WO2014031984A1

WO2014031984A1 - Immunological detection methods and compositions

Info

Publication number: WO2014031984A1
Application number: PCT/US2013/056434
Authority: WO
Inventors: Gerard Zurawski; Sandra Zurawski; Patrick LECINE
Original assignee: Baylor Research Institute
Priority date: 2012-08-24
Filing date: 2013-08-23
Publication date: 2014-02-27

Abstract

A flexible method for using bioluminescence to enhance the detection and sensitivity of immunoassays in the detection of any analyte. The method of the present invention comprises the use of antibodies attached to luciferase to enhance detection and sensitivity of immunoassays. Detection can be enhanced by use of methods that comprise incubation of a substrate or a set of beads coated with a dockerin or a cohesin protein with a cohesin or dockerin-antigen fusion proteins to attach the fusion protein essentially irreversibly by non- covalent interaction in addition to antibodies attached to luciferase.

Description

DESCRIPTION

IMMUNOLOGICAL DETECTION METHODS AND COMPOSITIONS

BACKGROUND OF THE INVENTION

[0001] The application claims priority to U.S. Provisional Patent Application 61/693,199 filed on August 24, 2012, which is hereby incorporated by reference.

[0002] The invention was made with government support under Grant No. 1U19AI057234-010000 awarded by the National Institutes of Health. The government has certain rights in the invention.

1. Field of the Invention

[0003] The present invention relates in general to the field of novel tools for molecular analysis, and more particularly, to the use of luciferase to improve and enhance the detection of any analyte for which a binding polypeptide is available and further enhance detection by use of cohesin and dockerin binding proteins.

2. Description of Related Art [0004] Affinity chromatography is one separation technique for isolating biologically active compounds. The separation is determined by a variety of factors including the binding constant, selectivity of the column, good column retention, the capacity of the column, and elution conditions. For example, a high affinity between the ligand and the biomolecule may require harsh elution conditions (such as low/high pH or very high salt concentrations), which may lead to unfolding or denaturation in the case of a recombinant protein, whereas an overly weak affinity may be insufficient for efficient retention on the column. For example, micro beads in distinct sets are coated with a reagent specific to a particular bioassay, allowing the capture and detection of specific analytes from a sample. The beads are directly chemically coupled to a micro bead and new antigens must be directly chemically coupled to new beads. [0005] For example, U.S. Patent Application Publication No. 2010/0062451, entitled

"Bead-Ligand-Nascent Protein Complexes" discloses bead-ligand-nascentprotein complexes, and method of creating and detecting a bead-ligand-nascent protein complexes, are described. PCR-amplified product which is attached to a surface, e.g. of a bead, is used to generate nascent protein, which in turn is captured on the bead and detected, e.g. by fluorescence. [0006] U.S. Patent Application Publication No. 2005/0106700 discloses use of C- terminal and N-terminal dockerin fusions in purification of target proteins on affinity columns.

[0007] International Patent Application No. WO 2009/028532 discloses purification systems and methods using a dockerin polypeptide characterized in that the amino acid at the 14-position in the second sub-domain of a dockerin originating from Clostridium josui is substituted by another amino acid.

[0008] U.S. Patent Application Publication No. 2011/0151538, entitled "Affinity Purification by Cohesin-Dockerin Interaction" discloses truncated dockerin polypeptides, recombinant polypeptides and affinity systems having the truncated dockerin polypeptide, methods of generating same, and methods of use thereof to purify, isolate, and detect molecules of interest, where the solid substrate is cellulose with carbohydrate -binding module (CBM) and the protein bound. The entire contents of this application are incorporated herein by reference. [0009] However, additional and improved methods and compositions are needed to detect a variety of analytes.

SUMMARY OF THE INVENTION

[0010] The present invention relates to using luciferase to improve and enhance the detection of any analyte. Accordingly, there are methods and compositions involving a luciferase immunoassay to provide enhanced sensitivity in the specific detection of any analyte.

[0011] In some embodiments, there are methods of detecting an analyte in a sample, comprising: (a) forming a first analyte complex comprising a Dockerin/Cohesin binding pair, (b) contacting the first analyte complex with an analyte-binding antibody, wherein the analyte-binding antibody is attached to a luciferase reporter, thereby forming a second complex; and (c) testing the second complex for luciferase activity, thereby detecting the presence of the analyte. In certain embodiments, the first analyte complex is bound to a solid support. In further embodiments, the Dockerin/Cohesin binding pair comprises Dockerin attached to a matrix binding domain and Cohesin attached to an analyte-binding polypeptide; alternatively, the Dockerin/Cohesin binding pair comprises Cohesin is attached to a matrix binding domain and Dockerin is attached to an analyte-binding polypeptide.

[0012] In some embodiments, there are methods of detecting an analyte in a sample, comprising : (a) contacting the sample with a solid matrix, wherein the matrix is bound to a Dockerin/Cohesin binding pair, and wherein the Dockerin is attached to a matrix binding domain and Cohesin is attached to an analyte-binding polypeptide; or wherein the Cohesin is attached to a matrix binding domain and Dockerin is attached to an analyte-binding polypeptide; thereby forming a first analyte complex; (b) contacting said first analyte -bound complex with an analyte-binding antibody, wherein the analyte-binding antibody is attached to a luciferase reporter, thereby forming a second complex; and (c) testing the second complex for luciferase activity, thereby detecting the presence of the analyte.

[0013] In some embodiments, methods include contacting of steps (a) and (b) that are performed in one step.

[0014] Further embodiments concern methods of detecting an analyte in a sample, comprising:

(a) contacting the sample with either: (i) a Cohesin fused to an analyte- binding polypeptide; or (ii) a Dockerin fused to an analyte-binding polypeptide, thereby forming a first analyte-bound complex;

(b) contacting the first analyte-bound complex with a solid matrix is bound by a matrix binding domain fused to the other member of the Cohesin/Dockerin binding pair to form an immobilized analyte-bound complex;

(c) contacting said immobilized analyte-bound complex with an analyte- binding antibody, wherein the analyte-binding antibody is fused to a luciferase reporter, thereby forming a further complex; and

(d) testing the second complex for luciferase activity, thereby detecting the presence of the analyte.

[0015] Analytes may include pathogen-specific antibodies present in human sera from infected individuals, environmental pathogens, circulating blood cell types or any analyte for which a binding polypeptide may have a bioluminescent protein attached. For example, an antibody fused to a luciferase protein (i.e. Renilla, Gaussia or Photinus) that recognizes a species specific IgG (or IgM) can be used in a diagnostic test to detect an analyte that includes, but is not limited to, a pathogen or infection (BioLuminescent Immuno Assay, BLIA).

[0016] Additionally, in some embodiments, methods involve a solid matrix support to enable purification of analyte/binding polypeptide complexes and enhance signal to noise ratio and signal fidelity of the detection assay. Solid matrix supports comprise any solid bead technologies, for example, cellulose beads. To this end, embodiments may take advantage of the properties of cohesin and dockerin and the essentially irreversible binding between cohesin and dockerin proteins to add the flexibility to the beads. In some embodiments, all beads in the set may be coated with a dockerin or cohesin domain containing protein. Then each bead set is incubated with cohesin or dockerin-antigen fusion proteins, which attach essentially irreversibly by non-covalent interaction. Thus, one dockerin or cohesin coated bead set can be used for multiple sets of cohesin or dockerin-antigens, obviating the need to directly chemically couple new antigens to new beads. [0017] The present invention also discloses a flexible method for making multiplexed assays for antigen-specific antibody responses by the use of one dockerin coated bead for multiple sets of cohesin-antigens, obviating the need to directly chemically couple new antigens to new beads and also incorporating bioluminescent detection methods. Thus, the present invention provides a very flexible method, and compositions for use in the same, for making multiplexed assays for antigen-specific antibody responses using bioluminescence as a signal readout.

[0018] In some embodiments, an analyte may be detected by contacting the sample suspected of containing the analyte with a solid matrix support, dockerin attached to a matrix binding domain and cohesin attached to a polypeptide capable of binding the analyte to be detected. The polypeptide capable of binding the analyte is attached to a luciferase reporter, forming a complex that can be detected via methods that can report luciferase activity. The solid matrix support may comprise plastic, cellulose, dextran, dextran cross linked with epichlorohydrin, agarose, acrylamide, glass, polystyrene, polyethylene glycol, Teflon, or nylon. The solid support may be in the form of beads, microspheres, or any support shape that is amenable to methods of separation such as centrifugation or filtration. [0019] In yet other embodiments, steps necessary to form the above mentioned complex can be carried out sequentially or can be carried out in a single step in a single vessel without separation.

[0020] In other embodiments, the analyte/solid matrix/cohesin/dockerin/ polypeptide/lucifease complex may be purified from the sample by centrifugation, ultracentrifugation, density grade centrifugation, size exclusion column chromatography or other methods of column chromatography capable of separating high molecular weight complexes.

[0021] In certain embodiments, the analyte may be an antibody and the analyte - binding polypepdtide may be an antigen recognized by the antibody analyte. In certain aspects the antigen may be attached to luciferase or may be recognized by a luciferase attached antibody.

[0022] In further embodiments the analyte may be a human antibody; in certain embodiments the analyte antibody may belong to the immunoglobulin group A, D, E, G or M. The antibody may be a non-human antibody in other embodiments. In addition, the antibody may be a chimeric antibody.

[0023] In still other embodiments the analyte may be a pathogen that may be selected from the list of, but is not restricted to, HIV, Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium knowlesi, dengue fever virus, Schistosomiasis mansoni, Schistosomiasis japonicum, Schistosomiasis haematobium, Streptococcus pneumonia, Trypanosoma cruzi, Mycobacterium Leprae, Vibrio cholerae, Bordetella pertussis, Shigella dysenteriae, Escherichia coli or Mycobacterium ulcerans. The analyte may comprise any surface molecule or complex that can be detected by antibodies, polypeptides or molecules capable of binding the a surface molecule or complex. [0024] In other embodiments the pathogen may comprise any genus of the following :

Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium, Enterococcus, Escherichia, Francisella, Haemophilus, Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema, Vibrio or Yersinia. [0025] In certain aspects, the environmental pathogen may be detected by binding of an antibody or binding polypeptide to a pathogen surface antigen. The surface antigen may be a protein, glycoprotein, polysaccharide, lipopolysaccharide or lipid. The surface antigen may belong to, but is not limited to, a component of the pathogen's coat, capsule, cell wall, flagella, fimbrae, toxin, pili, cytoplasmic membrane, outer membrane, peptidoglcan layer, periplasmic space, S-layer, capsid, protein coat, or envelope.

[0026] In certain embodiments the bioluminescent reporter may be a luciferase reporter. The luciferase reporter may comprise a Renilla, Gaussia, Photinus, or Cypridina luciferase. In other embodiments the luciferase may comprise a luciferase from one of the following organisms: Photinus pyralis, Luciola cruciata, Luciola italica, Luciola lateralis, Luciola mingrelica, Photuris pennsylvanica, Pyrophorus plagiophthalamus, Phrixothrix hirtus, Renilla reniformis, Gaussia princeps, Metridia longa or Oplophorus gracilorostris. In other embodiments the luciferase may be North American firefly luciferase, Japanese firefly (Genji-botaru) luciferase, Italian firefly Luciferase, Japanese firefly (Heike) luciferase, East European firefly luciferase, Pennsylvania firefly luciferase, Click beetle luciferase, Railroad worm luciferase, Renilla luciferase, Rluc8 (mutant of Renilla luciferase), Green Renilla luciferase, Gaussia luciferase, Gaussia-Dura luciferase, Cypridina luciferase, Cypridina (Vargula) luciferase, Metridia luciferase, Oplophorus luciferase (OLuc) or a bacterial luciferase. Substrates used in certain embodiments may include Luciferin, Coelenterazine, Vargulin or any compounds based on these substrates.

[0027] In some embodiments the luciferase may be a variant or derivative of a naturally occurring luciferase. For example, in some embodiments, there are reactions and assays involving modified, non-secreted forms of luciferases that are secreted in their native form. By way of example, a naturally secreted luciferase may be modified using standard molecular biological techniques by removal of signal sequences and/or fusion to an intracellular polypeptide such that the enzyme is no longer secreted but remains intracellular. Alternatively, or additionly, a number of other modifications well known to those in the art may be made, for example, to alter one or more amino acids in the luciferase polypeptide sequence to modulate expression and/or solubility of the enzyme in a cell culture system of choice. Such modulation may be an increase or decrease in expression and/or solubility, depending on the requirements of the particular application in which the luciferase, and the reagent compositions of the invention are to be employed. For example, it may be desirable to modify the luciferase by the introduction of one or more destabilising elements to destabilise the protein. Luciferases containing destabilising elements have shortened half- lives and are expressed at lower steady-state levels than luciferases which do not contain such elements. Suitable protein destabilising elements include PEST sequences (amino acid sequences enriched with the amino acids proline (P), glutamic acid (E), serine (S) and threonine (T)), a sequence encoding an intracellular protein degradation signal or degron, and ubiquitin. The enhanced sensitivity attained with reagent compositions of the present invention are particularly advantageous for use with destabilized luciferases given the lower steady state expression levels of such luciferases. Any suitable method for destabilising a protein is contemplated herein. Suitable methods are described, for example, in co-pending U.S. Patent Application Ser. No. 10/658,093 (the disclosure of which is incorporated herein by reference in its entirety). Expression of the luciferase may also be modified by, for example, the addition of sequences such as poly(A) tails, transcriptional or translational enhancers, and/or adapting codon usage in the encoding polynucleotide sequence for a particular expression system. For example, to optimise expression of the luciferase in insect cells or human cells, codon usage in the luciferase polynucleotide may be optimised for insect or human cells respectively. Approaches for codon usage adaptation and optimisation for different species are well known to those skilled in the art.

[0028] In other embodiments, an antigen binding polypeptides or antibodies may be attached to a luciferase enzyme at the antibody's light chain, heavy chain or both light chain and heavy chains. Attachment may be performed recombinantly, wherein the antibody is expressed either by hybridomas or heterologously and the luciferase coding sequence is fused to the antibody chain (light chain or heavy chain) coding sequence; generated antibodies would therefore be attached to luciferase. Luciferase and antibodies or binding polypeptides may also be attached by chemical means. In some embodiments, chemical coupling may be achieved through available primary amines. In certain other embodiments, click chemistry may be employed. In further embodiments, an antibody, Cohesin, Dockerin, antigen, or luciferase may be connected via a linker, which may be an amino acid linker or other chemical linker. [0029] In further embodiments the analyte binding polypeptide may be fused to one or more dockerin domains, either recombinantly or through chemical conjugation methods. Linkage of dockerin domains may be linear or branched. Luciferase reporter molecules attached to cohesin would be capable of attaching to dockerin domains and amplify the signal represented by the binding of a single polypedtide to an analyte.

[0030] In some embodiments the detection of human IgG can be compared between anti-human IgG horseradish peroxidase conjugated antibodies and anti-human IgG attached luciferase antibodies by plate well assays. In an example of a plate well assay, human IgG from serum can be linked to plate well by incubation of serum with acetate buffer. After fmalization of IgG linkage to plate wells by washing, anti-human IgG antibodies possessing either HRP or luciferase can be tested by exposure of antibodies to the wells and performing a colorimetric assay in the case of HRP or luminescence read in the case of luciferase. [0031] In yet other embodiments specific tuberculosis antigens may be detected via bioluminescent detection methods using a bioluminescent plate assay. In certain embodiments cellulose binding domain attached to dockerin may be used to coat the bioluminescent assay plate. Specific cohesin - tuberculosis antigen fusions include, but are not limited to, CFP10/Rv3874, LprG/Rvl41, Ag85B/Rvl886, HspX/Rv2031, ESAT6/Rv3875, ModD/Rvl860, Rv3878, GlcB/Rvl837, MPT51/Rv3803, Mtb48/Rv3881, 38 kDa/Rv0934, Rv2873,Rv0831, or Rv2658, which can be added to the coated wells. Sera from tuberculosis patients or control patients can be exposed to wells prepared with antigen complexes and subsequently queried with anti-human IgG luciferase antibodies. In other embodiments, cellulose beads may be used instead of plates to provide a solid matrix support that is amenable to purification or separation methods.

[0032] In additional embodiments CD4+ T cells can be detected and quantified using a bioluminescent readout. Anti-CD3 monoclonal antibody, such as can be derived from hybridoma OKT3 can be coated onto plates. Blood cells derived from circulation can be exposed to anti-CD3 prepared plates and CD4 detected with biotin labeled anti-CD4 antibody. The presence of the CD4 antigen can be detected with anti-biotin or strepavidin luciferase-attached binding polypeptides.

[0033] In one embodiment, the present invention comprises a method for detecting, isolating, or purifying one or more analytes in a sample, in a matrix, from a mixture, or any combinations thereof comprising: obtaining a solid substrate comprising a first member of a cohesin-dockerin binding pair, wherein the first member is attached to a protein, an antibody, an antigen, a peptide, a toxin, a cytokine, an enzyme, a structural protein, an extracellular matrix protein, a cell, or fragments thereof, wherein the solid substrate is selected from the group consisting of a bead, a cell, an extracellular matrix, a fibrous matrix, a container, an affinity column, or any combinations thereof; providing a second member of the cohesin- dockerin binding pair, wherein the second member is present in the sample, the matrix, the mixture or any combinations thereof, wherein the second member is capable of binding to one or more analytes to be detected, isolated, or purified from the sample, the matrix, the mixture or any combinations thereof; contacting the second member of the cohesin-dockerin binding pair with the sample, the matrix or the mixture suspected of having the analyte; and forming a complex on the substrate comprising the first and the second members of the cohesin-dockerin binding pairs and the analyte, wherein the presence of the analyte is detected. In one aspect, the method further comprises adding a detection reagent to the complex for determining presence or absence of the analyte, wherein the detection reagent comprises a secondary antibody, a radiolabel, a fiurophore, a colorimetric reagent, or any combinations thereof. In another aspect, the analyte comprises a nucleic acid (natural and unnatural, aptamers), carbohydrates, polysaccharides, peptides, proteins or peptoids (natural and unnatural), minerals, vitamins and lipids. In another aspect, the first member comprises a dockerin domain and the second member comprises a cohesin domain bound to the analyte. In another aspect, the first member comprises a cohesin domain and the second member comprises a dockerin domain bound to the analyte. In another aspect, the second member comprises a cohesin or a dockerin domain and the analyte forms a fusion protein with the cohesin or dockerin domain. In another aspect, the method is carried out in a container that comprises a beaker, a flask, a cylinder, a test tube, a centrifugation tube, a petri dish, a culture dish, or a multi-well plate. In another aspect, the substrate comprises one or more sets of beads, wherein the beads comprise nanospheres, nanoparticles, microspheres, microparticles, nanobeads, microbeads, beads, polystyrene beads, latex particles, latex beads, fluorescent beads, fluorescent particles, colored particles, or colored beads. In another aspect, the one or more sets of beads comprise polymeric beads, wherein the polymers are selected from the group consisting of carbohydrate -based polymers, polyaliphatic alcohols, poly(vinyl) polymers, polyacrylic acids, polyorganic acids, polyamino acids, co-polymers, block co- polymers, tertpolymers, polyethers, naturally occurring polymers, polyimids, surfactants, polyesters, branched polymers, cyclo-polymers, polyaldehydes and mixtures thereof, brominated polystyrene, polyacrylic acid, polyacrylonitrile, polyamide, polyacrylamide, polyacrolein, polybutadiene, polycaprolactone, polyester, polyethylene, polyethylene terephthalate, polydimethylsiloxane, polyisoprene, polyurethane, polyvinylacetate, polyvinylchloride, polyvinylpyridine, polyvinylbenzylchloride, polyvinyltoluene, polyvinylidene chloride, polydivinylbenzene, polymethylmethacrylate, polylactide, polyglycolide, poly(lactide-co-glycolide), polyanhydride, polyorthoester, polyphosphazene, polyphosophaze,poly-(styrene-co-vinylbenzyl chloride-co-acrylic acid) (85: 10:5 molar ratio), poly(styrene-co-acrylic acid) (99: 1 molar ratio), poly(styrene-co-methacrylic acid) (90: 10 molar ratio), poly(styrene-co-acrylic acid-co-m&p-divinylbenzene) (89: 10: 1 molar ratio), poly-(styrene-co-2-carboxy ethyl acrylate) (90: 10 molar ratio), poly(methyl methacrylate-co- acrylic acid) (70:30 molar ratio) and poly(styrene-co-butyl acrylate-co-methacrylic acid)(45:45:10 weight ratio) synthetic polymers polystyrene, polyacrylamide, polyacrylate, latex, and any combinations or modifications thereof. In another aspect, the one or more analytes comprise antigens, antibodies, autoantibodies, peptides, proteins, nucleic acid sequences, and/or enzymes, wherein the antigens comprise one or more bacterial, viral, fungal, mycoplasmal, rickettsial, chlamydial, or protozoal antigens. In another aspect, the dockerin may be a substituted dockerin, a truncated dockerin, a modified dockerin, or any combinations thereof. In another aspect, the cohesin may be a substituted cohesin, a truncated cohesin, a modified cohesin, or any combinations thereof. In another aspect, the cohesin is derived or isolated from Clostridium thermocellum, C. cellulolyticum, C. cellulovorans, C. papyrosolvens, and Clostridium cellobioparum, C. papyrosolvens, C. josui, Acetivibrio cellulolyticus, Bacteroides cellulosolvens, R. flavefaciens, Ruminococcus albus, Archaeoglobus fulgidus protein, cellulosomal cohesin domain.

[0034] In one embodiment, the present invention comprises a multiplex bead based method for detecting, isolating, or purifying one or more analytes in a sample, in a matrix, from a mixture, or any combinations thereof comprising: providing one, a plurality, or a set of beads comprising nanospheres, nanoparticles, microspheres, microparticles, nanobeads, microbeads, beads, polystyrene beads, latex particles, latex beads, fluorescent beads, fluorescent particles, colored particles, or colored beads; attaching a first member of a cohesin-dockerin binding pair to the beads, wherein the first member is attached to a protein, an antibody, an antigen, a peptide, a toxin, a cytokine, an enzyme, a structural protein, an extracellular matrix protein, a cell, or fragments thereof; providing at least one second member of the cohesin-dockerin binding pair attached to the second member is attached to the one or more analytes to be detected, isolated, or purified from the sample, the matrix, the mixture or any combinations thereof; contacting the first member of the cohesin-dockerin binding pair with the sample, the matrix or the mixture comprising the second cohesin- dockerin binding pair; and forming a complex comprising at least one second member of the cohesin-dockerin binding pair bound irreversibly in a non-covalent manner to the at least one first member of the cohesin-dockerin binding pair. In one aspect, the method further comprises adding a detection reagent to the complex for determining presence or absence of the analyte, wherein the detection reagent comprises a secondary antibody, a radiolabel, a flurophore, a colorimetric reagent, or any combinations thereof; releasing the second member comprising the analyte from the complex by one or more physical or chemical methods; and isolating the analyte from a mixture comprising cohesin, dockerin, proteins, antigens, peptides, antibodies, or any combinations thereof. In another aspect, the analyte comprises a nucleic acid (natural and unnatural, aptamers), carbohydrates, polysaccharides, peptides, proteins or peptoids (natural and unnatural), minerals, vitamins and lipids. In another aspect, the first member comprises a dockerin domain and the second member comprises a cohesin domain bound to the analyte. In another aspect, the first member comprises a cohesin domain and the second member comprises a dockerin domain bound to the analyte. In another aspect, the second member comprises a cohesin or a dockerin domain and the analyte forms a fusion protein with the cohesin or dockerin domain. In another aspect, the one or more sets of beads comprise polymeric beads, wherein the polymers are selected from the group consisting of carbohydrate -based polymers, polyaliphatic alcohols, poly(vinyl) polymers, polyacrylic acids, polyorganic acids, polyamino acids, co-polymers, block co- polymers, tertpolymers, polyethers, naturally occurring polymers, polyimids, surfactants, polyesters, branched polymers, cyclo-polymers, polyaldehydes and mixtures thereof, brominated polystyrene, polyacrylic acid, polyacrylonitrile, polyamide, polyacrylamide, polyacrolein, polybutadiene, polycaprolactone, polyester, polyethylene, polyethylene terephthalate, polydimethylsiloxane, polyisoprene, polyurethane, polyvinylacetate, polyvinylchloride, polyvinylpyridine, polyvinylbenzylchloride, polyvinyltoluene, polyvinylidene chloride, polydivinylbenzene, polymethylmethacrylate, polylactide, polyglycolide, poly(lactide-co-glycolide), polyanhydride, polyorthoester, polyphosphazene, polyphosophaze,poly-(styrene-co-vinylbenzyl chloride-co-acrylic acid) (85: 10:5 molar ratio), poly(styrene-co-acrylic acid) (99: 1 molar ratio), poly(styrene-co-methacrylic acid) (90: 10 molar ratio), poly(styrene-co-acrylic acid-co-m&p-divinylbenzene) (89: 10: 1 molar ratio), poly-(styrene-co-2-carboxy ethyl acrylate) (90: 10 molar ratio), poly(methyl methacrylate-co- acrylic acid) (70:30 molar ratio) and poly(styrene-co-butyl acrylate-co-methacrylic acid)(45:45:10 weight ratio) synthetic polymers polystyrene, polyacrylamide, polyacrylate, latex, or any combinations or modifications thereof. In another aspect, the one or more analytes comprise antigens, antibodies, autoantibodies, peptides, proteins, nucleic acid sequences, and/or enzymes, wherein the antigens comprise one or more bacterial, viral, fungal, mycoplasmal, rickettsial, chlamydial, and/or protozoal antigens. In another aspect, the dockerin is selected from a Domain I dockerin, a Domain II dockerin, a Domain III dockerin, a substituted dockerin, a truncated dockerin, a modified dockerin, or any combinations thereof. In another aspect, the cohesin may be a Type I cohesin, a Type II cohesin, a Type III cohesin, a substituted cohesin, a truncated cohesin, a modified cohesin, or any combinations thereof.

[0035] Another embodiment of the invention is an assay system comprising: a substrate and at least one attached or immobilized dockerin or cohesin binding domain bound to the substrate, and a dockerin or cohesin binding pair is attached to a protein, an antibody, an antigen, a peptide, a toxin, a cytokine, an enzyme, a structural protein, an extracellular matrix protein, a cell, or fragments thereof. In one aspect, the substrate comprises one or more beads. In another aspect, the substrate comprises nanospheres, nanoparticles, microspheres, microparticles, nanobeads, microbeads, beads, polystyrene beads, latex particles, latex beads, fluorescent beads, fluorescent particles, colored particles, and colored beads. In another aspect, the analyte comprises a nucleic acid (natural and unnatural, aptamers), carbohydrates, polysaccharides, peptides, proteins or peptoids (natural and unnatural), minerals, vitamins and lipids. In another aspect, the first member comprises a dockerin domain and the second member comprises a cohesin domain bound to the analyte. In another aspect, the first member comprises a cohesin domain and the second member comprises a dockerin domain bound to the analyte. In another aspect, the second member comprises a cohesin or a dockerin domain and the analyte forms a fusion protein with the cohesin or dockerin domain. [0036] In one embodiment, the present invention comprises a bead based assay system comprising one or more or a set of beads at least one attached or immobilized dockerin or cohesin binding domain, wherein the dockerin or cohesin binding domain may be attached to a protein, an antibody, an antigen, a peptide, a toxin, a cytokine, an enzyme, a structural protein, an extracellular matrix protein, a cell, or fragments thereof. In one aspect, the one or more beads comprise nanospheres, nanoparticles, microspheres, microparticles, nanobeads, microbeads, beads, polystyrene beads, latex particles, latex beads, fluorescent beads, fluorescent particles, colored particles, or colored beads. In another aspect, the analyte comprises a nucleic acid (natural and unnatural, aptamers), carbohydrates, polysaccharides, peptides, proteins or peptoids (natural and unnatural), minerals, vitamins or lipids. In another aspect, the first member comprises a dockerin domain and the second member comprises a cohesin domain bound to the analyte. In another aspect, the first member comprises a cohesin domain and the second member comprises a dockerin domain bound to the analyte. In another aspect, the second member comprises a cohesin or a dockerin domain and the analyte forms a fusion protein with the cohesin or dockerin domain.

[0037] In one embodiment, the present invention comprises a method for performing an immunoassay on a bead surface for the simultaneous detection of more than one analyte from a sample, a matrix, or a mixture comprising the steps of: providing one or more beads or sets of beads on a substrate, wherein the substrate is selected from the group consisting of a beaker, a flask, a cylinder, a test tube, a centrifugation tube, a petri dish, a culture dish, and a multi-well plate, or any combinations or modifications thereof; attaching or immobilizing at least two dockerin binding domain to a surface of the beads, wherein the dockerin binding domain may be attached to a protein, an antibody, an antigen, a peptide, a toxin, a cytokine, an enzyme, a structural protein, an extracellular matrix protein, a cell, or fragments thereof; contacting the beads with the attached or immobilized dockerin binding domains with the sample, the matrix or the mixture comprising the multiple analytes of interest, wherein the multiple analytes are attached to one or more cohesin fusion proteins; forming more than one complexes comprising the multiple cohesin fusion protein attached analyte with the multiple dockerin binding domains on the surface of the one or more beads; and adding multiple detecting reagents or labels to the beads, wherein each of the detecting regent or label is specific and binds to the multiple analytes suspected of being present in the sample, matrix, or the mixture, and the detection reagents comprise a secondary antibody, a radiolabel, a fluorophore, a colorimetric reagent, or any combinations thereof; and detecting the presence or absence of the multiple analytes be reading, monitoring, or measuring a signal emitted by the bound detection reagent or label and the analyte. In one aspect, the method further comprises the optional steps of: performing one or more wash steps with a suitable buffer or water at one or in between different steps of the immunoassay; generating a calibration curve or a standard curve for determination of a concentration or an amount of the multiple analytes in the sample, the matrix, or the mixture by performing the immunoassay using one or more pure analytes or standards; releasing the cohesin fusion protein attached to the analyte from the complex by one or more physical or chemical methods; and isolating the analyte from a mixture comprising cohesin, dockerin, proteins, antigens, peptides, antibodies, or any combinations thereof. In one aspect, the substrate comprises one or more sets of beads, wherein the beads comprise nanospheres, nanoparticles, microspheres, microparticles, nanobeads, microbeads, beads, polystyrene beads, latex particles, latex beads, fluorescent beads, fluorescent particles, colored particles, or colored beads. In another aspect, the one or more sets of beads comprise polymeric beads, wherein the polymers are selected from the group consisting of carbohydrate -based polymers, polyaliphatic alcohols, poly(vinyl) polymers, polyacrylic acids, polyorganic acids, polyamino acids, co-polymers, block copolymers, tertpolymers, polyethers, naturally occurring polymers, polyimids, surfactants, polyesters, branched polymers, cyclo-polymers, polyaldehydes and mixtures thereof, brominated polystyrene, polyacrylic acid, polyacrylonitrile, polyamide, polyacrylamide, polyacrolein, polybutadiene, polycaprolactone, polyester, polyethylene, polyethylene terephthalate, polydimethylsiloxane, polyisoprene, polyurethane, polyvinylacetate, polyvinylchloride, polyvinylpyridine, polyvinylbenzylchloride, polyvinyltoluene, polyvinylidene chloride, polydivinylbenzene, polymethylmethacrylate, polylactide, polyglycolide, poly(lactide-co-glycolide), polyanhydride, polyorthoester, polyphosphazene, polyphosophaze,poly-(styrene-co-vinylbenzyl chloride-co-acrylic acid) (85: 10:5 molar ratio), poly(styrene-co-acrylic acid) (99: 1 molar ratio), poly(styrene-co-methacrylic acid) (90: 10 molar ratio), poly(styrene-co-acrylic acid-co-m&p-divinylbenzene) (89: 10: 1 molar ratio), poly-(styrene-co-2-carboxy ethyl acrylate) (90: 10 molar ratio), poly(methyl methacrylate-co- acrylic acid) (70:30 molar ratio) and poly(styrene-co-butyl acrylate-co-methacrylic acid)(45:45:10 weight ratio) synthetic polymers polystyrene, polyacrylamide, polyacrylate, latex, and any combinations or modifications thereof. In another aspect, the beads may be free flowing beads or may be attached to the solid substrate. In another aspect, the one or more analytes comprise antigens, antibodies, autoantibodies, peptides, proteins, nucleic acid sequences, enzymes, or small molecules wherein the antigens comprise one or more bacterial, viral, fungal, mycoplasmal, rickettsial, chlamydial, or protozoal antigens. In another aspect, the dockerin may be a substituted dockerin, a truncated dockerin, a modified dockerin, or any combinations thereof. In another aspect, the cohesin may be a substituted cohesin, a truncated cohesin, a modified cohesin, or any combinations thereof. In another aspect, the cohesin is derived or isolated from Clostridium thermocellum, C. cellulolyticum, C. cellulovorans, C. papyrosolvens, and Clostridium cellobioparum, C. papyrosolvens, C. josui, Acetivibrio cellulolyticus, Bacteroides cellulosolvens, R. flavefaciens, Ruminococcus albus, Archaeoglobus fulgidus protein, cellulosomal cohesin domain. [0038] In one embodiment, the present invention comprises an affinity purification method utilizing one or more beads comprising the steps of: providing one or more beads or bead sets on a substrate, wherein the substrate is selected from the group consisting of a beaker, a flask, a cylinder, a test tube, a centrifugation tube, a petri dish, a culture dish, and a multi-well plate, an column and any combinations or modifications thereof; attaching or immobilizing at least one dockerin binding domain to a surface of the beads, wherein the dockerin binding domain may be attached to a protein, an antibody, an antigen, a peptide, a toxin, a cytokine, an enzyme, a structural protein, an extracellular matrix protein, a cell, or fragments thereof; contacting the beads by flowing or pumping a sample, a cellular mixture, a fermentation medium, a cell extract, or any combinations thereof comprising one or more analytes to be purified, wherein at least one of the analyte to be purified is coupled or attached to a cohesin fusion protein; binding the analyte comprising attached cohesin fusion protein with the one or more beads comprising at least one dockerin binding domain to form a complex, wherein the binding generates a flow through comprising one or more undesirable materials or materials from which an isolation of the analyte is desired; and releasing the desired analyte from the complex by one or a combination of physical or chemical methods, wherein the methods comprise a change in ionic strengths, addition of EDTA, removal of

Ca from the medium, pH, temperature, or any combinations thereof. In another aspect, the one or more beads are immobilized to a solid substrate or a column packing material. In another aspect, the one or more beads or bead sets are packed in a column. In another aspect, the one or more beads are polymeric beads and comprise nanospheres, nanoparticles, microspheres, microparticles, nanobeads, microbeads, beads, polystyrene beads, latex particles, latex beads, fluorescent beads, fluorescent particles, colored particles, or colored beads. In another aspect, the analyte to be purified comprises one or more of antigens, antibodies, autoantibodies, peptides, proteins, fusion proteins, nucleic acid sequences, or enzymes, or any combination thereof wherein the antigens comprise one or more bacterial, viral, fungal, mycoplasmal, rickettsial, chlamydial, or protozoal antigens. In another aspect, the dockerin may be a substituted dockerin, a truncated dockerin, a modified dockerin, or any combinations thereof. In another aspect, the cohesin may be a substituted cohesin, a truncated cohesin, a modified cohesin, or any combinations thereof. In another aspect, the cohesin is derived or isolated from Clostridium thermocellum, C. cellulolyticum, C. cellulovorans, C. papyrosolvens, and Clostridium cellobioparum, C. papyrosolvens, C. josui, Acetivibrio cellulolyticus, Bacteroides cellulosolvens, R. flavefaciens, Ruminococcus albus, Archaeoglobus fulgidus protein, or a cellulosomal cohesin domain. [0039] In some embodiments there is a system or kit comprising luciferase; an antibody with a reactive group for attaching to the luciferase;a dockerin polypeptide comprising a reactive group for attaching to a solid support;and a cohesin polypeptide comprising a reactive group for coupling to an antigen. In other embodiments there is a system or kit wherein the antibody and luciferase are coupled, or wherein the dockerin polypeptide is attached to a solid support; and/or a cohesin polypeptide attached, fused or coupled to an antigen. In other embodiments, one or more components described herein is provided as a nucleic acid encoding the polypeptide. The nucleic acid may be a vector or other expression construct. [0040] In certain embodiments, a kit or system further comprises an antibody attached, fused, or conjugated to one or more dockerin polypeptides, and luciferase attached, fused or conjugated to cohesin.

[0041] Any embodiment discussed herein with respect to a Dockerin-Cohesin binding pair, Dockerin can be switched with Cohesin and Cohesin switched with Dockerin. Moreover, in any embodiment concering Cohesin, one Cohesin polypeptide may be employed or multiple Cohesin polypeptides (1, 2, 3, 4, 5,6 ,7 ,8 ,9, 10 or more, or any range derivable therein) may be employed. Similarly, 1, 2, 3, 4 or more Dockerin polypeptides may be used instead of a single Dockerin polypeptide.

[0042] As used herein the specification, "a" or "an" may mean one or more. As used herein in the claim(s), when used in conjunction with the word "comprising", the words "a" or "an" may mean one or more than one.

[0043] The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." As used herein "another" may mean at least a second or more.

[0044] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

[0045] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [0046] Any embodiment described in the context of a particular method may be implemented in the context of any other method discussed herein. Additionally, any embodiment described in the context of a particular component, reagent, or system may be implemented in the context of any other component reagent, or system discussed herein. Moreover, any component, reagent, or system may be used in any method discussed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [0048] FIGS. 1A-B show that Cohesin and dockerin are protein modules from cellulose-degrading bacteria that bind non-covalently with very high affinity (K_D ~ 30 pM): FIG. 1A A set of up to 100 different fluorescent microspheres (e.g., Luminex® beads) are cross-linked chemically to a cellulose-binding domain fused to dockerin (CBD-Doc). Each cohesin-antigen fusion protein is mixed to a CBD-Doc beads of a particular color (e.g., bead region 21). The beads are washed and pooled with CBD-Doc beads similarly coated with other desired cohesin-antigen fusion proteins, FIG. IB The bead-antigen sets are incubated with a dilution series of each serum sample, washed, incubated with e.g., anti-human IgG-PE conjugate, washed again, then read in a flow cytometry instrument which measures fluorescence intensity associated with each bead color. [0049] FIG. 2 is a comparison of serum anti-Gag p24 assays using the bead-based assay and standard ELISA. The three upper panels are serial dilutions of pre -immune serum (gray) and immune serum (black) from three individuals assayed using Cohesin-Gag p24 coated beads. The three lower panels are the identical samples assayed using standard ELISA with Cohesin-Gag p24-coated plates. Consistently, the bead-based assay is 10-fold more sensitive. [0050] FIG. 3 represents a serum anti-Influenza antigen-specific antibody assay using the bead-based multiplexed assay of the present invention. Dilutions of serum samples from four normal donors. This is a multiplexed assay with beads displaying antigens Ml (PR8), HAl-1 (PR8) and HAl-1 (HA5, avian H1N1 and Flu NP, avian H1N1). Beads displaying Cohesin alone are included to define the non-specific background. None of the donors had detectable antibodies binding to H1N1 avian influenza HAl-1;

[0051] FIG. 4 represents reagents for capture and detection of antigen-specific B cells. Beads displaying a Cohesin-antigen fusion protein (Ag-1) can be used to enrich antigen-specific B cells from ex vivo samples, while antigen tetramers assembled via streptavidin-phycoerythrin (SA-PE) : biotin-CBD-Doc : Cohesin-antigen complex can be uses in flow cytometry to sort or quantify antigen- specific B cells. The antigen-bead complexes can also be assembled on biotin-coated Q dots of different diameters, permitting the possible development of multiplexed flow analysis of several antigen-specific B cell categories. Furthermore, the combinatorial strategy of mixing tetramers for each specificity but with two different colors [Newell et al, Nature Methods 6, 497 - 499 (2009)] can be readily applied to our antigen tetramers to reduce background staining and increase the number of antigens that can be tested simultaneously.

[0052] FIG. 5 is an example of a multiplex bead-based assay of antigen-specific serum antibodies from a Influenza-infected non-human primate. Three different HA antigens are represented, as well as Ml, M2 ectodomain, NP, and NA.

[0053] FIGS. 6A-C provide an example of a multiplex bead antigen-specific IgG assay: FIG. 6A a serum sample from an influenza-infected monkey was diluted and mixed with a bead set coated with cohesin fused to various Influenza proteins NP5, nuclear protein; HAl PR8, HAl domain from hemagglutinin of the PR8 strain; HAl SF, HAl domain from hemagglutinin of the swine flu strain (CAL04); HA3, HAl domain from hemagglutinin a H3 Influenza strain; M2e, the ectodomain of the M2 protein from PR8; Ml, matrix protein 1; Coh, Cohesin without antigen. FIGS. 6B and 6C compares the serum from the monkey before (FIG. 6B) and after vaccination (FIG. 6C) with a vaccine bearing HAl from PR8.

[0054] FIG. 7: Cohesin and dockerin are protein modules from cellulose-degrading bacteria that bind non-covalently with very high affinity (KD ~ 30 pM). Left panel. Cellulose-binding domain fused to dockerin (CBD-Doc) is bound to cellulose microspheres. Each cohesin-antigen fusion protein is mixed to a CBD-Doc bead. Right panel. The bead- antigen complex is incubated with a dilution series of each serum sample, washed, incubated with anti-human IgG-Renilla Luciferase fusion antibody, washed again, then read in a luminometer after substrate addition. [0055] FIG. 8A-B: Validation of anti-DCIR/RLuc antibodies. A) Schematic representation of the fusion proteins used in this assay. The Renilla Luciferase gene was fused to the C-terminus of the light or the heavy chain, as indicated. B) Comparison of Luciferase activity from 293T cells supernatants transfected with anti-DCIR/Rluc antibodies fused to the light chain, the heavy chain or both. [0056] FIG. 9: Comparison of a BioLuminiscent ImmunoAssay (BLIA) and an

Enzyme-Linked Immunosorbent Assay using an anti-DCIR/RLuc as a primary antibody. Cohesin alone or fused to DCIR ectodomain were coated on plastic. An anti-DCIR/RLuc antibody was used to detect the fusion protein, alone (BLIA, left panel) or with a secondary anti-hlgG/HRP (ELISA, right panel). In the later setting, bound antibody was revealed with HRP substrate.

[0057] FIG. 10: Comparison of BLIA and ELISA to detect Coh-DCIR ectodomain using our anti-DCIR/RLuc antibody. Coh-DCIR ectodomain was coated on 96-well plate and detection was made by using serial dilution of anti-DCIR/RLuc. For BLIA, detection was made using Colenterazyne h, the substrate of Renilla Luciferase. For ELISA, detection of anti-DCIR/RLuc was performed with a secondary antibody anti-human IgG linked to HRP. At low concentrations, BLIA still gives very good results compare to ELISA, which is close to background.

[0058] FIG. 11: PBMCs from a tuberculosis patient were incubated for 16h with Mycobacterium tuberculosis specific peptides in presence or absence of aCD28. Secreted IFNy, IP- 10 and IL-2 were quantified by Luminex.

[0059] FIG. 12: Renilla Luciferase was incubated with perm/wash solution or PBS and then coelenterazine h was added. Bioluminescence was measured for 2 seconds and plotted on the graph. [0060] FIG. 13: Environmental pathogens can be detected by using cellulose beads and Bioluminescence. By generating two antibodies that recognize two different extracellular proteins, any pathogen can be detected in any liquid sample.

[0061] FIG. 14: Detection of huma IgG within several dilutions of human sera with anti-human IgG fused to Gaussia Luciferase or anti-human IgG/HRP, narrow range. Comparison of anti-human IgG-HRP and anti-human IgG/Gaussia Luciferase in their ability to detect human IgG from serum (directly coated on plastic). This experiment shows that Luciferase-fused anti-human antibody is able to accurately detect human IgG from a wide range of concentrations and at very low concentrations compared to HRP-conjugated anti- human IgG. Furthermore, the detection is linear.

[0062] FIG. 15 : Detection of huma IgG within several dilutions of human sera with anti-human IgG/Gaussia Luciferase or anti-human IgG/HRP, wide range. Comparison of anti-human IgG-HRP and anti-human IgG/Gaussia Luciferase in their ability to detect human IgG from serum (directly coated on plastic). This experiment shows that Luciferase-fused anti-human antibody is able to accurately detect human IgG from a wide range of concentrations and at very low concentrations compared to HRP-conjugated anti-human IgG. Furthermore, the detection is linear.

[0063] FIG. 16: Anti-human IgG/Gaussia Luciferase detection of 16 antigens in sera dilutions from health control or active tuberculosispatient. [0064] FIG. 17: Anti-human IgG/HRP detection of 16 antigens in sera dilutions from health control or active tuberculosispatient.

[0065] FIG. 18: Detection of Ag-specific human IgG in serum from tuberculosis patients #3 to #9 and BD1863 with anti-human IgG/HRP with cellulose beads coated with CBD-Doc/Coh-Ag, 1/800 dilution of serum. [0066] FIG. 19: Detection of Ag-specific human IgG in serum from tuberculosis patients #3 to #9 and BD1868 with anti-human IgG/HRP, 1/400 dilution of serum, regular ELISA.

[0067] FIG. 20: Detection of increasing amount of purified CD4+ T cells using anti- CD3 coated plates and anti-CD4-biotin + Streptavidin HRP. [0068] FIG. 21: Detection of increasing amount of purified CD4+ T cells mixed to a constant amount of CD4-depleted PBMCs (200k) using anti-CD3 coated plates and anti- CD4-biotin + Streptavidin HRP.

[0069] FIG. 22: Schematic diagram illustrating direct attachment of luciferase reporter to mouse monoclonal antibody or amplification of luciferase reporter signal through multiple luciferase reporter molecule binding involving cohesin/dockerin implementation.

[0070] FIG. 23: Schematic diagram illustrating detection of serological IgG using direct luciferase attachment to antibodies or cohesin/dockerin luciferase amplification.

[0071] FIG. 24: Schematic diagram illustrating detection of antigens using direct luciferase attachment to antibodies or cohesin/dockerin luciferase amplification.

[0072] FIG. 25: Schematic diagram illustrating detection of pathogens using direct luciferase attachment to antibodies or cohesin/dockerin luciferase amplification.

[0073] FIG. 26: Schematic diagram illustrating detection of CD4 T-cells using direct luciferase attachment to antibodies or cohesin/dockerin luciferase amplification. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0074] While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.

[0075] To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as "a", "an" and "the" are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims. [0076] As used herein, the term "analogs" extends to any functional chemical or recombinant equivalent of the peptides of the present invention, characterized, in a most preferred embodiment, by their possession of at least one of the abovementioned activities. The term "analog" is also used herein to extend to any amino acid derivative of the peptides as described hereinabove. Generally, an analog will possess in one embodiment at least 70% sequence identity, other embodiments can have at least 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity. Percentage sequence identity can be determined by the skilled artisan.

[0077] As used herein, the term "epitope(s)" refer to a peptide or protein antigen that includes a primary, secondary or tertiary structure similar to an epitope located within any of a number of pathogen polypeptides encoded by the pathogen DNA or RNA. The level of similarity will generally be to such a degree that monoclonal or polyclonal antibodies directed against such polypeptides will also bind to, react with, or otherwise recognize, the peptide or protein antigen. [0078] As used herein, the term "recombinant polypeptide" refers to a polypeptide that has been produced in a host cell which has been transformed or transfected with a nucleic acid encoding the polypeptide, or produces the polypeptide as a result of homologous recombination.

[0079] As used herein, the term "dockerin-cohesin pair" is used to denote the binding of the dockerin domain and the cohesin domain and it is understood that the dockerin domain, the cohesin domain or both may be truncated, substituted, modified, or altered. In addition, the embodiments are described in various ways but it is to be understood that the dockerin- cohesin pair can also be considered a cohesin-dockerin pair. It is to be understood that either the dockerin or the cohesin may be bound to the substrate and the corresponding pair may be conjugated to the binding agent to form a dockerin-cohesin pair.

[0080] Microarray, micro-bead and bead technologies of the present invention can be used as tools to conduct biological, chemical or biochemical analyses in a parallel, massively parallel or multiplexed fashion because of the large number of different compounds or substances that can be fabricated or deposited on the microarray substrate or beads. Micro- bead technologies are analogous to microarrays except that the features are spatially segregated on different beads or particles. The analysis can be formatted like a microarray, for example, with the beads arrayed or embedded on the surface or in wells of a device such as a microscope slide or plate. The analysis can alternatively be performed with the beads suspended in a solution for example. The working density of features for micro-bead technologies is potentially far greater than for microarrays, depending primarily on the minimum usable bead size and maximum usable bead concentration or density.

[0081] As used herein, the term "nanospheres" is used to denote particles in sizes ranging from about 10 nanometers (nm) to about 100,000 nm in diameter. Optimally preferred diameters are within about 10 and 1,000 nm, preferably within 200 and 500 nm. Polymeric microspheres used in this invention as carrier particles to which nanospheres are bound normally range in size from 0.01 to 1000 micrometers in diameter. Even though the microparticle can be of any size, the preferred size is 0.1-500 micrometers, more preferably 1-200 micrometers, and even more preferably 2-12 micrometers. The particles can be uniform (being about the same size) or of variable size such that the differences can be determined by size-dependent properties such as light scattering or optical refraction. Particles are made of any regularly shaped material. The preferred shape is spherical, however, particles of any other shape can be employed since this parameter is immaterial to the nature of the invention.

[0082] Usually these nanospheres as well as carrier particles are made of the same material such as polystyrene or latex. However, other polymeric materials are acceptable including polymers selected from the chemical group consisting of carbohydrate-based polymers, polyaliphatic alcohols, poly(vinyl) polymers, polyacrylic acids, polyorganic acids, polyamino acids, co-polymers, block co-polymers, tertpolymers, polyethers, naturally occurring polymers, polyimids, surfactants, polyesters, branched polymers, cyclo-polymers, polyaldehydes and mixtures thereof. More specifically, brominated polystyrene, polyacrylic acid, polyacrylonitrile, polyamide, polyacrylamide, polyacrolein, polybutadiene, polycaprolactone, polyester, polyethylene, polyethylene terephthalate, polydimethylsiloxane, polyisoprene, polyurethane, polyvinylacetate, polyvinylchloride, polyvinylpyridine, polyvinylbenzylchloride, polyvinyltoluene, polyvinylidene chloride, polydivinylbenzene, polymethylmethacrylate, polylactide, polyglycolide, poly(lactide-co-glycolide), polyanhydride, polyorthoester, polyphosphazene, polyphosophaze, or combinations thereof are preferable. Representative combination polymers of which the polymeric particles are composed include for example poly-(styrene-co-vinylbenzyl chloride-co-acrylic acid) (85: 10:5 molar ratio), poly(styrene-co-acrylic acid) (99:1 molar ratio), poly(styrene-co- methacrylic acid) (90: 10 molar ratio), poly(styrene-co-acrylic acid-co-m&p-divinylbenzene) (89: 10:1 molar ratio), poly-(styrene-co-2-carboxyethyl acrylate) (90: 10 molar ratio), poly(methyl methacrylate-co-acrylic acid) (70:30 molar ratio) and poly(styrene-co-butyl acrylate-co-methacrylic acid)(45:45: 10 weight ratio). Most of beads formed from synthetic polymers such as polystyrene, polyacrylamide, polyacrylate, or latex are now commercially available from numerous sources such as Bio-Rad Laboratories (Richmond, Calif.) and LKB Produkter (Stockholm, Sweden). Beads formed from natural macromolecules and particles such as agarose, crosslinked agarose, globulin, deoxyribose nucleic acid, and liposomes are commercially available from sources such as Bio-Rad Laboratories, Pharmacia (Piscataway, N.J.), and IBF (France). Beads formed from copolymers of polyacrylamide and agarose are commercially available from sources such as IBF and Pharmacia.

[0083] In general, the present invention provides the production of a plurality of substrates (e.g., beads) with different features such as antibodies, markers, binding agents, probes, targets or analytes for example, that can serve as delivery agents, purification mechanisms, isolation mechanisms, affinity matrix, multiplex arrays or similar functions. The different feature substances attached to the beads are typically produced off-line and can then be bound to beads in separate reactors, in a mechanical process of mixing solutions containing to form the dockerin-cohesin binding pair. This can be done in separate test tubes, vials or wells of a microtiter plate for example.

[0084] The present invention provides an affinity matrix system having a dockerin- cohesin pair bound to a substrate and configured to bind an antigen or other composition. The present invention provides a flexible method for making multiplexed assays for antigen- specific antibody responses. A standard Luminex technology uses color-coded micro beads in up to 500 distinct sets are coated with a reagent specific to a particular bioassay, allowing the capture and detection of specific analytes from a sample. In contrast, the present invention provides beads in a set that are first coated with a dockerin domain containing protein. Then each bead set is simply incubated with cohesin-antigen fusion proteins, which attach essentially irreversibly by non-covalent interaction. Thus, one dockerin- or cohesin-coated bead set can be used for multiple sets of cohesin- or dockerin-antigens, obviating the need to directly chemically couple new antigens to new beads. In a similar process the present invention provides beads in a set that are first coated with a cohesin domain containing protein. Then each bead set is simply incubated with dockerin-antigen fusion proteins, which attach essentially irreversibly by non-covalent interaction. Thus, one cohesin- or dockerin- coated bead set can be utilized for multiple sets of dockerin- or cohesin-antigens, obviating the need to directly chemically couple new antigens to new beads. [0085] The present invention obviates the need for chemical cross-linking of antigens to beads and obviates the purification of the antigens from a recombinant expression system since the dockerin-beads can be used as an affinity matrix directly. The beads can be used as an affinity matrix directly resulting in beads ready for the assay. Also, this format presents the antigen in a configuration that does not obscure epitopes in contrast to chemical cross- linking techniques.

[0086] The present invention exploits the dockerin-cohesin pair interaction. The cohesin domains interact with small domains (e.g., 56 residues) called dockerins. These are

Ca containing structures with two-fold symmetry and they can bind to a cognate cohesin with various affinities. The interaction is non-covalent and is well defined (by structure analysis) for at least one dockerin-cohesin pair. Dockerins are designed to be domains linked to different domain (enzyme in nature), and cohesins are designed to function in linear arrays (either directly end-to-end, or joined by flexible PT-rich linkers of various sizes. It is known that a particular dockerin can have specificity for a particular cohesin (e.g., a dockerin- cohesin pair from one bacterial species may not be interchangeable with a dockerin-cohesin pair from a different species). This feature makes it is possible to ensure the specific and precise interaction of various fusion proteins with an engineered cohesin domains of various specificities.

[0087] Based on the crystal structure of the cohesin-dockerin complex it is apparent that one embodiment is an antigen-dockerin fusion proteins (i.e., antigen fused to the N- terminus of a dockerin). However, both from the structure and from the nature of cohesin domain organization within scaffolding, it is apparent that cohesins can be fused end-to-end, even without spacer sequences. Furthermore, it is apparent that well-described techniques are available to engineer miniaturized versions of the cohesin and dockerin domains.

[0088] Cellulosomes are multi-enzyme complexes that orchestrate the efficient degradation of cellulose and related plant cell wall polysaccharides. The complex is maintained by the high affinity protein-protein interaction between two complementary modules: the cohesin and the dockerin. Dockerin is a protein domain found in the cellulosome cellular structure. It is part of endoglucanase enzymes. The dockerin's binding partner is the cohesin domain. This interaction is essential to the construction of the cellulosome complex. The dockerin domain has two tandem repeats of a non-EF hand calcium binding motif characterized by a loop-helix structure. There are three types of Dockerin domains: I, II and III which bind to Cohesin Type I, Cohesin Type II and Cohesin Type III respectively.

[0089] A common feature of the cellulosomes is that they consist of a large number of catalytic components arranged around noncatalytic scaffolding proteins. These proteins fundamentally consist of repetitive noncatalytic domains of about 140 residues, termed cohesin domains, and a carbohydrate-binding module (CBM). For example, C. josui CipA N-terminus is composed of 3 CBMs followed by a hydrophilic domain and six cohesin domains; and C. thermocellum CipA contains a CBM between the second and third of nine repeated cohesin domains and a type II dockerin domain at its C terminus. The amino acid sequences of all the cohesin domains from these bacteria are in many cases highly homologous to each other, especially within the same species. Each cohesin domain is a subunit-binding domain that interacts with a docking domain, called dockerin, of each catalytic component. The dockerin domain contains two segments, also known as conserved

2__|_

duplicated regions (CDRs), each of which contains a Ca -binding loop and an alpha helix (namely, the calcium binding motif). An additional alpha helix intervenes between the two segments. The alpha helix in each duplicated sequence contains a conserved KR or K dipeptide. The species-specific attachment of the dockerin module to the cohesin module is

2__|_

mediated via a high affinity Ca -dependent interaction. The cellulosome contains a CBM and a series of cohesin modules that anchor the cellulosomal enzymes to the multienzyme complex. The various cellulosomal enzymes contain inter alia a conserved dockerin module that binds to the cohesin counterpart. Biochemical and structural studies on the cohesins- dockerin interaction from have shown that the dockerins can bind to each of the cohesins on the scaffoldin with a strong affinity constant. Binding is can be reversible by addition of divalent ion chelators such as EDTA. [0090] The present invention provides a cost-effective method and composition for connecting antigens to beads to obviate chemical cross-linking and purification of antigens from recombinant expression systems since the dockerin-beads can be used as an affinity matrix directly resulting in beads ready for the assay. Also, there is a need for an antigen configuration that does not obscure epitopes in contrast to chemical cross-linking techniques.

[0091] The solid substrate of methods and compositions of the present invention is, in another embodiment, a bead. In another embodiment, the solid substrate is a cell. In another embodiment, the solid substrate is an extracellular matrix. In another embodiment, the solid substrate is a fibrous matrix. In another embodiment, the solid substrate is a container. In another embodiment, the container is selected from the group consisting of a beaker, a flask, a cylinder, a test tube, a centrifugation tube, Petri dish, a culture dish, a multi-well plate or a chip. In another embodiment, the solid substrate is attached to or associated with an affinity column. Each possibility represents a separate embodiment of the present invention.

[0092] In another embodiment, an antibody-binding moiety is attached to a solid substrate of the present invention via fusion of the antibody-binding moiety to a truncated dockerin polypeptide. The truncated dockerin polypeptide is able to attach to a cohesin- containing protein bound to the solid substrate. [0093] In another embodiment, a solid substrate of methods and compositions of the present invention comprises cellulose, and the protein bound to the solid substrate further comprises a carbohydrate-binding module (CBM). In another embodiment, the means of attachment of the protein to the solid substrate is via interaction between the CBM and the cellulose. Each possibility represents a separate embodiment of the present invention. [0094] The molecule of interest of the methods and compositions of the present invention is any molecule that can be bound covalently, either directly or indirectly, to the truncated dockerin (e.g., truncated dockerin, substituted dockerin or modified dockerin) domain containing as disclosed herein. In various embodiments, the molecule of interest is any type of molecule which is desirable to purify or for which it is desirable to engineer an association with a solid substrate. In another embodiment , the molecule of interest of the methods and compositions of the present invention is any molecule that can be bound covalently, either directly or indirectly, to the cohesin (e.g., truncated cohesin, substituted cohesin or modified cohesin) domain. In various embodiments, the molecule of interest is any type of molecule which is desirable to purify or for which it is desirable to engineer an association with a solid substrate. [0095] In certain embodiments the molecule of interest is a peptide. In another embodiment, the molecule of interest is a protein. In another embodiment, the peptide is an enzyme. In another embodiment, the molecule is a peptide hormone. In another embodiment, the molecule is a recombinant peptide. In another embodiment, the molecule is a nucleic acid. In another embodiment, the molecule is a messenger. In another embodiment, the molecule is a drug. In another embodiment, the molecule is a cell receptor. In another embodiment, the molecule is a cell. In another embodiment, the molecule of interest is any other type of molecule for which it is desirable to purify or to engineer an association with a solid substrate. [0096] As provided herein, a variety of proteins can be successfully purified with high-efficiency under gentle conditions following fusion to dockerin domains of the present invention. Methods for identification of dockerin domains are well known in the art. The dockerin-cohesin pair utilized in methods and compositions of the present invention are, in another embodiment, from the same species. Dockerins have been shown to bind to each of the cohesins on the scaffolding; thus, any cohesin from a given species is expected to bind any dockerin from that species. Cohesin-dockerin interactions are not species-specific; however, in some cases the interaction may be species-specific.

[0097] In another embodiment, the K_a of the dockerin domain (or substituted or modified dockerin domain) with the wild-type cohesin, in the presence of EDTA is low enough to act as a reversible affinity tag. In another embodiment, the K_a of this combination is under 10⁷M^_1. In another embodiment, the K_a of this combination is under 3X10⁶ M^"1. In another embodiment, the K_a of this combination is under 10⁶M^_1. In another embodiment, the K_a of this combination is under 3X10⁵ M^"1. In another embodiment, the K_a of this combination is under 10⁵ M-l . In another embodiment, the K_a of this combination is under 3X10⁴ M^"1. In another embodiment, the K_a of this combination is under 10⁴ M^"1. In another embodiment, the 3 -1

K_a of this combination is under 5X10 M^" . In another embodiment, the K_a of this combination is under 2X10 5 M -^"3. In another embodiment, the K_a of this combination is under 10 3 M-^"1. In another embodiment, the 2 -1

K_a of this combination is under 5X10 M^" . In another embodiment, the nder 2X10 2 M -^"1

K_a of this combination is u . In another embodiment, the his combination is under 10 2 M -^"1

K_a of t . In another embodiment, the K_a of this combination is under 5X10¹ M^"1. In another embodiment, the K_a of this combination is under 2X10¹ M^"1. In another embodiment, the K_a of this combination is under 10¹ M^"1. Each possibility represents a separate embodiment of the present invention.

[0098] The cohesin domain of methods and compositions of the present invention is, in another embodiment, a Type-I cohesin domain. In another embodiment, the cohesin domain is a Type-II cohesin domain. In another embodiment, the cohesin domain is any other type of cohesin domain known in the art. Each possibility represents a separate embodiment of the present invention.

[0099] The present invention uses cohesin-dockerin from different species or sequences to make multivalent compositions. The invention includes the use of all cohesin- dockerin sequences from diverse cellulose degrading microbes, but describes the application of specific cohesin and dockerin and linker sequences from the microbe Clostridium thermocellum. The cohesin dockerin pairing exists in diverse cellulose degrading species. While they have sequence similarities, they can have specificities that do not cross between species. This affords an opportunity to build novel constructs with different specificities in a spatially and numerically controlled manner. By extension multiple cohesin-dockerin specificities can be used to make bivalent substrates with higher order antigen specificities. Cellulose degrading bacteria and similar organisms also use cellulose-binding domains to organize the degradation machinery. The invention encompasses the use of entities to assemble spatially and numerically ordered complexes and multi subunit receptors. [00100] In another embodiment, the cohesin domain is from a species selected from the group consisting of Clostridium thermocellum, C cellulolyticum, and C. cellulovorans. In another embodiment, the cohesin domain is from a species selected from the group consisting of Clostridium thermocellum, C papyrosolvens, and Clostridium cellobioparum. In another embodiment, the cohesin domain is from a species selected from the group consisting of Clostridium thermocellum, C cellulolyticum, C cellulovorans, C papyrosolvens, C josui, Acetivibrio cellulolyticus, Bacteroides cellulosolvens, R. flavefaciens, Ruminococcus albus, and Clostridium cellobioparum.

[00101] In another embodiment, the cohesin domain is a cohesin domain from a protein selected from CipA (or scaffoldin) of C. thermocellum, CipC of C. cellulolyticum, CbpA of C. cellulovorans, and CipA of C. josui. In another embodiment, the cohesin domain of methods and compositions of the present invention is from an Archaeoglobus fulgidus protein. In another embodiment, a cellulosomal cohesin domain is utilized in methods and compositions of the present invention. In another embodiment, a Type I cohesin domain from a cellulosomal protein is used. In another embodiment, a non-cellulosomal cohesin domain is utilized in methods and compositions of the present invention. [00102] In one non- limiting example, the substrate of the present invention is engineered to have one or more modular cohesin-dockerin protein domains for making specific and defined protein complexes. In some embodiments the cohesin or dockerin protein domains may be from different species to allow for specific pairing of species- specific cohesin-dockerin proteins. [00103] The substrates are alternately termed nanospheres, nanoparticles, microspheres, microparticles, nanobeads, microbeads, beads, polystyrene beads, latex particles, latex beads, fluorescent beads, fluorescent particles, colored particles and colored beads. The substrates serve as vehicles for molecular reactions. Illustrative microspheres and methods of manufacturing same are, for example, found in U.S. Patent No. 7,141,431, the contents of which are incorporated by reference.

[00104] The substrate will be used to carry a separate molecule, e.g., a bead, a peptide, protein, lipid, carbohydrate, nucleic acid (oligonucleotide, aptamer, vector with or without base or backbone modifications) or combinations thereof by binding that separate molecule to the complementary half of the cohesin: dockerin pair. For example, either the dockerin or cohesin may be made into a fusion protein or chemically bound to an antigen, a peptide, a protein, a toxin, a cytokine, an enzyme, a structural protein, an extracellular matrix protein, another antibody, a cell or fragments thereof.

[00105] The substrate may have one or more cohesin, dockerin or both cohesin and dockerin domains that allow the formation of a complex with one or more complementary cohesin/dockerin-molecules and a binding domain. The binding domain can be used to conjugate a protein, antibody, nucleic acid, dye, metal, label, active agent or any other composition. For example, the binding domain may be an antibody used to bind an antigen. In another embodiment the binding domain may be used to purify an antigen that binds to the antibody connected to the cohesin/dockerin-molecules and substrate. [00106] The biological sample to be tested using the instant invention, for example, includes plasma, serum, tears, mucus, saliva, urine, pleural fluid, spinal fluid, gastric fluid, sweat, semen, vaginal secretion, fluid from ulcers and/or other surface eruptions, blisters, abscesses, and/or extracts of tissues, such as biopsies of normal, malignant, and/or suspect tissues.

[00107] The analytes of interest for these bioassays include, for example, antigens, antibodies, autoantibodies, peptides, proteins, nucleic acid sequences, and/or enzymes. The antigenic analytes, for example, includes bacterial, viral, fungal, mycoplasmal, ridkettsial, chlamydial, and/or protozoal antigens.

[00108] Examples of antigens include tumor proteins, e.g., mutated oncogenes; viral proteins associated with tumors; and tumor mucins and glycolipids. The antigens may be viral proteins associated with tumors would be those from the classes of viruses noted above. Certain antigens may be characteristic of tumors (one subset being proteins not usually expressed by a tumor precursor cell), or may be a protein which is normally expressed in a tumor precursor cell, but having a mutation characteristic of a tumor. Other antigens include mutant variant(s) of the normal protein having an altered activity or subcellular distribution, e.g., mutations of genes giving rise to tumor antigens. Specific non- limiting examples of tumor antigens include: CEA, prostate specific antigen (PSA), HER- 2/neu, BAGE, GAGE, MAGE 1-4, 6 and 12, MUC (Mucin) (e.g., MUC-1, MUC-2, etc.), GM2 and GD2 gangliosides, ras, myc, tyrosinase, MART (melanoma antigen), Pmel 17(gpl00), GnT-V intron V sequence (N-acetylglucoaminyltransferase V intron V sequence), Prostate Ca psm, PRAME (melanoma antigen), beta-catenin, MUM-l-B (melanoma ubiquitous mutated gene product), GAGE (melanoma antigen) 1, BAGE (melanoma antigen) 2-10, C-ERB2 (Her2/neu), EBNA (Epstein-Barr Virus nuclear antigen) 1-6, gp75, human papilloma virus (HPV) E6 and E7, p53, lung resistance protein (LRP), Bcl-2, and Ki-67. In addition, the immunogenic molecule can be an autoantigen involved in the initiation and/or propagation of an autoimmune disease, the pathology of which is largely due to the activity of antibodies specific for a molecule expressed by the relevant target organ, tissue, or cells, e.g., SLE or MG. In such diseases, it can be desirable to direct an ongoing antibody-mediated (i.e., a Th2-type) immune response to the relevant autoantigen towards a cellular (i.e., a Thl- type) immune response. Alternatively, it can be desirable to prevent onset of or decrease the level of a Th2 response to the autoantigen in a subject not having, but who is suspected of being susceptible to, the relevant autoimmune disease by prophylactically inducing a Thl response to the appropriate autoantigen. Autoantigens of interest include, without limitation: (a) with respect to SLE, the Smith protein, RNP ribonucleoprotein, and the SS-A and SS-B proteins; and (b) with respect to MG, the acetylcholine receptor. Examples of other miscellaneous antigens involved in one or more types of autoimmune response include, e.g., endogenous hormones such as luteinizing hormone, follicular stimulating hormone, testosterone, growth hormone, prolactin, and other hormones. Antigens involved in autoimmune diseases, allergy, and graft rejection can be used in the compositions and methods of the invention. For example, an antigen involved in any one or more of the following autoimmune diseases or disorders can be used in the present invention: diabetes, diabetes mellitus, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), multiple sclerosis, myasthenia gravis, systemic lupus erythematosis, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), psoriasis, Sjogren's Syndrome, including keratoconjunctivitis sicca secondary to Sjogren's Syndrome, alopecia areata, allergic responses due to arthropod bite reactions, Crohn's disease, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drug eruptions, leprosy reversal reactions, erythema nodosum leprosum, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens- Johnson syndrome, idiopathic sprue, lichen planus, Crohn's disease, Graves opthalmopathy, sarcoidosis, primary biliary cirrhosis, uveitis posterior, and interstitial lung fibrosis. Examples of antigens involved in autoimmune disease include glutamic acid decarboxylase 65 (GAD 65), native DNA, myelin basic protein, myelin proteolipid protein, acetylcholine receptor components, thyroglobulin, and the thyroid stimulating hormone (TSH) receptor. Examples of antigens involved in allergy include pollen antigens such as Japanese cedar pollen antigens, ragweed pollen antigens, rye grass pollen antigens, animal derived antigens such as dust mite antigens and feline antigens, histocompatiblity antigens, and penicillin and other therapeutic drugs. Examples of antigens involved in graft rejection include antigenic components of the graft to be transplanted into the graft recipient such as heart, lung, liver, pancreas, kidney, and neural graft components. The antigen may be an altered peptide ligand useful in treating an autoimmune disease.

[00109] In one example, the present invention can be used in any system for multiplex detection of agents. For example, color-coded beads, pre-coated with analyte- specific capture dockerin or cohesin bound to the beads for the molecule of interest, are added. Either the cohesin or dockerin is bound to the bead, with the analyte bound to the other half of the cohesin-dockerin pair. Multiple analytes can be simultaneously detected in the same sample by binding to the dockerin or cohesin, e.g., as a fusion protein. The analyte (e.g., antigens, antibodies, autoantibodies, peptides, proteins, nucleic acids, enzymes, or small molecules) can be bound, loaded or linked (covalently or non-covalently) to the dockerin or cohesin matching pair on the beads.

[00110] Next, analyte-specific antibodies are added to the beads to capture the analyte of interest. In one embodiment, biotinylated detection antibodies specific to the analyte of interest are added and form an antibody-antigen sandwich.

[00111] In the case of a biotinylated agent, a detectable agent (e.g.,

Phycoerythrin (PE)) conjugated to Streptavidin is added. In another embodiment, the dockerin-cohesin pair can substitute for the biotin-streptavidin, wherein one portion of the binding pair is bound to a detectable label (fluorescent, magnetic, radioactive, electron-dense, enzymatic, and the like).

[00112] The beads are read using, e.g., a dual-laser flow-based detection instrument, such as the Luminex 200™ or Bio-Rad® Bio-Plex® analyzer. One laser classifies the bead and determines the analyte that is detected. The second laser determines the magnitude of the signal derived from the detectable label. The signal from the detectable label is generally proportional to the amount of bound analyte. For example, the analyte can be a lymphokine such as, e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL- 11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, and/or, IL-36, and variants thereof. Examples of cytokines and other agents that can be used as the analyte include, e.g., granulocyte-colony stimulating factor, macrophage-colony stimulating factor, granulocyte-macrophage colony stimulating factor, leukemia inhibitory factor, erythropoietin, granulocyte macrophage colony stimulating factor, oncostatin M, leukemia inhibitory factor, IFN-alpha, -beta, or -gamma, lymphotoxin -alpha or -beta, CD40 ligand, Fas ligand, CD27 ligand, CD30 ligand, 4-1BBL, TGF-alpha or -beta, IL-1 RA, macrophage inhibitory factor, and/or a mixture thereof. The beads can be color-coded or comprise a detectable label or bar code that identifies the bead. Different beads can be coated with either dockerin or cohesin and a specific analyte, bound to the other half of the dockerin-cohesin pair, is added to the coated bead to make the bead specific to a single analyte.

[00113] Example of sequence encoding Cl .C2.C3.Cn is taken from the public sequence >gi|50656899|gb|AAT79550.1 | of cellulosomal anchoring scaffoldin B precursor (Bacteroides cellulosolvens). Below in bold showing the leader secretion sequence and italics and bold-underlined highlighting various cohesin domains. Italics-underlined regions are linkers spacing some of the cohesin domains. The skilled artisan will easily be able to make one or more of these domains as individual domains, as concatamers, as multimers and to provide for isolation of the same, e.g., by adding recognition domains such as His-tags and the like. Nucleic acid sequences that encode the domains (cohesin or dockerin) can easily be synthesized and customized to the specific expression system used, e.g., bacterial, fungal, helminthic or mammalian.

[00114] MQSPRLKRKILSVILAVCYIISSFSIQFA^JPeWV/HG^eG/ G^

TVKVPINLQNVPEIGINNCDFTIKFDSDILDFNSVEAGDIVPLPVASFSSNNSKDIIKFLFSDA TQGNMPINENGLFA VISFKIKDNA QKGISNIKVSSYGSFSGMSGKEMQSLSPTFFSGSID VS D S7¾T LDVKVGNVEGIAGTEVNVPITFENVPDNGINNCNFTLSYDSNALEFLTTE AGNIIPLAIADYSSYRSMEGKIKFLFSDSSOGTRSIKNDGVFANIKFKIKGTV^/RDr YRIDLSELGSFSSKONNNLKSIATQFLSGSVNVKDIESSVSPTTSVHPTPTSVPPTPTKSSP GNKMKIQIGD VKANOGD TVIVPITFNE VP VMG VNNCNFTLA YDKNIMEFISADA GDIVTLP MANYSYNMPSDGL VKFL YNDQA QGAMSIKEDGTFANVKFKIKQSAAFGKYSVGIKAIGSIS ALSNSKLIPIESIFKDGSITVTNKPIVN iGK\K\KAGOKlK\r\ElKOIPSIG NCm TLKYNSNVLKYVSNEAGTIVPAPLANLSINKPDEGIIKLLFSDASOGGMPIKDNGI FVNLEFOAVNDANIGVYGLELDTIGAFSGISSAKMTSIEPQFNTVGS/^/ NS^Or K PSNTEVQTPTNTISVTPTNNSTPTNNSTPKPNPL YNLNVNIGEISGEA GG VIEVPIEFKNVPD FGINNCDFSVKYDKSIFEYVTYEA GSIVKDSIVNLA CMENSGIINLLFNDA TQSSSPIKNNG V FAKLKFKINSNAASGTYQINAEGYGKFSGNLNGKLTSINPIFENGIINIGNVTVKPTSTPADS STITPTA TPTA TPTIKGTPTVTPIYWMNVL1GNMNAA1GEE VWPIEFKNVPPFGINNC DFKLVYDSNALELKKVEAGDIVPEPLANLSSNKSEGKIOFLFNDASOGSMQIENG GVFAKITFKVKSTAASGIYNIRKDSVGSFSGLIDNKMTSIGPKFTDGSIWGrrTPr A TA TPSAIVTTITPTA TTKPIA TPTIKGTPTA TPMYWMNWIGKMNAEVGGEVWPIEFNNV PSFGINNCDFKL VYDA TALELKNVEA GDIIKTPLANFSNNKSEEGKISFLFNDASQGSMQIE NGG VFAKITFKVKSTTA TG VYDLRKDL VGSFSGLKDNKMTSIGAEFTNGSITVAA TAPTVTP TVNA TPSAA TPTVTPTA TA TPSVTIPTVTPTA TA TPSVTIPTVTPTA TA TPSAA TPTVTPTA TA T PSVTIPTVTPTVTA TPSDTIPTVTPTA TA TPSAIVTTITPTA TAKPIA TPTIKGTPTA 7PMYWM NWIGKMNAEVGGEVWPIEFKNVPSFGINNCDFKLVYDATALELKNVEAGDIIK TPLANFSNNKSEEGKISFLFNDASOGSMQIENGGVSAKITFKVKSTTAIGVYDIRK DLIGSFSGLKDSKMTSIGAEFTNGSI7>¾ TTAPTVTPTA TA TPSVTIPTVTPTA TA TPGTA TPGTA TPTA TA TPGAA TPTETA TPSVMIPTVTPTA TA TPTA TA TPTVKGTPTIKP VYKMNVVI GR VNWA GEEVVVP VEFKNIPAIG VNNCNFVLEYDANVLEVKKVDA GEIVPDALINFGSNN SDEGKVYFLFNDALQGRMQIANDGIFANITFKVKSSAAA GIYNIRKDSVGAFSGL VDKL VPI

^E r GfflSrE&^ TSTPTATATGTNVTPTVAATVTPTATPASTTPTATPTATSTVK GTPTATPLYSMNVIIGKVNAEASGEWVPVEFKDVPSIGINNCNFILEYDASALEL DSAEAGEIVPVPLGNFSSNNKDEGKIYFLFSDGTOGRMQIVNDGIFAKIKFKVKS TASDGTYYIRKDSVGAFSGLIEKKIIKIGAEFTDGSITVRSLTPTPTVTPNVASPTPT KWAEPTSNQPAGPGPITGTIPTATTTATATPTKASVATATPTATPIVWEPTIVRPGY NKDADLAVFISSDKSRYEESSIITYSIEYKNIGKVNATNVKIAAQIPKFTKVYDAAKGA VKGSEIVWMIGNLAVGESYTKEYKVKVDSLTKSEEYTDNTVTISSDQTVDIPENITTG NDDKSTIRVMLYSNRFTPGSHSSYILGYKDKTFKPKQNVTRAEVAAMFARIMGLTVK DGAKSSYKDVSNKHWALKYIEAVTKSGIFKGYKDSTFHPNAPITRAELSTVIFNYLHL NIAPSKVHFTDINKHWAKNYIEEIYRFKLIQGYSDGSFKP NNITRAEWTMINRML YRGPLKVKVGSFPDVSPKYWAYGDIEEASRNHKYTRDEKDGSEILIE (SEQ ID NO: 1) [00115] The cohesin domains (C) interact with small domains (e.g., 56 residues) called dockerins (D). These are Ca⁺⁺ containing structures with two-fold symmetry and they can bind to a cognate cohesin with various affinities (e.g., 6E6 M, 2E7M). Affinities between dockerin and multiple cohesins (as found on scaffoldins) can be much higher (e.g., >E9 M). The interaction is non-covalent and is well defined (by structure analysis) for at least one C-D pair. Dockerins are designed to be domains linked to different domain (enzyme in nature), and cohesins are designed to function in linear arrays (either directly end-to-end, or joined by flexible PT-rich linkers of various sizes (e.g., 12, 17, 25, 28, 36). It is known that a particular dockerin can have specificity for a particular cohesin (e.g., a C-D pair from one bacterial species may not be interchangeable with a C-D pair from a different species). This feature makes it is possible to ensure the specific and precise interaction of various D-antigen fusion proteins with an engineered mAb containing cohesin domains of various specificities.

[00116] Dockerin: GDVNDDGKVNSTDLTLLK YVLKAVSTLPSSKAEKNADVNRDGRVDVTILSRYLIR VIEKLPI (SEQ ID NO: 2).

[00117] In practice, this invention includes adapting C-D pairs known from the literature, newly gleamed from nature, or developed with new specificities using phage display technology. The latter technology can also be used to enhance ('mature') the affinity of a C-D interaction, should this be desired. Also, engineering cysteine residues at opposing faces of the C-D interaction (based on modeling from the published C-D structures) could be used to make a covalent bond between C-D to strengthen the interaction. Furthermore, the dimeric nature of the mAb (and therefore the linked C-domains) can be used to advantage for affinity enhancement purposes. In this embodiment, e.g., the D-antigen fusion protein is engineered either with a second identical dockerin domain (D-antigen-D, or D-D-antigen), or with a homodimerization domain. This configuration, provided the linkers between domains were not constraining, will result in the preferred simultaneous binding of both D domains to the same mAb, with greatly enhanced stability compared to the single interaction. [00118] Based on the crystal structure of the cohesin-dockerin complex (e.g., see Carvalho, et al., 2003), it is apparent that one embodiment is an antigen-dockerin fusion proteins (i.e., antigen fused to the N-terminus of a dockerin). However, both from the structure and from the nature of cohesin domain organization within scaffoldins, it is apparent that cohesins can be fused end-to-end, even without spacer sequences. Furthermore, it is apparent that well-described techniques are available to engineer miniaturized versions of the cohesin and dockerin domains (see for example, Starovasnik, et al, 1997).

[00119] It is recognized herein that the linker sequences have a propensity for

O-linked glycosylation resulting from ST richness. Also, both the C and D domains can have potential N-linked sites. These features can be advantageous in enhancing the solubility of the mammalian cell-expressed engineered mAb through decoration with carbohydrates. Of course, the consequences of glycosylation of the C domains needs to be check by function (binding to the cognate D), and if needed rectified by site directed mutagenesis. An attractive feature of this invention is that D-A can be expressed in whatever system is known to be best. The dockerin domain is below. [00120] The present inventors have developed a flexible, sensitive, accurate, high throughput, sample sparing, multiplexed bead-based assay for the simultaneous measurement of antibodies against multiple antigens. This is represented schematically in FIGS. 1A and IB.

[00121] FIGS. 1A and IB show that cohesin and dockerin are protein modules from cellulose-degrading bacteria that bind non-covalently with very high affinity (K_D ~ 30 pM). In FIG. 1A a set of up to 100 different fluorescent microspheres (e.g., Luminex® beads) are cross-linked chemically to a cellulose-binding domain fused to dockerin (CBD- Doc). Each cohesin-antigen fusion protein is mixed to CBD-Doc beads of a particular color (e.g., bead region 21). The beads are washed and pooled with CBD-Doc beads similarly coated with other desired cohesin-antigen fusion proteins. In FIG. IB the bead-antigen sets are incubated with a dilution series of each serum sample, washed, incubated with e.g., anti- human IgG-PE conjugate, washed again, then read in a flow cytometry instrument which measures fluorescence intensity associated with each bead color.

[00122] A sample protocol for confirmation of antibody coupling is presented herein below. Microspheres should be protected from prolonged exposure to light throughout this procedure, (i). Select the appropriate antibody-coupled microsphere sets.

(ii) . Resuspend the microspheres by vortex and sonication for approximately 20 seconds.

(iii) . Prepare a Working Microsphere Mixture by diluting the coupled microsphere stocks to a final concentration of 100 microspheres of each set/μΕ in PBS-1% BSA. (Note: 50 of Working Microsphere Mixture is required for each reaction. Either PBS-1% BSA or PBS-BN (PBS, 1 % BSA, 0.05% Azide, pH 7.4) may be used as Assay Buffer. Not using Azide.

(iv) . Prepare two-fold serial dilutions of phycoerythrin-labeled anti-species IgG detection antibody from 4 to 0.0625 μg/mL in PBS-1% BSA. (Note: 50 μΕ of diluted detection antibody is required for each reaction.)

(v) . Pre-wet a 1.2 μιη Millipore filter plate with 100 μΕΛνεΙΙ of PBS-1% BSA and aspirate by vacuum manifold.

(vi) . Aliquot 50 μΐ_^ of the Working Microsphere Mixture into the appropriate wells of the filter plate.

(vii) . Add 50 μί of the diluted detection antibody into the appropriate wells of the filter plate.

(viii) . Mix the reactions gently by pipetting up and down several times with a multi-channel pipettor. (ix) . Cover the filter plate and incubate for 30 minutes at room temperature on a plate shaker, (let go 1.5 hours)

(x) . Aspirate the supernatant by vacuum manifold

(xi) . Wash each well twice with 100 of PBS- 1% BSA and aspirate by vacuum manifold. (xii). Resuspend the microspheres in 100 μΙ_, of PBS- 1% BSA by gently pipetting up and down five times with a multi-channel pipettor.

(xiii). Analyze 50-75 on the Luminex analyzer according to the system manual.

[00123] This multiplex assay format described hereinabove is very robust.

Samples rerun independently give virtually identical results. Bead sets are monitored to ensure equivalent loading of each Cohesin-antigen fusion protein, thereby enabling direct comparison of titers against comparable antigens (e.g., HA-1 from PR8 vs. H1N1 swine flu). Data for cross-reactive antigens are equivalent if the related antigens are run separately, validating the use of the multiplexed format to derive this data.

[00124] Also, there is no detectable exchange of Cohesin-antigens between bead sets during the assay. The Dockerin-Cohesin binding requires 8M guanidine to disrupt the interaction and survives in strong acid and base conditions. The assay described herein is at least 10-fold more sensitive than a standard solid phase ELISA using the same antigen. FIG. 2 shows the comparison of serum HIV Gag p24-specific antibody titers in patients acquired by bead assay or conventional ELISA using commercial reagents. FIG. 3 shows the influenza antigen-specific IgG levels in healthy donors tested by the bead assay of the present invention. Typically serum dilution series start at 1 : 10 and use 20 μΐ total serum. Because the antigen is coupled to beads via the Dockerin-Cohesin interaction, all antigen epitopes are available for binding antibody, uncompromised by non-specific plate binding or chemical cross-linking effects. [00125] Recombinant human IgG-Cohesin fusion protein can be run as an internal standard. For e.g., to configure and execute antigen-specific assays for the Influenza antigens Ml (PR8), NP (PR8, H1N1 swine flu, H1N1 avian flu), M2e (PR8, H1N1 swine Flu), HA-1 (PR8, H1N1 avian flu, H1N1 swine flu, and seasonal flu strains). Pure proteins, and E. coli expression constructs or stably transfected CHO-S cell lines are already established along with validated purification protocols for all these antigens including HA-1 proteins from new seasonal flu strains. [00126] In another example, isotype-specific antigen-specific assay can be done in the context of the multiplexed bead assay. The current standard assay configuration uses a pan anti-IgG Fc reagent (couples to PE for fluorescent read-out) to detect bound antibodies. The present invention provides the capacity to detect Ig subclass-specific antibodies by adapting commercially available isotype-specific detection reagents (e.g., anti- IgM-PE, anti-IgA-PE). To this end, the present inventors have made a panel of recombinant antibodies by systematically grafting H chain constant regions of each isotype onto the same variable region specificity (e.g., from an existing anti-human CD40 antibody construct). These reagents permit the assembly of a fully matched antibody-coated multiplexed bead set for the same antigen-specificity (e.g., beads coated with Cohesin-CD40 ectodomain fusion protein and then bound to saturation with the anti-CD40 antibody of each isotype. This permits sensitive and accurate appraisal and quality assurance of new isotype-specific detection reagents. It has been found that commercially available isotype-specific detection reagents are of mixed quality, so the set-up of the present invention allows strict quality control of these reagents for cross-reactivity.

[00127] The present invention enables quantifying antigen specific blood B cells based on the same set of Cohesin-antigen and CBD-Dockerin reagents that have been successfully deployed hereinabove for antigen-specific multiplexed bead assay. This also obviates the need for making new protein reagents. [00128] Reagents for capture and detection of antigen-specific B cells are shown in FIG. 4. Beads displaying a Cohesin-antigen fusion protein (Ag-1) can be used to enrich antigen-specific B cells from ex vivo samples, while antigen tetramers assembled via streptavidin-phycoerythrin (SA-PE) : biotin-CBD-Doc : Cohesin-antigen complex can be uses in flow cytometry to sort or quantify antigen- specific B cells. The antigen-bead complexes can also be assembled on biotin-coated Q dots of different, permitting the possible development of multiplexed flow analysis of several antigen- specific B cell categories. Furthermore, the combinatorial strategy of mixing tetramers for each specificity but with two different colors (Newell, et ah, 2009) can be readily applied to our antigen tetramers to reduce background staining and increase the number of antigens that can be tested simultaneously.

[00129] FIG. 5 demonstrates the method of the present invention using tetramers made of Cohesin-Dockerin, which can recognize receptors expressed on cell surface. The plot shown in FIG. 5 is a multiplex bead-based assay of antigen-specific serum antibodies from a Influenza-infected non-human primate. Three different HA antigens are represented. As well as Ml, M2 ectodomain, NP, and NA.

[00130] FIG. 5 demonstrates the capacity of the multiplex bead-based assay to simultaneously measure both antigen-specific titers (by limiting dilution, or the highest dilution to give a measurable response, but also cross-reactivity. For example, this sera contains a high titer of antibodies specific to HAl (swine flu) shown in green squares (this animal was infected with swine flu and had no detectable anti-flu antigen antibodies before the infection), but a lower titer on antibodies that are cross-reactive to a different HAl shown in red. The FI plateaus also reveals the complexity of the antibody response (a more complex response reflects a larger number of epitopes on the antigen were bound).

[00131] FIGS. 6A-6C provides an example of a multiplex bead antigen-specific

IgG assay. In FIG. 6A a serum sample from an influenza-infected monkey was diluted and mixed with a bead set coated with cohesin fused to various Influenza proteins NP5, nuclear protein; HAl PR8, HAl domain from hemagglutinin of the PR8 strain; HAl SF, HAl domain from hemagglutinin of the swine flu strain (CAL04); HA3, HAl domain from hemagglutinin a H3 Influenza strain; M2e, the ectodomain of the M2 protein from PR8; Ml, matrix protein 1; Coh, Cohesin without antigen. FIGS. 6B and 6C compares the serum from the monkey before (FIG. 6B) and after vaccination (FIG. 6C) with a vaccine bearing HAl from PR8.

[00132] C191 Ecoli-pET28[6xHis-CBD-Dockerin]

[00133] ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCT GGTGCCGCGCGGCAGCCATATGGCTAGTGGCAATGCAACACCGACCAAGGGAGC AACACCAACAAATACAGCTACGCCGACAAAATCAGCTACGGCTACGCCCACCAG GCCATCGGTACCGACAAACACACCGACAAACACACCGGCAAATACACCGGTATC AGGCAATTTGAAGGTTGAATTCTACAACAGCAATCCTTCAGATACTACTAACTCA ATCAATCCTCAGTTCAAGGTTACTAATACCGGAAGCAGTGCAATTGATTTGTCCA AACTCACATTGAGATATTATTATACAGTAGACGGACAGAAAGATCAGACCTTCTG GTGTGACCATGCTGCAATAATCGGCAGTAACGGCAGCTACAACGGAATTACTTC AAATGTAAAAGGAACATTTGTAAAAATGAGTTCCTCAACAAATAACGCAGACAC CTACCTTGAAATAAGCTTTACAGGCGGAACTCTTGAACCGGGTGCACATGTTCAG ATACAAGGTAGATTTGCAAAGAATGACTGGAGTAACTATACACAGTCAAATGAC TACTCATTCAAGTCTGCTTCACAGTTTGTTGAATGGGATCAGGTAACAGCATACT TGAACGGTGTTCTTGTATGGGGTAAAGAACCCGGTGGCAGTGTAGTACCATCAAC ACAGCCTGTAACAACACCACCTGCAACAACAAAACCACCTGCAACAACAAAACC ACCTGCAACAACAATACCGCCGTCAGATGATCCGAATGCAGCTAGCAATTCTCCT CAAAATGAAGTACTGTACGGAGATGTGAATGATGACGGAAAAGTAAACTCCACT GACTTGACTTTGTTAAAAAGATATGTTCTTAAAGCCGTCTCAACTCTCCCTTCTTC CAAAGCTGAAAAGAACGCAGATGTAAATCGTGACGGAAGAGTTAATTCCAGTGA TGTCACAATACTTTCAAGATATTTGATAAGGGTAATCGAGAAATTACCAATATAA

(SEQ ID NO: 3)

[00134] MGSSHHHHHHSSGLVPRGSHMASGNATPT GATPTNTATPT S ATATPTRPSVPTNTPTNTPANTPVSGNL VEFYNSNPSDTTNSINPQF VTNTGSSAID LSKLTLRYYYTVDGOKDQTFWCDHAAIIGSNGSYNGITSNVKGTFVKMSSSTNNAD TYLEISFTGGTLEPGAHVQIQGRFAKNDWSNYTQSNDYSF SASQFVEWDQVTAYL NGVLVWGKEPGGSWPSTQPVTTPPATTKPPATTKPPATTIPPSDDPNAASNSPONEV LYGDVNDDGKVNSTDLTLLKRYVLKAVSTLPSSKAEKNADVNRDGRVNSSDVTI LSRYLIRVIEKLPI (SEQ ID NO: 4)

[00135] SEQ ID NO: 3 represents a cellulose binding domain - dockerin domain fusion protein that is efficiently expressed in E. coli [using the pET28 vector system] as an abundant and soluble intracellular protein. Underlined region is GENE ID: 4809951 Cthe_3077 | cellulosome anchoring protein, cohesin region [Clostridium thermocellum ATCC 27405] residues 322-561; Residues indicated in bold are sp|P0C2S4.1 |GUND_CLOTM RecName: FulHEndoglucanase D residues 551-625.

[00136] For production of this protein, pET28[6xHis-CBD-Dockerin] plasmid in a suitable E. coli strain is grown in L broth to mid-log phase, induced with IPTG for ~3 hours, then harvested by centrifugation. Cells are broken e.g., by sonication, in 50 mM Tris.HCl pH 7.5 buffer with 1 mM EDTA and a cocktail of standard protease inhibitors, then clarified by centrifugation (SS34 rotor, 14,500 r.p.m. 20 min). The supernatant is passed through Q sepharose, adjusted to 50 mM NaH₂P0₄, pH 8.0; 300 mM NaCl; 10 mM imidazole, and then loaded onto a Ni⁺⁺ charged metal chelating column, washed with 50 mM NaH₂P0₄, pH 8.0; 300 mM NaCl; 20 mM imidazole buffer containing 0.5% ASB14 to lower LPS levels, then eluted with gradient to 50 mM NaH²P0⁴, pH 8.0; 300 mM NaCl; 250 mM imidazole. Fractions containing eluted protein are pooled and buffer exchanged into 10 mM Borate pH 7.4. This protein retains both cohesin binding and (likely) cellulose binding activity.

[00137] The present invention can also be modified to multiplexed protein array format. For example, using a modification of a method described in Versatile protein microarray based on carbohydrate -binding modules (Ofir, et ah, 2005). Here the cohesin- antigen proteins can be simply spotted on a surface pre-coated with a dockerin domain- containing protein, which is chemically cross-linked to the surface, or if CBD.Doc, attached by non-covalent means to a cellulose coated surface.

[00138] Table 1 presents a list of examples of cohesin-antigen fusion proteins that were successfully expressed either in E. coli, or in mammalian cell systems by the present inventors. Since the cohesin portion is robust and can be functionally expressed as a secreted product in mammalian cells, or as a soluble, or refolded, product from E. coli this brings versatility since the best system for abundantly expressed and properly folded antigen fusion partner can be selected. The sequences corresponding to the cohesin-antigen fusion proteins are also presented herein below.

[00139] Table 1 : Cohesin- Antigen fusion proteins expressed either in E.coli or in mammalian cell systems.

NUMBER/

ANTIGEN CONSTRUCT NAME SEQ ID NO:

Cancer Ecoli-pET28 [Cohesin-hCyclinD 1 -6xHis] C515/13

Cancer Ecoli-pET28[Cohesin-hgpl00-PeptideA-6xHis] C180/ 14

Cancer Ecoli-pET28[Cohesin-hMART-l-PeptideB-6xHis] C181 / 15

Mam-cetHS-puro[SLAML-6xHis-Cohesin-hNY-ESO-

Cancer l-6xHis] C1179 / 16

Cancer Mam-cetHS-puro[SLAML-6xHis-Cohesin-hPSA] C1163/17

Cancer Mam-cetHS-puro[SLAML-Cohesin-hSurvivin-6xHis] C875/ 18

Cancer Mam-pCDM8 [SLAML-Cohesin-hgp 100] C294/ 19

Control Ecoli-pET28 [Cohesin-6xHis] C21 /20

Control Ecoli-pET28[Cohesin] C189/21

Hepatitis C Ecoli-pET28[6xHis-CthermoCohesin-HCV-NS5B- Virus (HCV) Palm] C1843 /22

Ecoli-pET28[6xHis-CthermoCohesin-HCV-

HCV NS5B(l+2)] C1641 /23

HCV Ecoli-pET28[6xHis-CthermoCohesin-hHCVElb] C1920/24

HCV Ecoli-pET28[6xHis-CthermoCohesin-hHCVE2] C1923 /25

Ecoli-pET28[6xHis-CthermoCohesin-

HCV ViralHCVprotease] C1662/26

HCV Ecoli-pET28[CthermoCohesin-ViralHCVhelicase] C1664/27

HIV Ecoli-pET28 [6xHis-Cohesin-Pep-gag 17] C1079/28

HIV Ecoli-pET28 [6xHis-Cohesin-Pep-gag253 ] C1080/29

HIV Ecoli-pET28[6xHis-Cohesin-Pep-nefl 16] C1078/30

HIV Ecoli-pET28[6xHis-Cohesin-Pep-nef66] C1077/31

HIV Ecoli-pET28 [6xHis-Cohesin-Pep-pol 158] C1081 /32

HIV Ecoli-pET28 [6xHis-Cohesin-Viralgag-p 17-6xHis] Cllll /33

HIV Mam-cetHS-puro[SLAML-Cohesin-Viralgag-6xHis] C492 / 34 NUMBER/

ANTIGEN CONSTRUCT NAME SEQ ID NO:

HIV Mam-cetHS-puro[SLAML-Cohesin-ViralNef-6xHis] C879 / 35

Human

Papilloma Virus

(HPV) Ecoli-pET28[6xHis-Cohesin-HPV16E6] C1435 / 36

HPV Ecoli-pET28[6xHis-Cohesin-HPV16E7] C1436 / 37

HPV Ecoli-pET28[6xHis-Cohesin-HPV16L2] C1775 / 38

HPV Ecoli-pET28[6xHis-Cohesin-HPV18E7] C1469 / 39

Ecoli-pET28[6xHis-CthermoCohesin-HPV16E6-

HPV HPV16E7] C1463 / 40

Ecoli-pET28[6xHis-CthermoCohesin-HPV18E6-

HPV HPV18E7] C1510 / 41

HPV Ecoli-pET28[6xHis-CthermoCohesin-HPV18E6] C1496 / 42

Influenza Ecoli-pET28 [6xHis-Cohesin-FluHA 1 - 1 c] C2096 / 43

Influenza Ecoli-pET28[6xHis-Cohesin-FluHA3-lk] C2039 / 44

Influenza Ecoli-pET28[6xHis-CthermoCohesin-FluNA-6xHis] C1699 / 45

Influenza Ecoli-pET28 [Cohesin-FluM 1 -6xHis] C32 / 46

Influenza Ecoli-pET28 [CthermoCohesin-FluHAl - 1 s-6xHis] C1887 / 47

Influenza Ecoli-pET28[CthermoCohesin-FluNP-5-6xHis] C943 / 48

Influenza Mam-cetHS-puro[6xHis-Cohesin-FluHAl-lu] C1996 / 49

Influenza Mam-cetHS-puro[6xHis-Cohesin-FluHA3-lk] C1998 / 50

Influenza Mam-cetHS-puro[6xHis-Cohesin-FluNP-ls] C2043 / 51

Mam-cetHS-puro[Cohesin-Flex-vl -FluM2- 1 s-M2e-

Influenza FluM2-l-M2e] C2254 / 52

Influenza Mam-cetHS-puro[HAl-0-S-Cohesin-6xHis] C1819 / 53

Influenza Mam-cetHS-puro[SLAML-6xHis-Cohesin-FluHAb- 1 ] C2297 / 54

Influenza Mam-cetHS-puro[SLAML-6xHis-Cohesin-FluM2- 1 s] C1734 / 55

Influenza Mam-cetHS-puro[SLAML-6xHis-Cohesin-M2e] C1977 / 56 NUMBER/

ANTIGEN CONSTRUCT NAME SEQ ID NO:

Influenza Mam-cetHS-puro[SLAML-Cohesin-FluHAl - 1 -6xHis] C489 / 57

Influenza Mam-cetHS-puro[SLAML-Cohesin-FluHA5-0-6xHis] C543 / 58

Influenza Mam-cetHS-puro[SLAML-Cohesin-FluHA5- 1 -6xHis] C490 / 59

SIV Mam-cetHS-puro[6xHis-Cohesin-ViralgagSIV-p 17] C1274 / 60

SIV Mam-cetHS-puro[6xHis-Cohesin-ViralgagSIV-p24] C1273 / 61

SIV Mam-cetHS-puro[6xHis-Cohesin-ViralnefSIV] C1275 / 62

Mam-cetHS-puro[SLAML-6xHis-Cohesin-

SIV ViralenvSIV-gp41 -6xHis] CI 193 / 63

Tuberculosis Ecoli-pET28 [6xHis-Cohesin-m.tbCFP 10] C1254 / 64

Tuberculosis Ecoli-pET28 [6xHis-Cohesin-m.tbRvO 125] C1334 / 65

Tuberculosis Ecoli-pET28[6xHis-Cohesin-m.tbRv0570] C1500 / 66

Tuberculosis Ecoli-pET28[6xHis-Cohesin-m.tbRv0577] C1335 / 67

Tuberculosis Ecoli-pET28[6xHis-Cohesin-m.tbRvl626] C1256 / 68

Tuberculosis Ecoli-pET28[6xHis-Cohesin-m.tbRv2875] C1310 / 69

Tuberculosis Ecoli-pET28[6xHis-Cohesin-m.tbRv3044] C1312 / 70

Tuberculosis Ecoli-pET28[6xHis-Cohesin-m.tbRv3478] C1308 / 71

Ecoli-pET28[6xHis-CthermoCohesin-

Tuberculosis m.tbAg85BDel41] C2200 / 72

Tuberculosis Ecoli-pET28[6xHis-CthermoCohesin-m.tbMtb72f] C2236 / 73

Tuberculosis Ecoli-pET28[6xHis-CthermoCohesin-m.tbRv0288] C2202 / 74

Tuberculosis Ecoli-pET28[6xHis-CthermoCohesin-m.tbRv0475] C2199 / 75

Tuberculosis Ecoli-pET28[6xHis-CthermoCohesin-m.tbRvl980] C2197 / 76

[00140] Autoantigen: Ecoli-pET28[Cohesin-hGadB-6xHis] C914 [00141] MDPKGSLSWRILLFLSLAFELSYGLDDLDAVRIKVDTVNAKPGDT VRIPVRFSGIPSKGIANCDFVYSYDPNVLEIIEIEPGDIIVDPNPDKSFDTAVYPDRKIIVF LFAEDSGTGAYAITKDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQF FDGGVNVGDTTEPATPTTPVTTPTTTDDLDALEASQIGNMISIWVSHSIHTGNQHQSE PISNTNFLTEKAVASVKLAGNSSLCPINGWAVYSKDNSIRIGSKGDVFVIREPFISCSHL ECRTFFLTQGALLNDKHSNGTVKDRSPHRTLMSCPVGEAPSPYNSRFESVAWSASAC HDGTSWLTIGISGPDNGAVAVLKYNGIITDTIKSWRNNILRTQESECACVNGSCFTVM TDGPSNGQASHKIFKMEKGKWKSVELDAPNYHYEECSCYPNAGEITCVCRDNWHG SNRPWVSFNQNLEYQIGYICSGVFGDNPRPNDGTGSCGPVSSNGAYGVKGFSFKYGN GVWIGRTKSTNSRSGFEMIWDPNGWTETDSSFSVKQDIVAITDWSGYSGSFVQHPEL TGLDCIRPCFWVELIRGRPKESTIWTSGSSISFCGVNSDTVGWSWPDGAELPFTIDKH HHHHH (SEQ ID NO: 5)

[00142] Bacterial:Ecoli-pET28[6xHis-Cohesin-Flgn-l-Flgn-2] C1036

[00143] MGSSHHHHHHSSGLVPRGSHMASIERLSSGLRINSAKDDAAGQAIA NRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQS ANSTNSQSDL DSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTL GLDSLNVQASQPELAEAAAKTTENPLQKIDAALAQVDALRSDLGAVQNRFNSAITNL GNT VNNL SE ARSRIED SD Y ATE VSNM SRAQILQ AS (SEQ ID NO: 6)

[00144] Bacterial toxin: Ecoli-pET28[Cohesin-P.aeruginosaPE38] C340 [00145] MDLDAVRIKVDTVNAKPGDTVNIPVRFSGIPSKGIANCDFVYSYDP

NVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIVFLFAEDSGTGAYAITKDGVFATIV AKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQFFDGGVNVGDTTEPATPTTPVTTP TTTDDLDAASEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVAL YLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQ GTGNDEAGAANGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQ AHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYG YAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTSLTLAAPEAAGEVERLIGHPLPLR LDAITGPEEEGGRLETILGWPLAERTWIPSAIPTDPRNVGGDLDPSSIPDKEQAISALP DYASQPGKPPREDLK (SEQ ID NO: 7) [00146] Cancer Ecoli-pET28[6xHis-CthermoCohesin-hADH3] C1328 [00147] MGSSHHHHHHSSGLVPRGSHMASMDLDAVRIKVDTVNAKPGDTV NIPVRFSGIPSKGIANCDFVYSYDPNVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIV FLFAEDSGTGAYAITKDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQ FFDGGVNVGDTTEPATPTTPVTTPTTTDDLDAASELEVRRVRQAFLSGRSRPLRFRLQ QLEALRRMVQEREKDILTAIAADLCKSEFNVYSQEVITVLGEIDFMLENLPEWVTAK PVKKNVLTMLDEAYIQPQPLGVVLIIGAWNYPFVLTIQPLIGAIAAGNAVIIKPSELSE NTAKILAKLLPQYLDQDLYIVINGGVEETTELLKQRFDHIFYTGNTAVGKIVMEAAA KHLTPVTLELGGKSPCYIDKDCDLDIVCRRITWGKYMNCGQTCIAPDYILCEASLQN QIVWKIKETVKEFYGENIKESPDYERIINLRHFKRILSLLEGQKIAFGGETDEATRYIAP TVLTDVDPKTKVMQEEIFGPILPIVPVKNVDEAINFINEREKPLALYVFSHNHKLIKRM IDETSSGGVTGNDVIMHFTLNSFPFGGVGSSGMGAYHGKHSFDTFSHQRPCLLKSLK REGANKLRYPPNSQSKVDWGKFFLLKRFNKEK (SEQ ID NO: 8)

[00148] Cancer: Ecoli-pET28[6xHis-CthermoCohesin- hHomosapienssyndecanbinding protein] CI 327 [00149] MGSSHHHHHHSSGLVPRGSHMASMDLDAVRIKVDTVNAKPGDTV

NIPVRFSGIPSKGIANCDFVYSYDPNVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIV FLFAEDSGTGAYAITKDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQ FFDGGVNVGDTTEPATPTTPVTTPTTTDDLDAASSLYPSLEDLKVDKVIQAQTAFSAN PANPAILSEASAPIPHDGNLYPRLYPELSQYMGLSLNEEEIRANVAWSGAPLQGQLV ARPSSINYMVAPVTGNDVGIRRAEIKQGIREVILCKDQDGKIGLRLKSIDNGIFVQLVQ ANSPASLVGLRFGDQVLQINGENCAGWSSDKAHKVLKQAFGEKITMTIRDRPFERTI TMHKDSTGHVGFIFKNGKITSIVKDSSAARNGLLTEHNICEINGQNVIGLKDSQIADIL STSGTWTITIMPAFIFEHIIKRMAPSIMKSLMDHTIPEV (SEQ ID NO: 9)

[00150] Cancer: Ecoli-pET28[6xHis-CthermoCohesin-hLOC552889] C1326 [00151] MGSSHHHHHHSSGLVPRGSHMASMDLDAVRIKVDTVNAKPGDTV

NIPVRFSGIPSKGIANCDFVYSYDPNVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIV FLFAEDSGTGAYAITKDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQ FFDGGVNVGDTTEPATPTTPVTTPTTTDDLDAASEEISLANLDTNKLEAIAQEIYVDLI EDSCLGFCFEVHRAVKCGYFYLEFAETGSVKDFGIQPVEDKGACRLPLCSLPGEPGN GPDQQLQRSPPEFQ (SEQ ID NO: 10)

[00152] Cancer: Ecoli-pET28[Cohesin-hCyclinBl-vl-6xHis] C708 [00153] MDLDAVRIKVDTVNAKPGDTVNIPVRFSGIPSKGIANCDFVYSYDP NVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIVFLFAEDSGTGAYAITKDGVFATIV AKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQFFDGGVNVGDTTEPATPTTPVTTP TTTDDLDAASLEPEPEPEPEPVKEEKLSPEPILVDTASPSPMETSGCAPAEEDLCQAFS DVILAVNDVDAEDGADPNLCSEYVKDIYAYLRQLEEEQAVRPKYLLGREVTGNMRA ILIDWLVQVQMKFRLLQETMYMTVSIIDRFMQNNCVPKKMLQLVGVTAMFIASKYE EMYPPEIGDFAFVTDNTYTKHQIRQMEMKILRALNFGLGRPLPLHFLRRASKIGEVDV EQHTLAKYLMELTMLDYDMVHFPPSQIAAGAFCLALKILDNGEWTPTLQHYLSYTE ESLLPVMQHLAKNWMVNQGLTKHMTVKNKYATSKHAKISTLPQLNSALVQDLAK AVAKVHHHHHH (SEQ ID NO : 11 )

[00154] Cancer: Ecoli-pET28[Cohesin-hCyclinBl-v4-6xHis] C736

[00155] MDLDAVRIKVDTVNAKPGDTVNIPVRFSGIPSKGIANCDFVYSYDP NVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIVFLFAEDSGTGAYAITKDGVFATIV AKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQFFDGGVNVGDTTEPATPTTPVTTP TTTDDLD AASLEMALRVTRNSKINAENKAKINMAGAKRVPTAPAATSKPGLRPRTA LGDIGNKVSEQLQAKMPMKKEAKPSATGKVIDKKLPKPLEKVPMLVPVPVSEPVPEP EPEPEPEPVKEEKLSPEPILVDTASPSPMETSGCAPAEEDLCQAFSDVILAVNDVDAED GADPNLCSEYVKDIYAYLRQLEEEQAVRPKYLLGREHHHHHH (SEQ ID NO: 12)

[00156] Cancer: Ecoli-pET28[Cohesin-hCyclinDl-6xHis] C515 [00157] MDLDAVRIKVDTVNAKPGDTVNIPVRFSGIPSKGIANCDFVYSYDP

NVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIVFLFAEDSGTGAYAITKDGVFATIV AKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQFFDGGVNVGDTTEPATPTTPVTTP TTTDDLD AASLEMEHQLLCCEVETIRRAYPDANLLNDRVLRAMLKAEETCAPSVSYF KCVQKEVLPSMRKIVATWMLEVCEEQKCEEEVFPLAMNYLDRFLSLEPVK SRLQL LGATCMFVASKMKETIPLTAEKLCIYTDNSIRPEELLQMELLLVNKLKWNLAAMTPH DFIEHFLSKMPEAEENKQIIRKHAQTFVALCATDVKFISNPPSMVAAGSWAAVQGL NLRSPNNFLSYYRLTRFLSRVIKCDPDCLRACQEQIEALLESSLRQAQQNMDPKAAEE EEEEEEEVDLACTPTDVRDVDIHHHHHH (SEQ ID NO: 13)

[00158] Cancer: Ecoli-pET28[Cohesin-hg l00-PeptideA-6xHis] C180 [00159] MDLDAVRIKVDTVNAKPGDTVNIPVRFSGIPSKGIANCDFVYSYDP NVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIVFLFAEDSGTGAYAITKDGVFATIV AKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQFFDGGVNVGDTTEPATPTTPVTTP TTTDDLDAARSAFTIMDQVPFSVSVSASRKGAAALEHHHHHH (SEQ ID NO: 14) [00160] Cancer: Ecoli-pET28[Cohesin-hMART-l-PeptideB-6xHis] C181

[00161] MDLDAVRIKVDTVNAKPGDTVNIPVRFSGIPSKGIANCDFVYSYDP NVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIVFLFAEDSGTGAYAITKDGVFATIV AKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQFFDGGVNVGDTTEPATPTTPVTTP TTTDDLDAARTAEELAGIGILTVILGASRKGAAALEHHHHHH (SEQ ID NO: 15) [00162] Cancer: Mam-cetHS-puro[SLAML-6xHis-Cohesin-hNY-ESO-l-6xHis]

C1179

[00163] LDITSHHHHHHDDLDAVRIKVDTVNAKPGDTVRIPVRFSGIPSKGIA NCDFVYSYDPNVLEIIEIEPGDIIVDPNPDKSFDTAVYPDRKIIVFLFAEDSGTGAYAIT KDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQFFDGGVNVGDTTEP ATPTTPVTTPTTTDDLDAASQTPTNTIS VTPTNNSTPTNNSNPKPNPASMQAEGRGTG GSTGDADGPGGPGIPDGPGGNAGGPGEAGATGGRGPRGAGAARASGPGGGAPRGP HGGAASGLNGCCRCGARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPLPVPG VLLKEFTVSGNILTIRLTAADHRQLQLSISSCLQQLSLLMWITQCFLPVFLAQPPSGQR RHHHHHH (SEQ ID NO: 16) [00164] Cancer: Mam-cetHS-puro[SLAML-6xHis-Cohesin-hPSA] CI 163

[00165] LDITSHHHHHHDDLDAVRIKVDTVNAKPGDTVRIPVRFSGIPSKGIA NCDFVYSYDPNVLEIIEIEPGDIIVDPNPDKSFDTAVYPDRKIIVFLFAEDSGTGAYAIT KDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQFFDGGVNVGDTTEP ATPTTPVTTPTTTDDLDAASDTTEPATPTTPVTTPTTTLLAPLILSRIVGGWECEKHSQ PWQVLVASRGRAVCGGVLVHPQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQV SHSFPHPLYDMSLLKNRFLRPGDDSSHDLMLLRLSEPAELTDAVKVMDLPTQEPALG TTCYASGWGSIEPEEFLTPKKLQCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGG KSTCSGDSGGPLVCNGVLQGITSWGSEPCALPERPSLYTKWHYRKWIKDTIVANP

(SEQ ID NO: 17) [00166] Cancer: Mam-cetHS-puro[SLAML-Cohesin-hSurvivin-6xHis] C875

[00167] LDDLDAVRIKVDTVNAKPGDTVRIPVRFSGIPSKGIANCDFVYSYD PNVLEIIEIEPGDIIVDPNPDKSFDTAVYPDR IIVFLFAEDSGTGAYAITKDGVFATIVA KVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQFFDGGVNVGDTTEPATPTTPVTTPT TTDDLDALEMGAPTLPPAWQPFLKDHRISTFKNWPFLEGCACTPERMAEAGFIHCPT ENEPDLAQCFFCFKELEGWEPDDDPIEEHK HSSGCAFLSVK QFEELTLGEFLKLDR ERAKNKIAKETNNKKKEFEETAK VRRAIEQLAAMDHHHHHH (SEQ ID NO: 18)

[00168] Cancer: Mam-pCDM8[SLAML-Cohesin-hg l00] C294

[00169] GLDDLD AVRIKVDT VN AKPGDT VRIP VRF S GIP SKGI ANCDFVY S Y DPNVLEIIEIEPGDIIVDPNPDKSFDTAVYPDRKIIVFLFAEDSGTGAYAITKDGVFATIV AKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQFFDGGVNVGDTTEPATPTTPVTTP TTTDDLDALEKVPRNQDWLGVSRQLRTKAWNRQLYPEWTEAQRLDCWRGGQVSL KVSNDGPTLIGANASFSIALNFPGSQKVLPDGQVIWVNNTIINGSQVWGGQPVYPQET DDACIFPDGGPCPSGSWSQKRSFVYVWKTWGQYWQVLGGPVSGLSIGTGRAMLGT HTMEVTVYHRRGSRSYVPLAHSSSAFTITDQVPFSVSVSQLRALDGGNKHFLRNQPL TFALQLHDPSGYLAEADLSYTWDFGDSSGTLISRAPWTHTYLEPGPVTAQWLQAA IPLTSCGSSPVPGTTDGHRPTAEAPNTTAGQVPTTEWGTTPGQAPTAEPSGTTSVQV PTTEVISTAPVQMPTAESTGMTPEKVPVSEVMGTTLAEMSTPEATGMTPAEVSIWLS GTTAAQVTTTEWVETTARELPIPEPEGPDASSIMSTESITGSLGPLLDGTATLRLVKRQ VPLDCVLYRYGSFSVTLDIVQGIESAEILQAVPSGEGDAFELTVSCQGGLPKEACMEIS SPGCQPPAQRLCQPVLPSPACQLVLHQILKGGSGTYCLNVSLADTNSLAWSTQLIVP GILLTGQEAGLGQ (SEQ ID NO: 19)

[00170] Control: Ecoli-pET28[Cohesin-6xHis] C21

[00171] MDLDAVRIKVDTVNAKPGDTVNIPVRFSGIPSKGIANCDFVYSYDP NVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIVFLFAEDSGTGAYAITKDGVFATIV AKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQFFDGGVNVGDTTEPATPTTPVTTP TTTDDLD AASMTGGQQMGRDPNS S S VDKLAAALEHHHHHH (SEQ ID NO: 20)

[00172] Control: Ecoli-pET28[Cohesin] CI 89 [00173] MDLDAVRIKVDTVNAKPGDTVNIPVRFSGIPSKGIANCDFVYSYDP NVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIVFLFAEDSGTGAYAITKDGVFATIV AKVKEGAPNGLSVIKFVEVGGFA NDLVEQKTQFFDGGVNVGDTTEPATPTTPVTTP TTTDDLDAASMTGGQQMGRDPNSSLRPHSSTTTTTTEIRLLTKPERKLSWLLPPLS N

(SEQ ID NO: 21)

[00174] Hepatitis C Virus: Ecoli-pET28[6xHis-CthermoCohesin-HCV-NS5B- Palm] CI 843

[00175] MGSSHHHHHHSSGLVPRGSHMASMDLDAVRIKVDTVNAKPGDTV NIPVRFSGIPSKGIANCDFVYSYDPNVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIV FLFAEDSGTGAYAITKDGVFATIVAKVKEGAPNGLS VIKFVEVGGFANNDLVEQKTQ FFDGGVNVGDTTEPATPTTPVTTPTTTDDLDAASVLDSHYQDVLKEVKAAASKVKA NALYDWSKLPLAVMGSSYGFQYSPGQRVEFLVQAWKSK TPMGFSYDTRCFDSTV TESDIRTEEAIYQCCDLDPQARVAIKSLTERLYVGRCRASGVLTTSCGNTLTCYIKAR AACRAAGLQDCTMLVCGDDLWICESAGVQEDAASLRAFTEAMTRYSAPPGDPPQP E YDLELIT AS (SEQ ID NO : 22)

[00176] Hepatitis C Virus: Ecoli-pET28[6xHis-CthermoCohesin-HCV- NS5B(l+2)] C1641

[00177] MGSSHHHHHHSSGLVPRGSHMASMDLDAVRIKVDTVNAKPGDTV NIPVRFSGIPSKGIANCDFVYSYDPNVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIV FLFAEDSGTGAYAITKDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQ FFDGGVNVGDTTEPATPTTPVTTPTTTDDLDAASSMSYSWTGALVTPCAAEAALVYS TTSRSACQRQK VTFDRLQVLDSHYQDVLKEVKAAASKVKANLLSVEEACSLTPPH SAKSKFGYGAKDVRCHARKAVNHINSVWKDLLEDSVTPIDTTIMAKNEVFCVQPEK GGRKPARLIVFPDLGVRVCEKMALYDVVSKLPLAVMGSSYGFQYSPGQRVEFLVQA WKSK TPMGFSYDTRCFDSTVTESDIRTEEAIYQCCDLDPQARVAIKSLTERLYVGGP LTNSRGENCGYRRCRASGVLTTSCGNTLTCYIKARAACRAAGLQDCTMLVCGDDLV VICESAGVQEDAASLRAFTEAMTRYSAPPGDPPQPEYDLELITSCSAS (SEQ ID NO: 23)

[00178] Hepatitis C Virus: Ecoli-pET28[6xHis-CthermoCohesin-hHCVElb] C1920 [00179] MGSSHHHHHHSSGLVPRGSHMASMDLDAVRIKVDTVNAKPGDTV NIPVRFSGIPSKGIANCDFVYSYDPNVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIV FLFAEDSGTGAYAITKDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQ FFDGGVNVGDTTEPATPTTPVTTPTTTDDLDAASVGQLFTFSPRRHWTTQDCNCSIYP GHITGHRMAWDMMMNWSPTTAWAQLLRIPQAILDMIAGAS (SEQ ID NO: 24)

[00180] Hepatitis C Virus: Ecoli-pET28[6xHis-CthermoCohesin-hHCVE2] C1923

[00181] MGSSHHHHHHSSGLVPRGSHMASMDLDAVRIKVDTVNAKPGDTV NIPVRFSGIPSKGIANCDFVYSYDPNVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIV FLFAEDSGTGAYAITKDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQ FFDGGVNVGDTTEPATPTTPVTTPTTTDDLDAASETHVTGGS AARTTAGLAGLFTPG AKQNIQLINTNGSWHINRTALNCNDSLNTGWVAGLFYYHKFNSSGCPERLASCRPLT DFDQGWGPISYANGSGPDQRPYCWHYPPKPCGIVPAKSVCGPVYCFTPSPWVGTTD RSGAPTYNWGENDTDVFVLNNTRPPLGNWFGCTWMNSTGFTKVCGAPPCVIGGVG NNTLHCPTDCFRKHPEATYSRCGSGPWITPRCLVDYPYRLWHYPCTINYTIFKIRMYV GGVEHRLEAACNWTRGERCDLEDRDRSELSPLLLSTTQWQVLPCSFTTLPALSTGLI HLHQNIVDVQYLYGVGSSIASWAIKWEYWLLFLLAS (SEQ ID NO: 25)

[00182] Hepatitis C Virus: Ecoli-pET28[6xHis-CthermoCohesin- ViralHCVprotease] cl662

[00183] MGSSHHHHHHSSGLVPRGSHMASMDLDAVRIKVDTVNAKPGDTV NIPVRFSGIPSKGIANCDFVYSYDPNVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIV FLFAEDSGTGAYAITKDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQ FFDGGVNVGDTTEPATPTTPVTTPTTTDDLDAASAPITAYAQQTRGLLGCIITSLTGR DKNQVEGEVQIVSTAAQTFLATCINGVCWTVYHGAGTRTIASPKGPVIQMYTNVDQ DLVGWPAPQGARSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGS SGGPLLCPAGHAVGIFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTDNSSPPAVPQS AS (SEQ ID NO: 26)

[00184] Hepatitis C Virus: Ecoli-pET28[CthermoCohesin-ViralHCVhelicase]

CI 664

[00185] MGSSHHHHHHSSGLVPRGSHMASMDLDAVRIKVDTVNAKPGDTV NIPVRFSGIPSKGIANCDFVYSYDPNVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIV FLFAEDSGTGAYAITKDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQ FFDGGVNVGDTTEPATPTTPVTTPTTTDDLDAASFQVAHLHAPTGSGKSTKVPAAYA AQGYKVLVLNPSVAATLGFGAYMSKAHGIDPNIRTGVRTITTGSPITYSTYGKFLAD GGCSGGAYDIIICDECHSTDATSILGIGTVLDQAETAGARLWLATATPPGSVTVPHP NIEEVALSTTGEIPFYGKAIPLEVIKGGRHLIFCHSKK CDELAAKLVALGINAVAYYR GLDVSVIPTSGVWWATDALMTGFTGDFDSVIDCNTCVTQTVDFSLDPTFTIETTTL PQDAVSRTQRRGRTGRGKPGIYRFVAPGERPSGMFDSSVLCECYDAGCAWYELTPA ETTVRLRAYMNTPGLPVCQDHLEFWEGVFTGLTHIDAHFLSQTKQSGENLPYLVAY QATVCARAQAPPPSWDQMWKCLIRLKPTLHGPTPLLYRLGAVQNEVTLTHPITKYIM TCMS ADLEVVTAAALEHHHHHH (SEQ ID NO : 27)

[00186] HIV: Ecoli-pET28[6xHis-Cohesin-Pep-gagl7] C1079

[00187] MGSSHHHHHHSSGLVPRGSHMASMDLDAVRIKVDTVNAKPGDTV NIPVRFSGIPSKGIANCDFVYSYDPNVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIV FLFAEDSGTGAYAITKDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQ FFDGGVNVGDTTEPATPTTPVTTPTTTDDLDAASEKIRLRPGGKK YKLKHIVAS

(SEQ ID NO: 28)

[00188] HIV: Ecoli-pET28[6xHis-Cohesin-Pep-gag253] C1080

[00189] MGSSHHHHHHSSGLVPRGSHMASMDLDAVRIKVDTVNAKPGDTV NIPVRFSGIPSKGIANCDFVYSYDPNVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIV FLFAEDSGTGAYAITKDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQ FFDGGVNVGDTTEPATPTTPVTTPTTTDDLDAASNPPIPVGEIYKRWIILGLNKIVRMY SPTSILDAS (SEQ ID NO: 29)

[00190] HIV: Ecoli-pET28[6xHis-Cohesin-Pep-nefl 16] C1078

[00191] MGSSHHHHHHSSGLVPRGSHMASMDLDAVRIKVDTVNAKPGDTV NIPVRFSGIPSKGIANCDFVYSYDPNVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIV FLFAEDSGTGAYAITKDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQ FFDGGVNVGDTTEPATPTTPVTTPTTTDDLDAASHTQGYFPDWQNYTPGPGVRYPLT FGWLYKLAS (SEQ ID NO: 30)

[00192] HIV: Ecoli-pET28[6xHis-Cohesin-Pep-nef66] C1077 [00193] MGSSHHHHHHSSGLVPRGSHMASMDLDAVRIKVDTVNAKPGDTV NIPVRFSGIPSKGIANCDFVYSYDPNVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIV FLFAEDSGTGAYAITKDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQ FFDGGVNVGDTTEPATPTTPVTTPTTTDDLDAASVGFPVTPQVPLRPMTYKAAVDLS HFLKEKGGL AS (SEQ ID NO : 31 )

[00194] HIV: Ecoli-pET28[6xHis-Cohesin-Pep-poll58] C1081

[00195] MGSSHHHHHHSSGLVPRGSHMASMDLDAVRIKVDTVNAKPGDTV NIPVRFSGIPSKGIANCDFVYSYDPNVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIV FLFAEDSGTGAYAITKDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQ FFDGGVNVGDTTEPATPTTPVTTPTTTDDLDAASAIFQSSMTKILEPFRKQNPDIVIYQ YMDDLYAS (SEQ ID NO: 32)

[00196] HIV: Ecoli-pET28[6xHis-Cohesin-Viralgag- l7-6xHis] CI 111

[00197] MGSSHHHHHHSSGLVPRGSHMASMDLDAVRIKVDTVNAKPGDTV NIPVRFSGIPSKGIANCDFVYSYDPNVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIV FLFAEDSGTGAYAITKDGVFATIVAKVKEGAPNGLS VIKFVEVGGFANNDLVEQKTQ FFDGGVNVGDTTEPATPTTPVTTPTTTDDLDAASLEMGARASILSGGELDRWEKIRL RPGGK KYKLKHIVWASRELERFAVNPGLLETSEGCRQILGQLQPSLQTGSEELRSLY NTVATLYCVHQRIEIKDTKEALDKIEEEQNKSVDHHHHHH (SEQ ID NO: 33)

[00198] HIV: Mam-cetHS-puro[SLAML-Cohesin-Viralgag-6xHis] C492 [00199] LDDLDAVRIKVDTVNAKPGDTVRIPVRFSGIPSKGIANCDFVYSYD

PNVLEIIEIEPGDIIVDPNPDKSFDTAVYPDRKIIVFLFAEDSGTGAYAITKDGVFATIVA KVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQFFDGGVNVGDTTEPATPTTPVTTPT TTDDLDALEVHQAISPRTLNAWVKWEEKAFSPEVIPMFSALSEGATPQDLNTMLNT VGGHQAAMQMLKETINEEAAEWDRVHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQ IGWMTNNPPIPVGEIYKRWIILGLNKIVRMYSPTSILDIRQGPKEPFRDYVDRFYKTLR AEQASQEVKNWMTETLLVQNANPDCKTILKALGPAATLEEMMTACQGVGHHHHH

H (SEQ ID NO: 34)

[00200] HIV: Mam-cetHS-puro[SLAML-Cohesin-ViralNef-6xHis] C879 [00201] LDDLDAVRIKVDTVNAKPGDTVRIPVRFSGIPSKGIANCDFVYSYD PNVLEIIEIEPGDIIVDPNPDKSFDTAVYPDR IIVFLFAEDSGTGAYAITKDGVFATIVA KVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQFFDGGANVGDTTEPATPTTPVTTPT TTDDLDAASMGGKWSKRSWGWPTVRERMRRAEPAADGVGAVSRDLEKHGAITSS NTAANNADCAWLEAQEEEEVGFPVRPQVPLRPMTYKGALDLSHFLKEKGGLEGLIY SQKRQDILDLWVYHTQGYFPDWQNYTPGPGIRYPLTFGWCFKLVPVEPEKVEEANE GENNSLLHPMSLHGMDDPEREVLVWKFDSRLAFHHMARELHPEYYKDCHHHHHH

(SEQ ID NO: 35)

[00202] Human Papilloma Virus: Ecoli-pET28[6xHis-Cohesin-HPV16E6] C1435 [00203] MGSSHHHHHHSSGLVPRGSHMASMDLDAVRIKVDTVNAKPGDTV

NIPVRFSGIPSKGIANCDFVYSYDPNVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIV FLFAEDSGTGAYAITKDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQ FFDGGVNVGDTTEPATPTTPVTTPTTTDDLDAASMHQKRTAMFQDPQERPRKLPQL CTELQTTIHDIILECVYCKQQLLRREVGDFAFRDLCIVYRDGNPYAVCDKCLKFYSKI SEYRHYCYSVYGTTLEQQYNKPLCDLLIRCINCQKPLCPEAS (SEQ ID NO: 36)

[00204] Human Papilloma Virus: Ecoli-pET28[6xHis-Cohesin-HPV16E7] C1436

[00205] MGSSHHHHHHSSGLVPRGSHMASMDLDAVRIKVDTVNAKPGDTV NIPVRFSGIPSKGIANCDFVYSYDPNVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIV FLFAEDSGTGAYAITKDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQ FFDGGVNVGDTTEPATPTTPVTTPTTTDDLDAASMHGDTPTLHEYMLDLQPETTDLY GYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKA (SEQ ID NO: 37)

[00206] Human Papilloma Virus: Ecoli-pET28[6xHis-Cohesin-HPV16L2] C1775

[00207] MGSSHHHHHHSSGLVPRGSHMASMDLDAVRIKVDTVNAKPGDTV NIPVRFSGIPSKGIANCDFVYSYDPNVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIV FLFAEDSGTGAYAITKDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQ FFDGGVNVGDTTEPATPTTPVTTPTTTDDLDAASSGTGGRTGYIPLGTRPPTATDTLA PVRPPLTVDPVGPSDPSIVSLVEETSFIDAGAPTSVPSIPPDVSGFSITTSTDTTPAILDIN NTVTTVTTHNNPTFTDPSVLQPPTPAETGGHFTLSSSTISTHNYEEIPMDTFIVSTNPNT VTSSTPIPGSRPVARLGLYSRTTQQVKWDPAFVTTPTKLITYDNPAYEGIDVDNTLY FSSNDNSINIAPDPDFLDIVALHRPALTSRRTGIRYSRIGNKQTLRTRSGKSIGAKVHY YYDLSTIDPAEEIELQTITPSTYTTTSHAASPTSINNGLYDIYAAS (SEQ ID NO: 38)

[00208] Human Papilloma Virus: Ecoli-pET28[6xHis-Cohesin-HPV18E7] C1469

[00209] MGSSHHHHHHSSGLVPRGSHMASMDLDAVRIKVDTVNAKPGDTV NIPVRFSGIPSKGIANCDFVYSYDPNVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIV FLFAEDSGTGAYAITKDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQ FFDGGVNVGDTTEPATPTTPVTTPTTTDDLDAASMHGPKATLQDIVLHLEPQNEIPVD LLGHGQLSDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKAS (SEQ ID NO: 39)

[00210] Human Papilloma Virus: Ecoli-pET28[6xHis-CthermoCohesin-HPV16E6- HPV16E7] C1463

[00211] GSSHHHHHHSSGLVPRGSHMASMDLDAVRIKVDTVNAKPGDTVN IPVRFSGIPSKGIANCDFVYSYDPNVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIVFL FAEDSGTGAYAITKDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQFF DGGVNVGDTTEPATPTTPVTTPTTTDDLDAASMHQKRTAMFQDPQERPRKLPQLCT ELQTTIHDIILECVYCKQQLLRREVGDFAFRDLCIVYRDGNPYAVCDKCLKFYSKISE YRHYCYSVYGTTLEQQYNKPLCDLLIRCINCQKPLCPEASMHGDTPTLHEYMLDLQP ETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKAS (SEQ ID NO: 40)

[00212] Human Papilloma Virus: Ecoli-pET28[6xHis-CthermoCohesin-HPVl 8E6- HPV18E7] C1510 [00213] GSSHHHHHHSSGLVPRGSHMASMDLDAVRIKVDTVNAKPGDTVN

IPVRFSGIPSKGIANCDFVYSYDPNVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIVFL FAEDSGTGAYAITKDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQFF DGGVNVGDTTEPATPTTPVTTPTTTDDLDAASMARFEDPTRRPYKLPDLCTELNTSL QDIEITCVYCKTVLELTEVGEFAFKDLFWYRDSIPHAACHKCIDFYSRIRELRHYSDS VYGDTLEKLTNTGLYNLLIRCLRCQKPLNPASMHGPKATLQDIVLHLEPQNEIPVDLL GHGQLSDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKAS (SEQ ID NO: 41)

[00214] Human Papilloma Virus: Ecoli-pET28[6xHis-CthermoCohesin-HPVl 8E6]

C1496 [00215] GSSHHHHHHSSGLVPRGSHMASMDLDAVRIKVDTVNAKPGDTVN IPVRFSGIPSKGIANCDFVYSYDPNVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIVFL FAEDSGTGAYAITKDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQFF DGGVNVGDTTEPATPTTPVTTPTTTDDLDAASMAPvFEDPTPvRPYKLPDLCTELNTSL QDIEITCVYCKTVLELTEVGEFAFKDLFWYRDSIPHAACHKCIDFYSRIRELRHYSDS VYGDTLEKLTNTGLYNLLIRCLRCQKPLNPAS (SEQ ID NO: 42)

[00216] Influenza: Ecoli-pET28[6xHis-Cohesin-FluHAl-lc] C2096

[00217] MGSSHHHHHHSSGLVPRGSHMASMDLDAVRIKVDTVNAKPGDTV NIPVRFSGIPSKGIANCDFVYSYDPNVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIV FLFAEDSGTGAYAITKDGVFATIVAKVKEGAPNGLS VIKFVEVGGFANNDLVEQKTQ FFDGGVNVGDTTEPATPTTPVTTPTTTDDLDAASDTTEPATPTTPVTTDTICIGYHAN NSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPE CESLSTASSWSYIVETSSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNH DSNKGVTAACPHAGAKSFYKNLIWLVK GNSYPKLSKSYINDKGKEVLVLWGIHHP STSDDQQSLYQNADAYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGD KITFEATGNLWPRYAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIQPI TIGKCPKYVKSTKLRL (SEQ ID NO: 43)

[00218] Influenza: Ecoli-pET28[6xHis-Cohesin-FluHA3-lk] C2039

[00219] MGSSHHHHHHSSGLVPRGSHMASMDLDAVRIKVDTVNAKPGDTV NIPVRFSGIPSKGIANCDFVYSYDPNVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIV FLFAEDSGTGAYAITKDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQ FFDGGVNVGDTTEPATPTTPVTTPTTTDDLDAASDTTEPATPTTPVTTATLCLGHHAV PNGTLVKTITDDQIEVTNATELVQSSSTGKICNNPHRILDGIDCTLIDALLGDPHCDVF QDETWDLFVERSKAFSNCYPYDVPDYASLRSLVALSGTLEFITEGFTWTGVTQNGGS NACKRGPGSGFFSRLNWLTKSGSTYPVLNVTMPNNDNFDKLYIWGVHHPSTNQEQT SLYVQASGRVTVSTRRSQQTIIPNIGSRPWVRGLSSRISIYWTIVKPGDVLVINSNGNLI APRGYFKMRTGKSSIMRSDAPTDTCISECITPNGSIPNDKPFQNVNKITYGACPKYVK QNTLKLA (SEQ ID NO: 44)

[00220] Influenza: Ecoli-pET28[6xHis-CthermoCohesin-FluNA-6xHis] CI 699 [00221] MGSSHHHHHHSSGLVPRGSHMASMDLDAVRIKVDTVNAKPGDTV NIPVRFSGIPSKGIANCDFVYSYDPNVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIV FLFAEDSGTGAYAITKDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQ FFDGGVNVGDTTEPATPTTPVTTPTTTDDLDAASQIGNMISIWVSHSIHTGNQHQSEPI SNTNFLTEKAVASVKLAGNSSLCPINGWAVYSKDNSIRIGSKGDVFVIREPFISCSHLE CRTFFLTQGALLNDKHSNGTVKDRSPHRTLMSCPVGEAPSPYNSRFESVAWSASACH DGTSWLTIGISGPDNGAVAVLKYNGIITDTIKSWR NILRTQESECACVNGSCFTVMT DGPSNGQASHKIFKMEKGKWKSVELDAPNYHYEECSCYPNAGEITCVCRDNWHGS NRPWVSFNQNLEYQIGYICSGVFGDNPRPNDGTGSCGPVSSNGAYGVKGFSFKYGN GVWIGRTKSTNSRSGFEMIWDPNGWTETDSSFSVKQDIVAITDWSGYSGSFVQHPEL TGLDCIRPCFWVELIRGRPKESTIWTSGSSISFCGVNSDTVGWSWPDGAELPFTIDKH HHHHH (SEQ ID NO: 45)

[00222] Influenza: Ecoli-pET28[Cohesin-FluMl-6xHis] C32

[00223] MDLDAVRIKVDTVNAKPGDTVNIPVRFSGIPSKGIANCDFVYSYDP NVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIVFLFAEDSGTGAYAITKDGVFATIV AKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQFFDGGVNVGDTTEPATPTTPVTTP TTTDDLDAASLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRP ILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREI TFHGAKEIALSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQHRSHRQ MVTTTNPLIRHENRMVLASTTAKAMEQMAGSSEQAAEAMDIASQARQMVQAMRTI GTHPSSSAGLKDDLLENLQAYQKRMGVQMQRFKLEHHHHHH (SEQ ID NO: 46)

[00224] Influenza: Ecoli-pET28[CthermoCohesin-FluHAl-ls-6xHis] C1887

[00225] HHHHHHSSGLVPRGSHMASMDLDAVRIKVDTVNAKPGDTVNIPV RFSGIPSKGIANCDFVYSYDPNVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIVFLFA EDSGTGAYAITKDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQFFDG GVNVGDTTEPATPTTPVTTPTTTDDLDAASDTLCIGYHANNSTDTVDTVLEKNVTVT HSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSS DNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKS FYKNLIWLVK GNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADTYV FVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFA MERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLAH HHHHH (SEQ ID NO: 47)

[00226] Influenza: Ecoli-pET28[CthermoCohesin-FluNP-5-6xHis] C943

[00227] MDLDAVRIKVDTVNAKPGDTVNIPVRFSGIPSKGIANCDFVYSYDP NVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIVFLFAEDSGTGAYAITKDGVFATIV AKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQFFDGGVNVGDTTEPATPTTPVTTP TTTDDLDAASMASQGTKRSYEQMETGGERQNATEIRASVGRMVSGIGRFYIQMCTE LKLSDYEGRLIQNSITIERMVLSAFDERRNRYLEEHPSAGKDPKKTGGPIYRRRDGKW VRELILYDKEEIRRIWRQANNGEDATAGLTHLMIWHSNLNDATYQRTRALVRTGMD PRMCSLMQGSTLPRRSGAAGAAVKGVGTMVMELIRMIKRGINDRNFWRGENGRRT RIAYERMCNILKGKFQTAAQRAMMDQVRESRNPGNAEIEDLIFLARSALILRGSVAH KSCLPACVYGLAVASGYDFEREGYSLVGIDPFRLLQNSQVFSLIRPNENPAHKSQLV WMACHSAAFEDLRVSSFIRGTRWPRGQLSTRGVQIASNENMEAMDSNTLELRSRY WAIRTRSGGNTNQQRASAGQISVQPTFSVQRNLPFERATIMAAFTGNTEGRTSDMRT EIIRMMESARPEDVSFQGRGVFELSDEKATNPIVPSFDMNNEGS YFFGDNAEEYDNH HHHHH (SEQ ID NO: 48)

[00228] Influenza: Mam-cetHS-puro[6xHis-Cohesin-FluHAl-lu] CI 996

[00229] LDITSHHHHHHDDLDAVRIKVDTVNAKPGDTVRIPVRFSGIPSKGIA NCDFVYSYDPNVLEIIEIEPGDIIVDPNPDKSFDTAVYPDRKIIVFLFAEDSGTGAYAIT KDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQFFDGGVNVGDTTEP ATPTTPVTTPTTTDDLDAASDTTEPATPTTPVTTDTICIGYHANNSTDTVDTVLEKNV TVTHSVNLLEDSHNGKLCRLKGIAPLQLGKCSIAGWILGNPECESLVSKKSWSYIAET PNSENGTCYPGYFADYEELREQLSSVSSFERFEIFPKERSWPKHNVTRGVTASCSHKG KSSFYRNLLWLTEKNGSYPNLSKSYVNNKEKEVLVLWGVHHPSNIEDQKTIYRKEN AYVSWSSNYNRRFAPEIAERPKVRGQAGRINYYWTLLEPGDTIIFEANGNLIAPWHA FALNRGFGSGIITSNASMDECDTKCQTPQGAINSSLPFQNIHPVTIGECPKYVRSTKLR MV (SEQ ID NO: 49)

[00230] Influenza: Mam-cetHS-puro[6xHis-Cohesin-FluHA3-lk] CI 998

[00231] LDITSHHHHHHDDLDAVRIKVDTVNAKPGDTVRIPVRFSGIPSKGIA NCDFVYSYDPNVLEIIEIEPGDIIVDPNPDKSFDTAVYPDRKIIVFLFAEDSGTGAYAIT KDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQFFDGGVNVGDTTEP ATPTTPVTTPTTTDDLDAASDTTEPATPTTPVTTATLCLGHHAVPNGTLVKTITDDQI EVTNATELVQSSSTGKICNNPHRILDGIDCTLIDALLGDPHCDVFQNETWDLFVERSK AFSNCYPYDVPDYASLRSLVASSGTLEFITEGFTWTGVTQNGGSNACKRGPGSGFFS RLNWLTKSGSTYPVLNVTMP NDNFDKLYIWGVHHPSTNQEQTSLYVQASGRVTVS TRRSQQTIIPNIGSRPWVRGLSSRISIYWTIVKPGDVLVINSNGNLIAPRGYFKMRTGK SSIMRSDAPIDTCISECITPNGSIPNDKPFQNVNKITYGACPKYVKQNTLKLA (SEQ ID NO: 50)

[00232] Influenza: Mam-cetHS-puro[6xHis-Cohesin-FluNP-ls] C2043 [00233] LDITSHHHHHHDDLDAVRIKVDTVNAKPGDTVRIPVRFSGIPSKGIA

NCDFVYSYDPNVLEIIEIEPGDIIVDPNPDKSFDTAVYPDRKIIVFLFAEDSGTGAYAIT KDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQFFDGGVNVGDTTEP ATPTTPVTTPTTTDDLDAASMASQGTKRSYEQMETGGERQDATEIRASVGRMIGGIG RFYIQMCTELKLSDYDGRLIQNSITIERMVLSAFDERRNKYLEEHPSAGKDPK TGGP IYRRVDGKWMRELILYDKEEIRRVWRQANNGEDATAGLTHIMIWHSNLNDATYQRT RALVRTGMDPRMCSLMQGSTLPRRSGAAGAAVKGVGTIAMELIRMIKRGINDRNFW RGENGRRTRVAYERMCNILKGKFQTAAQRAMMDQVRESRNPGNAEIEDLIFLARSA LILRGSVAHKSCLPACVYGLAVASGHDFEREGYSLVGIDPFKLLQNSQWSLMRPNE NPAHKSQLVWMACHSAAFEDLRVSSFIRGK VIPRGKLSTRGVQIASNENVETMDSN TLELRSRY WAIRTRS GGNTNQQKAS AGQI S VQPTF S VQRNLPFERAT VM AAF S GNNE GRTSDMRTEVIRMMESAKPEDLSFQGRGVFELSDEKATNPIVPSFDMSNEGSYFFGD NAEEYD S ASHHHHHH (SEQ ID NO: 51)

[00234] Influenza: Mam-cetHS-puro[Cohesin-Flex-vl -FluM2- 1 s-M2e-FluM2- 1 - M2e C2254 [00235] LDITSHHHHHHDDLDAVRIKVDTVNAKPGDTVRIPVRFSGIPSKGIA

NCDFVYSYDPNVLEIIEIEPGDIIVDPNPDKSFDTAVYPDRKIIVFLFAEDSGTGAYAIT KDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQFFDGGVNVGDTTEP ATPTTPVTTPTTTDDLDAASQTPTNTISVTPTNNSTPTNNSNPKPNPASLLTEVETPTR SEWECRCSDSSDPASLLTEVETPIRNEWGCRCNGSSDPAS (SEQ ID NO: 52) [00236] Influenza: Mam-cetHS-puro[HAl-0-S-Cohesin-6xHis] CI 819 [00237] DTLCIGYHANNSTDTVDTVLEKNVTVTHSWLLEDKHNGKLCKLR GVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETPSSDNGTCYPGDFIDYEELRE QLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPK LSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADTYVFVGSSRYSKKFKPEIAIRP KVRDQEGRMNYYWTLVEPGDKITFEATGNLWPRYAFAMERNAGSGIIISDTPVHDC NTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNIPSIQSRGLFGAIAG FIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQF TAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLY EKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDG VKLESTRIYQIASTTEPATPTTPVTTPTTTDDLDAVRIKVDTVNAKPGDTVNIPVRFSG IPSKGIANCDFVYSYDPNVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIVFLFAEDSG TGAYAITKDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQFFDGGVN VGDTHHHHHH (SEQ ID NO: 53)

[00238] Influenza: Mam-cetHS-puro[SLAML-6xHis-Cohesin-FluHAb-l] C2297 [00239] DITSHHHHHHDDLDAVRIKVDTVNAKPGDTVRIPVRFSGIPSKGIA

NCDFVYSYDPNVLEIIEIEPGDIIVDPNPDKSFDTAVYPDRKIIVFLFAEDSGTGAYAIT KDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQFFDGGVNVGDTTEP ATPTTPVTTPTTTDDLDAASDRICTGITSSNSPHWKTATQGEVNVTGVIPLTTTPTKS HFANLKGTETRGKLCPKCLNCTDLDVALGRPKCTGKIPSARVSILHEVRPVTSGCFPI MHDRTKIRQLPNLLRGYEHIRLSTHNVINAENAPGGPYKIGTSGSCPNITNGNGFFAT MAWAVPKNDKNKTATNPLTIEVPYICTEGEDQITVWGFHSDDETQMAKLYGDSKPQ KFTSSANGVTTHYVSQIGGFPNQTEDGGLPQSGRIWDYMVQKSGKTGTITYQRGIL LPQKVWCASGRSKVIKGSLPLIGEADCLHEKYGGLNKSKPYYTGEHAKAIGNCPIWV KTPLKLANGTK (SEQ ID NO: 54) [00240] Influenza: Mam-cetHS-puro[SLAML-6xHis-Cohesin-FluM2-ls] CI 734

[00241] GLDITSHHHHHHDDLDAVRIKVDTVNAKPGDTVRIPVRFSGIPSKGI ANCDFVYSYDPNVLEIIEIEPGDIIVDPNPDKSFDTAVYPDRKIIVFLFAEDSGTGAYAI TKDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQFFDGGVNVGDTTE PATPTTPVTTPTTTDDLDAASLLTEVETPTRSEWECRCSDSSDPAS (SEQ ID NO: 55)

[00242] Influenza: Mam-cetHS-puro[SLAML-6xHis-Cohesin-M2e] CI 977 [00243] GLDITSHHHHHHDDLDAVRIKVDTVNAKPGDTVRIPVRFSGIPSKGI ANCDFVYSYDPNVLEIIEIEPGDIIVDPNPDKSFDTAVYPDRKIIVFLFAEDSGTGAYAI TKDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQFFDGGVNVGDTTE PATPTTPVTTPTTTDDLDAASLLTEVETPIRNEWGCRCNGSSDPAS (SEQ ID NO: 56) [00244] Influenza: Mam-cetHS-puro[SLAML-Cohesin-FluHAl-l-6xHis] C489

[00245] MDPKGSLSWRILLFLSLAFELSYGLDDLDAVRIKVDTVNAKPGDT VRIPVRFSGIPSKGIANCDFVYSYDPNVLEIIEIEPGDIIVDPNPDKSFDTAVYPDRKIIVF LFAEDSGTGAYAITKDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQF FDGGVNVGDTTEPATPTTPVTTPTTTDDLDALDDTICIGYHANNSTDTVDTVLEKNV TVTHS VNLLEDSHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRS WS YIVE TPNSENGIC YPGDFID YEELREQLS S VS SFERFEIFPKES S WPNFINTNGVT AACSHEGK SSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENA YVSWTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYA FALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECPKYVRSAKLR MVHHHHHH (SEQ ID NO: 57)

[00246] Influenza: Mam-cetHS-puro[SLAML-Cohesin-FluHA5-0-6xHis] C543

[00247] LDDLDAVRIKVDTVNAKPGDTVRIPVRFSGIPSKGIANCDFVYSYD PNVLEIIEIEPGDIIVDPNPDKSFDTAVYPDRKIIVFLFAEDSGTGAYAITKDGVFATIVA KVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQFFDGGVNVGDTTEPATPTTPVTTPT TTDDLDALEDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKKHNGKLCDLDGV KPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEELKHL LSRINHFEKIQIIPKSSWSSHEASLGVSSACPYQGKSSFFRNWWLIKKNSTYPTIKRSY NNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNG QSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQ TPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRERRRKKRGLFGAIA GFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQ FEAVGREFNNLERRIENLNK MEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNL YDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEIH HHHHH (SEQ ID NO: 58) [00248] Influenza: Mam-cetHS-puro[SLAML-Cohesin-FluHA5-l-6xHis] C490 [00249] MDPKGSLSWRILLFLSLAFELSYGLDDLDAVRIKVDTVNAKPGDT VRIPVRFSGIPSKGIANCDFVYSYDPNVLEIIEIEPGDIIVDPNPDKSFDTAVYPDRKIIVF LFAEDSGTGAYAITKDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQF FDGGVNVGDTTEPATPTTPVTTPTTTDDLDALEDQICIGYHANNSTEQVDTIMEKNV TVTHAQDILEK HNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVE KANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSSHEASLGVSSACPYQG KSSFFRNWWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTT YISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAY KIVK GDSTIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLV LAHHHHHH (SEQ ID NO: 59)

[00250] SIV: Mam-cetHS-puro[6xHis-Cohesin-ViralgagSIV- l7] C1274

[00251] LDITSHHHHHHDDLDAVRIKVDTVNAKPGDTVRIPVRFSGIPSKGIA NCDFVYSYDPNVLEIIEIEPGDIIVDPNPDKSFDTAVYPDRKIIVFLFAEDSGTGAYAIT KDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQFFDGGVNVGDTTEP ATPTTPVTTPTTTDDLDAASMRVRNS VLSGKKADELEKIRLRPNGKK YMLKHWW AANELDRFGLAESLLENKEGCQKILSVLAPLVPTGSENLKSLYNTVCVIWCIHAEEKV KHTEEAAS (SEQ ID NO: 60)

[00252] SIV: Mam-cetHS-puro[6xHis-Cohesin-ViralgagSIV-p24] C1273

[00253] LDITSHHHHHHDDLDAVRIKVDTVNAKPGDTVRIPVRFSGIPSKGIA NCDFVYSYDPNVLEIIEIEPGDIIVDPNPDKSFDTAVYPDRKIIVFLFAEDSGTGAYAIT KDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQFFDGGVNVGDTTEP ATPTTP VTTPTTTDDLD AASKT SRPT AP S S GRGGN YP VQ QIGGN Y VHLPL SPRTLN A WVKLIEEKKFGAEWPGFQALSEGCTPYDINQMLNCVGDHQAAMQIIRDIINEEAAD WDLQHPQPAPQQGQLREPSGSDIAGTTSSVDEQIQWMYRQQNPIPVGNIYRRWIQLG LQKCVRMYNPTNILDVKQGPKEPFQSYVDRFYKSLRAEQTDAAVKNWMTQTLLIQN ANPDCKLVLKGLGVNPTLEEMLTACQGVGAS (SEQ ID NO: 61)

[00254] SIV: Mam-cetHS-puro[6xHis-Cohesin-ViralnefSIV] C1275

[00255] LDITSHHHHHHDDLDAVRIKVDTVNAKPGDTVRIPVRFSGIPSKGIA NCDFVYSYDPNVLEIIEIEPGDIIVDPNPDKSFDTAVYPDRKIIVFLFAEDSGTGAYAIT KDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQFFDGGVNVGDTTEP ATPTTPVTTPTTTDDLDAASLGEVEDGYSQSPGGLDKGLSSLSCEGQKYNQGQYMN TP WPJSiP AEEREKL A YR QNMDDIDEEDDDL VG VS VRPKVPLRTM S YKL AIDM SHFI KEKGGLEGIYYSARRHRILDIYLEKEEGIIPDWQDYTSGPGIRYPKTFGWLWKLVPVN VSDEAQEDEEHYLMHPAQTSQWDDPWGEVLAWKFDPTLAYTYEAYVRYPEEFGSK AS (SEQ ID NO: 62)

[00256] SIV: Mam-cetHS-puro[SLAML-6xHis-Cohesin-ViralenvSIV-gp41 -6xHis]

C1193

[00257] LDITSHHHHHHDDLDAVRIKVDTVNAKPGDTVRIPVRFSGIPSKGIA NCDFVYSYDPNVLEIIEIEPGDIIVDPNPDKSFDTAVYPDRKIIVFLFAEDSGTGAYAIT KDGVFATIVAKVKEGAPNGLS VIKFVEVGGFANNDLVEQKTQFFDGGVNVGDTTEP ATPTTPVTTPTTTDDLDAASAQSRTLLAGIVQQQQQLLDWKRQQELLRLTVWGTK NLQTRVTAIEKYLKDQAQLNAWGCAFRQVCHTTVPWPNASLTPKWNNETWQEWE RKVDFLEENITALLEEAQIQQEKNMYELQKLNSHHHHHHAS (SEQ ID NO: 63)

[00258] Tuberculosis: Ecoli-pET28[6xHis-Cohesin-m.tbCFP10] C1254 [00259] MGSSHHHHHHSSGLVPRGSHMASMDLDAVRIKVDTVNAKPGDTV

NIPVRFSGIPSKGIANCDFVYSYDPNVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIV FLFAEDSGTGAYAITKDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQ FFDGGVNVGDTTEPATPTTPVTTPTTTDDLDAASMAEMKTDAATLAQEAGNFERISG DLKTQIDQVESTAGSLQGQWRGAAGTAAQAAWRFQEAANKQKQELDEISTNIRQA GVQYSRADEEQQQALSSQMGF (SEQ ID NO: 64)

[00260] Tuberculosis: Ecoli-pET28[6xHis-Cohesin-m.tbRv0125] C1334

[00261] MGSSHHHHHHSSGLVPRGSHMASMDLDAVRIKVDTVNAKPGDTV NIPVRFSGIPSKGIANCDFVYSYDPNVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIV FLFAEDSGTGAYAITKDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQ FFDGGVNVGDTTEPATPTTPVTTPTTTDDLDAASMSNSRRRSLRWSWLLSVLAAVG LGLATAPAQAAPPALSQDRFADFPALPLDPSAMVAQVGPQWNINTKLGYNNAVGA GTGIVIDPNGWLTNNHVIAGATDINAFSVGSGQTYGVDWGYDRTQDVAVLQLRG AGGLPSAAIGGGVAVGEPWAMGNSGGQGGTPRAVPGRWALGQTVQASDSLTGA EETLNGLIQFDAAIQPGDSGGPWNGLGQWGMNTAASDNFQLSQGGQGFAIPIGQA MAIAGQIRSGGGSPTVHIGPTAFLGLGWDNNGNGARVQRWGSAPAASLGISTGDV ITAVDGAPINSATAMADALNGHHPGDVISVTWQTKSGGTRTGNVTLAEGPPA (SEQ ID NO: 65)

[00262] Tuberculosis: Ecoli-pET28[6xHis-Cohesin-m.tbRv0570] C1500

[00263] MGSSHHHHHHSSGLVPRGSHMASMDLDAVRIKVDTVNAKPGDTV NIPVRFSGIPSKGIANCDFVYSYDPNVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIV FLFAEDSGTGAYAITKDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQ FFDGGVNVGDTTEPATPTTPVTTPTTTDDLDAASVGVSWPAKVRRRDGTLVPFDIAR IEAAVTRAAREVACDDPDMPGTVAKAVADALGRGIAPVEDIQDCVEARLGEAGLDD VARVYIIYRQRRAELRTAKALLGVRDELKLSLAAVTVLRERYLLHDEQGRPAESTGE LMDRSARCVAAAEDQYEPGSSRRWAERFATLLRNLEFLPNSPTLMNSGTDLGLLAG CFVLPIEDSLQSIFATLGQAAELQRAGGGTGYAFSHLRPAGDRVASTGGTASGPVSFL RLYDSAAGWSMGGRRRGACMAVLDVSHPDICDFVTAKAESPSELPHFNLSVGVTD AFLRAVERNGLHRLVNPRTGKIVARMPAAELFDAICKAAHAGGDPGLVFLDTINRA NPVPGRGRIEATNPCGEVPLLPYESCNLGSINLARMLADGRVDWDRLEEVAGVAVR FLDDVIDVSRYPFPELGEAARATRKIGLGVMGLAELLAALGIPYDSEEAVRLATRLM RRIQQAAHTASRRLAEERGAFPAFTDSRFARSGPRRNAQVTSVAPTGTISLIAGTTAGI EPMFAIAFTRAIVGRHLLEVNPCFDRLARDRGFYRDELIAEIAQRGGVRGYPRLPAEV RAAFPTAAEIAPQWHLRMQAAVQRHVEAAVSKTVNLPATATVDDVRAIYVAAWKA KVKGIT VYRYGSREGQ VL S Y AAPKPLL AQ ADTEF S GGC AGRS CEF (SEQ ID NO: 66) [00264] Tuberculosis: Ecoli-pET28[6xHis-Cohesin-m.tbRv0577] C1335

[00265] MNRLTSSLLLLIVPAYVLSQVTLKESGPGILQPSQTLSLTCSFSGFSL STSGMGLSWIRQPSGKGLEWLAHIYWDDDKRYNPSLKSRLTISKDTSSNQVFLKITIV DTADAATYYCARSSHYYGYGYGGYFDVWGAGTTVTVSSAKTKGPSVFPLAPCSRST SESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMIS RTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVL HQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF SCSVMHEALHNHYTQKSLSLSLGKASQTPTNTISVTPTNNSTPTNNSNPKPNPASMPK RSEYRQGTPNWVDLQTTDQSAAKKFYTSLFGWGYDDNPVPGGGGVYSMATLNGEA VAAIAPMPPGAPEGMPPIWNTYIAVDDVDAWDKWPGGGQVMMPAFDIGDAGRM SFITDPTGAAVGLWQANRHIGATLVNETGTLIWNELLTDKPDLALAFYEAWGLTHS SMEIAAGQNYRVLKAGDAEVGGCMEPPMPGVPNHWHVYFAVDDADATAAKAAA AGGQVIAEPADIPSVGRFAVLSDPQGAIFSVLKPAPQQ (SEQ ID NO: 67)

[00266] Tuberculosis: Ecoli-pET28[6xHis-Cohesin-m.tbRvl626] C1256 [00267] MGSSHHHHHHSSGLVPRGSHMASMDLDAVRIKVDTVNAKPGDTV

NIPVRFSGIPSKGIANCDFVYSYDPNVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIV FLFAEDSGTGAYAITKDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQ FFDGGVNVGDTTEPATPTTPVTTPTTTDDLDAASMTGPTTDADAAVPRRVLIAEDEA LIRMDLAEMLREEGYEIVGEAGDGQEAVELAELHKPDLVIMDVKMPRRDGIDAASEI ASKRIAPIVVLTAFSQRDLVERARDAGAMAYLVKPFSISDLIPAIELAVSRFREITALE GEVATLSERLETRKLVERAKGLLQTKHGMTEPDAFKWIQRAAMDRRTTMKRVAEV VLETLGTPKDT (SEQ ID NO: 68)

[00268] Tuberculosis: Ecoli-pET28[6xHis-Cohesin-m.tbRv2875] C1310

[00269] MGSSHHHHHHSSGLVPRGSHMASMDLDAVRIKVDTVNAKPGDTV NIPVRFSGIPSKGIANCDFVYS YDPNVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIV FLFAEDSGTGAYAITKDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQ FFDGGVNVGDTTEPATPTTPVTTPTTTDDLDAASMKVKNTIAATSFAAAGLAALAV AVSPPAAAGDLVGPGCAEYAAANPTGPASVQGMSQDPVAVAASNNPELTTLTAALS GQLNPQVNLVDTLNSGQYTVFAPTNAAFSKLPASTIDELKTNSSLLTSILTYHVVAGQ TSPANWGTRQTLQGASVTVTGQGNSLKVGNADWCGGVSTANATVYMIDSVLMP PA (SEQ ID NO: 69)

[00270] Tuberculosis: Ecoli-pET28[6xHis-Cohesin-m.tbRv3044] C1312

[00271] MGSSHHHHHHSSGLVPRGSHMASMDLDAVRIKVDTVNAKPGDTV NIPVRFSGIPSKGIANCDFVYSYDPNVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIV FLFAEDSGTGAYAITKDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQ FFDGGVNVGDTTEPATPTTPVTTPTTTDDLDAASMRSTVAVAVAAAVIAASSGCGSD QPAHKASQSMITPTTQIAGAGVLGNDRKPDESCARAAAAADPGPPTRPAHNAAGVS PEMVQVPAEAQRIWLSGDQLDALCALGLQSRIVAAALPNSSSSQPSYLGTTVHDLP GVGTRSAPDLRAIAAAHPDLILGSQGLTPQLYPQLAAIAPTVFTAAPGADWENNLRG VGAATARIAAVDALITGFAEHATQVGTKHDATHFQASIVQLTANTMRVYGANNFPA SVLSAVGVDRPPSQRFTDKAYIEIGTTAADLAKSPDFSAADADIVYLSCASEAAAERA AVILDSDPWRKLSANRDNRVFWNDQVWQTGEGMVAARGIVDDLRWVDAPIN

(SEQ ID NO: 70)

[00272] Tuberculosis: Ecoli-pET28[6xHis-Cohesin-m.tbRv3478] C1308 [00273] MGSSHHHHHHSSGLVPRGSHMASMDLDAVRIKVDTVNAKPGDTV

NIPVRFSGIPSKGIANCDFVYSYDPNVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIV FLFAEDSGTGAYAITKDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQ FFDGGVNVGDTTEPATPTTPVTTPTTTDDLDAASWDFGALPPEINSARMYAGPGSA SLVAAAKMWDSVASDLFSAASAFQSWWGLTVGSWIGSSAGLMAAAASPYVAWM S VTAGQAQLTAAQVRVAAAAYETAYRLTVPPPVIAENRTELMTLTATNLLGQNTPAI EANQAAYSQMWGQDAEAMYGYAATAATATEALLPFEDAPLITNPGGLLEQAVAVE EAIDTAAANQLMNNVPQALQQLAQPAQGVVPSSKLGGLWTAVSPHLSPLSNVSSIA NNHMSMMGTGVSMTNTLHSMLKGLAPAAAQAVETAAENGVWAMSSLGSQLGSSL GSSGLGAGVAANLGRAASVGSLSVPPAWAAANQAVTPAARALPLTSLTSAAQTAPG HMLGGLPLGHS VNAGSGINNALRVPARAYAIPRTP AAG (SEQ ID NO : 71 )

[00274] Tuberculosis: Ecoli-pET28 [6xHis-CthermoCohesin-m.tbAg85BDel41 ]

C2200

[00275] MGSSHHHHHHSSGLVPRGSHMASMDLDAVRIKVDTVNAKPGDTV NIPVRFSGIPSKGIANCDFVYSYDPNVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIV FLFAEDSGTGAYAITKDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQ FFDGGVNVGDTTEPATPTTPVTTPTTTDDLDAASSRPGLPVEYLQVPSPSMGRDIKVQ FQSGGNNSPAVYLLDGLRAQDDYNGWDINTPAFEWYYQSGLSIVMPVGGQSSFYSD WYSPACGKAGCQTYKWETFLTSELPQWLSANRAVKPTGSAAIGLSMAGSSAMILAA YHPQQFIYAGSLSALLDPSQGMGPSLIGLAMGDAGGYKAADMWGPSSDPAWERND PTQQIPKLVANNTRLWVYCGNGTPNELGGANIPAEFLENFVRSSNLKFQDAYNAAG GHNAVFNFPPNGTHSWEYWGAQLNAMKGDLQSSLGAG (SEQ ID NO: 72)

[00276] Tuberculosis: Ecoli-pET28[6xHis-CthermoCohesin-m.tbMtb72f] C2236

[00277] MGSSHHHHHHSSGLVPRGSHMASMDLDAVRIKVDTVNAKPGDTV NIPVRFSGIPSKGIANCDFVYSYDPNVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIV FLFAEDSGTGAYAITKDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQ FFDGGVNVGDTTEPATPTTPVTTPTTTDDLDAASTAASDNFQLSQGGQGFAIPIGQA MAIAGQIRSGGGSPTVHIGPTAFLGLGWDNNGNGARVQRWGSAPAASLGISTGDV ITAVDGAPINSATAMADALNGHHPGDVISVTWQTKSGGTRTGNVTLAEGPPAEFMV DFGALPPEINSARMYAGPGSASLVAAAQMWDSVASDLFSAASAFQSWWGLTVGS WIGSSAGLMVAAASPYVAWMSVTAGQAELTAAQVRVAAAAYETAYGLTVPPPVIA ENRAELMILIATNLLGQNTPAIAVNEAEYGEMWAQDAAAMFGYAAATATATATLLP FEEAPEMTSAGGLLEQAAAVEEASDTAAANQLMNNVPQALQQLAQPTQGTTPSSKL GGLWKTVSPHRSPISNMVSMANNHMSMTNSGVSMTNTLSSMLKGFAPAAAAQAVQ TAAQNGVRAMSSLGSSLGSSGLGGGVAANLGRAASVGSLSVPQAWAAANQAVTPA ARALPLTSLTS AAERGPGQMLGGLPVGQMGARAGGGLSGVLRVPPRPYVMPHSPAA GDIAPPALSQDRFADFPALPLDPSAMVAQVGPQVVNINTKLGYNNAVGAGTGIVIDP NGWLTNNHVIAGATDINAFSVGSGQTYGVDWGYDRTQDVAVLQLRGAGGLPSA AIGGGVAVGEPWAMGNSGGQGGTPRAVPGRWALGQTVQASDSLTGAEETLNGLI QFDAAIQPGDSGGPWNGLGQWGMNTAAS (SEQ ID NO: 73) [00278] Tuberculosis: Ecoli-pET28[6xHis-CthermoCohesin-m.tbRv0288] C2202

[00279] MGSSHHHHHHSSGLVPRGSHMASMDLDAVRIKVDTVNAKPGDTV NIPVRFSGIPSKGIANCDFVYSYDPNVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIV FLFAEDSGTGAYAITKDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQ FFDGGVNVGDTTEPATPTTPVTTPTTTDDLDAASMSQIMYNYPAMLGHAGDMAGY AGTLQSLGAEIAVEQAALQSAWQGDTGITYQAWQAQWNQAMEDLVRAYHAMSST HEANTMAMMARDTAEAAKWGG (SEQ ID NO: 74)

[00280] Tuberculosis: Ecoli-pET28[6xHis-CthermoCohesin-m.tbRv0475] C2199

[00281] MGSSHHHHHHSSGLVPRGSHMASMDLDAVRIKVDTVNAKPGDTV NIPVRFSGIPSKGIANCDFVYSYDPNVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIV FLFAEDSGTGAYAITKDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQ FFDGG VN VGDTTEP ATPTTP VTTPTTTDDLD AAS C S CM AEN SNIDDIKAPLL AALG A ADLALATVNELITNLRERAEETRTDTRSRVEESRARLTKLQEDLPEQLTELREKFTAE ELRKAAEGYLEAATSRYNELVERGEAALERLRSQQSFEEVSARAEGYVDQAVELTQ EALGTVASQTRAVGERAAKLVGIELPKKAAPAKKAAPAKKAAPAKKAAAKKAPAK KAAAKKVTQKCSC (SEQ ID NO: 75)

[00282] Tuberculosis: Ecoli-pET28[6xHis-CthermoCohesin-m.tbRvl980] C2197 [00283] MGSSHHHHHHSSGLVPRGSHMASMDLDAVRIKVDTVNAKPGDTV NIPVRFSGIPSKGIANCDFVYSYDPNVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIV FLFAEDSGTGAYAITKDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQ FFDGGVNVGDTTEPATPTTPVTTPTTTDDLDAASAAPKTYCEELKGTDTGQACQIQM SDPAYNINISLPSYYPDQKSLENYIAQTRDKFLSAATSSTPREAPYELNITSATYQSAIP PRGTQAWLKVYQNAGGTHPTTTYKAFDWDQAYRKPITYDTLWQADTDPLPWFPI VQGELSKQTGQQVSIAPNAGLDPVNYQNFAVTNDGVIFFFNPGELLPEAAGPTQVLV PRSAIDSMLAEF (SEQ ID NO: 76)

[00284] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

[00285] It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims. [00286] All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. [00287] The use of the word "a" or "an" when used in conjunction with the term

"comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

[00288] As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[00289] The term "or combinations thereof as used herein refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, MB, BBC, AAABCCCC, CBBAAA, CAB ABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[00290] All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

EXAMPLES

[00291] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. Example 1 - Novel Diagnostic Tests Employing Renilla Luciferase Antibody

Fusion Proteins

Cohesin-Antigen/Dockerin-CBD system

[00292] Specific antibody responses in sera from healthy donors and patients can be measured using the Ag-Cohesin/Dockerin-CDB system. In this setting, the Dockerin-CBD fusion protein is chemically linked to Luminex beads. This multiplex assay format is very robust and harbors multiple features. First, cohesin-antigen fusion proteins can be produced at low cost in a soluble manner in high yield E. coli expression systems. Second, samples that are re-run in the bead assay independently give virtually identical results, ensuring reproducibility. Additionally, bead sets can monitored to ensure equivalent loading of each Cohesin-antigen fusion protein, thereby enabling direct comparison of titers against comparable runs. Furthermore, the assay is at least 10-fold more sensitive than a standard solid phase ELISA using the same antigen and typical serum dilution series start at 1 : 10 and use 20 μΐ total serum. Because the antigen is coupled to beads via the Dockerin-Cohesin interaction, all antigen epitopes are available for binding antibody, uncompromised by non- specific plate binding or chemical cross-linking effects. As a control, recombinant human IgG-Cohesin fusion protein can be run as an internal standard.

The Bioluminescent ImunoAssay (BLIA)

[00293] An antibody fused to any luciferase protein (i.e. Renilla, Gaussia or Photinus) that recognizes an species specific IgG (or IgM) can be used in a diagnostic test to detect an analyte that includes, but is not limited to, a pathogen or infection (BioLuminescent Immuno Assay, BLIA).

Methods

[00294] These methods describe a bioluminescent immunoassay to specifically detect pathogen- specific antibodies present in human sera from infected individuals. Alternatively, this assay could be used with animals. To this end, and as a proof of concept, we have developed a panel of more than 30 Cohesin-TB antigens capable of being simply produced as soluble proteins in high-yield E. coli expression systems. This accomplishment is of note given the difficulty in expressing M. tuberculosis antigens heterologously. These antigens were chosen among well-established antigenic proteins from M. tuberculosis, based on literature (Gideon et al., 2013; Ireton et al., 2010; Kunnath-Velayudhan et al., 2010; Steingart et al., 2009) (Table 1).

Table 1. Cohesin-fusion proteins that have been produced in E coli in large amounts and could be used for a TB

BLIA.

[00295] To translate this technology and establish a robust tuberculosistest, Cohesin-antigens are linked to cellulose beads through a CBD-Doc bridge as outlined in Figure 1, left panel. In this setting, we conserve all the benefits from the Luminex platform and by using cellulose beads, we decrease the price of each run. Furthermore, volumes larger than 20 μΐ can be used as Mtb antigen-specific antibodies will be "extracted" from the serum following binding to the cellulose beads-CBD-Doc/Coh- Antigen (up to 10 000 μΐ of diluted serum could be used and incubated with cellulose beads-CBD-Doc/Coh-Antigen). Antigen- specific antibodies present in low amount in serum may thus be detected.

[00296] To enable use as a Point Of Care test, i.e. the test will be simple, rapid, and highly protable. To achieve these goals, bioluminescence is used as a readout. To this end, Gaussia, Renilla or any Luciferase protein is fused to the C-terminal part of a mouse monoclonal antibody recognizing specifically human IgG (FIG 7, right panel).

Results

[00297] First the use of Renilla Luciferase as a read out is validated. To this end, a simple immunoassay was performed, using a recombinant monoclonal antibody directed against DCIR (Dendritic Cell ImmunoReceptor) fused to Renilla Luciferase (anti- DCIR/RLuc). The Renilla Luciferase gene was fused to the light or heavy or both chains of anti-DCIR antibody (FIG 8A). [00298] Supernatant from 293T cells expressing these constructs was assessed for Luciferase activity (FIG 8B). These results clearly show that the Renilla Lucif erase protein is still active following fusion with an antibody; addition of the Luciferase gene to both light and heavy chains gives the best result. [00299] Subsequently the ability of these antibodies to detect a Cohesin-DCIR ectodomain fusion protein was determined. The recombinant protein Cohesin-DCIR ectodomain was coated to 96-well plates and incubated with different concentrations of anti- DCIR/RLuc. Following washing steps, anti-DCIR/RLuc binding was detected directly by adding RLuc substrate or by incubating an anti-human IgG antibody couple to HRP as described in FIG 9.

[00300] In this experiment, different concentrations of anti-DCIR/RLuc antibody were used to assess the assay sensitivity. For high concentrations, there is no difference between the ELISA and BLIA (FIG 10). However, as the antibody concentration is decreased, the Luciferase assay demonstrates higher sensitivity compared to the ELISA approach (FIG 10). In part this is due to an RLU variation of 100 is still significant (background around 200 RLU) in comparison with HRP, signal around 0.09-0.099 with a background at 0.08 (FIG 10). The inventors were therefore able to specifically detect lower amount of Cohesin-DCIR by using a recombinant anti-DCIR antibody fused to Renilla Luciferase as a primary antibody in a BLIA compared to an ELISA with the same anti- DCIR/RLuc antibody and an HRP conjugated secondary antibody.

Example 2 - Dectection of intracellular IFNy with Bioluminescent ICS

Methods

[00301] In a standard IGRA assay IFNy secretion in T-cells is stimulated with peptides specific for a pathogen. Secreted IFNy or T-cells secreting IFNy in response to stimulation is subsequently measured. In a standard IGRA protocol, T-cells stimulation takes 16h to obtain enough secreted IFNy in the supernatant for adequate detection. Following this, an ELISA or an ELISPOT are performed to evaluate levels of cytokines production. As a result of the long stimulation period, the results can only be delivered the next day. To shorten this process, in the current protocol T-cells are stimulated with pools of peptides from ESAT6 or CFP10 in the presence of CD28 in order to increase the levels of secreted IFNy. Subsequently a recombinant anti-IFNy antibody fused to Renilla or Gaussia Luciferase is used to detect the cytokine. By doing so, it is possible to stimulate T-cells for only 6h and perform an Intra-Cellular Staining (ICS) with a Luciferase-fused antibody.

Results

[00302] First, the impact of aCD28 costimulation on IFNy, IP- 10 and IL-2 secretion, after peptide stimulation for 16 hours is analyzed (FIG 11).

[00303] To test whether paraformaldehyde might compromise the enzymatic activity of Renilla or Gaussia Luciferase during ICS due to the paraformaldehyde content of ICS buffer, incubation of recombinant Renilla Luciferase with ICS buffer (from BD in this experiment) or PBS was performed. As evidenced in FIG 12, the perm/wash solution has no negative effect on Renilla Luciferase and in contrast, the ICS solution enhances the Renilla Luciferase activity.

[00304] Stimulation with pathogen-derived peptides and aCD28 for 6h and detection using an anti-IFNy antibody fused to Renilla Luciferase could allow the detection intracellular IFNy in T-cells. The addition of a second biomarker (CD4 or CD3, for example) fused to the Firefly Luciferase would allow detection in the same assay.

Example 3 - Detection of environmental pathogens using Bioluminescent recombinant antibody.

Methods

[00305] To detect environmental pathogens, at least 2 monoclonal antibodies that recognize different extracellular proteins from the same pathogen can be used to perform a sandwich assay as described FIG 13. First, one of these antibodies has to be fused to the cohesin protein, and then linked to the beads, through an interaction with the Dockerin-CBD protein. Second, another antibody, recognizing a different extracellular protein, has to be fused to the Renilla or Gaussia or any other Luciferase. By incubating those cellulose beads with any liquid sample, only the presence of a specific pathogen would result in bioluminescence.

Example 4 - Detection of huma IgG within several dilutions of human sera with anti-human IgG/Gaussia Luciferase or anti-human IgG/HRP.

Methods [00306] Human IgG from serum BD1863 (control patient) was plated at 2 and 20 μ§/ι 1 (10 mg/ml of human IgG in serum, on average) overnight at 4°C in Acetate buffer pH 9.4. Subsequently, plates were washed 3 x 200 μΐ of PBS 0.05% T20 and blocked for lh at 37°C in PBS 0.05% T20 2% BSA. Following washes with 2 x 200 μΐ of PBS 0.05% T20, secondary antibodies (anti-human IgG fused to Gaussia Luciferase or conjugated to HRP ) were added (PBS 0.05%> T20 1% BSA). First concentration was 1/2200 and serial dilutions were of ¼. Following incubation at 37°C for 30 min reactions were washed 3 x 200 μΐ of PBS 0.05% T20. Finally, reactions were developed by addition of Coelenterazine-h, final concentration 5 μΜ and bioluminescence was read in the case of luciferase reactions. For HRP-conjugated antibody 100 μΐ of TMB was added and after 15 min 100 μΐ of HC1 1M was used to stop the reaction (FIG. 14, FIG 15).

Example 5 - Comparison of anti-human IgG/Gaussia Luciferase and anti-human IgG/HRP with 16 antigens and sera dilutions from health control or active tuberculosispatient.

Methods

[00307] CBD-Doc was coated onto ELISA and BLIA plates by diluting CBD-Doc to 66 nM in Acetate buffer pH9.4 and incubating for 2 h at 37°C (100 μΐ per well). Then, 3 x 200 μΐ washes of PBS 0.05% T20 and blocking for 2h at 37°C in PBS 0.05% T20 2% BSA followed. After a 1 x 200 μΐ wash of PBS 0.05% T20, Coh and Coh-Ag were then added in PBS Mg+ Ca+ 0.05 % T20 at a concentration of 160 nM and incubated for lh at 37°C. Subsequently 3 x 200 μΐ washes of PBS 0.05%> T20 were performed. Detection of Coh-Ag proteins was carried out on sera from a tuberculosis positive patient (#8) and healthy control (BD1863) diluted 1/400, 800, 1600, 3200 and 6400 in PBS 0.05 % T20 1% BSA. 100 μΐ of diluted sera was added to each well and incubated for 60 min at 37°C. After washing 3 x 200 μΐ with PBS 0.05% T20, anti-human HRP 1/10 000 or anti-human IgG/Gaussia Luciferase 1/20 000 in PBST 1% BSA was added (100 μΐ) and incubated 45 min at 37°C. Subsequently, 3 washes with 200 μΐ of PBS 0.05%> T20 were performed. The reactions were developed as follows. For ELISA, 100 μΐ of TMB was added and 15 min later 100 μΐ of HC1 1 M was added and absorbance measured at 450 nm (FIG 17). For BLIA, coelenterazine h was added to 5 μΜ and bioluminescence was read (FIG 16).

[00308] Comparing the ability of anti-IgG/HRP and anti-IgG/Gaussia Luciferase to detect TB-specific human IgG by doing an ELISA with CBD-Doc coated on plastic (ELISA plates or Luciferase assay plates, both maxisorb) by using 16 different antigens and 5 sera dilutions reveals that the luciferase-based assay is able to detect very low amount of human anti-TB antigen IgG, and the detection is linear relative to the HRP based assay (FIG 16, FIG 17).

Example 6 - Detection of Ag-specific human IgG in serum from tuberculosis # 3 to 9 and BD1863 with anti-human IgG/HRP with cellulose beads coated with CBD- Doc/Coh-Ag

Methods

[00309] To generate CBD-Doc/Coh coated beads, first, CBD-Doc/Coh complexes were formed in 1.5 ml tubes. To achieve this, 800 nM of CBD-Doc was added to 1600 nM of Coh in PBS Mg2+/Ca2+ 0.05 % T20 overnight at 4°C, with rotation. CBD-Doc/Coh complexes were then diluted to 400 nM with 50 μΐ of cellulose beads (25 μΐ bed volume) per 100 μΐ of complex solution of PBS Mg2+/Ca2+ at room temperature overnight, with agitation. For CBD-Doc & CDB-Doc/Coh, 250 μΐ in 500 μΐ, for other conditions, 400 μΐ in 800 μΐ. After 2 washes with 1 ml in PBS with Mg2+/Ca2+ and a wash with 1.5 ml PBS Mg2+/Ca2+, coated cellulose beads were blocked by incubating at RT for 60 min with 1000 μΐ of PBS 0.05 % T20 2% BSA with vigorous agitation. Detection of Coh-Ag proteins was achieved as follows. Sera from a tuberculosis patients #3-9 and BD1863+BD1575 (healthy patients) was diluted 1/800 in total volume of 10 ml (falcon tube) and then added to cellulose beads-CBD/Doc-Coh alone or Coh/ESAT6 or Coh/CFPIO (20 μΐ of beads, i.e. 10 μΐ of bed volume) in PBS 0.05% Tween 20 2% BSA and incubated lh at RT with rotation. After 3 washes with 10 ml PBS Mg2+/Ca2+ 0.05 % T20, anti-human HRP 1/25 000 in PBS Mg2+/Ca2+, 0.05% Tween 20 2% BSA was then added (1 ml) and incubated 30min at RT with vigorous agitation. After 3 washes with 10 ml with PBS Mg2+/Ca2+ 0.05 % T20 beads were resuspended in 50 μΐ of TMB and transferred into 96-well plate. 50 μΐ of TMB was used to rinse the falcon tube. Then 100 μΐ of HC1 1 M was added and 100 μΐ of the solution was used to read at 450 nm (FIG 18).

[00310] Detection of antigen specific human IgG in serum of tuberculosis patients and healthy controls as decribed above can carried out with luciferase attached antibodies or binding polypeptides to enhace sensitivity and signal fidelity. Example 7 - Detection of CD4+ T cells from PBMCs using anti-CD3 coated plates and anti-CD4-biotin + Streptavidin HRP

Methods [00311] CD4+ T cells were purified from PBMCs using CD4 microbeads following the Miltenyi and LS column protocol (Miltenyi Biotec). 25 x 10⁶ cells were thawed and 10 x 10⁶ recovered after thawing the cells. After purification yields were as follow: 4.6 x 10⁶ PBMCs depleted of CD4+ T cells (93.4% of CD4 negative cells) and 2.2 x 10⁶ CD4+ T cells (99.2% of CD4 positive cells). Cells were maintained at 2 x 10⁶ cells /ml in cRPMI overnight. To coat anti-CD3 OKT3 on plates OKT3 mAb was diluted at 2 μg/ml in Carbonate buffer pH 9.4 and 100 μΐ was added to each well and further incubated at 4°C overnight. After 3 washes 200 μΐ with PBS Mg2+/Ca2+ 0.05 % T20 wells were blocked by incubation at room temperature for 60 min with 200 μΐ of PBS 0.05 % T20 2% BSA with vigorous agitation. Subsequently 3 washes with 200 μΐ PBS Mg2+/Ca2+ were performed. Cells were added as indicated in Table 2 and incubated at 30 min at 4°C followed by 3 washes with 200 μΐ of PBS Mg2+/Ca2+. To detect CD4, anti-CD4-biotin was added at 0.125 μg per well as indicated above in PBS 3%> FCS and incubated for 20 min on ice. After 3 washes with 200 μΐ PBS Mg2+/Ca2+ cells were treated with PFA 1% in PBS to avoid cell lysis when TMB was added. Following 3 washes with 200 μΐ PBS Mg2+/Ca2+ strep-HRP at 1/ 10 000 was added in PBS 3% FCS for 30 min at 4°C. Reactions were washed with 200 μΐ PBS Mg2+/Ca2+ 3 times. 100 μΐ of TMB was added to each well. Once color appears, the reaction was stopped by transferring 50 μΐ of TMB solution from each well into new well containing 50 μΐ of HC1 1M. Results were read at 450 nm. [00312] Detection of CD4+ T cells as decribed above can carried out with luciferase attached antibodies or binding polypeptides to enhace sen^sitivity and signal fidelity.

* * *

[00313] All of the methods disclosed and claimed herein can be made and executed witho^ut undue experimentation in light of the present disclosure. While the comp°sitions and methods of this invention have been described in terms of prefer^red embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. REFERENCES

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

Carvalho, et al, Proc Natl Acad Sci USA. 100(24): 13809-14, 2003.

Gideon, et al, PLoS One. 8(8):e71351, 2013

International Patent Application No. WO 2009/028532

Ireton, et al, Clin Vaccine Immunol. 17(10): 1539-47, 2010.

Kunnath-Velayudhan, et al, Proc Natl Acad Sci USA. 107(33): 14703-8, 2010.

Newell, et al, Nature Methods. 6:497-99, 2009.

Ofir, et al, Proteomics. 5: 1806-14, 2005.

Starovasnik, et al, Proc Natl Acad Sci USA. 94(19): 10080-5, 1997.

Steingart, et al, Clin Vaccine Immunol. 16(2):260-76, 2009.

U.S. Patent Application No. 10/658,093

U.S. Patent No. 7,141,431

U.S. Publication No. 2005/0106700

U.S. Publication No. 2010/0062451

U.S. Publication No. 2011/0151538

Claims

WHAT IS CLAIMED IS:

1. A method of detecting an analyte in an ex vivo sample, comprising :

(a) contacting the sample with a solid matrix, wherein the matrix is bound to a Dockerin/Cohesin binding pair, and wherein the Dockerin is attached to a matrix binding domain and Cohesin is attached to an analyte-binding polypeptide; or

wherein the Cohesin is attached to a matrix binding domain and Dockerin is attached to an analyte-binding polypeptide; thereby forming a first analyte complex;

(b) contacting said first analyte-bound complex with an analyte-binding antibody, wherein the analyte-binding antibody is attached to a luciferase reporter, thereby forming a second complex; and

(c) testing the second complex for luciferase activity, thereby detecting the presence of the analyte.

2. The method of claim 1, wherein the contacting of steps (a) and (b) are performed in one step.

3. The method of claim 1 or 2, wherein the analyte is an antibody.

4. The method of claim 3, wherein the analyte-binding polypeptide is an antigen recognized by the antibody analyte.

5. The method of claim 3 or 4, wherein the analyte is a human antibody.

6. The method of claim 5, wherein the analyte is IgG, IgM, IgA, IgD or IgE.

7. The method of any of claims 1-4, wherein the analyte is an environmental pathogen.

8. The method of claim 7, wherein the environmental pathogen is detected by binding to a surface antigen.

9. The method of claim 8, wherein the surface antigen comprises a protein, glycoprotein, polysaccharide, lipopolysaccharide or lipid.

10. The method of claim 9, wherein the surface antigen comprises a component of the pathogen coat, capsule, cell wall, flagella, fimbrae or toxin.

11. The method of claim 7, wherein the environmental pathogen is HIV, Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium knowlesi, dengue fever virus, Schistosomiasis mansoni, Schistosomiasis japonicum, Schistosomiasis haematobium, Streptococcus pneumonia, Trypanosoma cruzi, Mycobacterium Leprae, Vibrio cholerae, Bordetella pertussis, Shigella dysenteriae, Escherichia coli or Mycobacterium ulcerans.

12. The method of claim 11, wherein the analyte is an antigen from the environmental pathogen.

13. The method of any of claims 1-12, wherein the analyte-binding antibody binds to IgG, IgM, IgA, IgD or lgE.

14. The method of any of claims 1-13, wherein the analyte-binding antibody is an antigen binding fragment of a recombinant monoclonal antibody.

15. The method of any of claims 1-14, wherein the analyte-binding antibody is attached to Renilla, Gaussia or Photinus luciferase.

16. The method of any of claims 1-15, wherein the luciferase reporter is attached to one or both of the light chains of the analyte-binding antibody.

17. The method of any of claims 1-16, wherein the luciferase gene is attached to a heavy chain or both light and heavy chain of the analyte-binding antibody.

18. The method of any of claims 1-17, wherein at least one dockerin domain is attached to the analyte-binding antibody.

19. The method of claim 18, wherein the luciferase reporter is attached to cohesin and binds to dockerin attached to the analyte-binding antibody.

20. The method of claim 19, wherein signal amplification is achieved by the binding of more than one cohesin/luciferase per analyte-binding antibody.

21. The method of any of claims 1-20, wherein the luciferase protein is detected by a luminometer.

22. The method of any of claims 1-21, wherein the sample is a biological sample from a subject.

23. The method of any of claims 1-22, wherein the sample is an environmental subject comprising a water sample, an air sample or a soil sample.

24. A method of detecting an analyte in an ex vivo sample, comprising:

(a) contacting the sample with either: (i) a Cohesin attached to an analyte - binding polypeptide; or (ii) a Dockerin attached to an analyte -binding polypeptide, thereby forming a first analyte-bound complex;

(c) contacting said immobilized analyte-bound complex with an analyte - binding antibody, wherein the analyte-binding antibody is attached to a luciferase reporter, thereby forming a further complex; and