WO2019197365A1 - Chemical and biological complexes and conjugates with multiple labels and applications thereof - Google Patents

Abstract

Disclosed herein are complexes comprising: (i) a targeting agent or binding agent; (ii) a first detection agent; and (iii) a second, different, detection agent. The first detection agent can be, for example, a direct detection agent such as a colorimetric detection agent. The second detection agent can be, for example, an indirect detection agent such as an enzymatic detection agent.Such complexes are useful, for example, the methods involving detection of either or both of the detection agents. The use of two different kinds of detection agents results in a greater dynamic range of detection in an assay using the complexes.

Description

CHEMICAL AND BIOLOGICAL COMPLEXES AND CONJUGATES WITH

MULTIPLE LABELS AND APPLICATIONS THEREOF

REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of the priority date of U.S. provisional application 62/654,601 , filed April 9, 2018, the contents of which are incorporated herein in their entirety.

BACKGROUND

[0002] Western blotting or immunoblotting is widely used to study the presence or absence of polypeptides in complex mixture of proteins. This involves electrophoresis of proteins in SDS-polyacrylamide gels and then transfer of proteins to a nitrocellulose or PVDF membrane, which is then probed with a primary antibody specific for the polypeptide of interest. A label whose purpose is to provide measurability is associated with the primary antibody, through either a covalent or a non-covalent interaction, the latter typically involving the binding of a labelled anti-species secondary antibody or a streptavidin conjugate.

[0003] Labels used in western blotting include colored particles, fluorescent dyes, and enzyme conjugates. Gold nanoparticles are readily visualized by eye and are convenient for detecting quantitatively major polypeptides in a mixture. Fluorescent dyes potentially provide lower limits of protein detection, but they require more complex equipment to excite the dye and to capture an image. Enzyme labels include horseradish peroxidase (HRP) and alkaline phosphatase which provide a range of options for detection, including visual, i.e., by depositing insoluble colored products on the membrane at the site of primary antibody binding; fluorescence, by conversion of non-fluorescent substrates into fluorescent products, or, more commonly, by chemiluminescence.

[0004] Because the amount of the polypeptide of interest in the sample is usually not known, it is difficult to know which method of detection should be used. A negative western blotting result raises the question of whether a positive might have been obtained had a different label been used. It is difficult and time-consuming to strip and re-probe a blot with a label that allows a greater level of sensitivity. Equally, it is not desirable to use enhanced chemiluminescence or fluorescence in all situations, as these techniques are more labor- intensive and, in many cases, simple visual detection with a colored substance would suffice.

[0005] Thus, there is a need for a single detection reagent or complex, comprising two or more detection agents, which reagent or complex provides high dynamic range of detection that can be exploited to measure both quantitatively major and minor antigens in Western blots and in related paper-based assay techniques

BRIEF DESCRIPTION OF THE DRAWINGS

[0006] 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:

[0007] FIG. 1 shows a characterisation of aminated-HRP gold. Solid line: Binary aminated HRP-gold complex from method 1 c, below. Dotted line: Uncoated gold

nanoparticles particles in water.

[0008] FIGs. 2A and 2B depicts a split-view comparison between a Western blot using the methods described herein and traditional Western blot detection methods.

[0009] FIG. 3 shows ability to detect protein with a ternary complex as disclosed herein. Amounts of protein above 1 ng can be detected visually while amounts of protein 1 ng and below can be detected using enzymatic detection agent.

SUMMARY

[00010] In one aspect provided herein is a method for detecting a target molecule in a sample comprising: (a) contacting the sample with a targeting agent that specifically binds to the target molecule to allow binding between the targeting agent and the target molecule; (b) associating a first detection agent and a second detection agent with the targeting agent, either covalently or non-covalently; and (c) detecting the binding through detection of the first detection agent or the second detection agent. In one embodiment the first detection agent and second detection agent are associated with the targeting agent before contacting. In another embodiment the first detection agent and second detection agent are associated with the targeting agent after contacting. In another embodiment the first detection agent and second detection agent are non-covalently associated with the targeting agent through binding of a ternary complex comprising (1 ) a binding agent that specifically binds the targeting agent and (2) the first and second detection agents. In another embodiment the targeting agent comprises a primary antibody and the complex comprises: (i) a binding agent comprising a secondary antibody; (ii) the first detection agent; and (iii) the second detection agent. In another embodiment the targeting agent comprises a tag and the binding agent specifically binds the tag. In another embodiment the first detection agent comprises a direct label and the second detection agent comprises an indirect label. In another embodiment the detection limit of the second detection agent is at least ten times, at least 100 times or at least 1000 times greater than the detection limit of the first detection agent. In another embodiment the method has a dynamic range of detection of at least 10³, at least 10⁴, at least 10⁵, or at least 10⁶. In another embodiment the first detection agent and second detection agents are differently selected from colorimetric, light-emitting, enzymatic and radioactive detection agents. In another embodiment one of the detection agents comprises a colorimetric detection agent. In another embodiment the colorimetric detection agent comprises a gold particle (e.g., colloidal gold), a carbon particle, a platinum particle, an organic dye, a glass particle, a plastic particle (e.g., polystyrene, polypropylene, latex) or a dye-modified sugar polymer. In another embodiment the colorimetric detection agent comprises a particle comprising a polymeric coating comprising a plurality of functional groups to facilitate multivalent attachment to the complex. In another embodiment the polymeric coating material comprises a sugar polymer or polypeptide. In other embodiments one of the detection agents comprises an enzymatic detection agent. In another embodiment the enzymatic detection agent comprises urease, b-galactosidase, b-glucuronidase, alkaline phosphatase, (horseradish) hydrogen peroxidase (HRP) or glucose oxidase. In another embodiment one of the detection agents comprises a light-emitting detection agent. In another embodiment the light-emitting detection agent comprises a fluorescent label or a bioluminescent label. In another embodiment the light-emitting detection agent comprises a bioluminescent label selected from a luciferase or a green fluorescent protein. In another embodiment one of the detection agents comprises a radioactive detection agent. In another embodiment the first detection agent comprises a colorimetric detection agent and the second detection agent comprises an enzymatic detection agent. In another embodiment the colorimetric detection agent comprises a gold particle and the enzymatic detection agent comprises horse radish peroxidase. In another embodiment the colorimetric detection agent has a detection limit of at least one nanogram of target and the enzymatic detection agent has a detection limit of at least one picogram of target. In another embodiment the first detection agent comprises a colorimetric detection agent and the second detection agent comprises a detection agent selected from a light-emitting detection agent and a radioactive detection agent. In another embodiment the targeting agent comprises an antibody. In another embodiment the targeting agent comprises a non-antibody protein, an aptamer or a nucleic acid. In another embodiment detecting comprises performing an immunoassay. In another embodiment the immunoassay uses a primary antibody and a secondary antibody, which secondary antibody is comprised in the complex. In another embodiment the immunoassay comprises a protein immune blot (“Western blot”) comprising: (I) separating proteins in the sample by gel electrophoresis (e.g., SDS PAGE or isoelectric focusing); (II) transferring separated proteins to a solid support (e.g., a membrane, e.g., made of nitrocellulose (NC) or polyvinylidene difluoride (PVDF)), e.g., by electroblotting; (III) contacting the transferred protein with a primary antibody to allow specific binding between the primary antibody and a target protein, and removing unbound primary antibody; (IV) contacting the bound primary antibody with the complex, wherein, the targeting agent comprises a secondary antibody, to allow specific binding between the secondary antibody and the primary antibody, and removing unbound secondary antibody; (V) detecting bound secondary antibody through detection of the first detection agent or the second detection agent. In another embodiment the first detection agent comprises a direct label and the second or detection agent comprises an indirect label, and, wherein, if the direct label is not visually detected, binding is determined through detection of the indirect label. In another embodiment the immunoassay comprises a sandwich immunoassay. In another embodiment detecting comprises performing an oligonucleotide probe hybridization assay. In another embodiment the hybridization comprises a nucleic acid blot selected from a DNA blot (“Southern blot”) and an RNA blot (“northern blot”) comprising: (I) separating nucleic acids in the sample by gel electrophoresis; (II) transferring separated nucleic acids to a solid support; (III) contacting the transferred nucleic acid with the complex, wherein, the targeting agent comprises a nucleic acid probe that hybridizes with a target nucleic acid, to allow specific binding between the nucleic acid probe and the target nucleic acid, and removing unbound nucleic acid probe; (V) detecting bound target nucleic acid through detection of the first detection agent or the second detection agent. In another embodiment the first detection agent comprises a direct label and the second or detection agent comprises an indirect label, and, wherein, if the direct label is not visually detected, binding is determined through detection of the indirect label. In another embodiment the complex further comprises (iv) one or more other detection agents. In another embodiment the method further comprises, after forming the targeting agent/target combination, washing to remove unbound material.

[00011] In another aspect provided herein is a complex comprising: (i) a targeting agent; (ii) a first detection agent; and (iii) a second, different, detection agent. In one embodiment the first detection agent comprises a direct label and the second detection agent comprises an indirect label. In another embodiment the detection limit of the second detection agent is at least ten times, at least 100 times or at least 1000 times greater than the detection limit of the first detection agent. In another embodiment the first detection agent and second detection agents are differently selected from colorimetric, light-emitting, enzymatic and radioactive detection agents. In another embodiment one of the detection agents comprises a colorimetric detection agent. In another embodiment the colorimetric detection agent comprises a gold particle (e.g., colloidal gold), a carbon particle, a platinum particle, an organic dye, a glass particle, a plastic particle (e.g., polystyrene, polypropylene, latex) or a dye-modified sugar polymer. In another embodiment the enzymatic detection agent comprises urease, b-galactosidase, b-glucuronidase, alkaline phosphatase, (horseradish) hydrogen peroxidase (HRP) or glucose oxidase. In another embodiment the first detection agent comprises a colorimetric detection agent and the second detection agent comprises an enzymatic detection agent. In another embodiment the colorimetric detection agent comprises a gold particle and the enzymatic detection agent comprises horse radish peroxidase. In another embodiment the colorimetric detection agent has a detection limit of at least one nanogram of target and the enzymatic detection agent has a detection limit of at least one picogram of target. In another embodiment the targeting agent comprises an antibody. In another embodiment the antibody is an anti-immunoglobulin antibody (e.g., anti- IgG). In another embodiment the first detection agent comprises a gold nanoparticle and the second detection agent comprises an enzyme, e.g., horse radish peroxidase, and further wherein the gold nanoparticle and the enzyme are covalently attached through amide bond, and further wherein the covalently bound first and second detection agents are bound to a polypeptide binding agent through a thiol group. In another embodiment the targeting agent comprises a non-antibody protein, an aptamer or a nucleic acid.

[00012] In another aspect provided herein is a kit comprising: (a) a container containing a complex comprising: (i) a targeting agent; (ii) a colorimetric detection agent; and (iii) and enzymatic detection agent comprising an enzyme; and (b) a container containing a substrate for the enzyme wherein, the product of the enzymatic reaction produces a detectable signal.

[00013] In another aspect provided herein is a kit comprising: (a) a container containing a ternary complex comprising: (i) a binding agent, wherein the binding agent binds a tag; (ii) a first detection agent; and (iii) a second, different, detection agent; (b) a second container containing the tag. In another embodiment the kit further comprises: (c) a third container containing reagents for conjugating the tag to a targeting agent.

[00014] In another aspect provided herein is a method of making a complex, wherein the complex comprises: (i) a targeting agent; (ii) a first detection agent; and (iii) a second, different, detection agent; wherein the method comprises: (a) coupling the first detection agent to the targeting agent; and (b) coupling the second detection agent to the targeting agent or to the first detection agent. In one embodiment the method comprises coupling the second detection agent to the targeting agent. In another embodiment the method comprises coupling the second detection agent to the first detection agent. In another embodiment the first detection agent comprises a visually detectable particle. In another embodiment the particle comprises a plurality of functional groups, e.g., amines, amides, carboxyls, and coupling comprises reacting the functional groups with a functional group on the targeting agent (e.g., coupling the second detection agent to the targeting agent. In another embodiment the visually detectable particle comprises a gold particle (e.g., colloidal gold), a carbon particle, a platinum particle, an organic dye, a glass particle, a plastic particle (e.g., polystyrene, polypropylene, latex) or a dye-modified sugar polymer. In another embodiment the visually detectable particle comprises a particle comprising a polymeric coating comprising a plurality of functional groups to facilitate multivalent attachment to the complex. In another embodiment the method comprises coupling the second detection agent to the targeting agent.

[00015] In another aspect provided herein is a method for detecting a target molecule in a mixture involving the binding of a complex comprising (i) a first detection agent, (ii) a second detection agent, (iii) a targeting agent, and (iv) optionally, one or more other detection agents; further involving, after washing, the measurement of the first detection agent and, optionally, measurement of the second detection agent. In one embodiment the target molecule is a protein or a nucleic acid. In another embodiment the first or second detection agent has a polymeric coating material. In another embodiment the targeting agent is an antibody, or other binding protein, or a small molecule capable of binding to a biomolecule.

In another embodiment at least one of the detection agents is a colored substance or an enzyme capable of generating a detectable substance. In another embodiment the colored substance is a particle. In another embodiment the detectable substance is a product or by- product of enzyme action. In another embodiment the particle is inorganic or organic in nature. In another embodiment the particle is gold or colored latex or fluorescent latex. In another embodiment the enzyme product is colored or can be made to emit light after supplying external energy. In another embodiment the by-product of the reaction is light. In another embodiment the enzyme is horse radish peroxidase (HRP) or alkaline phosphatase. In another embodiment the polymeric coating material is a sugar polymer or polypeptide. In another embodiment the polymer is a detection agent. In another embodiment the target molecule(s) are immobilized on a surface.

[00016] In another aspect provided herein is a complex comprising at least two different detection agents and a targeting or binding agent. In one embodiment the detection agents are two or more of the following: gold particles, carbon particles, platinum particles, organic dyes, dyed latex particles, dye-modified sugar polymers, radiolabels, chemiluminescent molecules, fluorescent molecules, or enzymes capable of generating chemiluminescent molecules or fluorescent molecules. In another embodiment the binding or targeting agent is one of the following: antibodies, antibody fragments, nanobodies, other binding proteins, polypeptides and fragments; small molecules including peptides, metal ions (attached to chelators), streptavidin, biotin and nucleic acids. [00017] In another aspect provided herein is a kit for detecting a target protein comprising a complex as described herein wherein the binding agent recognizes a tag, together with a conjugation kit for attaching the tag to an antibody that recognizes the target protein; mixing said complex with the tagged antibody, and incubating the resulting reagent with the target protein.

DETAILED DESCRIPTION

I. Introduction

[00018] Disclosed herein are covalently linked ternary conjugates comprising a targeting agent (sometimes referred to herein as a binding agent) (B) and two or more detection agents (labels). The targeting agent can be any agent that preferentially or specifically binds a target, such as an antibody or a polynucleotide probe. One of the detection agents, referred to herein as a“direct label” (L1 ), is detectable by the human eye without intermediate intervention or indirect detection. The other detection agent, referred to herein as an“indirect label” (L1 ) is either invisible to the human eye and requires detection by a machine, or is becomes visible to the human eye after physical, chemical or biochemical intervention.

[00019] In one aspect a method for detecting a target molecule in a sample comprises:

(a) contacting the sample with a targeting agent that specifically binds to the target molecule to allow binding between the targeting agent and the target molecule; (b) associating a first detection agent and a second detection agent with the targeting agent, either covalently or non-covalently; and (c) detecting the binding through detection of the first detection agent or the second detection agent.

[00020] In certain embodiments, a ternary complex as disclosed herein comprises a targeting agent that directly binds the target molecule. In such a case, the first detection agent and second detection agent are associated with the targeting agent before contacting with the target molecule.

[00021] In other embodiments, a ternary complex as disclosed herein comprises a binding agent that binds a targeting molecule which, itself, is directed against the target molecule. In this case, the ternary complex can associate with the targeting molecule before or after the targeting molecule binds a target molecule. For example, the targeting molecule can be brought into contact with, and bind to, the target molecule. Then, afterwards, the ternary complex can be brought into contact with, and bind to, the targeting molecule.

Alternatively, the ternary complex and the targeting molecule can be brought into contact such that the ternary complex binds to the targeting molecule. This secondary complex can then be brought into contact with, and bind to, the target molecule.

[00022] More broadly, this invention can be readily applied to applications beyond polypeptides and immunoblotting, such as nucleic acid detection using complementary DNA or RNA as the targeting agent.

II. Ternary Conjugates

A. Targeting Agents

[00023] As used herein, the terms“targeting agent” and“binding agent” refer to agents that specifically bind a target molecule. Targeting agents include, without limitation, antibodies, non-antibody proteins (e.g., protein receptors, aptamers (peptide or nucleic acid) and nucleic acids (e.g., DNA or RNA oligonucleotide probes). Binding or targeting agents include antibodies, antibody fragments, nanobodies, other binding proteins; small molecules including peptides, metal ions (attached to chelators), streptavidin and biotin. Targeting agents further including binding fragments of any of the above. In certain instances, for example when the two terms are used together the term“targeting agent” refers to an agent that is directed to a target molecule, and the term“binding agent” refers to an agent that binds to a targeting agent.

[00024] A targeting agent may be any agent that selectively or specifically binds a target of interest. A targeting agent specifically binds a target molecule if the targeting 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. The binding agent can be selected to bind to the target analyte with an affinity of at least 10³ M, 10⁴ M, 10 ⁵ M, 10⁶ M, 10 ⁷ M, 10 ⁸ M, 10⁹ M, 10 ¹° M, 10 ¹¹ M or 10 ¹² M.

[00025] In one preferred embodiment, B is an antibody. The antibody may be from any species and of any class or subclass. It may be a whole molecule or fragment derived therefrom, either enzymatically or by expression after cloning of the binding domain, along with some or none of the other elements found in a whole antibody.

[00026] As used herein, the term“antibody” refers to a polypeptide comprising a framework region from an immunoglobulin gene, that specifically bind and recognize an antigen. Typically, the“variable region” contains the antigen-binding region of the antibody (or its functional equivalent) and is most critical in specificity and affinity of binding. An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one“light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The term“antibody” encompasses (1 ) whole immunoglobulins (two light chains and two heavy chains, e.g., a tetramer), (2) an

immunoglobulin polypeptide (a light chain or a heavy chain), (3) an antibody fragment, such as Fab (V_LC_L V_HC_H), F(ab')₂ , Fv (V_LV_H), scFv (single chain Fv), (scFv)₂, sc(Fv)₂, bispecific SC(FV)₂, bispecific (scFv)₂, minibody (intact immunoglobulin without CH₂ region), triabody, Fd (portion of the heavy chain contained in the Fab fragment) and dAb (diabody), and (4) a fusion protein comprising a binding portion of an immunoglobulin fused to another amino acid sequence (such as a fluorescent protein), all retaining antigen binding activity. The term includes but is not limited to polyclonal or monoclonal antibodies of the isotype classes IgA, IgD, IgE, IgG, and IgM, derived from human or other mammalian cells. The term also includes natural or genetically modified forms such as humanized, human, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, grafted, and in vitro generated antibodies.

[00027] In another preferred embodiment, B is a non-antibody binding agent, which includes proteins, glycoproteins, other biomolecules or small molecules. In the last- mentioned category the following are examples: peptides, other small molecules that may have a receptor site on a larger biomolecule, and chelating groups.

[00028] Receptors include non-antibody proteins that specifically bind targets. Receptors are frequently found on the cell surface.

[00029] In another embodiment, the targeting agent comprises a nucleic acid. This includes DNA, RNA and artificial molecules such as peptide nucleic acids. Typically, nucleic acid probes are oligonucleotides, e.g., no more than about 200, no more than about 150, no more than about 100, no more than about 50, no more than about 40, no more than about 30 or no more than about 20 nucleotides long.

[00030] Finally, B may recognise a molecular tag previously introduced into antibodies or other binding proteins by chemical means to allow subsequent detection with an anti-tag ternary complex. In this way one ternary complex can be used with multiple tagged primary antibodies. Tags include biotin, digoxigenin or fluorescein, which may be attached to primary antibodies using methods well known in the art. Alternatively, streptavidin can be used. Where the ternary complex is configured to bind a tag, the binding agent can be, for example, streptavidin (for binding biotin) or anti-digoxigenin antibody for digoxigenin. [00031] B may be attached to L1 or L2. Alternatively, L2 may act as a bridge between B and L1. In another aspect of the invention L2 had dual functionality, acting both as a coating material and as a detection agent.

B. Detection Agents

[00032] As used herein, the term“detection agent” (sometimes referred to as a“label”) refers to a composition producing a detectable signal, such as a colorimetric, light-emitting, enzymatic or radioactive signal. Light-emitting agents include, for example, fluorescent, bioluminescent, chemiluminescent and phosphorescent agents. These terms are not meant to be mutually exclusive. For example, bioluminescent agents, such as fluorescent proteins, generate fluorescence. Detection agents can be detected by visual, spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means.

[00033] Detection agents can be classified as directly detectable labels (“direct labels”) (L1 ) and indirectly detectable labels (“indirect labels”) (L2). Direct labels are detectable by the human eye without intervention of a machine or chemical reaction to render the agent detectable. Indirect labels are those that require intervention of a machine or chemical reaction to render the agent detectable. In certain embodiments, a ternary complex comprises a direct label and an indirect label.

[00034] Detection agents for attachment to targeting agents can be any of those known in the art. Examples are provided in the following references: Armstrong et al., Diagnostic Imaging, 5^th Ed., Blackwell Publishing (2004); Torchilin, V. P., Ed., Targeted Delivery of Imaging Agents, CRC Press (1995); Vallabhajosula, S., Molecular Imaging:

Radiopharmaceuticals for PET and SPECT, Springer (2009); C. Kessler, ed.

Nonradioacticve Labeling and Detection of Biomolecules, Springer-Verlag, 1992; and G.C. Howard, ed., Methods in Nonradioactive Detection, Appleton & Lange, 1993. A detection agent can be detected in a variety of ways, including as an agent providing and/or enhancing a detectable signal.

[00035] 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). [00036] In a particularly preferred embodiment, chelators on L1 or L2 are bound to metal ions typically used in the technique of immobilised metal ion chromatography; such metals are well-known in the art and include Ni²⁺, and Co²⁺. As used herein, the term“labeled” molecule (e.g., antibody, protein or nucleic acid) refers to a molecule that is bound to a detection agent, either covalently, through a linker or a chemical bond, or noncovalently, through ionic, van der Waals, electrostatic, or hydrogen bonds, such that the presence of the molecule may be detected by detecting the presence of the detection agent bound to the molecule.

[00037] Secondary binding ligands include, e.g., biotin and avidin or streptavidin compounds as known in the art.

1. Direct Labels

[00038] Direct labels detectable by the human eye include colorimetric labels, that is, labels that reflect or absorb light to render a color. Such labels do not, themselves, generate light, such as fluorescent labels do.

[00039] In one embodiment, one of the labels (L1 ) allows detection of binding events by eye, e.g., reflection of natural light from gold particles (e.g., colloidal gold), carbon particles, platinum particles, organic dyes, dyed latex particles, dye-modified sugar polymers and the like. Other such colorimetric labels include without limitation, colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.).

[00040] In one aspect of the invention, the minimally ternary complex comprises an inorganic or organic core particle (L1 ), e.g., gold or latex which imparts colour to the complex, further comprising a polymer surface coat bearing functional groups which facilitates the covalent attachment of other agents.

[00041] The nature of the polymeric coating material is not particularly limiting, but it will preferably have a plurality of functional groups to facilitate multivalent attachment to the core, creating a stable coated product. More than one type of functional group may be present on the coating material, but generally only one type will be used for anchoring to the core material.

[00042] In a preferred embodiment, the core particle is a gold nanoparticle, between 10 and 100nm in diameter, with a functionalised organic coating layer. The available functional groups include, but are not limited to, carboxyl, amine, or maleimide groups, or combinations thereof.

[00043] In one embodiment, L1 is a coated gold particle, e.g., Innovacoat GOLD™ (available from Expedeon Ltd.). [00044] The coated particle is covalently attached to another label, L2, and/or to binding entity, B, which is capable of binding to a polypeptide or other biomolecule or substance.

2. Indirect Labels

[00045] In one embodiment, one of the labels is an indirect label (L2) requiring special equipment to detect, for example, the emission of light arising from chemical reactions (chemiluminescence) (e.g. HRP with luminol substrate) or electronic decay processes after laser excitation of fluorescent molecules or particles (fluorescence).

[00046] Indirect labels include, for example, light-emitting labels (e.g., fluorescent labels, bioluminescent, chemiluminescent labels or phosphorescent labels), radioactive labels and enzymatic labels.

a) Light-emitting Labels

(1) Fluorescent Labels

[00047] Fluorescent labels can include a variety of organic and/or inorganic small molecules that, when excited, fluoresce. Fluorescent labels include, without limitation, cyanines, phthalocyanines, porphyrins, indocyanines, rhodamines, phenoxazines, phenylxanthenes, phenothiazines, phenoselenazines, fluoresceins, benzoporphyrins, squaraines, dipyrrolo pyrimidones, tetracenes, quinolines, pyrazines, corrins, croconiums, acridones, phenanthridines, acridines, anthraquinones, chalcogenopyrylium analogues, chlorins, naphthalocyanines, methine dyes, indolenium dyes, azo compounds, azulenes, azaazulenes, triphenyl methane dyes, indoles, benzoindoles, indocarbocyanines, benzoindocarbocyanines, phycoerythrin, isothiocyanate, texas red and BODIPY™

derivatives. Fluorescent dyes are discussed, for example, in U.S. Pat. No. 4,452,720, U.S. Pat. No. 5,227,487, and U.S. Pat. No. 5,543,295.

[00048] Fluorescence can be detected when a fluorescent molecule (fluorophore) is excited with light of specific wavelength. Fluorescence can be measured by, for example, a photomultiplier tube, CMOS, CCD etc.

(2) Bioluminescent Labels

[00049] The detection agent can comprise a bioluminescent protein, such as luciferase or a fluorescent protein, such as, green fluorescent protein, red fluorescent protein, or yellow fluorescent protein. Luciferases (which also may be classified as enzymatic labels) are available from, for example, Thermo Fisher, Promega and Active Motif. Green fluorescent proteins are available from, for example, Sigma Aldrich and Thermo Fisher. [00050] Light produced by bioluminescence or chemiluminescence can be measured using a detector, such as a photomultiplier tube, CMOS, CCD etc.

b) Radioactive Labels

[00051] Other detection agents useful as labels in the invention include radiolabels, e.g., biomolecules labelled with a radioactive material, such as, ³⁵S-methionine or with ³⁵S-, ³³P-, or ³²P-nucleotides combined with autoradiographic detection.

[00052] The label can be a radioisotope, e.g., radionuclides that emit gamma rays, positrons, b and alpha particles, and X-rays. Suitable radionuclides include but are not limited

6⁸Ga, ³

4⁷Sc, ¹⁵³Sm, ⁸⁹Sr, ^99mTc, ⁸⁸Y and ⁹⁰Y. In some embodiments, radioactive agents can include ¹¹¹ln-DTPA, ^99mT C(CO)₃-DTPA, ^99mTc(CO)₃-ENPy2 , ^62/64/67Cu-TETA, ^99mTc(CO)₃-IDA, and "^mTc(CO)₃triamines (cyclic or linear). In some embodiments, the agents can include DOTA and its various analogs with ¹¹¹ln, ¹⁷⁷Lu, ¹⁵³Sm, ^88/90Y, ^62/64/67Cu, or ^67/68Ga. In some embodiments, a nanoparticle can be labeled by incorporation of lipids attached to chelates, such as DTPA-lipid, as provided in the following references: Phillips et al., Wiley

Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology, 1 (1 ): 69-83 (2008);

Torchilin, V.P. & Weissig, V., Eds. Liposomes 2nd Ed. : Oxford Univ. Press (2003);

Elbayoumi, T.A. & Torchilin, V.P., Eur. J. Nucl. Med. Mol. Imaging 33:1 196-1205 (2006); Mougin-Degraef, M. et al., Int’l J. Pharmaceutics 344:1 10-1 17 (2007).

c) Enzymatic Labels

[00053] In one embodiment of the invention L2 is an enzyme. This can be covalently attached to the surface coat, or directly to a gold particle by passive means, and optionally to another component in the ternary complex. Horseradish peroxidase (“HRP”) and alkaline phosphatase are preferred examples of L2.

[00054] HRP is particularly preferred as a label but one of its disadvantages is the relatively low number of available lysines through which covalent attachment to other molecules can be made. HRP has only six lysines and, of these, only one or two are readily accessible.

[00055] In one embodiment of the invention HRP is reacted with surface carboxyls on the surface of a coated gold nanoparticle. In a particularly preferred embodiment HRP is first modified to incorporate extra amines providing additional points of attachment. Amines may be added to Glu and Asp amino acid side chains on HRP using a diamine compound e.g. ethylene diamine, together with a carbodiimide. Alternatively, the diamine may be reacted with periodate oxidised sugar residues on HRP using methods well known in the art.

[00056] In some embodiments, the detection agent can be an enzyme that produces a detectable signal when an enzyme substrate is put into contact with it. Such labels are used in ELISA assays. The enzyme can be, for example, urease, b-galactosidase, b- glucuronidase, alkaline phosphatase, (horseradish) hydrogen peroxidase (HRP) or glucose oxidase.

C. Embodiments

[00057] In one embodiment, the targeting agent or binding agent comprises an antibody, the first detection agent comprises a direct detection agent and the second detection agent comprises an indirect detection agent. More specifically, the direct detection agent can comprise a gold particle and the indirect detection can comprise an enzyme, for example, horse radish peroxidase.

III. Methods of Making

[00058] In another preferred embodiment surface maleimide groups are attached to the surface of L1 or L2, introduced by reacting surface amines with a heterobifunctional reagent, such as SMCC (sulfo-succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate). A wide range of reagents with similar functionality to SMCC (i.e. amine and thiol reactive) are well known, any of which may be used to facilitate the attachment of thiolated molecules.

[00059] Thiolated forms of B that may be attached to maleimides include Ni-NTA derivatives, e.g., to create a ternary complex with his6-tag binding properties. This complex can be used to probe, for example, crude extracts of expressed proteins to assess the level of expression of his-tagged proteins. Thiolated antibodies may also be attached.

[00060] In yet another embodiment, L2 is attached to the gold surface following the introduction of sulfur-containing ligands using methods well known in the art. For example, amine-reactive thiolation reagents, e.g., 2-iminothiolane or SPDP [succinimidyl 3-(2- pyridyldithio) propionate] may be used to introduce thiol groups, or protected thiol groups (which require reduction), respectively. Reagents that can be reacted with carboxyl groups in the presence of carbodiimide may also been used, e.g., cystamine plus EDC [1-Ethyl-3-(3- dimethylaminopropyl) carbodiimide], which introduces a reducible disulphide.

[00061] In a particularly preferred embodiment, HRP is modified with a mixture of two diamine compounds to introduce amines and reducible disulphide bonds e.g. by reaction with EDBA [2,2'(ethylenedioxy)bis-ethylamine] and cystamine. Following reduction, the aminated and thiolated enzyme binds strongly to gold via thiolate groups and the amines provide additional points of attachment of other molecules. By varying the ratio of the two diamines, the proportions of amines and thiols (after reduction) in the enzyme can be controlled.

[00062] Where L2 is thiolated and attached to gold, the thiol groups not directly involved in dative bonding to the metal surface may be used to bind molecules of B which have been derivatised with maleimides, or with other thiol-reactive functional groups.

[00063] In another embodiment, L2 is directly attached to the gold surface by passive (non-covalent) means. Successful attachment of proteins is readily determined by addition of salts, e.g. sodium chloride which cause aggregation of uncoated gold particles.

[00064] In a particularly preferred embodiment aminated HRP is bound to gold, optionally with thiol functions to enhance binding, and the non-occluded functional groups (amines or thiols, or both) are used for covalent attachment of other biomolecules or molecules, either directly to these functional groups or after modification to e.g. maleimide, bromoacetyl groups, epoxide. Methods of carrying out such modifications are well known in the art.

[00065] It has been found that aminated HRP affords a more stable interaction with gold nanoparticles than unmodified HRP. Coordination of gold with oxygen and nitrogen atoms, or interactions with other elements of the EDBA linker, may explain this behaviour, allowing HRP to be both a surface coat and a label. It will be obvious that other linkers with different lengths and composition but with the same general features may be used to enhance affinity of HRP or other molecules to gold surfaces.

[00066] A protein, such as an antibody, can be conjugated to 40nm InnovaCoat® GOLD (Innova Biosciences, code 230-0005) according to the manufacturer’s instructions. Briefly, 1- 2 pg of antibody in 45 pi is added to a mini-vial and the reaction is quenched after 15 minutes.

IV. Methods of Use

[00067] Provided herein are methods of detecting an analyte in a sample. Detecting includes both detecting the presence of, and the absence of, the target analyte. Accordingly, detecting the target analyte includes determining that the analyte is not present or is below the detection level of the assay. Detecting includes determining a quantitative measure of the analyte. The quantitative measure can be on a discrete or continuous range. For example, the measure could be binary, such as“detectable” or“undetectable”. Alternatively, the measure could be a discrete number on a numeric scale. Alternatively, the measure could be a number on a continuous scale. The measure could be an absolute or relative amount of the analyte, for example, number of nanograms. Alternatively, measure could be an amount of signal produced by a direct or indirect label.

[00068] The use of two different kinds of labels, each with a different range of detection, produces an assay with a very wide dynamic range. So, for example, a direct label, such as a gold particle, can detect analyte at nanogram quantity levels. An indirect label, such as an enzymatic label, can detect analyte at picogram quantity levels, but might be ineffective at accurately measuring amounts in the nanogram range. Accordingly, by using both types of labels the dynamic range of detection of an assay can span at least three orders of magnitude, for orders of magnitude, five orders of magnitude, six orders of magnitude, or seven orders of magnitude.

[00069] According to certain methods of this disclosure, detection of target analytes using complex of this invention involve, first, associating the complex through the targeting agent directly or indirectly with the target analyte. Then, a signal from the direct label is detected by eye to detect the target analyte. The method may stop there. However, if a signal from the direct label cannot be detected by eye (or even if it can be detected by eye) a subsequent step involves detecting a signal from the indirect label the target analyte. Thus, in one embodiment, if a signal cannot be detected from the direct label by eye, a signal from the indirect label is detected.

A. Immunoassay

[00070] Immunoassays use antibodies to detect and/or quantify one or more analytes in the sample. Immunoassays include noncompetitive immunoassays and competitive immunoassays.

[00071] In a noncompetitive assay the antibody associates with the analyte by binding directly or by binding to an intermediate compound bound to the analyte, such as another antibody. Noncompetitive immunoassays include sandwich immunoassays in which the analyte is immobilized on a solid support by binding to an immobilized antibody and then detected with a labeled antibody. In one embodiment of the sandwich immunoassay, wells of a microtiter plate are coated with an antibody against a target molecule. In another embodiment the antibody is provided attached to a solid particle, such as, for example, a magnetically attractable particle. In this way, the particles can be immobilized in a well of a microtiter plate, for example, by application of magnetic force.

[00072] In a competitive immunoassay and unlabeled analyte competes with a labeled analyte for binding with an antibody. [00073] In one method, a first antibody directed to a target protein comprises a tag. A ternary complex of this disclosure comprises a second antibody directed to the tag. The first antibody and the ternary complex can be combined to form a reagent. The reagent can be contacted with a sample comprising the target. Upon binding to the target, the target can be detected using the detection agents. Alternatively, the first antibody can bind to the target before exposure to the ternary complex.

B. Immunoblotting

[00074] Another noncompetitive immunoassay is a blot assay in which an analyte or target molecule is bound to a solid support, such as a nitrocellulose filter, and detected, through direct or indirect binding, with a labeled antibody. One such blot assay is a dot blot assay in which a sample containing an analyte is dotted at an addressable location on a solid support. Another blot assay is the Western blot. In a Western blot, proteins in a mixture are separated by electrophoresis. Electrophoresis can be, for example, gel electrophoresis (e.g., polyacrylamide gel electrophoresis) or agarose electrophoresis. The gel can be a native gel, or an SDS gel, which separates proteins based on size. Alternatively, the gel can separate proteins by isoelectric focusing which separates based on isoelectric point.

Separated proteins are blotted onto a solid support, such as a nitrocellulose filter. This can be done, for example, by electroblotting. Blotted proteins can be detected either by direct binding with the complex of this disclosure or by indirect binding in which, for example, the blot is contacted with a primary antibody directed against the target analyte, which is allowed to bind with the target. Typically, the blot is washed, to remove unbound antibody. Then, the target analyte is detected using a labeled antibody (typically referred to as a secondary antibody), such as complex of this disclosure, directed against the primary antibody or a tag attached to the primary antibody.

[00075] In an immunoblotting embodiment, these complexes or conjugates comprising L1 and L2 often simplify western blotting of samples containing unknown amounts of the target polypeptide, as the more complex L2-based detection procedure involving, for example, chemiluminescence or fluorescence is deployed only if the polypeptide of interest is not first detected by eye using L1.

C. Nucleic Acid Blotting

[00076] In another embodiment, the essay comprises nucleic acid blotting. This includes both DNA and RNA blotting. One form of nucleic acid blotting is the dot blot in which a sample comprising nucleic acid is dotted onto a spot of a solid support. Another form of DNA blotting is the Southern blot. In the Southern blot, DNA fragments of different sizes are separated on a gel, such as an agarose gel. The separated fragments are blotted onto a solid support, such as a nitrocellulose filter. Fragments having target nucleotide sequences are detected using a complex of this disclosure that includes a polynucleotide probe as the targeting agent.

[00077] One form of RNA blotting is the Northern blot. Northern blots are performed in a similar way as Southern blot’s except that the target molecules in the sample comprises RNA.

V. Kits

[00078] 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., complexes of this disclosure, reagents, buffers, 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.

[00079] Kits provided herein can comprise elements for conjugating detection agents to targeting agents, such as antibodies, gold particles, and enzymes.

[00080] In one embodiment, a kit of this disclosure comprises a first container containing a ternary complex as disclosed herein comprising an enzyme as one of the detection agents, and a second container containing a substrate for an enzyme. The kit also can include a container containing buffer, such as wash buffer for removing unbound complex. The kit also can include a solid support for assaying a target, such as a nitrocellulose filter or a multiwell plate, e.g., 96-well plate.

[00081] In another embodiment, a kit of this disclosure comprises a first container containing a tag, such as biotin or digoxigenin. A second container contains a ternary complex as disclosed herein comprising binding agent that binds to the tag. The kit can include a third container comprising reagents to attach the tag to a targeting agent, such as an antibody. VI. Systems

[00082] Also provided herein are systems and an illuminator for exciting a signal from an indirect a detection agent or a reader (e.g., a camera) for detecting a signal from an indirect detection agent, and a solid support comprising a target attached thereto and a complex as disclosed herein associated with the target. For example, the reader can be a fluorescence detection reader.

EXAMPLES

1. Production of antibody/gold nanoparticle/HRP ternary complexes.

Method (a)

0.2ml of aminated HRP (0.175 mg/ml) or HRP (0.175 mg/ml) was mixed with 0.95 ml of 40 nm carboxyl gold nanoparticles (40 OD/ml; Expedeon code 340-0005) in a final volume of 18ml 50mM MES pH 5 containing 0.1 mM EDC. After 15 minutes the reaction was quenched with 50 mM Tris buffer and the conjugate was centrifuged in a microfuge at 7000rpm for 10 min. The pellet washed twice by resuspension with 50mM Hepes pH 7.4 followed by centrifugation. The resulting conjugate was reacted with 4mM sulfo-succinimidyl 4-(N- maleimidomethyl)-cyclohexane-1-carboxylate (sulfo-SMCC)(Pierce code 22322) for 30 min and washed as above.

Goat anti-rabbit IgG was reacted with a 200-fold molar excess of 2-iminothiolane for 30 min and then desalted on a PD10 column (GE Healthcare). An excess of thiolated antibody was reacted with the gold-HRP-maleimide conjugate from above for 1 hour in 50mM Hepes pH 7.4. Free thiols were blocked by addition of N-ethyl maleimide to 1 mM concentration. After 30 minutes the ternary conjugate was centrifuged, and the pellet resuspended in 50% glycerol.

Method (b). 0.2ml of aminated HRP (0.175 mg/ml) or HRP (0.175 mg/ml) was mixed with 0.2ml of goat anti-rabbit IgG antibody (0.025 mg/ml) and combined with 1 ml 40 nm carboxyl gold nanoparticles (40 OD/ml; Expedeon code 340-0005) in 50mM MES pH 5 in the presence of 0.1 mM EDC. After 15 minutes the reaction was quenched with 50 mM Tris buffer. Conjugates were pelleted by centrifugation, washed and resuspended in 50% glycerol.

Method (c). 1 ml of aminated HRP (0.8mg/ml) or HRP (0.8mg/ml) was mixed with 200ul of 10 OD gold nanoparticles [Expedeon product code 210-0100] in 10mM Hepes, pH 7.4. After 30 minutes, tubes were centrifuged for 10 min at 7000 rpm. The non-aminated sample aggregated during centrifugation and the particles could not be dispersed. The aminated HRP-gold conjugate was reacted with 4mM sulfo-SMCC for 30 min at room temperature and washed as above. Antibodies were reacted with 2 mM 2-iminothiolane for 30 min in 50mM Hepes pH 7.4, then desalted into the same buffer and added in excess to the gold-HRP- maleimide conjugate. After 1 h ternary complexes were centrifuged and resuspended in 50mM Hepes, pH 7.4.

Characterisation of aminated-HRP gold

The binary aminated HRP-gold complex from method 1 c in 100 mM Hepes pH 7.4 was scanned at 400nm-700 nm. Uncoated gold nanoparticles particles in water were used as a reference. The uncoated nanoparticles display a characteristic peak which is right shifted for the aminated gold, consistent with an interaction of aminated HRP with the gold surface. The non-aminated HRP conjugate could not be scanned as it aggregated during conjugate wash steps.

Production of ternary complex containing Ni-NTA.

Aminated HRP-gold complexes prepared by either method 1 a or 1 c were suspended in 50mM Hepes pH 7.4 and incubated with 4 mM sulfo-SMCC. After 30 minutes the conjugate was centrifuged to remove excess SMCC and washed with 50mM Hepes pH 7.4. The conjugate was reacted with N-[Na, Na-Bis(carboxymethyl)-L-lysine]-12- mercaptododecanamide (Sigma Code 38152) (1 mM) for 1 hour at room temperature, after which the reaction was brought to 10mM NiS0₄ concentration from 100mM stock. After 15 min, the N,²⁺-charged complex was desalted on a PD10 column (GE healthcare) into 50mM Hepes pH 7.4

2. Detection of polypeptide on western blots using HRP-Gold-anti-rabbit ternary

complex.

Transferrin (TF) in varying amounts was run on a 4-12% Bis-Tris gel (Expedeon,

NBT41212), under non-reducing conditions for 40’ at 180V in 1x MOPS (Expedeon, NXB75500) and transferred onto nitrocellulose (Expedeon, NXA19020), using 1x Tris- Glycine buffer (Expedeon, NXB86500) + 10% methanol. Membranes were incubated with blocking buffer (1x TBS, 0.05% Tween20, 5% BSA) for 1 hour at RT and then for 18 hours with anti-transferrin rabbit polyclonal (Abeam, ab82411 ) diluted in blocking buffer with gentle shaking. Membranes were washed 3 times with 1x TBS, 0.05% Tween20 and incubated for one hour with HRP-Gold-Goat anti-rabbit IgG ternary complex diluted to 2 OD units/ml in blocking buffer. For comparison, GAR-HRP conjugate diluted 1 :25.000 in blocking buffer was also tested. Membranes were washed 3 times as before, photographed, and then developed with an enhanced chemiluminescent reagent ECL Pico (Expedeon, ECLP0250) with image capture at: 3s, 10s, 30s, & 60s. Note in FIG. 3 that M is a marker protein pre-stained with a red dye. Using

chemiluminescence, around 5pg of transferrin can be detected with both Goat anti-rabbit- HRP and the HRP-Gold-Goat anti-rabbit IgG ternary complex. For transferrin amounts above 500pg, the protein is easily detected by the gold nanoparticle component of the ternary complex, and chemiluminescent detection is not required.

[00083] 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.” The term“any of between a modifier and a sequence means that the modifier modifies each member of the sequence. So, for example, the phrase“at least any of 1 , 2 or 3” means“at least 1 , at least 2 or at least 3”. The term "consisting essentially of" refers to the inclusion of recited elements and other elements that do not materially affect the basic and novel characteristics of a claimed combination. In certain embodiments the term“comprising” can be replaced with the term“consisting essentially of”.

[00084] 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 method for detecting a target molecule in a sample comprising:

(a) contacting the sample with a targeting agent that specifically binds to the target molecule to allow binding between the targeting agent and the target molecule;

(b) associating a first detection agent and a second detection agent with the targeting agent, either covalently or non-covalently; and

(c) detecting the binding through detection of the first detection agent or the second detection agent.

2. The method of claim 1 , wherein the first detection agent and second detection agent are associated with the targeting agent before contacting.

3. The method of claim 1 , wherein the first detection agent and second detection agent are associated with the targeting agent after contacting.

4. The method of claim 3, wherein the first detection agent and second detection agent are non-covalently associated with the targeting agent through binding of a ternary complex comprising (1 ) a binding agent that specifically binds the targeting agent and (2) the first and second detection agents.

5. The method of claim 4, wherein the targeting agent comprises a primary antibody and the complex comprises:

(i) a binding agent comprising a secondary antibody;

(ii) the first detection agent; and

(iii) the second detection agent.

6. The method of claim 4, wherein the targeting agent comprises a tag and the binding agent specifically binds the tag.

7. The method of any of claims 1-5, wherein the first detection agent comprises a direct label and the second detection agent comprises an indirect label.

8. The method of any of claims 1-5, wherein the detection limit of the second detection agent is at least ten times, at least 100 times or at least 1000 times greater than the detection limit of the first detection agent.

9. The method any of claims 1-5, having a dynamic range of detection of at least 10³, at least 10⁴, at least 10⁵, or at least 10⁶.

10. The method of any of claims 1-5, wherein the first detection agent and second detection agents are differently selected from colorimetric, light-emitting, enzymatic and radioactive detection agents.

11. The method of any of claims 1-5, wherein one of the detection agents comprises a colorimetric detection agent.

12. The method of claim 11 , wherein the colorimetric detection agent comprises a gold particle (e.g., colloidal gold), a carbon particle, a platinum particle, an organic dye, a glass particle, a plastic particle (e.g., polystyrene, polypropylene, latex) or a dye-modified sugar polymers.

13. The method of claim 11 , wherein the colorimetric detection agent comprises a particle comprising a polymeric coating comprising a plurality of functional groups to facilitate multivalent attachment to the complex.

14. The method of claim 12, wherein the polymeric coating material comprises a sugar polymer or polypeptide.

15. The method of any of claims 1-5, wherein one of the detection agents comprises an enzymatic detection agent.

16. The method of claim 15, wherein the enzymatic detection agent comprises urease, b-galactosidase, b-glucuronidase, alkaline phosphatase, (horseradish) hydrogen peroxidase (HRP) or glucose oxidase.

17. The method of any of claims 1-5, wherein one of the detection agents comprises a light-emitting detection agent.

18. The method of claim 17, wherein the light-emitting detection agent comprises a fluorescent label or a bioluminescent label.

19. The method of claim 18, wherein the light-emitting detection agent comprises a bioluminescent label selected from a luciferase or a green fluorescent protein.

20. The method of any of claims 1-5, wherein one of the detection agents comprises a radioactive detection agent.

21. The method of any of claims 1-5, wherein the first detection agent comprises a colorimetric detection agent and the second detection agent comprises an enzymatic detection agent.

22. The method of claim 21 , wherein the colorimetric detection agent comprises a gold particle and the enzymatic detection agent comprises horse radish peroxidase.

23. The method of claim 21 , wherein the colorimetric detection agent has a detection limit of at least one nanogram of target and the enzymatic detection agent has a detection limit of at least one picogram of target.

24. The method of any of claims 1-5, wherein the first detection agent comprises a colorimetric detection agent and the second detection agent comprises a detection agent selected from a light-emitting detection agent and a radioactive detection agent.

25. The method of any of claims 1-5, wherein the targeting agent comprises an antibody.

26. The method of any of claims 1-5, wherein the targeting agent comprises a non-antibody protein, an aptamer or a nucleic acid.

27. The method of any of claims 1-5, wherein detecting comprises performing an immunoassay.

28. The method of claim 27, wherein the immunoassay uses a primary antibody and a secondary antibody, which secondary antibody is comprised in the complex.

29. The method of claim 27, wherein the immunoassay comprises a protein immune blot (“Western blot”) comprising:

(I) separating proteins in the sample by gel electrophoresis (e.g., SDS PAGE or isoelectric focusing);

(II) transferring separated proteins to a solid support (e.g., a membrane, e.g., made of nitrocellulose (NC) or polyvinylidene difluoride (PVDF)), e.g., by electroblotting;

(III) contacting the transferred protein with a primary antibody to allow specific binding between the primary antibody and a target protein, and removing unbound primary antibody;

(IV) contacting the bound primary antibody with the complex, wherein, the targeting agent comprises a secondary antibody, to allow specific binding between the secondary antibody and the primary antibody, and removing unbound secondary antibody;

(V) detecting bound secondary antibody through detection of the first detection agent or the second detection agent.

30. The method of claim 29, wherein the first detection agent comprises a direct label and the second or detection agent comprises an indirect label, and, wherein, if the direct label is not visually detected, binding is determined through detection of the indirect label.

31. The method of claim 27, wherein the immunoassay comprises a sandwich immunoassay.

32. The method of any of claims 1-5, wherein detecting comprises performing an oligonucleotide probe hybridization assay.

33. The method of claim 32, wherein the hybridization comprises a nucleic acid blot selected from a DNA blot (“Southern blot”) and an RNA blot (“northern blot”) comprising:

(I) separating nucleic acids in the sample by gel electrophoresis;

(II) transferring separated nucleic acids to a solid support;

(III) contacting the transferred nucleic acid with the complex, wherein, the targeting agent comprises a nucleic acid probe that hybridizes with a target nucleic acid, to allow specific binding between the nucleic acid probe and the target nucleic acid, and removing unbound nucleic acid probe;

(V) detecting bound target nucleic acid through detection of the first detection agent or the second detection agent.

34. The method of claim 33, wherein the first detection agent comprises a direct label and the second or detection agent comprises an indirect label, and, wherein, if the direct label is not visually detected, binding is determined through detection of the indirect label.

35. The method of any of claims 1-5, wherein the complex further comprises (iv) one or more other detection agents.

36. The method of any of claims 1-5, further comprising, after forming the targeting agent/target combination, washing to remove unbound material.

37. A complex comprising:

(i) a targeting agent;

(ii) a first detection agent; and

(iii) a second, different, detection agent.

38. The complex of claim 32, wherein the first detection agent comprises a direct label and the second detection agent comprises an indirect label.

39. The complex of claim 32, wherein the detection limit of the second detection agent is at least ten times, at least 100 times or at least 1000 times greater than the detection limit of the first detection agent.

40. The complex of claim 32, wherein the first detection agent and second detection agents are differently selected from colorimetric, light-emitting, enzymatic and radioactive detection agents.

41. The complex of claim 32, wherein one of the detection agents comprises a colorimetric detection agent.

42. The complex of claim 41 , wherein the colorimetric detection agent comprises a gold particle (e.g., colloidal gold), a carbon particle, a platinum particle, an organic dye, a glass particle, a plastic particle (e.g., polystyrene, polypropylene, latex) or a dye-modified sugar polymers.

43. The complex of claim 15, wherein the enzymatic detection agent comprises urease, b-galactosidase, b-glucuronidase, alkaline phosphatase, (horseradish) hydrogen peroxidase (HRP) or glucose oxidase.

44. The complex of claim 32, wherein the first detection agent comprises a colorimetric detection agent and the second detection agent comprises an enzymatic detection agent.

45. The complex of claim 44, wherein the colorimetric detection agent comprises a gold particle and the enzymatic detection agent comprises horse radish peroxidase.

46. The complex of claim 45, wherein the colorimetric detection agent has a detection limit of at least one nanogram of target and the enzymatic detection agent has a detection limit of at least one picogram of target.

47. The complex of claim 32, wherein the targeting agent comprises an antibody.

48. The complex of claim 47, wherein the antibody is an anti-immunoglobulin antibody (e.g., anti-lgG).

49. The complex of claim 32, wherein the first detection agent comprises a gold nanoparticle and the second detection agent comprises an enzyme, e.g., horse radish peroxidase, and further wherein the gold nanoparticle and the enzyme are covalently attached through amide bond, and further wherein the covalently bound first and second detection agents are bound to a polypeptide binding agent through a thiol group.

50. The complex of claim 32, wherein the targeting agent comprises a non- antibody protein, an aptamer or a nucleic acid.

51. A kit comprising:

(a) a container containing a complex comprising:

(i) a targeting agent;

(ii) a colorimetric detection agent; and

(iii) and enzymatic detection agent comprising an enzyme; and

(b) a container containing a substrate for the enzyme wherein, the product of the enzymatic reaction produces a detectable signal.

52. A kit comprising:

(a) a container containing a ternary complex comprising:

(i) a binding agent, wherein the binding agent binds a tag;

(ii) a first detection agent; and

(iii) a second, different, detection agent;

(b) a second container containing the tag.

53. The kit of claim 52, further comprising:

(c) a third container containing reagents for conjugating the tag to a targeting agent.

54. A method of making a complex, wherein the complex comprises:

(i) a targeting agent;

(ii) a first detection agent; and

(iii) a second, different, detection agent;

wherein the method comprises:

(a) coupling the first detection agent to the targeting agent; and

(b) coupling the second detection agent to the targeting agent or to the first detection agent.

55. The method of claim 54, comprising coupling the second detection agent to the targeting agent.

56. The method of claim 54, comprising coupling the second detection agent to the first detection agent.

57. The method of claim 54, wherein the first detection agent comprises a visually detectable particle.

58. The method of claim 57, wherein the particle comprises a plurality of functional groups, e.g., amines, amides, carboxyls, and coupling comprises reacting the functional groups with a functional group on the targeting agent (e.g., coupling the second detection agent to the targeting agent.

59. The method of claim 57, wherein the visually detectable particle comprises a gold particle (e.g., colloidal gold), a carbon particle, a platinum particle, an organic dye, a glass particle, a plastic particle (e.g., polystyrene, polypropylene, latex) or a dye-modified sugar polymers.

60. The method of claim 57, wherein the visually detectable particle comprises a particle comprising a polymeric coating comprising a plurality of functional groups to facilitate multivalent attachment to the complex.

61. The method of claim 54, comprising coupling the second detection agent to the targeting agent.

62. A method for detecting a target molecule in a mixture involving the binding of a complex comprising (i) a first detection agent, (ii) a second detection agent, (iii) a targeting agent, and (iv) optionally, one or more other detection agents; further involving, after washing, the measurement of the first detection agent and, optionally, measurement of the second detection agent.

63. The method of claim 62 where the target molecule is a protein or a nucleic acid.

64. The method according to claims 62-63, whereby the first or second detection agent has a polymeric coating material.

65. The method of claims 62-64 wherein the targeting agent is an antibody, or other binding protein, or a small molecule capable of binding to a biomolecule.

66. The method of claims 62 -65 wherein at least one of the detection agents is a colored substance or an enzyme capable of generating a detectable substance.

67. The method of claim 66 wherein the colored substance is a particle.

68. The method of claim 67 wherein the detectable substance is a product or by- product of enzyme action.

69. The method of claim 67 wherein the particle is inorganic or organic in nature.

70. The method of claim 69 wherein the particle is gold or colored latex or fluorescent latex.

71. The method of claim 68 wherein the enzyme product is colored or can be made to emit light after supplying external energy.

72. The method of claim 68 wherein the by-product of the reaction is light.

73. The method of claim 66 wherein the enzyme is HRP or alkaline phosphatase.

74. The method of claim 64 wherein the polymeric coating material is a sugar polymer or polypeptide.

75. The method according to claim 74 wherein the polymer is a detection agent.

76. The method according to claim 62 wherein the target molecule(s) are immobilized on a surface.

77. A complex comprising at least two different detection agents and a targeting or binding agent.

78. The complex of claim 77 wherein the detection agents are two or more of the following: gold particles, carbon particles, platinum particles, organic dyes, dyed latex particles, dye-modified sugar polymers, radiolabels, chemiluminescent molecules, fluorescent molecules, or enzymes capable of generating chemiluminescent molecules or fluorescent molecules.

79. The complex of claim 77 or 78 wherein the binding or targeting agent is one of the following: antibodies, antibody fragments, nanobodies, other binding proteins, polypeptides and fragments; small molecules including peptides, metal ions (attached to chelators), streptavidin, biotin and nucleic acids.

80. A kit for detecting a target protein comprising a complex of claims 77-79 wherein the binding agent recognizes a tag, together with a conjugation kit for attaching the tag to an antibody that recognizes the target protein; mixing said complex with the tagged antibody, and incubating the resulting reagent with the target protein.