WO2003104424A2 - Novel molecular conjugates for signal amplification - Google Patents

Abstract

The present invention relates to molecular conjugates that provide enhanced signal amplification in analytical studies using analyte-specific binding reagents that are associated with detectable label. The invention also includes kits and methods for use of the claimed molecular conjugates.

Description

NOVEL MOLECULAR CONJUGATES FOR SIGNAL

AMPLIFICATION

CROSS-REFERENCE TO RELATED APPLICATIONS

[01] This application claims the benefit of U.S. Provisional Application No.60/388,115 filed June 10, 2002, herein incorporated by reference.

FIELD OF THE INVENTION

[02] The present invention relates to molecular conjugates that provide enhanced signal amplification in analytical studies using analyte-specific binding reagents that are associated with a detectable label. The invention also includes kits and methods for use ofthe claimed molecular conjugates.

BACKGROUND OF THE INVENTION

[03] Since the introduction of indirect enzyme immunoassays (EIA) nearly 30 years ago as a novel methodology to determine the affinity distribution of antibodies with limiting amounts of serum or culture supernatants, application ofthe technique has dramatically increased. (Kemeny et al., in: ELISA and other solidphase immunoassays: Theoretical and practical aspects; John Wiley & Sons Ltd. (1988)). This technique has now been successfully applied to a wide range of disciplines. The major advantage of this technique is that it is possible to substitute analytical reagents used in the assay with alternative components at every step in the general assay procedure to achieve an optimized system, and providing enormous flexibility to customize the assay for desired usage.

[04] In addition to common laboratory techniques used in immunology, EIA and Western blot analyses are the most widely used serological tests by blood banks to detect blood borne infections. Serological tests include those for human immunodeficiency virus (HIV), human T lymphotropic virus (HTLV), hepatitis, and many others (Sarkodie et al., Vox Sang., 80:142- 147 (2001); Cameiro-Proietti et al., Haemophilia, 4:47-50 (1998); Bonacim et al., JAcquir Immune Defic Syndr., 26:340-344 (2001); Baumeister et al, JMed Virol, 60:256-263 (2000); Heim et al., JMed Virol, 58:291-295 (1999).). These infections may be transmitted by sexual contacts, exposure of infected blood or blood components, and by infected mother to the fetus. For example, an estimated 36 million individuals are currently infected with HJN worldwide and nearly 15,000 new infections occur every day, making HIN a major health concern worldwide (Ogwannv et al., J Acquir Immune Defic Syndr., 29:184-190 (2002)). Reagents that increase the sensitivity or speed of diagnosis of HIN and other infectious diseases would therefore have significant health benefits in the control and treatment of disease.

SUMMARY OF THE INVENTION

[05] This invention relates to novel molecular conjugates that are capable of recognizing specific analytes in a complex mixture and producing a detectable signal that is an order of magnitude or greater in strength than that produced by conventional conjugates for analyte detection.

[06] Signal amplification is accomplished in one embodiment by coupling a binding partner to a plurality of labels. For example, by coupling the binding partner to the N- terminal α amine of one or more polypeptides, and coupling a plurality of labels to side chains ofthe one or more polypeptides. As a consequence of this coupling strategy, specific recognition of an analyte by the binding partner results in association ofthe analyte with the plurality of labels. This provides enhancement of signal production over that normally accompanying conventional labeling strategies.

[07] The coupling strategy allows for several different approaches to joining the various components ofthe invention. For example, in one aspect, the binding partner is covalently coupled by a chemical linker to the N-terminal α amine of each ofthe one or more polypeptides. A preferred chemical linker for accomplishing the coupling has the structure:

[08] where A is the binding partner, B is the polypeptide, N' is a nitrogen of an ε amine of a lysine residue ofthe binding partner, N" is a nitrogen ofthe N-terminal amine ofthe polypeptide, and R is H or a substituted or unsubstituted alkyl group. Shorter chemical linkers also work, such as:

[09] where A is the binding partner, B is the polypeptide, S is a sulfur of a cysteine residue ofthe binding partner, N is a nitrogen ofthe N-terminal α amine ofthe polypeptide, and R is H or a substituted or unsubstituted alkyl group.

[10] In another aspect, microparticles are used as the molecular conjugate core through which binding partners and different multi-label scaffolds may be coupled. In this aspect, the linkers used can appear quite complex. For example a linker ofthe following structure may be formed using a microparticle with amine surface chemistry:

[11] where A is the binding partner, B is a multi-valent microparticle, C is the polypeptide,

N' is a nitrogen of an ε amine of a lysine residue ofthe binding partner, N" is a nitrogen of the N-terminal α amine ofthe polypeptide, and R is H or a substituted or unsubstituted alkyl group.

[12] An alternative aspect is molecular conjugates comprising binding partners coupled to multi-label scaffolds, the coupling comprising an amino particle and having the structure:

[13] where A is the binding partner, B is a multi-valent microparticle, C is the polypeptide, N' is a nitrogen of an N-terminal amine ofthe binding partner, N" is a nitrogen ofthe N- terminal α amine ofthe polypeptide, and R is H or a substituted or unsubstituted alkyl group. [14] Still another aspect ofthe invention comprises a molecular conjugate including binding partners and one or more polypeptides acting as multi-label scaffolds. In this aspect, both the binding partner and the polypeptides are modified through coupling to a biotin moiety, with the polypeptides being coupled to the biotin moiety at an N-terminal α amine of each polypeptide. The molecular conjugate further comprises a streptavidin or avidin-coated microparticle. Coupling ofthe binding partners and the polypeptides is achieved by contacting the binding partners and the polypeptides with the microparticle creating an affinity complex between the biotin label ofthe binding partners and the polypeptides and the streptavidin-coated microparticle. A plurality of labels are then coupled to the polypeptide multi-label scaffold.

[15] Another aspect ofthe invention comprises a protein A-coated microparticle and immunoglobulin binding partners. The immunoglobulin binding partners are specifically recognized by the protein A coating the microparticle and are coupled when contacted with it.

The polypeptides serving as multi-label scaffolds are covalently coupled to the Protein A coating the microparticle through one ofthe desired reactive groups discussed herein.

Finally, a plurality of labels are coupled to the polypeptide multi-label scaffold. A variation of this aspect substitutes protein G for protein A.

[16] A further aspect of this invention is that each polypeptide forming a multi-label scaffold contains at least one lysine. In one variation of this aspect, the labels are coupled to the existing lysine residues ofthe polypeptide through the ε amine ofthe lysine by a chemical linkage having the structure:

R^^NH-R- where R is the lysine, N is a nitrogen of an ε amine ofthe lysine, and R' is the label. In other aspects ofthe invention the labels are an enzyme.

[17] Some compositions ofthe invention have a binding partner that is a protein, nucleic acid, carbohydrate, glycoprotein, nucleoprotein, lipid, or lipoprotein. When the mult-label scaffold comprises a polypeptide, the polypeptide may be polylysine.

[18] Another embodiment ofthe invention is a molecular conjugate for signal amplification comprising one or more binding partners, one or more labels having a primary amine, and a multivalent microparticle. In this embodiment, the binding partners and the labels are coupled to the microparticle. In some aspects each valency ofthe microparticle comprises an amine.

[19] Some aspects ofthe invention have one or more labels covalently coupled to the microparticle through a chemical linkage having the structure;

where A is the label, B is the microparticle, N' is a nitrogen ofthe amine ofthe label, and N' is a nitrogen ofthe amine ofthe microparticle. [20] Other aspects have one or more binding partners covalently coupled to the microparticle through a chemical linkage having the structure;

where A is the binding partner, B is the microparticle, N' is a nitrogen of an amine ofthe binding partner, and N" is a nitrogen ofthe amine ofthe microparticle.

[21] Still other aspects have one or more binding partners covalently coupled to the microparticle through a chemical linkage having the structure;

where A is the binding partner, B is the microparticle, N' is a nitrogen of an N-terminal α amine ofthe binding partner, and N" is a nitrogen ofthe amine ofthe microparticle. [22] Other embodiments ofthe invention include methods for producing an amplified signal in response to the presence of an analyte. One such embodiment involves obtaining a sample including the analyte, immobilizing the analyte to a coupling surface, contacting the analyte with a molecular conjugate having a binding partner coupled to an N-terminal α amine of one or more polypeptides, and a plurality of labels where each label is coupled to an amino acid side chain of one ofthe polypeptides. At least one ofthe binding partners recognizes the analyte and binding ofthe analyte by a binding partner results in association of the analyte with the plurality of labels. Lastly, label ofthe molecular conjugate is detected. The presence ofthe analyte is indicated by a signal produced by a plurality of unit labels for each unit of analyte, with association ofthe labels and the analyte being mediated by a molecular conjugate ofthe present invention.

[23] In some aspects ofthe embodiment the analyte is a protein and the coupling surface is nitrocellulose. In other aspects the analyte is a protein and the coupling surface is a plastic. In still other aspects the analyte is an immunocomplex and the coupling surface is a plastic. Yet other aspects have a nucleic acid analyte and the coupling surface is nylon. Additional aspects have an the analyte that is an antigen and the coupling surface is an antibody-coated plastic. [24] Further embodiments ofthe invention include kits for detecting an analyte. Kits include components for producing an amplified signal to an analyte by supplying a molecular conjugate having a binding partner coupled to an N-terminal α amine of one or more polypeptides; and a plurality of labels each label coupled to an amino acid side chain of one ofthe polypeptides. At least one binding partner specifically recognizes the analyte. Coupling the binding partner with the analyte associates the analyte with the plurality of labels. The kits also contain directions for use ofthe molecular conjugate and any additional components ofthe kit.

[25] Additional embodiments include a molecular conjugate for signal amplification having one or more binding partners, one or more nucleic acids having a ribosyl residue with an α carbon at the 5' end. Each of these nucleic acids is covalently bound through its nucleic acid residues to a number of labels, with a single label being bound to a single base. The molecular conjugate ofthe embodiment also includes a multivalent microparticle to which both the binding partners and the nucleic acids may be coupled through a chemical linkage. The chemical linkage between the nucleic acid and the microparticle has the structure:

where A is the α carbon ofthe ribosyl residue at the 5' end ofthe nucleic acid, N' is a nitrogen of an amine on the surface ofthe microparticle, and B is the microparticle.

BRIEF DESCRIPTION OF THE DRAWINGS

[26] Figure 1 illustrates a rapid ELISA-type assay utilizing magnetic microparticles and multi-label conjugates ofthe present invention.

[27] Figure 2 illustrates a covalent coupling of a microparticle and a protein through an amide bond.

[28] Figure 3 is a schematic depiction of a microparticle-mediated rapid immunoassay.

[29] Figure 4 illustrates chemical structures of various detector microparticle immunoconjugates.

[30] Figure 5 illustrates construction of antibody or antigen-poly-enzyme-microparticle immunoconjugates using streptavidin polystyrene microparticles.

[31] Figure 6 illustrates construction of antibody or antigen-poly-enzyme-microparticle immunoconjugates using amino polystyrene microparticle.

[32] Figure 7 illustrates construction of antibody or antigen-poly-enzyme-microparticle immunoconjugates using lysine polypeptide coupled with multiple HRP molecules and amino polystyrene microparticle.

[33] Figure 8 depicts a schematic chemical structure of various antibody-HRP conjugates.

[34] Figure 9 illustrates the construction of antibody-HRP and antibody-poly-HRP conjugates.

[35] Figure 10 (i) illustrates chemical modification ofthe primary amine groups of an amino polystyrene microparticle using SATA. Figure 10(ii) illustrates the chemistry involved in derivatizing the 5' end of an oligonucleotide using a phosphoramadite, sulfo-

SMCC and NaSO₃ prior to coupling with a derivatized microparticle. Figure lθ(iii) depicts the final reaction mixture for creation of one embodiment ofthe present invention comprising the derivatized oligonucleotide, the chemically modified microparticle and a bromoacetylated polylysine, the oligonucleotide and polylysine components being present, as an example, in a

1:5 ratio. Once formed, the conjugate is reacted with label (e.g., acridinium biotin, fluorescein. etc.), covalently coupling the label to ε amine groups ofthe lysyl side chains of the polylysine. The boxed composition is the final product ofthe reactions, representing an embodiment ofthe invention.

[36] Figure 11 illustrates components of a microparticle-mediated immunodetection system ofthe invention.

[37] Figure 12 depicts models of immune complex formation. [38] Figure 13 is an illustration of detection of HIN-1 protein bands by Western blot analysis of low HIN-1 antibody positive plasma sample using antibody-enzyme conjugates and antibody-poly-enzyme conjugates.

[39] For the purposes of describing this invention the following terms will be helpful and will have the following meanings:

DEFINITIONS [40] For purposes of this invention, the terms, "absorbance" and "O.D." are used synonymously and refer to a logarithmic function ofthe percent transmission of light of a given wavelength through a liquid or gas.

[41] The term "affinity" refers to the strength of noncovalent chemical binding between two substances as measured by the dissociation constant ofthe complex. In terms of antigen/antibody interactions, affinity is a thermodynamic expression ofthe strength of interaction between a single antigen binding site and a single antigenic determinant (and thus ofthe stereochemical compatibility between them), most accurately applied to interactions among simple, uniform antigenic determinants such as haptens. Expressed as the association constant that, owing to the heterogeneity of affinities in a population of antibody molecules of a given specificity, actually represents an average value (mean intrinsic association constant).

[42] The term "affinity complex" refers to the preferential interaction of two or more components, each component having a greater predilection toward combining with at least one other component ofthe interaction than with other matter.

[43] The term "alkyl group" refers to a monovalent radical, such as ethyl or propyl, having the general formula C_nH_2n+ι. Alkyl groups may be substituted or unsubstituted. Substituted alkyls are characterized in having more than one methyl group (-CH₃), whereas unsubstituted alkyls have only a single methyl group in their structure. The term "alkyl" also includes derivatives such as haloalkyls, metalloalkyls, and the like.

[44] The term "amine" refers to any of a group of organic compounds of nitrogen, such as ethylamine, C₂H₅NH₂, that may be considered ammonia derivatives in which one or more hydrogen atoms have been replaced by a hydrocarbon radical.

[45] The term "amino acid side chain" refers to any molecular moiety R in a compound with the structure

where B may be a hydroxyl, a salt, a nitrogen or a carbon, and A may be a hydrogen atom, a salt, or a carbon, including a carbon ofthe R group for example forming a pyrolidine ring such as that found in the amino acid proline.

[46] The term "amplified signal" or "signal amplification" refers to an increase in intensity or duration of a detectable characteristic, i.e. the signal.

[47] The term "unit of refers to the indivisible part that has all ofthe characteristics ofthe substance, whether the substance be atomic, molecular, crystalline, amorphous or cellular. [48] The term "analyte" refers to a substance being measured in an analytical procedure. [49] The term "binding" refers to the adherence of molecules to one another, for example, enzymes to substrates, antibodies to antigens, DNA strands to their complementary strands. [50] The term "binding partner" refers to any substance possessing an inherent affinity to another substance. Exemplary binding partners include but are not limited to antigen and antibody, ligand and receptor, enzyme and substrate, and hybridizing nucleic acid strands. [51] The terms "biotin" and "biotin moiety" refer to a small molecule having the structure:

For purposes of this invention biotin may be conjugated to other molecules and acts as a covalent binding partner having high affinity binding to avidin and streptavidin. [52] The term "carbohydrate" refers to compounds, usually an aldehyde or ketone derivative of a polyhydric alcohol, particularly ofthe pentahydric and hexahydric alcohols. They are so named because the hydrogen and oxygen are usually in the proportion to form water with the general formula C_n(H₂O)_n. The most important carbohydrates are the starches, sugars, celluloses and gums. They are classified into mono, di, tri, poly and heterosaccharides. The smallest are monosaccharides like glucose whereas polysaccharides such as starch, cellulose or glycogen can be large and indeterminate in length. [53] The term "chemical linker" refers to any group of atoms joining one or more substances together. The bonds forming the attachment between the chemical linker and the substances being joined may be covalent or non-covalent in nature. The bonds between the atoms forming the linker may also take any form, being either covalent or non-covalent in nature. The bonds must be stable under the conditions the chemical linker is employed, such that both the linker and the substances being joined maintain their association, with the exception of specific circumstances where separating the substances is desired. [54] The terms "coupled" and "coupling" refer to joining one or more substances together. The bonds forming the coupling may be covalent or non-covalent in nature. The bonds must be stable under the conditions, such that the substances being joined maintain their association, with the exception of specific circumstances where separating the substances is desired. Preferred couplings include: streptavidin- or avidin- to biotin interaction; hydrophobic interaction; magnetic interaction (e.g. using functionalized Dynabeads); polar interactions; formation of a covalent bond, such as an amide bond, disulfide bond, thioether bond, or via crosslinking agents; and via an alkali or acid-labile linker. In a preferred embodiment for coupling nucleic acids to beads, the coupling introduces a variable spacer between the beads and the nucleic acids. In another prefened embodiment, the coupling is photocleavable (e.g. streptavidin- or avidin- to biotin interaction can be cleaved by a laser, for example for mass spectrometry).

[55] The term "coupling surface" refers to any surface capable of immobilizing a substance. Coupling surfaces may be fabricated from both virgin materials and materials that have been specially coated or otherwise treated to give them binding capacity for the substance to be immobilized. Coupling surfaces may take a variety of forms including sheets, particles, or be molecular, such as the surface of a protein. Bonding to the coupling surface may be non-specific or specific to the substance to be imobilized. The forces imobilizing the substance to the coupling surface may be ionic (e.g., nylon and DEAE-treated cellulose), hydrophobic (e.g., polyvinyldifluoride membranes), or covalent. [56] The term "covalently coupled" refers to the joining of two molecules by at least one interatomic bond characterized by the sharing of 2, 4, or 6 electrons between the atoms forming the bond. Covalent couplings include molecular linkers between two molecules, where at least one atomic strand between the coupled molecules consists of atoms that are themselves joined by covalent couplings. An atomic strand is a sequence of atoms and the bonds joining them, regardless of source. The atoms of a strand may have a diversity of bonds to other atoms. However, at most, only two ofthe bonds of any atom of a strand form a part ofthe strand, and only the atoms sharing two such bonds are part ofthe strand. [57] The term "enzyme" refers to a protein molecule that catalyzes chemical reactions of other substances without being destroyed itself or altered upon completion ofthe reactions. Enzymes are divided into six main groups, oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases.

[58] The term "imobilizing" refers to holding, fastening or binding in a fixed position. The forces causing the holding, fastening or binding may be covalent or non-covalent in nature. [59] The term "glycoprotein" refers to a protein with covalently attached sugar units, either bonded via the OH group of serine or threonine, (O glycosylated) or through the amide NH2 of asparagine (N glycosylated). Typical sugar units include: mannose, N acetyl glucosamine, N acetyl galactosamine, galactose, fucose and sialic acid. [60] The term "label" refers to a substance that is either directly detectable, such as a radionuclide, or indirectly detectable, such as an enzyme activity. Labels are generally coupled to other substances, allowing the presence ofthe other substance to be detected by the association with the label.

[61] The term "lipid" refers to any of a heterogeneous group of fats and fatlike substances characterised by being water insoluble and being extractable by nonpolar solvents such as alcohol, ether, chloroform, and benzene. All lipids contain aliphatic hydrocarbons as a major constituent. By way of example, lipids include fatty acids, neutral fats, waxes and steroids. Compound lipids comprise the glycolipids, lipoproteins and phospholipids. [62] The term "lipoprotein" refers to any of a group of conjugated proteins in which at least one ofthe components is a lipid. Lipoproteins, classified according to their densities and chemical qualities, are the principal means by which lipids are transported in the blood. [63] The term "microparticle" refers to a spheroid particle of a core composition that is insoluble in aqueous solution and usually capable of forming aqueous suspensions. Exemplary microparticle core compositions include various organic polymers (e.g., plastics and latex) polysaccharides, proteins, metals, carbon, and silica. Microparticles can range in size from 0.01 μm to lOμm in diameter, preferably between 0.05μm and 3μm in diameter, more preferably between 0.1 μm and 1 μm in diameter. The term "microparticle" further includes coatings made on the core composition, whether bonded covalently or non- covalently. Exemplary coatings include protenacious deposits, metallic films, nucleic acids, carbohydrates and polysaccharides, and lipid layers.

[64] The term "molecular conjugate" refers to a combination of normally disparate molecules into an association. The association is may be facilitated by a synthetic linkage, which may be covalent or non-covalent in nature, and may contain molecular material to facilitate the association in addition to the molecules being combined. [65] The terms "multi-valent" and "valency" refer to the number of binding sites (usually specific) for molecules, possessed by another molecule, such as an antibody or antigen. More generally, the terms refer to the capacity of something to unite, react, or interact with something else. [66] The term "N-terminal α amine" refers generally refers to the amine located in the α position in the structure of an amino acid. For example, in the generalized amino acid structure R-CHNH₂-COOH, the N-terminal α amine is the -NH2 radicle radical bound to the central substituted carbon. With regard to peptides, polypeptides and proteins, the N-terminal α amine is the free amine.

[67] The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, including analogs, such as phosphorothioates, phosphorarnidates, methyl phosphonates, chiral-methyl phosphonates, 2- O-methyl ribonucleotides, peptide-nucleic acids (PNAs). Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al, Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al, J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al, Mol. Cell. Probes, 8:91-98 (1994)). The term "nucleic acid" is used interchangeably with the terms "gene", "cDNA", "mRNA", "oligonucleotide", and "polynucleotide". [68] The term "nucleoprotein" refers to any of a group of complexes composed of protein and nucleic acid and found in the nuclei and cytoplasm of all living cells, as in chromatin and ribosomes, and in viruses.

[69] The term "polylysine" refers to a polypeptide that is comprised of at least 75% lysine by residue, more preferably 90% lysine by residue, most preferably 100% lysine by residue. [70] The term "polypeptide" refers to a polymer of amino acid residues, such as a small protein, containing between 10 and 200 amino acids, more typically between 15 and 100 amino acids.

[71] The term "protein" refers to any of a group of complex organic macromolecules that contain carbon, hydrogen, oxygen, nitrogen, and usually sulfur and are composed of one or more chains of amino acids. Protein also refers to complexes of two or more polypeptides, whether bound together by covalent or non-covalent forces.

[72] The term "specifically recognize" in the context of proteins, refers to an inherent property to bind to certain proteins with a higher affinity than they bind to other proteins to the extent that the binding reaction is determinative ofthe presence ofthe recognized protein, in a heterogeneous population of proteins and other biologies, i the context of nucleic acids, the phrase "specifically recognize" refers to the binding, duplexing, or hybridizing of a nucleic acid only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (e.g., total cellular or library DNA or RNA).

[73] In reference to immunoaffmity interactions, the phrase to "specifically recognize" an antibody or "specifically (or selectively) immunoreactive with", refers to specified antibodies binding to a particular protein, typically with the reaction being at least twice background signal or noise and more typically more than 10 to 100 times background, and do not substantially bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. Solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow and Lane, Antibodies, A Laboratory Manual (1988) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). [74] The term "streptavidin" refers to a protein isolated from Streptomycetes avidinii that has a high affinity for biotin and is used commonly used to detect biotin-conjugates. [75] The term "streptavidin-coated microparticle" refers to microparticles than have strepavidin protein coupled to their surface. The protein may be covalently coupled to the microparticle, or may remain fixed by noncovalent forces, normally predominated by ionic interactions between charged side chain residues ofthe protein and the surface ofthe microparticle, although several types of microparticles are constructed from hydrophobic material in which case the protein and microparticle remain coupled predominantly through hydrophobic interactions.

DETAILED DESCRIPTION

I. Introduction

[76] The present invention describes novel molecular conjugates for signal amplification in analyte detection systems. The invention uses multi-label scaffolds that allow a plurality of labels and/or label types to be coupled to a binding partner. By coupling multiple labels to each binding partner ofthe molecular conjugate, the present invention results in increased sensitivity and signal amplification that is at least an order of magnitude greater than that found for conventionally singly-labeled detection reagents. Preferably the signal amplification is at least ten times greater than that produced by binding partners coupled to a single (unit) label. More preferably the signal amplification is at least 15 times, still more preferably at least 20 times and even more preferably at least 25 times greater. [77] Binding partners ofthe present invention may be any molecule capable of recognizing, preferably specifically recognizing, another molecule. Prefened binding partners are proteins, nucleic acids and polysaccharides. In addition to recognition properties, binding partners also possess the capacity to be coupled to other molecules having one ofthe desired reactive groups ofthe invention, namely, amine, carboxyl, hydroxyl and sulfhydryl reactive groups.

[78] Suitable labels of the present invention may be any system having a detectable physical or chemical property and must have the capacity to be coupled to a molecule having one ofthe desired reactive groups noted above. Exemplary labels include enzymes producing detectable products, isotopes, fluorophores, phosphorescent compounds, chromgenic compounds and the like.

[79] The present invention provides multi-label scaffolds. Multi-label scaffolds may take a variety of forms from multi-valent microparticles consisting of synthetic and/or natural components to polypeptides and nucleic acids or any combination of two or more ofthe above. Multiple labels may be coupled directly to a microparticle that is in turn coupled to binding partner resulting in enhanced signal amplification. Alternatively, label may be coupled to the side-chain residues of a polypeptide, or the bases of a nucleic acid. The polypeptide or nucleic acid is then coupled directly to a binding partner or through a multivalent microparticle. The latter alternative is a preferred embodiment, having the capacity for signal amplification several orders of magnitude greater than that seen in conventional, single-label reagents. This is illustrated in Figure 13, where the signal in the right-most pair of lanes represents an assay carried out with a conjugate ofthe present invention and the left-most two lanes represents an assay carried out under identical conditions using a conventional, unitary-label reagent.

[80] An important aspect of multi-label scaffolds comprised of enzyme-linked polypeptides or nucleic acids is the differential coupling ofthe polypeptide or nucleic acid to a binding partner or microparticle. Polypeptides couple to the binding partner or microparticle are only coupled through the N-terminal aα-amino group. Similarly, nucleic acids are only coupled to a binding partner or microparticle through a 5' or 3' end, preferably the 5' end. By employing this coupling strategy, the present invention ensures that each polypeptide or nucleic acid, and in turn the plurality of labels coupled to it, are coupled to only a single binding partner or microparticle. This arrangement permits the maximal degree of signal amplification obtainable for the number of reactive groups present in the multi-label scaffold.

[81] The conjugates ofthe present invention have numerous uses in analytical investigation. For example, the conjugates may be used as detectable molecular binding reagents in ELISA, FACS analysis, Southern/Western/Nortern blotting studies, chromosome painting (mapping), determination of gene expression and biomolecule localization in histochemical studies.

[82] Kits including the present invention are also contemplated. Typically such kits will include instructions for use and optionally buffers, enhancing reagents and other components directly associated in generating a detectable signal in response to an analyte, when the kit is used.

II. Suitable binding partners

[83] Suitable binding partners ofthe present invention identify a desired component in a complex mixture and contain, or are conducive to modification to contain, at least one reactive NH₂, OH, CO H and/or SH radical, thereby allowing the binding partner to couple to other components ofthe molecular conjugate described herein. Reagents must be able to discriminate between different components ofthe mixture, at least to the extent that the reagent may identify the desired component. Binding partners with these properties are typically proteins, nucleic acids, a modified protein or nucleic acid species, or a complex containing one or more of these types of molecules.

A. Proteins

[84] Proteins suitable for use as binding partners ofthe present invention include antigens, antibodies, receptors and receptor ligands. Receptors by definition specifically recognize a receptor ligand or a specific group of ligands. Ligands in turn are recognized by a receptor or class of receptors. It is this reciprocal relationship that defines a set of binding partners. Thus any receptor may be used as a binding partner ofthe present invention to identify its corresponding binding partner ligand, the latter being present in a complex mixture. Conversely, embodiments ofthe present invention using a receptor ligand as the binding partner allows for the recognition ofthe presence ofthe ligand's receptor in a complex mixture. [85] Antibody/antigen interactions represent a special kind of receptor ligand relationship that is characterized by both highly specific recognition between the binding partners and high affinity coupling. Antigens are molecular moieties, frequently found within larger moieties, although small antigenic molecules have been isolated.

1. Antibodies [86] Methods of producing polyclonal and monoclonal antibodies that react specifically with antigens of interest, are known to those of skill in the art (see, e.g., Coligan, Current

Protocols in Immunology (1991); Harlow & Lane, supra; Goding, Monoclonal Antibodies:

Principles and Practice (2d ed. 1986); and Kohler & Milstein, Nature, 256:495-497 (1975).

Such techniques include antibody preparation by selection of antibodies from libraries of recombinant antibodies in phage or similar vectors, as well as preparation of polyclonal and monoclonal antibodies by immunizing mammals (see, e.g., Huse et al, Science, 246:1275-

1281 (1989); Ward et al, Nature, 341:544-546 (1989)).

Monoclonal antibodies [87] In some instances, it is desirable to prepare monoclonal antibodies from various mammalian hosts, such as mice, rodents, cows, sheep, goats, donkeys, primates, humans, etc.

Description of techniques for preparing such monoclonal antibodies may be found in, e.g.,

Stites, et al. (eds.) Basic and Clinical Immunology (4 ed.), Lange Medical Publications, Los

Altos, Calif, and references cited therein; Harlow and Lane (1988) Antibodies: A Laboratory

Manual, CSH Press; Goding (1986) Monoclonal Antibodies: Principles and Practice (2d ed.)

Academic Press, New York; and particularly in Kohler & Milstein, Eur. J. Immunol, 6:511-

519 (1976), which discusses one method of generating monoclonal antibodies. Each of these references is incorporated herein by reference.

[88] Other suitable techniques involve in vitro exposure of lymphocytes to the antigenic polypeptides or alternatively to selection of libraries of antibodies in phage or similar vectors.

See, Huse, et al, Science, 246:1275-1281 (1989); and Ward et al, Nature, 341:544-546

(1989), each of which is hereby incorporated herein by reference.

[89] Monoclonal antibodies and polyclonal sera are collected and titered against the immunogen protein in an immunoassay, for example, a solid phase immunoassay with the immunogen immobilized on a solid support. Typically, polyclonal antisera with a titer of 10⁴ or greater are selected and tested for cross reactivity against the antigen using a competitive binding immunoassay. Specific polyclonal antisera and monoclonal antibodies will usually bind with a kDa of at least about 1 mM, more usually at least about 300 μM, preferably at least about 3 μM or better, and most preferably, 0.03 μM or better. [90] Antibody titers of ascites or hybridoma culture fluids are determined by various serological or immunological assays which include, but are not limited to, precipitation, passive agglutination, enzyme-linked immunosorbent antibody (ELISA) technique and radioimmunoassay (RIA) techniques. Similar assays are used to detect the presence of OFP in culture media or tissue and cell extracts.

Polyclonal antibodies [91] Methods of production of polyclonal antibodies are known to those of skill in the art.

An exemplary method involves immunizing an inbred strain of mice (e.g., BALB/C mice) or rabbits with the protein using a standard adjuvant, such as Freund's adjuvant, and a standard immunization protocol. The animal's immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to the protein. When appropriately high titers of antibody to the immunogen are obtained, blood is collected from the animal and antisera are prepared. Further fractionation ofthe antisera to enrich for antibodies reactive to the protein can be done if desired (see, Harlow & Lane, supra).

[92] For a review of immunological and immunoassay procedures, see Basic and Clinical

Immunology (Stites & Terr eds., 7^th ed. 1991). Moreover, the immunoassays ofthe present invention may be performed in any of several configurations, which are reviewed extensively in Enzyme Immunoassay (Maggio, ed., 1980); and Harlow & Lane, supra.

B. Nucleic acids

[93] Nucleic acids are capable of serving at least two roles in the present invention. First, they may perform as binding partners for homologous nucleic acids. Second, they may serve as a multi-label scaffold for coupling multiple labels to the conjugate compositions ofthe invention, hi this section, the first role played by nucleic acids, as binding partners, is discussed.

[94] As a binding partner, the nucleic acids ofthe present invention will generally specifically recognize homologous nucleic acid sequences under stringent conditions. Typically the nucleic acids ofthe invention specifically recognize a sequence having at least 65% identity to the nucleic acid serving as the binding partner; preferably at least 75% identity; more preferably 85% identity; and most preferably greater than 95% identity. [95] Prefened nucleic acids for use as binding partners are derivatized to contain at least one reactive moiety for coupling to labelling components ofthe molecular conjugates ofthe present invention. Preferably the reactive moiety is at the 3' or 5' end. The coupling need not be direct, and may be through a multi-label scaffold, such as a microparticle. Any moiety capable of coupling to the binding partner and a detectable label may serve as multi-label scaffold. Exemplary multi-label scaffolds include nucleic acids, microparticles, micelles and polypeptides. A preferred multi-label scaffold is polylysine.

[96] Alternative methods for creating reactive moieties for coupling include synthesizing a nucleic acid with a modified base. Modification ofthe sugar moiety of a nucleotide at positions other than the 3' and 5' position is also possible through conventional methods. Also, nucleic acid bases may be modified, e.g., by using N7- or N9-deazapurine nucleosides or by modification of C-5 of dT (deoxy-thymidyl) with a linker arm, e.g., as described in F. Eckstein, ed., "Oligonucleotides and Analogues: A Practical Approach," IRL Press (1991). Alternatively, backbone-modified nucleic acids (e.g., phosphoroamidate DNA) may be used so that a reactive group may be attached to the nitrogen center provided by the modified phosphate backbone.

[97] i prefened embodiments, modification of a nucleic acid, e.g., as described above, does not substantially impair the ability ofthe nucleic acid or nucleic acid sequence to hybridize to its homologue. Thus, any modification should preferably avoid substantially modifying the functionalities ofthe nucleic acid(s) responsible for Watson-Crick base pairing.

III. Suitable label systems

[98] The particular label or detectable group used should not significantly interfere with the specific binding ofthe binding partner used in the molecular conjugate. The detectable group may be any material having a detectable physical or chemical property. Such detectable labels have been well-developed in the field of immunoassays and, in general, most any label useful in such methods may be applied to the present invention. Thus, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels in the present invention include magnetic beads (e.g., DYNABEADS™); fluorescent dyes and techniques capable of monitoring the change in fluorescent intensity, wavelength shift, or fluorescent polarization (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like); radiolabels (e.g., ³H, ¹²⁵1, ³⁵S, ¹⁴C, or ³²P); enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA); and colorimetric labels such as colloidal gold or colored glass or plastic beads (e.g., polystyrene, polypropylene, latex, etc.), or lipid micelles that bind or compartmentalize a detectable marker. For exemplary methods for incorporating such labels, see U.S. Pat. Nos. 3,940,475; 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.

[99] The label may be coupled directly or indirectly to the desired multi-label scaffold according to methods well known in the art and described herein. As indicated above, a wide variety of labels maybe used, with the choice of label depending on sensitivity required, ease of coupling to the multi-label scaffold, stability requirements, available instrumentation, and disposal provisions. Prefened labels ofthe present invention are enzymes, biotin, or fluorophores.

[100] Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidases, particularly peroxidases. Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc. Small fluorescent proteins, such as those isolated from marine creatures are particularly useful labels (See e.g., Chalfie et al, Science, 263:802 (1994); Prasher, Trends in Genetics, 11:320 (1995); WO 95/07463; Heim et al, Proc. Natl. Acad. Sci. USA, 91:12501 (1994)). Chemiluminescent compounds include luciferin, and 2,3-dihydrophthalazinediones, e.g., luminol. For a review of various labeling or signal producing systems that may be used, see, U.S. Patent No. 4,391,904.

[101] Means of detecting labels are well known to those of skill in the art. Thus, for example, where the label is a radioactive label, means for detection include a scintillation counter or photographic film as in autoradiography. Where the label is a fluorescent label, it may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence may be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like. Similarly, enzymatic labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product. Finally, simple colorimetric labels may be detected simply by observing the color associated with the label. Thus, in various dipstick assays, conjugated gold often appears pink, while various conjugated beads appear the color ofthe bead. IV. Suitable microparticles

[102] Microparticles serve as particularly good multi-label scaffolds due to their optimal surface area and potential for multi-valent character. Appropriate "microparticles" for use in the instant invention include any three dimensional structure that can be coupled to nucleic acids and/or proteins and is of such a size as to easily form suspensions in a liquid. Preferable microparticle diameters are given in the definitions section supra. Exemplary microparticles can be made of virtually any insoluble or solid material. For example, the microparticle can be comprised of silica gel, glass (e.g. controlled-pore glass (CPG)), nylon, Wang resin, Merrifield resin, Sephadex, Sepharose, cellulose, magnetic microparticles, Dynabeads, a metal surface (e.g. steel, gold, silver, aluminum, silicon and copper), a plastic material (e.g., polyethylene, polypropylene, polyamide, polyester, polyvinylidenedifluoride (PVDF)) and the like. Microparticles may be swellable, e.g., polymeric microparticles such as Wang resin, or non-swellable (e.g., CPG). Suitable materials for microparticles also include those capable of partitioning from the general solvent in which the microparticles are to be used. For example, in the context of several embodiments ofthe present invention, lipid globules and micelles that stably partition from the aqueous solution ofthe typical immunodetection assay and comprise functional groups reactive with NH₂, OH, CO H and SH radicals ofthe present invention are considered microparticles, as are living cells and viral particles.

[103] When absent, microparticles lacking the appropriate surface chemistry for coupling proteins and/or nucleic acids, may be coated with biological or synthetic materials providing the necessary reactive groups. For example, microparticles may be coated with protein compositions, such as BSA, or with nucleic acid compositions isolated from bacteria. Microparticles may also be coated with polysaccharides containing any or all ofthe desired functional groups.

[104] Microparticles may also be coated with compositions having specific or semi-specific recognition properties. For example, microparticles coated with protein A or G can specifically recognize and bind immunoglobulins non-covalently, while also providing the surface chemistries allowing for covalent attachment of other proteins and nucleic acids. Microparticles coated with avidin or strepavidin will specifically recognize biotin and biotinylated molecules. Biotinylated binding partners and biotinylated multi-label scaffolds, such as polypeptides, nucleic acids and microparticles will interact with the avidin or strepavidin microparticle, resulting in an embodiment ofthe present invention based on non- covalent couplings.

[105] Methods for coating microparticles with compounds to place reactive NH₂, OH, CO₂H and SH radicals or compositions with specific recognition properties on the surface of the microparticles are well known in the art. By way of example, microparticles are incubated with 1-10 microgram per milliliter solution of protein in 50 mM bicarbonate buffer, pH 9 for 18 h at 4°C. The microparticles are then washed with PBS, incubated with Pierce SuperBlock solution for 2 h at room temperature, washed, and stored at 4°C until used. Methods for coating microparticles include both non-covalent (e.g., the avidin/biotin system discussed supra) and covalent attachments, such as those utilizing the desired reactive groups discussed herein.

V. Multi-label scaffolds

[106] A prefened aspect ofthe present invention is the capacity to amplify the signal produced by analyte-specific detection reagents by an order of magnitude or more. This signal amplification is a consequence of associating a plurality of labels with each analyte- specific binding partner. The association of a plurality of labels with each analyte-specific binding partner is accomplished by linking the labels to the binding partner(s) through a multivalent moiety, or multi-label scaffold. The multi-label scaffold may take a variety of forms, including polypeptides, nucleic acids, synthetic micelles, microparticles, and cells, both prokaryotic and eukaryotic. The common feature of all of these multi-label scaffolds is the presence of reactive groups, typically -NH₂, -OH, -CO₂H and -SH radicals or chemical groups that can be modified to the same or similar reactive groups, that allow for coupling of proteins, polypeptides, sugars, nucleic acids and various types of labels as described herein. [107] By way of example, microparticles are generally constructed of materials that either inherently posses the desired reactive groups, or may be coated with substances that possess the desired reactive groups. Gold microparticles may for example be coated with protein or nucleic acid by techniques well known in the art, to supply the desired reactive groups to the microparticle as described supra.

A. Polypeptides

[108] Many biological materials comprise molecules that inherently have the desired reactive groups. For example, polypeptides contain amino acid resides with side chains that possess the desired reactive groups. For purposes of this invention, a suitable polypeptide will possess at least two amino acid residues or derivatives having one or more ofthe desired reactive groups. Preferably the polypeptide will preferably have 2 to about 250, more preferably 5 to about 100 desired reactive groups, more preferably between 10 and 50 desired reactive groups and most preferably between 15 and 30 desired reactive groups. Commensurate with the number of desired reactive groups, preferable multi-label scaffolds will comprise polypeptides preferably of between 2 to about 250 amino acids, more preferably 5 and 100 amino acids, more preferably 10-50 amino acids and most preferably between 15 and 30 amino acids.

[109] A particularly favorable reactive group is the amine radical, -NH , as this group is both an efficient site for coupling itself, and is easily modified chemically to produce other desired reactive groups. Polypeptides possessing this reactive group are also prefened, with the ε amino of lysyl residues being a particularly desired reactive group. Consequently a particularly preferred polypeptide is polylysine.

B. Nucleic Acids

[110] Nucleic acids also make effective mult-label scaffolds, with primary amino groups on adenylyl, guanylyl and cytosyl all providing the desired reactive primary amine group. As with polypeptides, the nucleic acids will preferably have 2 to about 250 desired reactive groups, more preferably 2 to about 100 desired reactive groups, more preferably between 10 and 50 desired reactive groups and most preferably between 15 and 30 desired reactive groups. Commensurate with the with the number of desired reactive groups, preferable multi-label scaffolds will comprise nucleotides preferably of between 2 to about 250 bases, more preferably 2 and 150 bases, still more preferably 10-75 bases and most preferably between 15 and 45 bases. Both single and double stranded nucleic acids, and nucleic acids taking secondary structural forms (loops and junctions) function as effective multivalent scaffolds.

[Ill] Nucleotides are modified either at the 3', 5', or in the middle ofthe sequence to contain amine groups, sulfhydryl groups, halide groups, digoxigenin, biotin phosphoramidite, Psoralen phosphoramidite (to introduce photoreactive groups), acridine phosphoramiditefluorescem phosphoramidite, phosphate group, or introduction of spacer arm using spacer phosphoramidite, etc. The custom synthesis of modified nucleotides is provided by Qiagen Operon (www . operon . com) or by other established vendors. [112] Micelles, and various cellular and viral moieties also function as effective multi-label scaffolds. All of these various bodies, both natural and synthetic are broadly categorized as microparticles due to their small size and their functional similarity, in the context of this invention, to classic microparticles. Micelles, cells and viral moieties all possess desired reactive groups on their surfaces making them desired multi-label scaffolds.

VI. Constructing nucleic acids and polypeptides as multi-label scaffolds A. Synthetic peptide synthesis

[113] Polypeptides may be chemically synthesized by methods well known in the art, e.g., using 9-fluorenylmethoxycarbonyl (Fmoc) chemistry (Atherton, E. et al., "Sheppard, R.c, In Solid Phase Peptide Synthesis -A Practical Approach;" IRL Press at Oxford University Press: Oxford, U.K., (1989); Merrifield, R.B. "Solid-phase peptide synthesis. In: Gutte B, ed. Peptides— Synthesis, structures and applications" San Diego, CA: Academic Press, pp 93- 169 (1995)). Alternatively, TBoc chemistry may also be used, hi a single synthesis of a peptide, amino acids are simultaneously coupled to a chemically functionalized solid support. Typically, an N-protected form ofthe carboxyl terminal amino acid, e.g. a t-butoxycarbonyl protected (Boc-) amino acid, is reacted with the chloromethyl residue of a chloromethylated styrene divinylbenzene copolymer resin to produce a protected amino acyl derivative ofthe resin, the amino acid being coupled to the resin as a benzyl ester. This derivative is deprotected and reacted with a protected form ofthe next required amino acid thus producing a protected dipeptide attached to the resin. The amino acid will generally be used in activated form, e.g. a carbodiimide or active ester. The addition step is repeated and the peptide chain grows one residue at a time by condensation ofthe required N-protected amino acids at the amino terminus until the required peptide has been assembled on the resin. The peptide-resin is then treated with anhydrous hydrofluoric acid to cleave the ester linking the assembled peptide to the resin and liberate the required peptide. The protecting groups on side chain functional groups of amino acids that were blocked during the synthetic procedure, using conventional methods, may also be removed. This entire procedure may be automated. Multiple peptides or oligonucleotides may be synthesized. [114] One such methodology for peptide synthesis is disclosed in Geysen, et al. International Publication Number WO 90/09395, hereby incorporated by reference. Geysen's method involves functionalizing the termini of polymeric rods and sequentially immersing the termini in solutions of individual amino acids. Geysen's approach has proven to be impractical for commercial production of peptides since only very minute quantities of polypeptides may be generated. In addition, this method is extremely labor intensive. [115] U.S. Pat. No. 5,143,854 to Pirrung et al., hereby incorporated by reference, discloses another method of peptide or oligonucleotide synthesis. This method involves sequentially using light for illuminating a plurality of polymer sequences on a substrate and delivering reaction fluids to said substrate. A photochemical reaction takes place at the point where the light illuminates the substrates. Reaction at all other places on the substrate is prevented by masking them from the light. A wide range of photochemical reactions may be employed in this method, including addition, protection, deprotection, and so forth, as are well known in the art. This method of synthesis has numerous drawbacks, however, including the fact that the products are non-cleavable and that the process produces large numbers, but only minute quantities, of products.

[116] A further method and device for producing peptides or oligonucleotides is disclosed in European Patent No. 196174. The disclosed apparatus is a polypropylene mesh container, similar to a tea-bag, which encloses reactive particles. The containers, however, are not amenable to general organic synthesis techniques.

B. Solid phase nucleic acid synthesis

[117] For nucleic acids, sizes are given in either kilobases (Kb) or base pairs (bp). These are estimates derived from agarose or acrylamide gel electrophoresis, from sequenced nucleic acids, or from published DNA sequences. For proteins, sizes are given in kilodaltons (kDa) or the number of amino acid residues. Proteins sizes are estimated from gel electrophoresis, from automated protein sequencing, from derived amino acid sequences, or from published protein sequences.

[118] Oligonucleotides that are not commercially available may be chemically synthesized according to the solid phase phosphoramidite triester method first described by Beaucage & Caruthers, Tetrahedron Letts., 22:1859-1862 (1981), using an automated synthesizer, as described in Van Devanter et. al, Nucleic Acids Res., 12:6159-6168 (1984). Purification of oligonucleotides is by either native acrylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson & Reanier, J. Chrom., 255:137-149 (1983). [119] The sequence ofthe cloned genes and synthetic oligonucleotides may be verified after cloning using, e.g., the chain termination method for sequencing double-stranded templates of Wallace etal, Gene, 16:21-26 (1981). C. Natural sources

[120] In addition to synthetic molecules, polypeptides and nucleic acids from virtually any biological source may be used as multi-label scaffolds or binding partners ofthe present invention. For multi-label scaffolds, the molecules must be functionally capable of coupling to at least two label moieties. Structurally, the principle limitation on the multi-label scaffolds is that they do not take on structural conformations that either interfere with binding partner interactions or interfere with label detection.

VII. Coupling Chemistries

[121] Coupling chemistries are critical in joining components ofthe invention described above into a functional molecular conjugate. Appropriate coupling agents for use in the invention include those capable of reacting with a functional group present on a surface of a microparticle, a protein or polypeptide, polysaccharide and/or nucleic acid, with a functional group present on another microparticle, a protein or polypeptide, polysaccharide and/or nucleic acid. Reagents capable of such reactivity include homo- and heterobifunctional reagents, many of which are known in the art. Heterobifunctional reagents are prefened. A prefened bifunctional coupling agent is N-succinimidyl(4-iodoacetyl) aminobenzoate (SIAB). However, other coupling agents, including, without limitation, dimaleimide, dithio- bis-nitrobenzoic acid (DTNB), N-succinimidyl-S-acetyl-thioacetate (SAT A), N- succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl 4-(N- maleimidomethyl)cyclohexane-l -carboxylate (SMCC), 6-hydrazinonicotimide (HYNTC) and;

1. Homobifunctional Cross-linking Reagents

(A) Amine-reactive: BS³ (Bis(sulfosuccnimidyl)-suberate; DSS (Disuccnimidyl suberate); DST (Disuccnimidyl tartarate); EGS (Ethylene glycol bis- (succnimidylsuccinate); Sulfo-EGS (Ethylene glycol bis-(sulfo- succnimidylsuccinate Sulfhydryl-reactive: BMB (1,4-Bis-maleimidobutane); BMH (1.6-Bis-Maleidohexane); BM[PEO]₃ (1,8- Bis-Maleimidotriethylene glycol)

(B) Photoreactive: BASED (Bis-beta-[4-azidosalicylamido)-butylamine)

2. Heterobifunctional Cross-linking Reagents (A) Amine- and Sulfhydryl-reactive: EMCS (N-epsilon-Maleidocaproyloxy) succinimide ester; Sulfo-EMCS (N-epsilon-Maleidocaproyloxy) sulfosuccinimide ester; SMCC (Succinimidyl 4-(N- maleimidomethyl)cyclohexane-l -carboxylate; Sulfo-SMCC (Sulfo- succinimidyl 4-(N-maleimidomethyl)cyclohexane-l-carboxylate); MBS (m- Maleimidobenzoyl-N-hydroxysuccinimide ester); SMPT (4-succinimidyloxy- carbonyl-methyl-alpha-(2-pyridyldithio)toluene) ; Sulfo-SMPT (4-sulfo- succinimidyloxy-carbonyl-methyl-alpha-(2-pyridyldithio)toluene); SPDP (N- Succinimidyl 3-(2-pyridyldithio)propionate); Sulfo-SPDP (N-Sulfo- succinimidyl 3-(2-pyridyldithio)propionate)

(B) Amine- and Photo-reactive: ANB-NOS (N-5-Azido-2-nitrobenzoyloxy- succinimide); SASD (Sulfosuccinimidyl 2-(p-azido-salicylamido)ethyl 1, 3'- dithio-proprionate); NHS-ASA (N-Hydroxysuccinimidyl-4-azidosalicylic acid); Sulfo-NHS-LC-ASA (N-Hydroxy sulfo-succinimidyl-4-azidosalicylic acid)

(C) Carbohydrate-reactive: ABH (p-azidobenzoyl) hydrazide; EMCH (N-epsilon- Maleimidocaproic acid) hydrazide); MPBH ( 4-(4-N-Maleimidophenyl)- butyric acid hydrazide.HCl); MPBA (3-Maleimidophenyl boronic acid); PDPH (3-(2-Pyridyldithio)-proprionyl hydrazide)

(D) Iodinatable/Photoreactive: ASBA (4-(p-Azidosalicylamido)-butylamine; NHS-ASA (N-Hydroxysuccinimidyl-4-azidosalicylic acid); Sulfo-NHS-LC- ASA (Sulfosuccinimido)hexanoate; SASD (Sulfosuccinimidyl 2-(p-azido- salicylamdo)ethyl l,3'-dithioproprionate); APDP (N-(4-[p- Azidosalicylamido]-butyl)-3 ' (2' -pyridyldithio)-proprionamide

(E) Sequential Amine-to- Amine-reactive: MSA (Methyl N-succinimidyl adipate); SATA (N-Succinimidyl S-acetylthioacetate)

(F) Sequential Sulfhydryl to Amine-reactive: BMPA (N-beta-maleimidopropionic acid); EMCA (N-epsilon-Maleimidocaproic acid); KMUA (N-k- Maleimidoundecanoic acid) hydrazide)

(G) Protein-DNA Cross-linking: SPB (Succinimidyl-(4-psoralen-8- yloxy)butyrate)

(H) Other Functional Group Reactive:- Arginine-Reactive/Photoreactive: APG (p- Azidophenyl glyoxal monohydrate); -COOH-Reactive/Photoreactive: ASBA (4-p-Azidosalicylamido)-butylamine; Sequential Hydroxyl to Sulfhydryl Coupling: PMPI (N-(p-Maleimidophenyl) isocyanate)

(I) Amine-Reactive and maleimide/ Alkyl Halide/Ninyl Sulfone-Reactive: SATA (Ν-Succinimidyl S-acetylthioacetate); SATP (Ν-Succinimidyl S- acetylthiopropionate

(J) Amine- and Carboxyl-Reactive: AEDP (3-([2-Aminoethyl] dithio)-propionic acid.HCl)

3. Trifunctional Cross-linking Reagents

Sulfo-SBED (amine-reactive, photoreactive, avidin Binding); TSAT (amine- reactive); TMEA (Sulfhydryl-reactive)

[122] In certain embodiments, the cross-linking agent may be selected to provide a selectively cleavable bond. For example, a photolabile cross-linker such as 3-amino-(2- nitrophenyl)propionic acid (Brown et al. (1995) Molecular Diversity 4-12 and Rothschild et al (1996) Nucleic Acids Res. 24:351-66) may be employed to provide a means for cleaving the binding partner and/or multi-label scaffold components from the microparticle, if desired. For further examples of cross-linking reagents, see, e.g., S. S. Wong, "Chemistry of Protein Conjugation and Cross-Linking," CRC Press (1991), and G. T. Hermanson, "Bioconjugate Techniques," Academic Press (1995).

[123] In one prefened embodiment, a covalent amide bond is formed between a microparticle and the binding partner and multi-label scaffold components of a molecular conjugate ofthe present invention by reacting a carboxyl-functionalized microparticle with the amino-functionalized binding partner and multi-label scaffold components (e.g., by reacting a carboxyl-functionalized Wang resin with an amino-functionalized polypeptide). Alternatively, carboxyl-functionalized binding partner and multi-label scaffold components may be reacted with an amino-functionalized microparticle, taking advantage of an acid- cleavable bifunctional trityl protection scheme usually employed for nucleic acid attachment. A bifunctional trityl linker can also be attached to the 4-nitrophenyl active ester on a microparticle (e.g. Wang resin) by an amino group as well as from a carboxy group of either the binding partner or multi-label scaffold component.

[124] As pointed out above, the microparticle may also be coupled to the binding partner and multi-label scaffold components by non-covalent interactions. For example, the microparticle may be provided with an ionic or hydrophobic moiety, which can associate with, respectively, an ionic or hydrophobic moiety ofthe binding partner and multi-label scaffold components. Alternatively, a bead may be provided with a member of a specific binding pair, and be coupled to the binding partner and multi-label scaffold components via a complementary binding moiety. For example, a bead coated with avidin or streptavidin may be bound to a binding partner or multi-label scaffold component coated with biotin or derivatives of biotin such as imino-biotin. Other specific binding pairs contemplated for use in the invention include hormone-receptor, enzyme-substrate, nucleic acid-complementary nucleic acid, antibody-antigen and the like. Also clathrate compounds (host-guest) of relatively small complexes in relatively large cavities in the macromolecule are possible.

A. Specific couplings for Non-proteins (e.g., polysaccharides and nucleic acids)

[125] Non-protein components ofthe present invention may be coupled after, for example, derivatization with SATA and other S-acetyl derivatives. A useful deblocking mode for SATA and other S-acetyl derivatives, which contain no other alkali labile groups (e.g., many non-protein binding partners), utilizes rapid to nearly instantaneous generation of thiols with 0.01 to 0.1M NaOH or other alkalis in the presence of DTP A and nitrogen to exclude oxygen (deblocking of SATA derivatives with 0.01M NaOH for 1/2 hr has been reported and probably results in considerable thiol oxidation even under exclusion of oxygen). [126] Upon completion of deblocking, the thiol is protected from oxidation and prepared for conjugation by addition of a weak acid (e.g., acetic acid) to adjust the pH to 6-8 prior to conjugation. Again, a highly effective chelant such as DTPA is needed, particularly during the high pH step, to minimize thiol oxidation.

[127] In addition to faster deblocking and anticipated higher deblocking yields in alkali, this alternative NaOH procedure has the added advantages of flexibility in the choice ofthe conjugation pH for greater selectivity and non-reactivity ofthe neutralized deblocking reagent. This minimizes potential side reaction with the thiol-reactive group and other groups on the substrate that may occur with HA and even CMA.

B. Coupling reactions for antibodies

[128] Antibodies are a particularly prefened class of component in the present invention, as these proteins may serve as both binding partners and as part of coupling strategies for other components when forming the molecular conjugates ofthe present invention. In addition to the general coupling chemistries described above for use with proteins, a second possibility of forming antibody conjugates starts with a gentle reduction of the disulfide bridges ofthe immunoglobulin molecule. In this step, the more sensitive disulfide bridges ofthe H-chains ofthe antibody molecule are cleaved while the S--S- linkages ofthe antigen-binding region remain intact so that there is practically no diminution ofthe binding affinity and specificity ofthe antibody (Biochem. 18:2226, 1979, Handbook of Experimental Immunology, vol. 1, Second Edition, Blackwell Scientific Publications, Lindon 1973, chapter 10). These free sulfhydryl groups ofthe intra-H-chain regions are then reacted with suitable functional groups of multi-label scaffolds to form a prefened embodiment of the present invention. This is generally accomplished by reacting multi-label scaffolds that have been haloacetylated (e.g., polypeptides bromoacetylated at the N-terminal amino group) with the reduced immunoglobulin. Briefly by way of example, 2-MEA reduced IgG is incubated with 5- bromoacetylated polylysine chain in bicarbonate buffer under nitrogen. At the end of incubation, unbound bromoacetylated polypeptide is removed by gel filtration chromatography. Immunoglobulin-polylysine complex is then conjugated to label, e.g., HRP. Haloacetylation of biomolecules, particularly peptides and other primary amine-containing molecules, is well known in the art. (see e.g., Boykins, RA. et al., Peptides, 21:9-17 (2000), which is incorporated herein by reference).

[129] A method well suited for the production of molecular conjugates comprising antibodies as well as antibody fragments is the coupling to microparticles. For the oxidation in the Fc portion ofthe antibody, the column must be protected from the effect of light by providing coverage; for the reduction of disulfide bridges (for example in the generation of Fab fragments) the process must be performable under argon as a protective gas. The actual coupling step then takes place as follows: After washing ofthe microparticles with a suitable buffer, a solution is used that produces a desired reactive group(s) on the antibody (for example, periodate solution for the production of aldehyde groups in the Fc portion of monoclonal antibodies or mercaptoethylamine solution for the production of sulfhydryl groups in fragments). Finally, the microparticles are washed with a buffer solution to remove unreacted material.

VIII. Constructing molecular conjugates

[130] Conjugates of the present invention may be made with any combination of couplings utilizing the desired primary amine, hydroxyl, sulfhydryl and carboxylate reactive groups described herein or in the art. Similarly, the ability to incorporate diverse binding partners allows the practitioner ofthe present invention to recognize virtually every class of analyte important to biochemical and forensic analysis, among others. The conjugates ofthe present invention also allow coupling of a range of labels that may be mixed and matched to form combinations of conjugates allowing for multiplex analysis that is only limited in the number of analytes that can be simultaneously detected by the sophistication ofthe detection and analysis apparatus available to the art.

[131] By way of example, figures 1 through 12 illustrate a fraction ofthe conjugates that comprise the present invention. In illustrating the conjugates ofthe figures, exemplary coupling chemistries and uses ofthe present invention will be described. One of skill in the art will recognize that there exists many additional conjugates and uses ofthe present invention, as well as additional combinations of coupling chemistries, available in the public domain and described herein, that may be employed in construction ofthe conjugates. [132] Figure 1 depicts the steps of a rapid ELISA-type assay utilizing magnetic microparticles and multi-label conjugates ofthe present invention. Viral nucleic acid is first released from the viral capsid. The nucleic acid is then cleaved into smaller oligonucleotides, preferably of between 10 and 10,000 base pairs, more preferably 20 to 5,000, still more preferably 50 to 1,000 base pairs in size. The resulting oligonucleotides are contacted with microparticle-based conjugates of two types: The first type is a capture particle comprising a capturing microparticle coupled to a binding partner specifically recognizing subsequences found in the viral nucleic acid. The capturing microparticle has a physical property allowing it to be isolated from any mixture brought in contact with it. For example, the capturing microparticle may be magnetic, or coupled to an inert solid support. [133] The second microparticle-based conjugate is a composition ofthe present invention that comprises a binding partner that specifically recognizes subsequences ofthe viral nucleic acid coupled to a microparticle. Also coupled to the microparticle is a multilabel scaffold, e.g., a polypeptide or polynucleotide capable of binding a plurality of labels. To the multilabel scaffold is coupled a plurality of label.

[134] Contacting the capture antibody with the viral oligonucleotides allows the olignucleotides to be immobilized using the physical property unique in the mixture to the capturing microparticle. Other components ofthe mixture may then be removed, the viral oligonucleotides being specifically retained with the capture microparticles. [135] Contacting the conjugate ofthe present invention to the viral oligonucleotides, either before or after association ofthe oligonucleotides with the capture microparticles, leads to coupling ofthe oligonucleotides to the plurality of labels. Once other components ofthe mixture are removed, the labels may be detected, e.g. by addition of substrate when the labels are enzymes, visually where the labels are chromophores. The entire assay procedure is explained in additional detail in example 6, using detection of HIV nucleic acid as an illustration.

[136] Figure 2 illustrates a covalent coupling of a microparticle and a protein through an amide bond. This type of coupling may be formed between any two components used in forming a composition ofthe present invention where one ofthe components has a desired amine reactive group and the other component has a carboxylate group, for example a polysaccharide binding partner and a polypeptide acting as a multi-label scaffold. [137] . Figure 3 is a schematic of a microparticle-mediated rapid immunoassayusing an antigen as the binding partner. A capture microparticle is first constructed in the manner described above, with the exception that an antigen is coupled to the capture microparticle instead of an antibody. The antigen-capture microparticle is then contacted ti a complex mixture containing an antibody that specifically recognizes the capture antigen. A conjugate ofthe present invention is then contacted to the mixture. This conjugate may have an antigen binding partner that is specifically recognized by the same antibody recognizing the capture microparticle binding partner, or the conjugate may comprise a binding partner that is an antibody (or protein A or G) that specifically recognizes the same antibody recognizing the capture microparticle. Once the components ofthe mixture, including uncomplexed conjugates, are washed away from the resulting immunocomplex, the label associated with the conjugate ofthe present invention may be detected as described above. [138] Figure 4 demonstrates exemplary couplings for joining binding partners and multi- label scaffolds to microparticles. Conjugate A demonstrates the use of specific non-covalent couplings in forming conjugates ofthe present invention, hi figure 4, the coupling is a specific recognition of a strepavidin-coated microparticle by a binding partner (antibody or antigen) and a mutli-label scaffold (polylysine) that have been biotinylated. One of skill in the art will recognize that other non-covalent coupling systems comprising components that specifically recognize each other may be used, for example receptor/ligand, enzyme/substrate, and antigen/antibody binding pairs. Conjugates B and C both illustrate covalent coupling of both binding partners and label components ofthe conjugate to a microparticle. In conjugate B, the label is covalently coupled directly to the microparticle, i.e., the microparticle itself is the multi-label scaffold. In conjugate C, the multilabel scaffold is a polylysine. The polylysine is covalently coupled to the microparticle via the N-terminal α-amino group ofthe polylysine. Label is in turn coupled to the ε- amino group ofthe lysyl residues. Figure 5 schematically depicts the chemical steps in constructing the conjugate A alternatives of figure 4. In this example, the final coupling reaction comprises binding partner and polylysine multi-label scaffold in a 1:5 ratio. One of skill in the art will recognize that other ratios may be used. Figures 6 and 7 are similar detail depictions of Figure 4 conjugates B and C, respectively.

[139] Figure 8 details exemplary chemistries for covalently coupling label to antibodies. Panels A and C depict coupling of label to antibody through a primary amine ofthe antibody. Panels B and D depict covalent coupling of label via sulfhydryls ofthe antibody heavy chain. As known in the art, certain sulfhydryl reducing agents, such as 2-DEA, reduce the dicysteine couplings between the heavy chains of antibodies. Once exposed, the sulfhydryls may be covalently coupled as shown in figure 8. Panels A and B of figure 8 illustrate coupling of single labels to the antibody binding partners, with panels C and D depicting the coupling of multiple labels to the binding partner via a multi-label scaffold, in this case polylysine. [140] Figure 9 illustrates the construction of antibody-HRP and antibody-poly-HRP conjugates. In panel A, label is coupled to SATA-modified IgG. Briefly, binding partner (antibody) is modified with SATA to convert amines into free sulfhydryl groups followed by covalent coupling with maleimide-activated label. In panel B, Label is coupled to 2- MEA EDTA-reduced IgG. label conjugates of IgG with reduced indigenous disulfide groups in the hinge region are prepared for example by reducing IgG with 2-mercaptothylamine (2- MEA) in the presence of EDTA to generate free sulfhydryl groups. These free sulfhydryl groups are then coupled with maleimide-activated label to generate antibody-enzyme conjugate as shown inside the box. Panel C depicts construction of IgG-enzyme conjugate containing multiple labels. SATA-modified IgG is reacted with bromoacetylated lysine polypeptide containing 20 lysine residues to introduce additional primary amines for label conjugation as described in herein. This complex is then conjugated with, for example, Pierce EZ-link plus peroxidase. The intermediate Schiff base is reduced with NaCNBH₃ to generate the antibody-poly-label conjugate shown inside the box. Panel D depicts construction of antibody-poly-label conjugate using 2-MEA/EDTA-reduced IgG. HRP conjugates of IgG with indigenous disulfide reduced with 2-MEA/EDTA in the hinge region prior to maleimide-activated enzyme. The free sulfhydryl groups thus generated are reacted with bromoacetylated lysine polypeptide containing lysine residues to introduce additional primary amine on IgG for conjugation with multiple labels as described in herein. The structure of antibody-poly-label conjugate is shown inside the box. [141] Figure 10 (i) illustrates chemical modification ofthe primary amine groups of an amino polystyrene microparticle using SATA. Figure 10( ii) illustrates the chemistry involved in derivatizing the 5' end of an oligonucleotide using a phosphoramadite, Sulfo- SMCC and NaSO₃ prior to coupling with a derivatized microparticle. Figure 10(iii) depicts the final reaction mixture for creation of one embodiment ofthe present invention comprising the derivatized oligonucleotide, the chemically modified microparticle and a bromoacetylated polylysine, the oligonucleotide and polylysine components being present, as an example, in a 1 :5 ratio. Once formed, the conjugate is reacted with label, covalently coupling the label to ε amine groups ofthe lysyl side chains ofthe polylysine. The boxed composition is the final product ofthe reactions, representing an embodiment ofthe invention. [142] Figure 11 illustrates the components of microparticle-mediated immunodetection system. Panel A, Antibody capturing system consisting of 5 μm magnetic carboxyl polystyrene microparticles covalently conjugated with antigen(s). Panel B, Detector system consisting of smaller (0.2-4 μm) non-magnetic polystyrene microparticles to which either a secondary antibody or a secondary antigen together with a label (such as HRP in the present study) was attached in a 1 :5 ratio (as shown in a). Microparticle containing secondary antibody or a secondary antigen together with multiple HRP molecules on a polypeptide chain containing 20 lysine residues (1:5) is shown in b. Open arrows show the amplified view of these detector microparticle immunoconjugates.

[143] Figure 12 illustrates model immunocomplexes comprising conjugates ofthe present invention. Panel A, Antibodies captured by magnetic microparticles bound detector conjugate containing a secondary antibody and multiple units of label. Panel B, Antibody captured by magnetic microparticles bound the detector conjugate containing secondary antigen and multiple units of label. The large aggregates of immune complexes thus formed produce amplified detection signals at least an order of magnitude greater than prior art reagents coupling unitary labels directly to the binding partner without using the multi-label scaffolds ofthe present invention. <

[144] Many ofthe couplings described herein may be purchased directly or kits for their use may be purchased directly from commercial vendors. For example, protein-based components may be coupled via aldehyde groups using EZ-Link plus activated peroxidase kit (Pierce) and purified using Pierce' s FreeZyme kit. Blocking of non-specific reactive groups may be done with SuperBlock (Pierce). Antibodies are available from a variety of vendors, including Sigma Chemical Corporation. Free sulfhydryl groups ofthe antibodies maybe exposed and conjugated to labels having a desired amine reactive group using EZ-Link Maleimide activated horseradish peroxidase kit (Pierce). Biotinylation of components may be done using EZ-link sulfo-NHS-LC-biotin (Pierce), and label, for example HRP coupled to polypeptide using Pierce EZ-Link HRP conjugation kit (Pierce).

IX. Uses for the signal amplification conjugate

[145] Most conventional labelled conjugates for analyte analysis consist of a single label coupled to a single binding partner. The molecular conjugates ofthe present invention overcome several limitations found in conjugates cunently in use. First, the conjugates ofthe present invention may be constructed with a monovalent binding partner, allowing for a one binding partner per cell ratio. This property enhances sensitivity because multiple selectable markers are not occupied with a single conjugate, allowing more conjugate to bind per selectable marker. Second, the present conjugate allows numerous label moieties to be coupled to a single binding partner, again increasing sensitivity. Third, conjugates ofthe present invention allow coupling of labels with different chemical and physical properties to the same binding partner. By allowing combinations of diverse labels to be coupled to a single binding partner, the present invention facilitates a multiplex approach to analyte detection on a scale previously unheard of. This is particularly true when analyte analysis with the present invention is combined with computer analysis. Diverse physical and chemical properties, or combinations ofthe same, of conjugates comprising a multiplicity of labels can be mapped to variations in pattern or color for output in a common format. [146] The conjugates ofthe present invention may be used to detect a multitude of markers regardless of type or location. Extracellularly, molecular conjugates ofthe present invention may detect cell surface antigens in a manner similar to conventional reagents. By using molecular conjugates with binding partners that specifically recognize different molecules, and altering the label population to provide association of labels with cummulatively distinct chemical and physical properties, FACS analysis can be done on multiple markers in a single sort in multiplex fashion.

[147] Intracellularly, the present invention may be used to sort cells base on gene expression, protein (antigen) content or the presence of a particular gene or chromosomal arrangement using techniques commonly known in the art.

[148] Histochemical techniques provide additional utility similar to that noted above for FACS analysis. Using nucleic acids as binding partners, molecular conjugates ofthe present invention may be used in chromosomal mapping (painting), and gene expression determinations. Using antibodies as the binding partner, the molecular conjugates function as efficient ELISA reagents capable of detecting protein, nucleic acid and polysaccharide antigens. By way of example, HIN-1 antigens may be detected by first contacting a sample containing the antigen to a coupling surface comprised of a substrate capable of binding the antigen. Once bound to the substrate, the antigen may be recognized by a molecular conjugate ofthe invention comprising a binding partner that specifically recognizes the antigen. Subsequent methodology to develop the label may be carried out in a manner identical to traditional ELISA analysis and is commonly known in the art. [149] It will also be readily apparent to those of skill in the art that the conjugates ofthe present invention may be used in any assay requiring detection of specific analytes, or classes of analytes, in a complex mixture. Such assays include northern and Southern blotting for detection of specific nucleic acid sequences and Western blotting for particular epitopes. Further, specific receptors can be detected using the respective ligand as a binding partner in a conjugate ofthe invention. Conversely, the presence of specific ligands, such as hormones, can be detected in a complex mix by the present invention using the appropriate receptor molecule as the binding partner. Protein protein, nucleic acid/nucleic acid and protein/nucleic acid interactions of other types are also readily amenable to study using the present invention, again using methodology analogous to, if not identical to, methods well known to those of skill in the art. For example, Western blot (immunoblot) analysis using the conjugates ofthe present invention may be carried out by separating sample proteins by gel electrophoresis on the basis of molecular weight; transferring the separated proteins to a suitable solid support, (such as a PNDF membrane, a nitrocellulose filter, a nylon filter, or derivatized nylon filter), and incubating the sample with conjugates ofthe invention comprising antibodies that specifically recognize the antigen of interest. HIN-1 antigen detection may be carried out using a Bio-Rad HIN-1 Western blot kit according to manufacturer's instructions, except that instead of antibody-enzyme conjugate and substrate provided in the kit, HRP-conjugated goat anti-human IgG or poly-HRP-conjugated goat anti- human IgG ofthe present invention is used for detection. When results using the present invention are compared with those produced using conventionally prepared immuno-reagent, the increase in sensitivity offered by the present invention is dramatic (e.g., see fig 13, comparing lanes A and B with lanes C and D, the latter two lanes produced using the present invention as a substitute for conventionally prepared immuno-reagent). [150] Kits comprising the molecular conjugates ofthe present invention together with instructions for use are an additional embodiment ofthe present invention. Optionally, such kits may comprise additional reagents, such as buffers, coupling surfaces and label development and/or enhancement tools in addition to other reagents and consumables used to cany out the analysis for which the particular kit was developed. [151] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated as incorporated by reference. [152] Although the foregoing invention has been described in some detail by way of illustration and example for clarity and understanding, it will be readily apparent to one of ordinary skill in the art in light ofthe teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit and scope ofthe appended claims.

[153] The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of non-critical parameters that could be changed or modified to yield essentially similar results. EXAMPLES

Example 1: Construction of an immunoglobulin-based detection reagent for HIN-1 antibodies, and having enhanced signal amplification compared to conventional detection reagents

[154] This example describes how to construct a poly-label binding reagent having at least 10-20x the sensitivity of conventionally-labelled antibodies. Like conventional antibodies, the immunoaffinity binding reagent described herein may be used routinely in detection assays common in the art, such as blotting procedures and ELISA' s.

Materials

[155] Purified goat anti-human IgG (Fc-specific), Tween-20, orthophenyldiamine (OPD), 4- chloro-1-naphthol substrate, and Sephadex G-25 were obtained from Sigma Chemical Co. (St. Louis, MO). RecombinantHIN-1 gp41 was obtained from The Binding Sites, Inc. (San Diego, CA). Maleimide activated HRP, E-Z Link Maleimide activated peroxidase kit, with Ν-succinimidyl-S-acetylthioacetate (SATA), and FreeZyme conjugate purification kit is available from Pierce Chemical Co. (Rockford, IL). Immulon-1 microtiter plates were obtained from Dynex Technologies, Inc. (Chantilly, NA). Western blot kit was from Bio- Rad Laboratories, Redmond, WA. Amino acids with side chain protected groups were obtained from Νovabiochem (La Jolla, CA). All other reagents were of analytical grade.

Solid phase synthesis of HIV-1 gp41 peptide and lysine polypeptide chain

[156] An antigenic polypeptide from HIN gp41 conesponding to amino acids 585-607 of the transmembrane region having the amino acid sequence: (Seq. ID No: 1)

AVERYLKDQQLLGIWGCSGKLIC

[157] was synthesized on an ABI Model 430 peptide synthesizer using 9- fluorenylmethoxycarbonyl (Fmoc) chemistry (Atherton et al., In Solid Phase Peptide Synthesis-A Practical Approach; IRL Press at Oxford University Press: Oxford, U.K. (1989); Merrifield, Solid-phase peptide synthesis. In: Gutte B, ed. Peptides— Synthesis, structures and applications. San Diego, CA: Academic Press, pp. 93-169 (1995)).

[158] A polylysine peptide consisting of 20 lysine residues was synthesized on a Renin Symphony Quarted peptide synthesizer (Protein Technologies, Inc., Tuscan, AZ) using Fmoc chemistry mediated by 2-[l-H-Benzotriazole-l-yl]-1.13.3-tetramethyluronium hexafluorophosphate (HBTU) activation on the Rink Amide [4,2', 4' Dimethoxyphenyl- Fmoc-aminomethyl] phenoxyacetamido-norleucyl-MBHA resin (0.72 mmol/g resin substitution) (Novabiochem, La Jolla, CA). The N-terminal ofthe lysine polypeptide was bromoacetylated with a bromoacetyl group as previously described (Boykins et al., Peptides, 21:9-17 (2000)). Following RP-HPLC purification, identity ofthe peptide was confirmed by amino acid compositional analysis and plasma desorption mass spectroscopic analysis. All peptides were lyophilyzed and stored at -70°C until used.

SATA modification of IgG

[159] Briefly, 0.2 ml of 5 mg/ml goat anti-human IgG solution was mixed with 4 ul of 10 mg/ml SATA solution prepared in dimethyl formamide and incubated for 30 min at room temperature. The SATA-IgG solution was then deacetylated by adding 20 ul solution of freshly prepared hydroxylamine hydrochloride in conjugation buffer at a concentration of 5 mg/ml and incubating the reaction mixture at room temperature for 2 h. The deacetylated IgG derivative was separated from hydroxylamine and by-products with the desalting column (Pierce) pre-equilibrated with maleimide conjugation buffer and 0.2 ml fractions were collected. The fractions were monitored at 280 nm and protein-containing fractions were pooled. The concentration of IgG was determined by A₂₈o measurement.

Coupling of N-terminal bromoacetylated polylysine peptide and HRP conjugation to IgG

[160] One-half milligram of SATA-modified goat anti-human IgG was incubated with 5 mg of bromoacetylated polylysine (Methods for bromoacetylating polypeptides are known by those with skill in the art) in 0.25 ml of 10 mM bicarbonate buffer, pH 8 for 2 h under nitrogen. At the end of incubation, unbound bromoacetylated polypeptide was removed using a Sephadex G-25 column pre-equilibrated in bicarbonate buffer. Fractions containing immunoglobulin-polylysine complex were pooled and conjugated to HRP using EZ-Link plus activated peroxidase kit (Pierce) and purified using the Pierce FreeZyme kit. The poly-HRP- antibody conjugates were stored at -20°C after adding glycerol to a final concentration of 50%.

Determination of protein concentrations in immunoconjugates

[161] Total protein concentration in fractions containing poly-label binding reagent was measured using the Pierce BCA protein assay. An extinction coefficient for HRP of A o₃-2.1 (0.1% solution) was used to calculate the HRP concentration in the same solution. Concentration of IgG was determined by subtracting HRP concentration from the total protein concentration determined from the Pierce BCA protein assay.

Example 2: Detection of HIV antibodies by ELISA using a poly-label binding reagent

[162] Poly-label binding reagent synthesized according to the method described in example 1 was tested for efficiency of enzyme conjugation by reacting them with OPD substrate as described in example 2. To accurately determine the specific activity of poly-HRP-IgG conjugates, conventionally prepared HRP-antibody conjugates were first diluted to yield low OD values (less than 0.5) at 490 nm upon reaction with OPD substrate. Then poly-HRP- antibody conjugates were diluted to the same IgG concentration, mixed with OPD substrate, and the color intensity was determined spectrophotometrically at 490 nm. The poly-label binding reagent exhibited approximately 15 -fold increase in reactivity with OPD as compared to conventionally prepared IgG conjugates.

Example 3: Determining the level of signal amplification produced by the poly- label binding reagent

[163] The functional capacity of a poly-label binding reagent synthesized according to the protocol in example 1 was examined by testing the ability ofthe reagent to detect HIN-1 antibody by ELISA assay. The ELISA assay was carried out in a 96-well plate coated with rgp41 plus a gp41 synthetic peptide as described below. The analytical sensitivity ofthe assays was determined by measuring the signal generated by the reagent when contacted with a panel of HIN-1 antibody-positive control samples with low, medium, and high reactivity, prepared by spiking normal human plasma with known amounts of HIN antibody positive control plasma specimens. The same samples served as internal controls for samples containing unknown amounts of HIN-1 antigen.

[164] Briefly, ninety-six well Immunol-2 microtiter plates (Dynex Technologies, Inc., Chantilly, NA) were coated with recombinant HIN-1 gp41 protein plus synthetic HIN-1 gp41 peptide at a concentration of 1 μg/ml in 50 mM bicarbonate buffer, pH 9.6 at 4°C for 18 h. The plates were washed six times with PBS, pH 7.4 containing 0.5% Tween-20 (PBST) and blocked with SuperBlock solution (Pierce) for 2 h at 4°C followed by washing six times with PBST. A lOOμl aliquot of each antibody positive control sample was diluted 1:100, added to a microwell, and incubated at for 2 h 37°C. After washing the plates six times with PBST, 100 μl solution of HRP-conjugated goat anti-human IgG or poly-label binding reagent was added and incubated for an additional period of 1 h at 37°C. At the end of incubation, plates were washed with PBST and 100 μl of OPD substrate solution was added for color development at room temperature. The reaction was stopped by addition of a 100 μl solution of 2M sulfuric acid after 10 min of incubation and absorbance of developed color was determined spectrophotometrically at 490 nm on an ELISA reader (Molecular Devices, Sunnyvale, CA).

Example 4: Synthesizing a poly-label binding reagent using a microparticle core and a biotin/strepavidin coupling system.

Biotinylation of goat anti-human IgG [165] Goat anti-human IgG and polylysine were biotinylated using EZ-link sulfo-ΝHS-LC- biotin (Pierce). Briefly, protein solution (1 mg/ml) was prepared in 10 mM bicarbonate buffer, pH 9, and incubated with EZ-link sulfo-ΝHS-LC-biotin at room temperature for 1 h.

At the end of incubation, the reaction mixture was dialyzed against PBS, pH 7.2 at 4°C for 24 h with several changes of PBS. Biotinylated protein was stored at 4°C until used.

Conjugation of biotinylated or bromoacetylated lysine polypeptide with horseradish peroxidase (HRP) [166] Amino groups of biotinylated polylysine were covalently conjugated with HRP using

Pierce EZ-Link HRP conjugation kit (Pierce). Briefly, 100 ul of EZ-link HRP Plus Activated

Peroxidase solution in water was added to 0.5 ml of polylysine solution prepared in bicarbonate conjugation buffer (Pierce) at a concentration of 2 mg/ml and incubated for 1 h at room temperature. The reaction was stopped by addition of 20 ul of 3M ethanolamine, pH 9, quenching buffer and incubating for 15 min at room temperature. The Schiff base conjugate was reduced by addition of 10 ul of 5M NaCNBH₃ reductant solution after conjugation with microparticles as described below.

Preparation of antibody-HRP conjugate detector system [167] Goat anti-human IgG and HRP-conjugated biotinylated polylysine were coupled with non-magnetic 0.44 um streptavidin-polystyrene microparticles (Spherotech). Briefly, 10⁹ streptavidin-polystyrene microparticles were incubated with a mixture of biotinylated goat anti-human IgG (2 mg/ml) and HRP-conjugated biotinylated polylysine Schiff base (0.8 mg/ml) mixture in a 1 :5 ratio prepared in 1 ml of 10 mM bicarbonate buffer, pH 9, for 30 min at 37°C. At the end of incubation, conjugate microparticles were washed with PBS to remove unbound reactants. Similarly, 10⁹ N-succinimidyl-S-acetylthioacetate (SATA)-activated 0.29 um polystyrene microparticles (Spherotech) were incubated for 30 minutes at room temperature with a mixture of sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-l- carboxylate (sulfo-SMCC)-activated goat anti-human IgG (2 mg/ml) and maleimide-activated

HRP (0.8 mg/ml) and α amino-bromoacetylated polylysine polypeptide conjugated with HRP

(Schiff base; 0.8 mg/ml) as a ratio of 1 :5 prepared in 1 ml of 10 mM bicarbonate buffer, pH

9. The optimal concentrations of IgG and polylysine-HRP were determined by their binding to streptavidin and SATA-modified amine microparticles, respectively. At the end of incubation, the conjugated microparticles were washed to remove unbound reactants, resuspended in bicarbonate buffer containing 10 μl of 5M NaCNBH₃ reductant solution, and incubated for an additional 15 min at room temperature. The microparticle conjugates were washed with PBS and stored at 4°C until use.

Example 5: Synthesizing a poly-label binding reagent having a nucleic acid binding partner

[168] Amino microparticles are activated with SATA to introduce free sulfhydryl groups on their surface (Step (i) in Figure B). Oligonucleotide(s) is modified by aminolink phosphoramidite to introduce primary amine group at the 5' end. The amine-containing oligonucleotide is treated with a bifunctional cross-linker sulfo-SMCC to create sulfhydryl- reactive groups [Step (ii) in Figure B]. hi the next step, sulfo-SMCC-modified oligonucleotide is combined with bromoacetylated polylysine in 1:5 ratio and reacted with SATA-modified (SH-containing) microparticles to prepare oligonucleotide-polyamine microparticle conjugate, followed by labeling of oligonucleotide-polyamine microparticle conjugate with acridinium ester to generate oligonucleotide-polyacridinium microparticle conjugate [Step (iii) in Figure B]. Example 6: Assay for the detection of HIV-1 nucleic acids

[169] Nucleic acid detection reagent ofthe present invention used in this assay is shown in Figure 1 and is synthesized according to example 5. Briefly, viral RNA is released by addition of lysis buffer (Qiagen) containing RNAase inhibitors. The viral nucleic acid (before or after reverse transcription) is then hybridized with multiple HIN-1 specific oligonucleotide capturing probes having the sequence:

5' 3'

SEQ ID. NO. 2: HIN-1 envelope capturing probe attccatgtgtacattgtactgtgctgaca SEQ ID. NO. 3: HIN-1 gag capturing probe ctccctgacatgctgtcatcatttcttc SEQ ID. NO. 4: HIV-1 pol capturing probe gactacagtctacttgtccatgcatggcttc

[170] conesponding to envelope, gag, and pol regions of HIV-1, respectively, covalently conjugated to (5 μm) magnetic microparticles. The viral nucleic acid is also hybridized to capture oligonucleotides after PCR amplification. After washing, the nucleic acid-bound magnetic microparticles are hybridized with poly-label conjugates ofthe present invention synthesized according to example 5 and having the following oligonucleotides as binding partners:

5' 3'

SEQ ID. NO. 5: HIV-1 envelope capturing probe ctgccatttaacagcagttgagttgatac SEQ ID. NO. 6: HIV-1 gag capturing probe ttcgcattttggaccaacaagg SEQ ID. NO. 7: HIV-1 pol capturing probe ttccttctaaatgtgtacaatcta conesponding to envelope, gag, and pol regions ofthe HIV-1 virus, respectively. The microparticles used to construct the poly-label conjugates comprise a non-magnetic microparticle (0.29 μm) in conjunction with multiple units of acridinium label coupled to polylysine. After hybridization, the complex is washed, incubated with a signal inducer, and chemiluminescent signal emitted by the acridinium label is detected. SEQUENCE LISTING

SEQ ED. NO. 1: HEV-1 antigenic polypeptide AVERYLKDQQLLGIWGCSGKLIC

SEQ ED. NO. 2: HEV-1 envelope capturing probe attccatgtgtacattgtactgtgctgaca

SEQ ED. NO. 3: HEV-1 gag capturing probe ctccctgacatgctgtcatcatttcttc

SEQ ED. NO. 4: HEV-1 pol capturing probe gactacagtctacttgtccatgcatggcttc

SEQ ED. NO. 5: HEV-1 envelope ctgccatttaacagcagttgagttgatac

SEQ ED. NO. 6: HEV-1 gag capturing probe ttcgcattttggaccaacaagg

SEQ ED. NO. 7: HEV-1 pol capturing probe ttccttctaaatgtgtacaatcta

Claims

CLAIMSWhat is claimed is:

1. A molecular conjugate for signal amplification comprising: a) a binding partner coupled to an N-terminal amine of one or more polypeptides; and, b) a plurality of labels, each label coupled to an amino acid side chain of one ofthe polypeptides, wherein the binding of an analyte by one ofthe binding partners results in association ofthe analyte with the plurality of labels.

2. The molecular conjugate of claim 1, wherein the binding partner is covalently coupled by a chemical linker to the N-terminal α amine of each ofthe one or more polypeptides.

3. The molecular conjugate of claim 2, wherein the chemical linker has the structure:

where

A is the binding partner,

B is the polypeptide,

N' is a nitrogen of an ε amine of a lysine residue ofthe binding partner,

N" is a nitrogen ofthe N-terminal α amine ofthe polypeptide, and

R is H or a substituted or unsubstituted alkyl group.

4. The molecular conjugate of claim 2, wherein the chemical lin ker has the structure:

where

A is the binding partner, B is the polypeptide, S is a sulfur of a cysteine residue ofthe binding partner,

N is a nitrogen ofthe N-terminal α amine ofthe polypeptide, and

R is H or a substituted or unsubstituted alkyl group.

5. The molecular conjugate of claim 1 , wherein the binding partner is coupled by a chemical linker to the N-terminal α amine of each ofthe one or more polypeptides, the chemical linker having the structure:

where

A is the binding partner,

B is a multi-valent microparticle,

C is the polypeptide,

N' is a nitrogen of an ε amine of a lysine residue ofthe binding partner,

N" is a nitrogen ofthe N-terminal α amine ofthe polypeptide, and

R is H or a substituted or unsubstituted alkyl group.

6. The molecular conjugate of claim 1 , wherein the binding partner is coupled by a chemical linker to the N-terminal α amine of each ofthe one or more polypeptides, the chemical linker having the structure:

where

A is the binding partner,

B is a multi-valent microparticle,

C is the polypeptide,

N' is a nitrogen of an N-terminal amine ofthe binding partner,

N" is a nitrogen ofthe N-terminal α amine ofthe polypeptide, and R is H or a substituted or unsubstituted alkyl group.

7. The molecular conjugate of claim 1 , wherein the binding partner and the one or more polypeptides include a modification througli coupling to a biotin moiety, the one or more polypeptides being coupled to the biotin moiety at an N-terminal α amine of each polypeptide; and, further comprising a streptavidin-coated microparticle whereby contacting the binding partners and the polypeptides with the microparticle creates an affinity complex wherein the biotin label ofthe binding partners and the polypeptides binds to the streptavidin-coated microparticle, thereby coupling the binding partner to the one or more polypeptides.

8. The molecular conjugate of claim 1, wherein each polypeptide contains at least one lysine.

9. The molecular conjugate of claim 8, wherein one ofthe plurality of labels is coupled to a lysine of one polypeptide, the label being coupled to an ε amine ofthe lysine by a

R- ^^NH-R' chemical linkage having the structure: where

R is the lysine,

N is a nitrogen of an ε amine ofthe lysine, and

R' is the label.

10. The molecular conjugate of claim 1 , wherein the plurality of labels are an enzyme.

11. The molecular conjugate of claim 1 , wherein the binding partner is selected from the group consisting of proteins, nucleic acids, carbohydrates, glycoproteins, nucleoproteins, lipids, and lipoproteins.

12. The molecular conjugate of claim 8, wherein the polypeptide is polylysine.

13. A molecular conjugate for signal amplification comprising: a) one or more binding partners, b) one or more labels having a primary amine, and c) a multivalent microparticle wherein the binding partners and the labels are coupled to the microparticle.

14. The molecular conjugate of claim 13, wherein each valency ofthe microparticle comprises an amine.

15. The molecular conjugate of claim 14, wherein the one or more labels are covalently

coupled to the microparticle through a chemical linkage having the structure; where

A is the label,

B is the microparticle,

N' is a nitrogen ofthe amine ofthe label, and

N" is a nitrogen ofthe amine ofthe microparticle.

16. The molecular conjugate of claim 14, wherein the one or more binding partners are covalently coupled to the microparticle through a chemical linkage having the structure;

where

A is the binding partner,

B is the microparticle,

N' is a nitrogen of an amine ofthe binding partner, and

N" is a nitrogen ofthe amine ofthe microparticle.

17. The molecular conjugate of claim 13, wherein the one or more binding partners are covalently coupled to the microparticle through a chemical linkage having the structure;

where A is the binding partner,

B is the microparticle,

N' is a nitrogen of an N-terminal α amine ofthe binding partner, and

N" is a nitrogen ofthe amine ofthe microparticle.

18. A method for producing an amplified signal in response to the presence of an analyte, the method comprising: a) obtaining a sample including the analyte; b) immobilizing the analyte to a coupling surface; c) contacting the analyte with the molecular conjugate of claim 1, wherein at least one binding partner specifically recognizes the analyte; and, d) detecting the label ofthe molecular conjugate wherein the presence ofthe analyte is indicated by a plurality of unit labels for each unit of analyte.

19. The method of claim 18, wherein the analyte is a protein and the coupling surface is nitrocellulose.

20. The method of claim 18, wherein the analyte is a protein and the coupling surface is a plastic.

21. The method of claim 20, wherein the analyte is an immunocomplex and the coupling surface is a plastic.

22. The method of claim 18, wherein the analyte is a nucleic acid and the coupling surface is nylon.

23. The method of claim 18, wherein the analyte is an antigen and the coupling surface is an antibody-coated plastic.

24. A kit for detection an analyte by producing an amplified signal, the kit comprising the molecular conjugate of claim 1 wherein at least one binding partner specifically recognizes the analyte, and directions for using the molecular conjugate.

25. A molecular conjugate for signal amplification comprising: a) one or more binding partners, b) one or more nucleic acids having a ribosyl residue with an α carbon at the 5' end, each nucleic acid covalently bound at nucleic acid base residues to a plurality of labels; and, c) a multivalent microparticle wherein the one or more binding partners and the one or more nucleic acids are coupled to the microparticle through a chemical linkage, the chemical linkage between the nucleic acid and the microparticle having the structure:

where

A is the α carbon ofthe ribosyl residue at the 5' end ofthe nucleic acid,

N' is a nitrogen of an amine on the surface ofthe microparticle; and,

B is the microparticle.