comprises a sample receiving area comprising a porous material which conducts lateral flow of a liquid sample, in lateral flow contact with the solid porous material.

[000118] 24. A kit comprising: (a) a test strip comprising a solid porous material that conducts lateral flow of a liquid, wherein the solid porous material comprises: (i) a capture area comprising an immobilized complex capture agent that specifically binds a first tag, and (ii) a control area comprising an immobilized detector binding agent that binds a control visualization agent, either directly or through a second tag attached to the control visualization agent; (b) a first conjugate comprising an analyte capture agent attached to the first tag, wherein the analyte capture agent specifically binds an analyte; (c) a second conjugate comprising an analyte detection agent attached to a detectable test agent, wherein the analyte detection agent specifically binds the analyte; and (d) control visualization agent optionally attached to a second tag.

[000119] 25. The kit of embodiment 24, wherein the analyte capture agent and/or the analyte detection agent each comprises an antibody.

[000120] 26. The kit of embodiment 24, wherein the detector binding agent does not specifically bind the analyte detection agent.

[000121] 27. The kit of embodiment 24, wherein the detector binding agent is not an anti- species antibody. [000122] 28. The kit of embodiment 24, wherein the complex capture agent is not a biotin binding protein.

[000123] 29. The kit of embodiment 24, wherein the complex capture agent does not target a peptide.

[000124] 30. The kit of embodiment 24, wherein the test strip further comprises: (a) (iii) a second capture area comprising an immobilized complex capture agent that specifically binds a third tag; and wherein the kit further comprises: (e) a third conjugate comprising a second analyte capture reagent attached to the third tag, where in the second analyte capture reagent specifically binds a second, different analyte; and (f) a fourth conjugate comprising a second analyte detection agent attached to a second detectable test agent, wherein the second analyte detection agent specifically binds a second analyte.

[000125] 31. An article comprising: a) a test strip comprising a solid porous material that conducts lateral flow of a liquid, wherein the solid porous material comprises: (i) a capture area comprising an immobilized complex capture agent that specifically binds a first tag, and (ii) a control area comprising an immobilized detector binding agent that binds a control visualization agent, either directly or through a second tag attached to the control visualization agent; b) a complex comprising: (i) an analyte; (ii) a first conjugate comprising an analyte capture agent attached to the first tag, wherein the analyte capture agent is bound to the analyte; (iii) a second conjugate comprising an analyte detection agent attached to the detectable test agent, wherein the analyte detection agent is bound to the analyte; wherein the complex is bound to the complex capture agent on the test strip through the first tag; and c) a control visualization agent bound to the detector binding agent on the test strip, optionally through the second tag, wherein the second tag is not the analyte detection agent.

[000126] 32. The article of embodiment 32, wherein the test strip comprises a sample pad comprising an absorbent material upstream of and in lateral flow contact with the solid porous material, and an absorbent pad downstream of and in lateral flow contact with the solid porous material.

[000127] 33. The article of embodiment 32, wherein the first tag is protease resistant.

[000128] 34. An article comprising: a) a test strip comprising a solid porous material that conducts lateral flow of a liquid, wherein the solid porous material comprises: (i) a capture area comprising an immobilized complex capture agent that specifically binds a small molecule not substantially found in blood, saliva, urine, sweat, breast milk, cerebrospinal fluid or lymphatic fluid, and (ii) a control area comprising an immobilized detector binding agent that specifically binds a small molecule.

[000129] 35. The article of embodiment 32, wherein the complex capture agent specifically binds digoxigenin.

[000130] 36. The article of embodiment 32, wherein the complex capture agent comprises an anti-digoxigenin antibody.

[000131] 37. The article of embodiment 32, wherein the complex capture agent is not targeted to a peptide.

[000132] 38. The article of embodiment 32, wherein the detector binding agent specifically binds biotin.

[000133] 39. The article of embodiment 32, wherein the detector binding agent comprises streptavidin.

[000134] 40. The article of embodiment 32, wherein the test strip is housed in a case comprising one or more windows over the capture area and control area and in window over sample application area.

[000135] 41. A method comprising: a) forming a mixture comprising: (i) a sample comprising an analyte; (ii) a first conjugate comprising an analyte capture agent attached to a first tag, wherein the analyte capture agent specifically binds the analyte; (iii) a second conjugate comprising an analyte detection agent attached to a detectable test agent, wherein the analyte detection agent specifically binds the analyte; and (iii) a control visualization agent optionally attached to a second tag; and (iv) an analyte capture agent attached to a first tag, wherein the analyte capture agent specifically binds the analyte; wherein the analyte, the first conjugate and the second conjugate form a complex through binding of the analyte detection agent and the analyte capture agent with the analyte; and b) performing lateral flow on the mixture on a test strip, wherein the test strip comprises a solid porous material that conducts lateral flow of a liquid, wherein the solid porous material comprises: (i) a capture area comprising an immobilized complex capture agent that specifically binds a first tag, and (ii) a control area comprising an immobilized detector binding agent that binds a control visualization agent, either directly or through a second tag attached to the control visualization agent; c) capturing, at the capture area, the complex through binding of the first tag with the capture reagent; and d) capturing, at the control area, the control visualization agent, directly or through a second tag, by binding with the detector binding agent.

[000136] 42. The method of embodiment 40, wherein the first tag comprises digoxigenin.

[000137] 43. The method of embodiment 40, wherein the sample is substantially free of the first tag.

[000138] 44. The method of embodiment 40, wherein the first tag is protease resistant.

[000139] 45. The method of embodiment 44, wherein the sample comprises proteases.

[000140] 46. The method of embodiment 40, wherein the sample comprises a raw biological sample.

[000141] 47. The method of embodiment 40, wherein forming a mixture comprises mixing the sample comprising the analyte, the first conjugate and the second conjugate to allow formation of a complex and then adding the control visualization agent optionally attached to the second tag.

[000142] 48. The method of embodiment 40, wherein the test strip comprises a conjugation pad comprising absorbent material upstream of and in lateral flow contact with the solid porous material, and, a sample pad comprising an absorbent material upstream of and in lateral flow contact with the conjugation pad, wherein the conjugation pad comprises the first conjugate, the second conjugate and the control visualization agent, and wherein the method comprises applying the sample to the sample pad and contacting the analyte with the first conjugate in the second conjugate by flowing the analyte from the sample pad into the conjugate pad.

[000143] 49. The method of embodiment 40, wherein performing lateral flow chromatography comprises inserting the test strip into a container containing the mixture.

[000144] 50. The method of embodiment 40, wherein performing lateral flow chromatography comprises depositing the mixture onto a sample application area of the test strip. [000145] 51. The method of embodiment 40, further comprising: e) detecting the complex captured at the capture area and the control visualization agent captured at the control area.

[000146] 52. The method of embodiment 40, wherein the detector binding agent binds a tag not comprised in the second conjugate and/or does not bind the second conjugate.

[000147] 53. A system comprising the kit of embodiment 1 , and a fluorescence detection reader.

EXAMPLES

[000148] The invention is further described below by way of illustrative examples and with reference to accompanying drawings.

[000149] Example 1. Production of digoxigenin conjugates [000150] Dextran derivatization

[000151] A carboxymethyl (CM) derivative of dextran (~500kDa, Pharmacosmos) was prepared by adding 5g to a freshly prepared solution of 50ml 1 M bromoacetic acid/2M NaOH in a polypropylene tube. The sample was vigorously shaken for 1 min and then subjected to gentle mixing for 24 hours at 22-25oC. The resulting CM dextran was desalted on Sephadex G-25 columns into 50mM MES, pH 6.0 and its optical rotation (OR) measured in a 10cm polarimeter cell and adjusted to a value of 4.27. Ethylene diamine was added to give a final concentration of 1 M, and EDC to a final concentration of 100mM and reacted at 22-25oC overnight. Two further additions of EDC were made 6 hours apart, each addition corresponding to 25mM final concentration. After overnight incubation, the resulting AM-dextran was exchanged into 1 mM sodium phosphate pH 7.2 and adjusted to 1.42 OR.

[000152] Reaction with digoxigenin/SMCC

[000153] Aminated dextran from 1 (a) was reacted with digoxigenin NHS ester (Enzo, code ENZ-45022- 0001 ) as follows: the NHS ester was dissolved to 5mM concentration with DMSO and 300μΙ of stock was added to 3ml of AM-dextran. After 1 hour at 25oC, sulfo-SMCC was added to 16mM final concentration. After 1 hour the derivatized dextran was desalted on Sephadex G25 (1.1 ml applied per column) into 10mM sodium phosphate pH 5.8. Peak fractions were collected and pooled to give 2.5ml of the digoxigenin- and maleimide-derivatized dextran. [000154] Preparation of digoxigenin-tagged conjugates

[000155] To the digoxigenin- and maleimide-derivatized dextran was added trehalose stock (1g + 2ml water) (100μΙ per ml of dextran) and 2-iminothiolane to 4.7 mM final concentration, giving dextran OR value of 0.48. Aliquots (34.4μΙ) of the derivatized dextran were freeze-dried or used immediately. To initiate conjugation reactions 100μΙ antibody was added at a final concentration of 1 mg/ml in 200mM Hepes/1 mM EDTA (from 10X stock pH 7.5). After 3hr or overnight incubation the reaction was quenched with 10μΙ of 500 mM glycine, pH 7.5.

[000156] Example 2. Preparation of detection conjugates.

[000157] The detection antibody was conjugated to 40nm InnovaCoat® GOLD (Innova Biosciences, code 230-0005) according to the manufacturer's instructions. Briefly, 1 -2 μg of antibody in 45 μΙ was added to a mini-vial and the reaction was quenched after 15 minutes.

[000158] Example 3. Preparation of universal test strips

[000159] T line reagent - Goat anti-digoxigenin antibody (Vector Labs, MB-7000) was mixed with isopropanol (5% v/v final concentration).

[000160] C line reagent - Streptavidin was made up at 2mg/ml in PBS containing 5% isopropanol.

[000161] A Biojet dispenser (ZX1010) (Biodot Inc.) was primed and T line and C line reagents were dispensed onto Millipore HF090 cards at set positions, 6mm and 1.3cm from the end of the nitrocellulose area at a rate of 0.8μΙ/αη. Cards were then dried at 37°C before being assembled with a glass fiber sample pad and a wicking pad. Fully assembled cards were cut using a Bioject CM4000 (Biodot Inc.) into 5mm wide strips which were stored in sealed pouches with desiccant.

[000162] Example 4. CRP assay using universal LFA method

[000163] The capture and detection anti CRP antibodies (Hytest, C2 and C7 respectively) were conjugated to digoxigenin (Example 1 , c) and 40nm InnovaCoat® GOLD (Example 2).

[000164] Recombinant human CRP (R&D Systems) was spiked in CRP-depleted serum (BBI) from 360ng/ml to 4.4ng/ml. 75μΙ of spiked serum was mixed with 5μΙ 0μg/ml C2-digoxigenin conjugate, 5μΙ 6 OD C7-lnnovaCoat® GOLD and 5μΙ 1 OD Biotin InnovaCoat® GOLD. After incubating for 5 minutes, 80μΙ of the mixture was loaded on each strip and read using

ESEQuant reader (Qiagen) after 20 minutes.

[000165] Figure 2 shows the measured values for the T Line (full) and C line (dotted). Note that the C line signal intensities are not significantly affected by serum. Where a reader is not available a score is assigned to the T line signal, aided by a scoring card, also illustrated in Figure 2.

[000166] The specific implementation of the universal LFA test using the above CRP antibodies is shown schematically in Figure 3.

[000167] Example 5. Universal lateral flow kit

[000168] The universal kit comprises the following reagents and allows methods of the present invention to be carried out by any competent technician with no experience of LFA tests: three freeze dried vials of digoxigenin- and maleimide-derivatized material (Example 1 c) suitable for reaction with 100μg of antibody; three mini-vials of 40nm InnovaCoat® GOLD (Example 2) which provide enough material for 100 LFA tests; 100 Universal LFA strips (Example 3); 2 cryovials of 10x Universal Running Buffer (0.5M Tris, 1.5M NaCI, 10% Tween 20); 1 x vial 40nm lnnovaCoat®GOLD-Biotin, 10 OD for visualizing the C line; 1 x scoring card; 2 x 96-well clear low binding plates.

[000169] The 10x Universal Running Buffer is diluted 1 :10 with distilled water and BSA is added as a blocking agent (0.1 % final concentration) to obtain 1x Universal Running Buffer + BSA. To optimize the amount of digoxigenin-conjugated capture antibody, initial tests use between 10μg and 150μg antibody per ml diluted in Universal Running Buffer + BSA. Once the amount of capture reagent has been optimized the amount of gold conjugate may be varied as required.

[000170] Kit components and samples are further prepared as follows:

[000171] Dilute the InnovaCoatOGOLD-detection antibody to 6 OD concentration in 1x Universal Running Buffer + BSA

[000172] Dilute the 40nm lnnovaCoat®GOLD-Biotin 1 OD in 1x Universal Running Buffer + BSA (1 :10 dilution) [000173] Dilute sample containing analyte in 1x Universal Running Buffer + BSA

[000174] Each sample is tested in duplicate or triplicate. For a single strip, prepare the following mix:

[000175] 5μΙ diluted capture digoxigenin-conjugated antibody 5μΙ 6 OD 40nm

InnovaCoatOGOLD-detection antibody 5μΙ 1 OD 40nm InnovaCoatOGOLD-Biotin

[000176] 75μΙ_ analyte solution

[000177] Incubate the mixture for 5 minutes In a 96 well plate, load 80μΙ of the mixture and insert one Universal LFA strip in each well. Handle the strip by the wicking pad to avoid touching the nitrocellulose, and make sure the sample pad (thinner and longer pad, made of glass fiber) is dipped into the well.

[000178] Leave the strip for 20 minutes.

[000179] Compare the T-line color intensity with the scoring card or use an LFA reader. If the C-line is not visible the test is not valid and should be repeated.

[000180] As used herein, the following meanings apply unless otherwise specified. The word "may" is used in a permissive sense (i.e., meaning having the potential to), rather than the mandatory sense (i.e., meaning must). The words "include", "including", and "includes" and the like mean including, but not limited to. The singular forms "a," "an," and "the" include plural referents. Thus, for example, reference to "an element" includes a combination of two or more elements, notwithstanding use of other terms and phrases for one or more elements, such as "one or more." The term "or" is, unless indicated otherwise, non-exclusive, i.e., encompassing both "and" and "or."

[000181] It should be understood that the description and the drawings are not intended to limit the invention to the particular form disclosed, but to the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present invention as defined by the appended claims. Further modifications and alternative

embodiments of various aspects of the invention will be apparent to those skilled in the art in view of this description. Accordingly, this description and the drawings are to be construed as illustrative only and are for the purpose of teaching those skilled in the art the general manner of carrying out the invention. It is to be understood that the forms of the invention shown and described herein are to be taken as examples of embodiments. Elements and materials may be substituted for those illustrated and described herein, parts and processes may be reversed or omitted, and certain features of the invention may be utilized independently, all as would be apparent to one skilled in the art after having the benefit of this description of the invention. Changes may be made in the elements described herein without departing from the spirit and scope of the invention as described in the following claims. Headings used herein are for organizational purposes only and are not meant to be used to limit the scope of the description.

Claims

WHAT IS CLAIMED IS:

1. A kit comprising:

a) one or more test strips, each test strip comprising a solid porous material that conducts lateral flow of a liquid, wherein the solid porous material comprises:

(i) a capture area comprising an immobilized complex capture agent that specifically binds a first tag, and

(ii) a control area comprising an immobilized detector binding agent that specifically binds a control visualization agent, either directly or through a second tag attached to the control visualization agent; b) a container containing the first tag;

c) a container containing the control visualization agent optionally attached to a second tag; and

d) a container containing a test visualization agent.

2. The kit of claim 1 , wherein the control visualization agent is attached to a second tag, and wherein the detector binding agent binds the second tag.

3. The kit of claim 2, wherein the second tag comprises biotin.

4. The kit of claim 3, wherein the detector binding agent comprises streptavidin.

5. The kit of claim 1 , wherein the second tag, if present, is not an antibody.

6. The kit of claim 1 , wherein the first tag comprises digoxigenin.

7. The kit of claim 1 , wherein the first tag is not biotin.

8. The kit of claim 1 , wherein the first tag is protease resistant.

9. The kit of claim 1 , wherein the detector binding agent does not specifically bind an antibody.

10. The kit of claim 1 , further comprising:

e) one, two or three elements selected from:

i) chromatography running buffer,

ii) one or more multi-well plates, and iii) one or more scoring cards.

11. The kit of claim 1 , further comprising:

e) conjugation reagents for conjugating the first tag to an analyte capture agent and/or for conjugating the test visualization agent to an analyte detection agent.

12. The kit of claim 11 , further comprising a quenching agent.

13. The kit of claim 1 , wherein the solid porous material comprises nitrocellulose or cellulose acetate.

14. The kit of claim 1 , wherein the capture area and/or the control area each are in the form of a line.

15. The kit of claim 1 , wherein the test strip comprises a sample application area positioned downstream of the capture area, and the control area is positioned upstream of the capture area.

16. The kit of claim 1 , wherein the test strip further comprises a solid support to which the solid porous material is attached.

17. The kit of claim 1 , wherein the test strip further comprises: (1 ) an absorbent material for sample application located upstream of the capture and control areas and (2) an absorbent material for wicking located downstream of the capture and control areas, wherein each is in lateral flow contact with the solid porous material.

18. The kit of claim 1 , wherein the test visualization agent and/or the control visualization agent each comprises a colored particle or a fluorescent material.

19. The kit of claim 18, wherein the test visualization agent and/or the control visualization agent each comprises a gold particle, e.g., with a diameter less than about 200 nm e.g. less than about 100 nm or between about 20 nm and 80 nm.

20. The kit of claim 18, wherein the test visualization agent and/or the control visualization agent each comprises a latex particle.

21. The kit of claim 18, wherein where in test visualization agent and/or the control visualization agent each comprises a fluorescent material comprising a fluorescent dye or a fluorescent protein.

22. The kit of claim 18, wherein where in the control visualization agent comprises a gold-biotin conjugate.

23. The kit of claim 1 , wherein each test strip further comprises a sample receiving area comprising a porous material which conducts lateral flow of a liquid sample, in lateral flow contact with the solid porous material.

24. A kit comprising:

(a) a test strip comprising a solid porous material that conducts lateral flow of a liquid, wherein the solid porous material comprises:

(ii) a control area comprising an immobilized detector binding agent that binds a control visualization agent, either directly or through a second tag attached to the control visualization agent;

(b) a first conjugate comprising an analyte capture agent attached to the first tag, wherein the analyte capture agent specifically binds an analyte;

(c) a second conjugate comprising an analyte detection agent attached to a detectable test agent, wherein the analyte detection agent specifically binds the analyte; and

(d) control visualization agent optionally attached to a second tag.

25. The kit of claim 24, wherein the analyte capture agent and/or the analyte detection agent each comprises an antibody.

26. The kit of claim 24, wherein the detector binding agent does not specifically bind the analyte detection agent.

27. The kit of claim 24, wherein the detector binding agent is not an anti-species antibody.

28. The kit of claim 24, wherein the complex capture agent is not a biotin binding protein.

29. The kit of claim 24, wherein the complex capture agent does not target a peptide.

30. The kit of claim 24, wherein the test strip further comprises:

(a) (iii) a second capture area comprising an immobilized complex capture agent that specifically binds a third tag;

and wherein the kit further comprises:

(e) a third conjugate comprising a second analyte capture reagent attached to the third tag, where in the second analyte capture reagent specifically binds a second, different analyte; and

(f) a fourth conjugate comprising a second analyte detection agent attached to a second detectable test agent, wherein the second analyte detection agent specifically binds a second analyte.

31. An article comprising:

a) a test strip comprising a solid porous material that conducts lateral flow of a liquid, wherein the solid porous material comprises:

b) a complex comprising:

(i) an analyte;

(ii) a first conjugate comprising an analyte capture agent attached to the first tag, wherein the analyte capture agent is bound to the analyte;

(iii) a second conjugate comprising an analyte detection agent attached to the detectable test agent, wherein the analyte detection agent is bound to the analyte;

wherein the complex is bound to the complex capture agent on the test strip through the first tag; and

c) a control visualization agent bound to the detector binding agent on the test strip, optionally through the second tag, wherein the second tag is not the analyte detection agent.

32. The article of claim 32, wherein the test strip comprises a sample pad comprising an absorbent material upstream of and in lateral flow contact with the solid porous material, and an absorbent pad downstream of and in lateral flow contact with the solid porous material.

33. The article of claim 32, wherein the first tag is protease resistant.

34. An article comprising:

(i) a capture area comprising an immobilized complex capture agent that specifically binds a small molecule not substantially found in blood, saliva, urine, sweat, breast milk, cerebrospinal fluid or lymphatic fluid, and

(ii) a control area comprising an immobilized detector binding agent that specifically binds a small molecule.

35. The article of claim 32, wherein the complex capture agent specifically binds digoxigenin.

36. The article of claim 32, wherein the complex capture agent comprises an anti- digoxigenin antibody.

37. The article of claim 32, wherein the complex capture agent is not targeted to a peptide.

38. The article of claim 32, wherein the detector binding agent specifically binds biotin.

39. The article of claim 32, wherein the detector binding agent comprises streptavidin.

40. The article of claim 32, wherein the test strip is housed in a case comprising one or more windows over the capture area and control area and in window over sample application area.

41. A method comprising:

a) forming a mixture comprising: (i) a sample comprising an analyte;

(ii) a first conjugate comprising an analyte capture agent attached to a first tag, wherein the analyte capture agent specifically binds the analyte;

(iii) a second conjugate comprising an analyte detection agent attached to a detectable test agent, wherein the analyte detection agent specifically binds the analyte; and

(iii) a control visualization agent optionally attached to a second tag; and

(iv) an analyte capture agent attached to a first tag, wherein the analyte capture agent specifically binds the analyte;

wherein the analyte, the first conjugate and the second conjugate form a complex through binding of the analyte detection agent and the analyte capture agent with the analyte; and

b) performing lateral flow on the mixture on a test strip, wherein the test strip comprises a solid porous material that conducts lateral flow of a liquid, wherein the solid porous material comprises:

c) capturing, at the capture area, the complex through binding of the first tag with the capture reagent; and

d) capturing, at the control area, the control visualization agent, directly or through a second tag, by binding with the detector binding agent.

42. The method of claim 40, wherein the first tag comprises digoxigenin.

43. The method of claim 40, wherein the sample is substantially free of the first tag.

44. The method of claim 40, wherein the first tag is protease resistant.

45. The method of claim 44, wherein the sample comprises proteases.

46. The method of claim 40, wherein the sample comprises a raw biological sample.

47. The method of claim 40, wherein forming a mixture comprises mixing the sample comprising the analyte, the first conjugate and the second conjugate to allow formation of a complex and then adding the control visualization agent optionally attached to the second tag.

48. The method of claim 40, wherein the test strip comprises a conjugation pad comprising absorbent material upstream of and in lateral flow contact with the solid porous material, and, a sample pad comprising an absorbent material upstream of and in lateral flow contact with the conjugation pad, wherein the conjugation pad comprises the first conjugate, the second conjugate and the control visualization agent, and wherein the method comprises applying the sample to the sample pad and contacting the analyte with the first conjugate in the second conjugate by flowing the analyte from the sample pad into the conjugate pad.

49. The method of claim 40, wherein performing lateral flow chromatography comprises inserting the test strip into a container containing the mixture.

50. The method of claim 40, wherein performing lateral flow chromatography comprises depositing the mixture onto a sample application area of the test strip.

51. The method of claim 40, further comprising:

e) detecting the complex captured at the capture area and the control visualization agent captured at the control area.

52. The method of claim 40, wherein the detector binding agent binds a tag not comprised in the second conjugate and/or does not bind the second conjugate.

53. A system comprising the kit of claim 1 , and a fluorescence detection reader.