Site-specific protein labeling using oligonucleotide tags

Abstract

Protein conjugates and kits for detecting proteins and antigens of interest in cell and tissue samples are described. Compositions and methods to tag an unmodified full-length antibody or antigen binding protein with an oligonucleotide are described. Fusion proteins comprising a protein and a site-specific relaxase domain which forms a covalent adduct with an oligonucleotide tag are also described. In vitro methods of detecting a target antigen in a sample using the compositions and conjugates are also described. Methods of assembling a labelled conjugate are further described.

Description

[0001] SITE-SPECIFIC PROTEIN LABELING USING OLIGONUCLEOTIDE TAGS

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority to Indian Provisional Application No. IN202441047066, filed June 19, 2004. The entire contents of the aforementioned application are incorporated by reference herein.

[0004] FIELD

[0005] This disclosure concerns labelled protein conjugates and use of the conjugates for detection of target antigens in cell or tissue samples.

[0006] SEQUENCE LISTING

[0007] The application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on May 29, 2025, is named “TP384974WOl.xml” and is 62,183 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety.

[0008] INCORPORATION BY REFERENCE

[0009] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0010] BACKGROUND

[0011] Studies to examine cellular functions involve various biochemical assays including, for example, protein or antigen tagging, molecular assays such as next-generation sequencing, qPCR, and tracking and imaging of proteins in cells and tissues. To examine protein interactions, spatial protein organization in tissues is interrogated using, for example, immunocytochemical approaches with antibodies conjugated to enzymes or dyes. To understand the function of cellular interactions in a tissue context, multiple analytes need to be simultaneously assayed, such as with mass spectrometry or antibody-based assays. However, currently available techniques for labelling proteins such as ligands, antigens, antibodies and antigen binding proteins have several limitations. The primary methodology for labelling proteins includes chemical coupling using reactive chemical linkers, which are non-selective and limited in terms of reaction specificity. Furthermore, methods of tagging proteins such as antibodies with dyes in a specific manner often require antibody engineering. Thus, a need exists for improved methods of labeling proteins and detecting proteins and antigens of interest in cells and tissues.

[0012] SUMMARY

[0013] Described herein are compositions and methods to tag a protein with an oligonucleotide using a relaxase domain fused to the protein. Fusion proteins comprising a protein and a sitespecific relaxase domain which forms a covalent adduct with an oligonucleotide tag are also provided in the present disclosure.

[0014] Provided herein are conjugates comprising a protein chosen from a ligand, a protein domain, an antigen, an antigen binding protein, an antibody, an antibody fragment, a singledomain antibody, a VHH antibody, or a protein comprising an Fc binding domain; a relaxase domain; and an oligonucleotide covalently attached to the relaxase domain. In some aspects, the conjugates further comprise a linker positioned between the protein and the relaxase domain. In some aspects, the linker of the conjugate comprises any one of SEQ ID NO: 37-41. In some aspects, the conjugates further comprise a purification tag. In some aspects, the purification tag is attached to the N-terminus of the protein. In some aspects, the protein is an Fc binding domain which is a bacterially derived or a synthetically prepared immunoglobulin binding domain or a Fc binding protein (FcBP). In some aspects, the bacterially derived immunoglobulin binding domain comprises a Z domain or a variant thereof that retains the capacity to bind an antibody Fc region. In some aspects, the amino acid sequence of the Z domain comprises SEQ ID NO: 1, or the amino acid sequence of the variant Z domain comprises SEQ ID NO: 2. In some aspects, the bacterially derived immunoglobulin binding domain comprises the C2 domain of Streptococcal Protein G or a variant thereof that retains the capacity to bind an antibody Fc region. In some aspects, the amino acid sequence of the C2 domain comprises SEQ ID NO: 3. In some aspects, the amino acid sequence of FcBP comprises any one of SEQ ID NO: 4-16. In some aspects, the relaxase domain is obtained from Porcine circovirus 2 (PCV2), Wheat dwarf virus (WDV) ox Agrobacterium tumefaciens. In some aspects, the relaxase domain is obtained from VirD2 protein. In some aspects, the relaxase domain comprises SEQ ID NO: 17 or 18. In some aspects, the relaxase domain obtained from VirD2 protein comprises SEQ ID NO: 19. In some aspects, the nucleic acid sequence of the oligonucleotide comprises any one of SEQ ID NO: 20-36. In some aspects, the oligonucleotide is about 10 to about 150 nucleotides in length, about 20 to about 125 nucleotides in length, or about 50 to about 100 nucleotides in length. In some aspects, the oligonucleotide comprises a qPCR handle. In some aspects, the qPCR handle further comprises a PCR priming site, a label, a barcode, or an RNA polymerase binding site. In some aspects, the oligonucleotide comprises a label. In some aspects, the label is directly or indirectly detectable. In some aspects, the label comprises a fluorescent agent, a hapten, biotin, a nucleic acid analog, or a digoxygenin label. In some aspects, the oligonucleotide is a fold-back probe. In some aspects, the fold-back probe comprises any one of SEQ ID NO: 24-36. In some aspects, the fold-back probe comprises one or more fluorescent agent. In some aspects, the fluorescent agent is chosen from a xanthene, a fluorescein, a rhodamine, a rhodol, a roseamine, a carbopyranone, an indole, an indacene, a borapolyazaindacene, a furan, a benzofuran, a cyanine, a benzocyanine, a benzopyrilium, a pyrene, a coumarin, a styryl, a squarine, a resorufin, an anthraquinone, an acridine and a benzophenoxazine. In some aspects, the conjugate further comprises an immunoglobulin molecule, wherein the Fc region of the immunoglobulin molecule is non-covalently bound to the Fc binding domain. In some aspects, the immunoglobulin molecule is an IgG, or the immunoglobulin molecule is a F(ab), a F(ab’)2, a single-domain VHH antibody, or a single-chain variable fragment (scFv).

[0015] In some aspects, the conjugate comprises an Fc binding domain, a relaxase domain, a linker positioned between the Fc binding protein and the relaxase domain, and an oligonucleotide comprising a label, wherein the oligonucleotide is covalently attached to the relaxase domain. In some aspects, the conjugate further comprises an immunoglobulin molecule, wherein the Fc region of the immunoglobulin molecule is non-covalently bound to the Fc binding domain. In some aspects, the conjugate further comprises a purification tag attached to the N-terminus of the Fc binding domain.

[0016] Provided herein are methods comprising contacting a sample with a conjugate of the present disclosure; incubating the sample with the conjugate for an amount of time sufficient to allow the conjugate to bind to a target within the sample; and detecting the conjugate. Also provided herein are methods comprising contacting a sample with the conjugate of the present disclosure, wherein the antigen binding protein, antibody, antibody fragment, or immunoglobulin molecule of the conjugate is specific for an antigen present in the sample; and detecting the conjugate. Also provided herein are methods comprising contacting a sample with an antibody, antibody fragment, or immunoglobulin molecule specific for an antigen present in the sample; incubating the sample with the antibody, antibody fragment, or immunoglobulin molecule for an amount of time sufficient to allow the antibody, antibody fragment, or immunoglobulin molecule to bind to the antigen forming an antigen complex; adding a conjugate of the present disclosure and incubating for an amount of time sufficient to allow the Fc binding domain to non-covalently bind to the Fc region of the antibody, antibody fragment, or immunoglobulin molecule; and detecting the conjugate. In some aspects, the sample is chosen from live cells, intracellular fluids, extracellular fluids, sera, biological fluids, biological fermentation media, environmental samples, industrial samples, viruses, proteins, peptides, buffer solutions, blood cells, immune cells, cultured cells, cellular extracts, tissue, muscle tissue, neurons, extracellular vesicles, vascular tissue, blood fluids, saliva, urine, water, soil, wastewater, sea water, pharmaceuticals, foodstuffs, and beverages. In some aspects, the sample is immobilized on a polymeric membrane, within a polymeric gel, on a microparticle, on a microarray, on a silicon chip, on a glass slide, on a microwell plate, and on a microfluidic chip. In some aspects, the tissue is chosen from tumor tissue, epidermal tissue, muscle tissue, bone marrow tissue, neural tissue, brain tissue, organ tissue, and human biopsy tissue. In some aspects, the tissue is a formalin-fixed paraformaldehyde-embedded tissue section or a fresh frozen tissue section. In some aspects, the detecting step comprises immunohistochemistry, fluorescence microscopy, flow cytometry, Western blot, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction, qPCR, immunoPCR, rolling circle amplification, next generation sequencing (NGS) using NGS-enabled barcoded oligonucleotides, or nicking enzyme signal amplification. In some aspects, the methods further comprise exposing the sample to low pH, high temperature and / or high salt concentration to release the Fc binding domain from the antibody, antibody fragment, or immunoglobulin molecule. In some aspects, the sample is exposed to pH 2 to pH 4, optionally wherein the sample is exposed to a composition comprising 10 mM glycine adjusted to pH 2-pH 4. In some aspects, the methods further comprise contacting the sample with a second conjugate of the present disclosure wherein the second conjugate has a different detectable label than the first conjugate.

[0017] Provided herein are kits comprising one or more of the conjugates according to the present disclosure and instructions for use according to any of the methods described herein. In some aspects, the kits further comprise at least one of the following: a buffering agent, a purification medium, a vial comprising the sample, and an organic solvent. In some aspects, the kits comprise a fusion protein comprising an antigen binding protein and a relaxase domain, and an oligonucleotide comprising a label. In some aspects, the kits comprise a fusion protein comprising an Fc binding protein and a relaxase domain, and an oligonucleotide comprising a label. In some aspects, the kits further comprise an antibody, antibody fragment, or immunoglobulin molecule. In some aspects, the fusion protein further comprises a purification tag. In some aspects, the purification tag is attached to the N-terminus of the fusion protein. In some aspects, the Fc binding domain is a bacterially derived or a synthetically prepared immunoglobulin binding domain or a Fc binding protein (FcBP). In some aspects, the bacterially derived immunoglobulin binding domain comprises a Z domain or a variant thereof that retains the capacity to bind an antibody Fc region. In some aspects, the amino acid sequence of the Z domain comprises SEQ ID NO: 1; or the amino acid sequence of the variant Z domain comprises SEQ ID NO: 2. In some aspects, the bacterially derived immunoglobulin binding domain comprises the C2 domain of Streptococcal Protein G or a variant thereof that retains the capacity to bind an antibody Fc region. In some aspects, the amino acid sequence of the C2 domain comprises SEQ ID NO: 3. In some aspects, the amino acid sequence of FcBP comprises any one of SEQ ID NO: 4-16. In some aspects, the relaxase domain is obtained from Porcine circovirus 2 (PCV2), Wheat dwarf virus (WDV) ox Agrobacterium tumefaciens. In some aspects, the relaxase domain is obtained from VirD2 protein. In some aspects, the relaxase domain comprises SEQ ID NO: 17 or 18. In some aspects, the relaxase domain obtained from VirD2 protein comprises SEQ ID NO: 19. In some aspects, the nucleic acid sequence of the oligonucleotide comprises any one of SEQ ID NO: 20-36. In some aspects, the oligonucleotide is about 10 to about 150 nucleotides in length, about 20 to about 125 nucleotides in length, or about 50 to about 100 nucleotides in length. In some aspects, the oligonucleotide comprises a qPCR handle. In some aspects, the qPCR handle further comprises a PCR priming site, a label, a barcode, or an RNA polymerase binding site. In some aspects, the oligonucleotide comprises a label. In some aspects, the label is directly or indirectly detectable. In some aspects, the label comprises a fluorescent agent, a hapten, biotin, a nucleic acid analog, or a digoxygenin label. In some aspects, the oligonucleotide is a fold-back probe. In some aspects, the fold-back probe comprises any one of SEQ ID NO: 24-36. In some aspects, the fold-back probe comprises one or more fluorescent agent. In some aspects, the kit further comprises instructions for use according to any of the methods described herein.

[0018] Provided herein are methods comprising contacting a sample with a conjugate of the present disclosure; incubating the sample with the conjugate for an amount of time sufficient to allow the conjugate to bind to a target within the sample; and detecting the conjugate. In some aspects, the sample comprises a cell population. In some aspects, the cell population comprises B cells. In some aspects, the conjugate comprises a ligand, an antigen, or a protein domain. In some aspects, the conjugate comprises a fold-back probe. In some aspects, the method further comprises selecting or enriching for B-cells that are bound to the conjugate.

[0019] The foregoing and other features of this disclosure will become more apparent from the following detailed description of several aspects which proceeds with reference to the accompanying figures.

[0020] BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1A depicts a schematic of a fusion protein comprising a protein, a relaxase domain and an oligonucleotide tag covalently bound to the relaxase domain (herein termed a “PRO- tag”) according to an embodiment of the present disclosure. In this example, the PRO-tag comprises a fusion protein of an antibody binding protein (Z domain represented by the rectangular “Zdom”) and a bacterial relaxase domain (represented by the oval “VirD2”) covalently attached to an oligonucleotide (a VirD2 recognition oligonucleotide represented by the curved line).

[0022] FIG. IB shows a Coomassie stained gel of the protein-relaxase domain fusion protein (Zdom-VirD2) depicted in FIG. 1A expressed and purified from E. coli. Size exclusion chromatography elution fractions were run in an SDS-PAGE gel under reducing and nonreducing conditions.

[0023] FIG. 1C shows a western blot of the results of an oligonucleotide conjugation with the Zdom-VirD2 fusion protein. “Zdom-VirD2-oligo adduct” is the Zdom-VirD2 fusion protein conjugated to the oligonucleotide (the “PRO-tag”) and “Zdom-VirD2” is the unreacted fusion protein (fusion protein without an attached oligonucleotide).

[0024] FIG. 2A is a schematic of a protein-relaxase domain fusion protein comprising a His- tag, thrombin cleavage site, Z-domain, a linker (such as GGGAS, SEQ ID NO: 37), and a PCV2 relaxase domain.

[0025] FIG. 2B is a Coomassie stained gel showing supernatants (S) and pellets (P) of cell extracts of E. coli BL21(DE3) strain either induced with 1 mM IPTG at 37°C or 18°C to express the Z-domain-PCV2 fusion protein, or uninduced (UI). The dotted box indicates the Z-domain- PCV2 fusion protein which has an expected molecular weight of ~26 kDa.

[0026] FIG. 2C is a western blot of the gel shown in FIG. 2B probed with an anti-6xHIS antibody confirming the expression of the Z-domain-PCV2 fusion protein in the E. coli BL21 (DE3) strain induced at 37°C and 18°C.

[0027] FIG. 2D is a chromatograph of the fractions from a size exclusion column (SEC) of the pooled supernatant fraction affinity purified from Ni-NTA resin.

[0028] FIG. 2E is a Coomassie stained gel of the eluted SEC fractions showing the fractions that contain the purified Z-domain-PCV2 fusion protein.

[0029] FIG. 3A is a schematic of the conjugation of a biotinylated oligonucleotide to a Z- domain-PCV2 fusion protein (a biotinylated PRO-tag), followed by confirmation by western blot using a mouse anti-His HRP antibody .

[0030] FIG. 3B is a western blot probed with an anti-6xHIS antibody showing successful covalent assembly of the biotinylated PRO-tag (“Zdom-PCV2-Biotin Oligo”).

[0031] FIG. 4A is a schematic of the conjugation of an Alexa Fluor™ 647-labeled oligonucleotide to a Z-domain-PCV2 fusion protein (a fluorescently-labeled PRO-tag), followed by confirmation by western blot using a mouse anti-His primary antibody and an antimouse Alexa Fluor™ Plus 800 (AFP800) secondary antibody.

[0032] FIG. 4B shows an SDS-PAGE gel shift analysis of the Alexa Fluor™ 647-labeled oligo (“Oligo-AF647”) conjugated to the Z-domain-PCV2 fusion protein at four different concentrations (left) and a western blot probed with an anti-6xHIS antibody and an Alexa Fluor™ 800-labeled secondary antibody (right) showing successful covalent assembly of the PRO-tag (Zdom-PCV2- Oligo-AF647).

[0033] FIG. 4C shows a multiplex overlay of the signal from the AF647 and AF800 channels shown in FIG. 4B where the PRO-tag (Zdom-PCV2-Oligo-AF647) is the top band of the 50, 100, 200 and 300 pmol lanes (right), and a silver-stained gel showing the PRO-tag (Zdom- PCV2-Oligo-AF647) and the unconjugated oligo. FIG. 5A is a schematic of the workflow used to detect recombinant IL-6 using an anti- IL-6 antibody conjugated with a PRO-tag according to an embodiment of the methods described herein.

[0034] FIG. 5B is a graph showing the results of an ELISA assay using an anti-IL6 antibody conjugated to a PRO-tag or an anti-ATG4B antibody conjugated to a PRO-tag to detect IL-6 (dotted line) or ATG4B (solid line), respectively.

[0035] FIG. 6A is a schematic of a workflow used in an immunoprecipitation assay using an antibody conjugated with a PRO-tag according to an embodiment of the methods described herein.

[0036] FIG. 6B shows a series of western blots showing the antibody-PRO-tag complex binds to its desired antigen in vitro in an immunoprecipitation assay performed using the workflow outlined in FIG. 6A. The top panel was probed with a mouse anti-IL-6 antibody used for detection (IL-6 was the antigen used in the assay). The middle panel was probed with a mouse anti-His-HRP antibody which recognizes the PRO-tag. The bottom panel was probed with a goat-anti-rabbit HRP secondary antibody (GAR-HRP) which recognizes the rabbit anti-IL-6 antibody that was conjugated to the PRO-tag. The band corresponding to the anti-IL-6 antibody heavy chain is indicated in the boxed region.

[0037] FIG. 7 A is a schematic of the workflow used in an immuno-PCR assay using an antibody conjugated with a PRO-tag according to an embodiment of the methods described herein.

[0038] FIG. 7B is a graph showing the results of an immuno-PCR assay using an anti-IL6 antibody conjugated to a PRO-tag or an anti-ATG4B antibody conjugated to a PRO-tag to detect IL-6 (dotted line) or ATG-4 (solid line), respectively and used in the method outlined in FIG. 7A.

[0039] FIG. 8A is a schematic of a workflow used in a dual antigen immuno-PCR assay using two separate antibodies each conjugated with a unique PRO-tag according to an embodiment of the methods described herein. FIG. 8B is a graph showing the results of a dual antigen immuno-PCR assay using the workflow outlined in FIG. 8A, wherein each PRO-tag conjugated antibody was titrated as a function of antigen concentration, IL-6 (grey bars) and ATG4B (black bars).

[0040] FIG. 9 A is a schematic of a workflow used for on-demand labeling of antibodies using a PRO-tag comprising a fold-back oligonucleotide according to an embodiment of the methods described herein.

[0041] FIG. 9B shows an exemplary fold-back oligo (SEQ ID NO: 46) with fluorescently labeled dNTPs using the Klenow fragment that can be attached to a protein, such as an antibody, using a relaxase domain (top panel). The bottom panel shows western blots of anti- ATG4B antibodies (bottom left) or anti-IL-6 antibodies (bottom right) labeled with an Alexa Fluor™ 647-labeled fold -back PRO-tag and detected either by direct visualization of the Alexa Fluor™ 647 label (1stand 3rdpanel) and compared with an Alexa Fluor™ 800-labeled goat- anti-rabbit secondary antibody (2ndand 4thpanel).

[0042] FIG. 10A is a schematic of a workflow used to test stability of PRO-tag under assay conditions involving one or more analytes in a mixed antibody solution.

[0043] FIG. 10B shows western blots of the experiment outlined in FIG. 10A demonstrating stability of a PRO-tag in an assay with two analytes. Rabbit anti-transferrin antibodies were labeled with an Alexa Fluor™ 647-labeled PRO-tag and were co-incubated with unlabeled rabbit anti-IL-6 antibodies. The left panel shows a blot showing the Alexa Fluor™ 647 labeling of only the anti-transferrin antibody (TF) whereas the right panel shows a blot that was probed using an Alexa Fluor™ 800-labeled secondary antibody (GAR-AF800) that binds to both the anti-transferrin antibody (TF) and the anti-IL-6 antibody (IL). The band in the TF lane is labeled with both the Alexa Fluor™ 647 and the Alexa Fluor™ 800-labeled secondary antibody (multiplex), however, the band in IL-6 lane is labeled with only Alexa Fluor™ 800-labeled secondary antibody demonstrating the stability of the PRO-tag and absence of dissociation in a mixed antibody solution with multiple analytes.

[0044] FIG. IOC depicts western blots showing the PRO-tag is stable in the presence of multiple analytes and a mixed antibody solution.

[0045] FIG. 11 shows a schematic of a PRO-tag that can bind to antibody fragments (e.g. Fab) or single-chain antibodies (a “VHH-PRO-tag”). In this example, the PRO-tag comprises a VHH antibody-relaxase domain fusion protein that is covalently attached to a detectably labeled oligonucleotide. Also shown is an antigen bound to the VHH portion of the PRO-tag.

[0046] FIG. 12A shows a schematic of a recombinant fusion of a ligand and a relaxase domain that is covalently linked to a labeled oligonucleotide (a “ligand-PRO-tag”). In this example, multiple ligand-PRO-tags are each labeled with a different dye and bind to their respective receptor on a single cell.

[0047] FIG. 12B shows a schematic depicting multiple ligand-PRO-tags each labeled with a different fold-back probe comprising a unique barcode sequence and a nicking sequence.

[0048] FIG. 12C shows a schematic of a recombinant fusion of an antigen and a relaxase domain that is covalently linked to a dye-labeled fold-back probe. In this example, two different antigen-PRO-tags are depicted binding to their respective B-cells and can be used for B-cell selection in a cell population.

[0049] FIG. 12D shows a schematic depicting a biotinylated antigen-PRO-tag. In this example, a specific B-cell can be pulled down using streptavidin-beads that bind the biotinylated fold-back probe, when the antigen-PRO-tag is bound to its specific B-cell receptor (BCR).

[0050] FIG. 12E shows a schematic for using PRO-tags for domain- specific enrichment of B cells. The left diagram depicts a receptor / protein- 1 that has four domains: I, II, III and IV. The right diagram depicts fusion proteins comprising a single domain of receptor / protein- 1 (e.g., domain I, II, III or IV) with a relaxase domain covalently bound to unique oligo tags which are designed to bind to a B-cell population (I-R-oligo, II-R-oligo, III-R-oligo, IV-R-oligo).

[0051] SEQUENCES

[0052] The nucleic acid and amino acid sequences are shown using standard letter abbreviations for nucleotide bases, and single letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. DETAILED DESCRIPTION

[0053] I. Abbreviations

[0054] FcBP Fc binding protein

[0055] PCV2 Porcine circo virus 2

[0056] VHH single domain antibody containing the variable domain of the heavy chain antibody

[0057] WDV Wheat dwarf virus

[0058] II. Summary of Terms

[0059] Unless otherwise noted, technical terms are used according to conventional usage. Definitions of many common terms in molecular biology may be found in Krebs et al. (eds.), Lewin ’s genes XII, published by Jones & Bartlett Learning, 2017. As used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term “an antigen” includes singular or plural antigens and can be considered equivalent to the phrase “at least one antigen.” As used herein, the term “comprises” means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various aspects, the following explanations of terms are provided:

[0060] Antibody or immunoglobulin: A polypeptide ligand that includes at least one variable region that recognizes and binds (such as specifically recognizes and specifically binds) an epitope of an antigen, such as an intracellular protein of interest. Mammalian immunoglobulin molecules are composed of two heavy (H) chains and two light (L) chains, each of which has a variable region, termed the variable heavy (VH) region and the variable light (VL) region, respectively. Together, the VH region and the VL region are responsible for binding the antigen recognized by the antibody. The heavy and light chains of mammalian immunoglobulins also include constant regions. For IgA, IgD, and IgG, the heavy chain constant region includes the CHI, CH2 and CH3 domains, while the light chain constant region includes the CL domain.

[0061] There are five main heavy chain classes (or isotypes) of mammalian immunoglobulin, which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Antibody isotypes not found in mammals include IgX, IgY, IgW and IgNAR. IgY is the primary antibody produced by birds and reptiles and is functionally similar to mammalian IgG and IgE. IgW and IgNAR antibodies are produced by cartilaginous fish, while IgX antibodies are found in amphibians.

[0062] Antibody variable regions contain framework regions (FR) and hypervariable (HV) regions, known as “complementarity determining regions” or “CDRs.” The CDRs are primarily responsible for binding to an epitope of an antigen. The framework regions of an antibody serve to position and align the CDRs in three-dimensional space. The amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well- known numbering schemes, including those described by Kabat et al. Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991; the “Kabat” numbering scheme), Chothia et al. (see Chothia and Lesk, J Mol Biol 196:901-917, 1987; Chothia et al., Nature 342:877, 1989; and Al-Lazikani et al., JMB 273,927-948, 1997; the “Chothia” numbering scheme), Kunik et al. (see Kunik et al., PLoS Comput Biol 8:el002388, 2012; and Kunik et al., Nucleic Acids Res 40(Web Server issue):W521-524, 2012; “Paratome CDRs”) and the ImMunoGeneTics (IMGT) database (see, Lefranc, Nucleic Acids Res 29:207- 9, 2001; the “IMGT” numbering scheme). The Kabat, Paratome and IMGT databases are maintained online.

[0063] A “monoclonal antibody” is an antibody produced by a single clone of lymphocytes or by a cell into which the coding sequence of a single antibody has been transfected. Monoclonal antibodies include humanized monoclonal antibodies.

[0064] A “chimeric antibody” has framework residues from one species, such as human, and CDRs (which generally confer antigen binding) from another species.

[0065] A “humanized” antibody is an immunoglobulin including a human framework region and one or more CDRs from a non-human (for example a camel, llama, mouse, rabbit, rat, shark or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDRs is termed a “donor,” and the human immunoglobulin providing the framework is termed an “acceptor.” Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, such as at least about 85-90%, such as about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions.

[0066] In some aspects herein, the antibody is an antigen-binding fragment of an immunoglobulin molecule. Examples of antibody fragments include but are not limited to Fab, Fab', F(ab')2 and single-chain antibody molecules (e.g., scFv, VHH). Antibody fragments include antigen binding fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies (see, e.g., Kontermann and Diibel (Eds.), Antibody Engineering, Vols. 1-2, 2nded., Springer- Verlag, 2010).

[0067] A Fab is an antibody fragment that contains a monovalent antigen-binding fragment of an antibody molecule, and can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain. A Fab' is a fragment of an antibody molecule obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule. A (Fab' , is an antibody fragment that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds.

[0068] A single-chain antibody (scFv) is a genetically engineered molecule containing the VH and VL domains of one or more antibody(ies) linked by a suitable polypeptide linker as a genetically fused single chain molecule (see, for example, Bird et al., Science, 242(4877):423-426, 1988; Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85(16):5879-5883, 1988; Ahmad et al., Clin. Dev. Immunol., 2012, doi: 10.1155 / 2012 / 980250; Marbry and Snavely, IDrugs, 13(8):543-549, 2010). The intramolecular orientation of the VH domain and the VL domain in a scFv is typically not decisive for scFvs. Thus, scFvs with both possible arrangements (VH domain-linker domain-Vi. domain; VL domain-linker domain-Vn domain) may be used. A “single-domain antibody” (also referred to as a “nanobody” or “VHH antibody” or “VHH antibody”) refers to an antibody having a single domain (a variable domain) that is capable of specifically binding an antigen, or an epitope of an antigen, in the absence of an additional antibody domain. Single-domain antibodies include, for example, VH domain antibodies, VNAR antibodies, camelid VHH antibodies, and VL domain antibodies. VNAR antibodies are produced by cartilaginous fish, such as nurse sharks, wobbegong sharks, spiny dogfish and bamboo sharks. Shark VNAR are comprised of the following regions (N-terminal to C-terminal): FRl-CDRl-FR2-HV2-FR3a-HV4-FR3b-CDR3-FR4. The positions of CDR1 and CDR3 of VNAR antibodies can be determined, for example, using IMGT. HV2 and HV4 can be determined, for example, using annotation described in Stanfield et al. Science 305: 1770-1773, 2004) and Fennell et al. (J Mol Biol 400: 155-170, 2010). Camelid VHH antibodies are produced by several species including camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain antibodies that are naturally devoid of light chains. Camelid VHH are comprised of the following regions (N-terminal to C-terminal): FR1-CDR1- FR2-CDR2-FR3-CDR3-FR4. Camel VHH CDR residues can be determined, for example, according to IMGT, Kabat or Paratome.

[0069] Conservative variant: A protein containing conservative amino acid substitutions that do not substantially affect or decrease the binding properties or activity of the protein. For example, an Fc binding domain, FcBP, Z domain or C2 domain disclosed herein can include at most about 1, at most about 2, at most about 3, at most about 4, at most about 5, or at most about 10, conservative substitutions and retain its ability to mediate to an antibody Fc region. The term “conservative variant” also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid. Non-conservative substitutions are those that reduce an activity or binding properties of a protein.

[0070] Conservative amino acid substitution tables providing functionally similar amino acids are well known. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another:

[0071] 1) Alanine (A), Serine (S), Threonine (T);

[0072] 2) Aspartic acid (D), Glutamic acid (E);

[0073] 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K);

[0074] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0075] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0076] Contacting: Placement in direct physical association; includes both in solid and liquid form.

[0077] Detect: To determine if a particular antigen, agent, analyte, or signal is present or absent, and in some examples further includes quantification of the antigen, agent, analyte, or signal if detected. In some examples, the signal detected is fluorescence or enzymatic activity.

[0078] Detectable label: A detectable compound or composition that is conjugated directly or indirectly to another molecule, such as an antibody, antibody fragment, protein, peptide, oligonucleotide, nucleic acid, or peptide nucleic acid (PNA), to facilitate detection of that molecule. Exemplary detectable labels include, but are not limited to ligands, radionuclides, fluorescent dyes, chemiluminescent agents, microparticles, nanoparticles (e.g., a gold nanoparticle), enzymes, colorimetric labels, magnetic labels, small molecules (e.g., biotin), streptavidin, haptens, allophycocyanin (APC), oligonucleotides, and peptide tags (e.g., Myc tag, His tag, or FLAG tag). Specific, non-limiting examples of labels include fluorescent dyes, enzymatic labels, and radioactive isotopes. In some aspects herein, the label is a fluorescent label (e.g., an ALEXA FLUOR dye, FITC, TRITC, Rhodamine, Texas red, or Qdot) or a non- fluorescent label (such as horseradish peroxidase (HRP), beta-galactosidase, luciferase, or alkaline phosphatase).

[0079] Fc binding domain: Any domain of a polypeptide or protein conjugate (such as a conjugate disclosed herein) that is capable of specifically binding an Fc region of an antibody. In some examples, the Fc binding domain is an Fc binding protein (FcBP), such as an FcBP having the amino acid sequence of any one of SEQ ID NOs: 4-16, or a variant thereof having at least 80% identity to any one of SEQ ID NOs: 4-16, as set forth herein. In other examples, the Fc binding domain is from a bacterial immunoglobulin binding protein, such as protein A or protein G. For example, the Fc binding domain may include the Z domain from protein A (or a portion or variant thereof), the G domain from protein G (or a portion or variant thereof), or the C2 domain of protein G (or a portion or variant thereof). Exemplary Z domain sequences are set forth herein as SEQ ID NOs: 1-2 and an exemplary C2 domain sequence is set forth herein as SEQ ID NO: 3. In other examples, the Fc binding domain is an Fc binding peptide. Exemplary Fc binding peptides include cyclic or branched peptides, such as PAM, Fc-III, FcBP-1, FcBP-2, FC-III-4C, and FcRM (see, e.g., Choe et al., Materials Basel 9:994, 2016).

[0080] Fc region: The constant region of an antibody excluding the first heavy chain constant domain. The “Fc region” generally refers to the last two heavy chain constant domains of IgA, IgD, and IgG, and the last three heavy chain constant domains of IgE and IgM. An Fc region may also include part or all of the flexible hinge N-terminal to these domains. For IgA and IgM, an Fc region may or may not include the tailpiece and may or may not be bound by the J chain. For IgG, the Fc region is typically understood to include immunoglobulin domains Cy2 and Cy3 and optionally the lower part of the hinge between Cyl and Cy2.

[0081] Fluorescent label: A molecule that is attached to aid in the detection of a biomolecule such as a protein, antibody, or amino acid. Fluorescent labels are also referred to as fluorophores, fluorescent tags, fluorescent dyes, or fluorescent probes. A fluorescent label may be a naturally occurring fluorescent protein (e.g., phycoerythrin, PE), a derivative thereof (e.g., PE-Cy7), a tandem dye, a polymer dye, a single molecule dye, an organic dye, a fluorescent nucleic acid, a fold-back oligonucleotide probe with a complementary 3' end for fluorescent dye incorporation, or a scaffold-based fluorescent label, for example a nucleic acid nanostructure including fluorescent DNA nanostructures such as PHTION nucleic acid nanostructures, including NOVAFEUOR dyes (Thermo Fisher Scientific, Waltham, MA).

[0082] Fold-back probe: As used herein, a fold-back probe (also referred to as a fold-back oligo) is a single- stranded oligonucleotide in which one or more portions of the single- stranded oligonucleotide form base pairs with one or more other portions of the same single- stranded oligonucleotide. When the two portions are base paired to form a double-stranded portion of the oligonucleotide, the double-stranded portion may be referred to as a hairpin. In some aspects, the Tmof the bases forming a hairpin is greater than 45°C. In some aspects herein, the oligonucleotide comprises a detectable label attached to the 3 ’ end or one or more detectable labels are covalently attached to the 3’ end or one or more detectable labels extend from the 3’ end. In some examples herein, the fold-back probe has the nucleic acid sequence of any one of SEQ ID NO: 24-34 or a variant thereof having at least 80% identity. An exemplary fold- back probe is illustrated in FIG. 9B. Fusion protein: A protein comprising at least a portion of two different (heterologous) proteins. In some aspects herein, a fusion protein includes an Fc binding domain, a linker peptide, and a relaxase domain.

[0083] Heterologous: Originating from a separate genetic source or species.

[0084] Relaxase domain: As used herein, a relaxase domain, also known as a relaxase endonuclease domain, is a protein domain derived from the relaxase endonuclease family (including Rep proteins, relaxases and transposons) that catalyzes cleavage and ligation of single-stranded DNA at a specific sequence. Relaxase domains contain a conserved HUH or “Histidine-hydrophobic amino acid(s)-Histidine” motif which coordinates a divalent metal ion and one or more catalytic tyrosine residue which attacks the phosphate backbone to nick the DNA. The 5’ end of the cleaved DNA is covalently attached to the tyrosine residue of the relaxase domain. In some aspects, the relaxase domain is obtained from Porcine circovirus 2 (PCV2), Wheat dwarf virus (WDV) or Agrobacterium tumefaciens. In some aspects, the relaxase domain is obtained from the VirD2 protein of Agrobacterium tumefaciens. In some examples herein, the relaxase domain has the amino acid sequence of SEQ ID NO: 17-19, or a variant thereof having 80% identity.

[0085] Hybridization: Oligonucleotides (such as PNA, RNA or DNA) and their analogs hybridize by hydrogen bonding, which includes Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary bases. Generally, nucleic acid consists of nitrogenous bases that are either pyrimidines (cytosine (C), uracil (U), and thymine (T)) or purines (adenine (A) and guanine (G)). These nitrogenous bases form hydrogen bonds between a pyrimidine and a purine, and the bonding of the pyrimidine to the purine is referred to as “base pairing.” More specifically, A will hydrogen bond to T or U, and G will hydrogen bond to C. “Complementary” refers to the base pairing that occurs between two distinct nucleic acid sequences or two distinct regions of the same nucleic acid sequence. In the context of the present disclosure, a first oligonucleotide that is “complementary to a second oligonucleotide” refers to an oligonucleotide that is sufficiently complementary to the second oligonucleotide such that the two molecules hybridize. The term “complementary to” does not require 100% complementarity.

[0086] Immunoglobulin binding domain: A domain of a protein that mediates binding of the protein to an immunoglobulin, typically an immunoglobulin constant region, such as an antibody Fc region (e.g., an Fc region of an IgG antibody). Exemplary immunoglobulin domains include, but are not limited to, the Z domain of Staphylococcus Protein A (or a variant thereof that retains the capacity to bind an antibody Fc region) and the C2 domain of Streptococcal Protein G (or a variant thereof that retains the capacity to bind an antibody Fc region).

[0087] Linker: A bi-functional molecule (such as a peptide) that can be used to link two molecules into one contiguous molecule, for example, to link two heterologous proteins or protein domains. In some aspects, the conjugates disclosed herein include a peptide linker between a Fc binding domain and a relaxase domain. Non-limiting examples of peptide linkers include glycine, serine, and glycine-serine linkers. In one example, the linker has the amino acid sequence of SEQ ID NO: 37-41, or a variant thereof having 80% identity.

[0088] Low pH: In the context of the present disclosure, “low pH” refers to a pH of 4.0 or less, such as a pH of 2.0 to 4.0.

[0089] PRO-tag: In the context of the present disclosure, a “PRO-tag” refers to a fusion protein comprising a protein and a relaxase domain and an oligonucleotide tag covalently bound to the relaxase domain. The protein can be a ligand, a protein domain, an antigen, an antigen binding protein, an antibody, an antibody fragment, a single-domain antibody, a VHH antibody or a protein comprising an Fc binding domain.

[0090] Purification tag: A heterologous peptide appended to a protein to assist with purification of the protein. Purification tags include, but are not limited to, His {e.g., 6XHis), glutathione S-transferase (GST), hemagglutinin (HA), V5, FLAG and Myc.

[0091] Purified: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified protein (such as a purified fusion protein or conjugate) preparation is one in which the protein is more enriched than the protein is in its environment, such as within a cell. In one aspect, a protein is purified such that the protein represents at least 50% of the total protein content of the preparation. Substantial purification denotes purification from other proteins or cellular components. A substantially purified protein is at least 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9% or 99.99% pure. In one specific, non-limiting example, a substantially purified protein is 90% free of other proteins or cellular components. Recombinant: A recombinant nucleic acid or protein (such as a fusion protein) is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques.

[0092] III. PRO-Tag Conjugates

[0093] Oligonucleotide-tagged antibodies have been used as single purpose reagents in molecular amplification and detection in NGS or qPCR. The present disclosure provides a dye incorporation process that can be used in a dual use context for both quantification as well as imaging. The compositions and methods provided herein enable both imaging a set of target proteins as well as signal quantification of those targets using qPCR for detection.

[0094] The compositions and methods provided herein enable rapid, enzymatic tagging of antigen binding proteins or antibodies to oligonucleotides for multiplexing across spectral imaging or flow cytometry, cell-based profiling and quantitative multiplex detection in a sitespecific manner. In some aspects, the present disclosure provides compositions and methods to tag an unmodified full-length antibody or antigen binding protein with an oligonucleotide. In some aspects, the present disclosure provides a scale-able approach to generate site-specific antibodies tags (through a fusion protein) with designed oligonucleotides. In some aspects, the sequences of the oligonucleotide are designed to incorporate detectable labels in a ‘programmed’ fashion. The compositions and methods disclosed herein provide a stoichiometry of the oligonucleotide bound to the full-length antibody is 2: 1; which allows for more accurate profiling of proteins in cells and tissues. The use of a site-specific cleavage site in the oligonucleotide sequence allows the release of specific oligonucleotide tags for qPCR or NGS profiling in a multiplex assay, following antigen binding and or imaging. Oligonucleotide tags or handles used for immuno-PCR (“qPCR handles”) are synthetic and each qPCR handle has unique 5 ’ and 3 ’ priming sites for PCR. These handles can also be used in applications such as proximity ligation assays (PLA), where two proteins that are in proximity can be detected and / or quantified. The qPCR handle length can vary from 80 base pairs (bp), 100 bp, 120 bp or 150 bp in length. Such qPCR handles can also be used in NGS profiling and can be of lengths such as 8 bp, 12 bp, 16 bp or 20 bp in length. In some aspects, the present disclosure provides a fusion protein comprising an antibody binding domain that is selective to the CH2-CH3 region of a full length antibody and a site-specific relaxase domain which forms a covalent adduct such as a phospho tyro sine adduct, with an oligonucleotide through a rapid reaction between the polypeptide chain and the nucleic acid phosphodiester backbone. The reaction is sequence specific with a transfer time of about 15-30 minutes, and the antibody binding domain enables defined stoichiometry in a 2: 1 ratio for tagging oligo to antibody. For ease of reference, the protein-relaxase domain fusion protein comprising an oligonucleotide tag is referred to herein as a “PRO-tag”.

[0095] In some aspects, the oligonucleotide is a fold-back self-priming oligonucleotide sequence (“fold back probe”) that can be used to incorporate a detectable label in a pre-defined configuration to modulate the spectra and / or the number of labels incorporated. In some examples, the fold-back probe is constructed using a polymerase, dye labeled dNTP’s and ddNTP’s which are designed for on-demand labelling and to control the number of dyes used in the probe. In some aspects, the oligonucleotide provided herein includes a site specific nicking site in the oligonucleotide, for selective release of specific tags for qPCR analysis or NGS profiling. In some aspects, the oligonucleotide provided herein includes unique 5’ and 3’ priming sites for PCR.

[0096] One of the advantages of the compositions and methods of the present disclosure is the efficient, rapid and scale-able site-specific tagging of proteins (e.g., cell ligands, antigens, and antibodies) with designed oligonucleotide sequences that enable easy antibody conjugate development with few components: an unmodified full-length antibody, a fusion chimera, and a designed oligonucleotide sequence, handle, or barcode. This enables large combinatorial flexibility and on-demand manufacturing to specific end goals without stocking conjugates as finished good inventory.

[0097] Another advantage of the compositions and methods of the present disclosure is the ability to define the stoichiometry of labeled oligonucleotide tags to antibody at a 2: 1 ratio and ensure site specific tagging for unmodified full-length antibodies which is critical for quantitative determination of analyte abundance. The methods and compositions disclosed herein provide an alternative for low variance in tag-antibody stoichiometry that is difficult to control with the chemical conjugation methods currently available. The compositions and methods provided herein provide the advantage of low batch variance across lots and more accurate estimation of counts leading to robust data analysis. Additionally, the present disclosure provides fold-back probes which enable preprogrammed and modulated labelling using incorporation of detectable labels, defined by the design of the oligonucleotide sequence using standard enzymatic components. An important design feature of the fold-back probe is the hairpin self-priming 3 ’ sequences that incorporate dNTP-labels with standard components (dATP, dCTP, dGTP, dTTP, dUTP and ddATP, ddGTP, ddCTP, ddTTP, ddUTP) and a polymerase. The sequence of the template strand defines the complementary labeled nucleotide base count, and the label-nucleotide base spectra defines the spectral properties. The combination of dye-nucleotides introduced in the reaction can provide novel spectral channels. Introducing a specific nicking site in the oligonucleotide sequence allows the selective release of labelled oligonucleotide tags for cyclic imaging.

[0098] Additionally in some aspects, the compositions and methods of the present disclosure provide for on-demand site-specific release of the oligonucleotide tag for qPCR, NGS quantification or release of labelled oligonucleotide for spectral channel re-use in multiplex imaging. In some aspects, the release of the oligonucleotide tag is achieved using low-pH to release the PRO-tag from the antibody. In other aspects, the oligonucleotide tag is detached from the antibody-PRO-tag for qPCR and NGS profiling using a site specific nickase / endonuclease.

[0099] Provided herein are conjugates that include an antigen binding protein, an antibody or an antibody fragment, or a protein comprising an Fc binding domain, a relaxase domain, and an oligonucleotide. In some aspects, the conjugate further includes a linker positioned between the antigen binding protein, antibody or antibody fragment, or protein comprising an Fc binding domain and the relaxase domain.

[0100] In some aspects of the conjugates, the Fc binding domain is a bacterially derived immunoglobulin binding domain. In some examples, the bacterially derived immunoglobulin binding domain includes a Z domain (of Staphylococcus Protein A) or a variant thereof that retains the capacity to bind an antibody Fc region. In particular examples, the amino acid sequence of the Z domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1. In a specific non-limiting example, the amino acid sequence of the Z domain includes or consists of SEQ ID NO: 1. In other particular examples, the amino acid sequence of the Z domain variant is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2. In specific non-limiting examples, the amino acid sequence of the variant Z domain includes or consists of SEQ ID NO: 2. In other examples, the bacterially derived immunoglobulin binding domain includes a C2 domain (of Streptococcus Protein G) or a variant thereof that retains the capacity to bind an antibody Fc region. In particular examples, the amino acid sequence of the C2 domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 3. In a specific nonlimiting example, the amino acid sequence of the C2 domain includes or consists of SEQ ID NO: 3.

[0101] In other aspects of the conjugates, the Fc binding domain is a Fc binding protein (FcBP). In some examples, the amino acid sequence of the FcBP is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 4-16. In a specific non-limiting example, the amino acid sequence of the FcBP includes or consists of any one of SEQ ID NOs: 4-16.

[0102] In some aspects of the conjugates, the relaxase domain is an endonuclease protein, or a portion of a relaxase endonuclease protein, obtained from Porcine circovirus 2 (PCV2), Wheat dwarf virus (WDV) or Agrobacterium tumefaciens. In some aspects of the conjugates, the relaxase domain is a VirD2 protein or a portion of VirD2 protein. In some examples, the amino acid sequence of the relaxase domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 17-19. In a specific non-limiting example, the amino acid sequence of the relaxase domain includes or consists of any one of SEQ ID NOs: 17-19.

[0103] In some aspects of the conjugates, the linker is a peptide of about 4 to about 12 amino acids in length, such as about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11 or about 12 amino acids in length. In some examples, the linker is a flexible glycine- serine rich linker. In particular examples, the amino acid sequence of the linker is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 37-41. In specific non-limiting examples, the amino acid sequence of the linker includes or consists of any one of SEQ ID NO: 37-41.

[0104] In some aspects of the conjugates, the relaxase domain is covalently attached to an oligonucleotide. In particular examples, the oligonucleotide is about 10 to about 150 nucleotides in length, or about 20 to about 125 nucleotides in length, or about 50 to about 100 nucleotides in length. In select examples, the oligonucleotide is about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, or about 150 nucleotides in length. In particular examples, the nucleic acid sequence of the oligonucleotide is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 20-36. In specific non-limiting examples, the nucleic acid sequence of the oligonucleotide includes or consists of any one of SEQ ID NOs: 20-36.

[0105] In some examples, the oligonucleotide includes a qPCR handle which is designed artificially. In some examples, each qPCR handle has unique 5’ and 3’ priming sites for applications such as PCR and PLA. In some examples, the qPCR handle length is about 80 base pairs in length, about 90 base pairs in length, about 100 base pairs in length, about 110 base pairs in length, about 120 base pairs in length, about 130 base pairs in length, about 140 base pairs in length, or about 150 base pairs in length. In some examples, the qPCR handle can be used in NGS profiling when used with a single priming site and is about 8 base pairs in length, about 10 base pairs in length, about 12 base pairs in length, about 14 base pairs in length, about 16 base pairs in length, about 18 base pairs in length, or about 20 base pairs in length. In some particular examples, the qPCR handle further includes a label, a barcode, or an RNA polymerase binding site. In some examples, the oligonucleotide includes a detectable label. In particular examples, the label is directly or indirectly detectable. In particular examples, the label includes a fluorescent agent, a hapten, biotin, a nucleic acid analog, or a digoxygenin label. In particular examples, the label is a fold-back probe. In particular examples, the nucleic acid sequence of the fold-back probe is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 24-36. In specific non-limiting examples, the nucleic acid sequence of the fold-back probe includes or consists of any one of SEQ ID NOs: 24-36. In particular examples, the fold-back probe includes one or more fluorescent agent. In particular examples, the fluorescent agent is a xanthene, a fluorescein, a rhodamine, a rhodol, a roseamine, a carbopyranone, an indole, an indacene, a borapolyazaindacene, a furan, a benzofuran, a cyanine, a benzocyanine, a benzopyrilium, a pyrene, a coumarin, a styryl, a squarine, a resorufin, an anthraquinone, an acridine or a benzophenoxazine.

[0106] In some aspects, the conjugate further includes an immunoglobulin molecule. The Fc region of the immunoglobulin molecule is non-covalently bound to the Fc binding domain. In some examples, the immunoglobulin molecule is an IgG, such as a human IgG of any isotype (e.g., IgGl, IgG2, IgG3 or IgG4). In other aspects, the immunoglobulin molecule is an IgA, IgD, IgE or IgM, such as a human IgA, IgD, IgE or IgM. In other aspects, the immunoglobulin molecule is from a non-human species, such as rabbit, mouse, rat or non-human primate.

[0107] In some aspects, the conjugate further includes a purification tag. In some examples, the purification tag is a His tag, a Myc tag or a FLAG tag, or another purification tag known in the art. A skilled person is capable of selecting an appropriate purification tag. In some examples, the purification tag is attached to the N-terminus of the Fc binding domain.

[0108] In specific non-limiting examples, the conjugate includes an Fc binding domain, a relaxase domain, a linker positioned between the Fc binding domain and the relaxase domain, an oligonucleotide covalently attached to the relaxase domain, and an immunoglobulin molecule, wherein the Fc region of the immunoglobulin molecule is non-covalently bound to the Fc binding domain. In some aspects, the conjugate further includes a purification tag, such as a purification tag attached to the N-terminus of the Fc binding domain.

[0109] IV. Kits

[0110] Further provided herein are kits that include one or more of the conjugates disclosed herein. In some aspects, the kit includes a fusion protein that includes a ligand, an antigen, an antigen binding protein, an antibody or an antibody fragment, or a protein comprising an Fc binding domain, a relaxase domain, and an oligonucleotide. In some aspects, the kit further includes a linker positioned between the antigen binding protein, antibody or antibody fragment, or protein comprising an Fc binding domain and the relaxase domain.

[0111] In some aspects of the kits, the Fc binding domain of the fusion protein is a bacterially derived immunoglobulin binding domain. In some examples, the bacterially derived immunoglobulin binding domain includes a Z domain (of Staphylococcus Protein A) or a variant thereof that retains the capacity to bind an antibody Fc region. In particular examples, the amino acid sequence of the Z domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1. In a specific nonlimiting example, the amino acid sequence of the Z domain includes or consists of SEQ ID NO: 1. In other particular examples, the amino acid sequence of the Z domain variant is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2. In specific non-limiting examples, the amino acid sequence of the variant Z domain includes or consists of SEQ ID NO: 2. In other examples, the bacterially derived immunoglobulin binding domain includes a C2 domain (of Streptococcus Protein G) or a variant thereof that retains the capacity to bind an antibody Fc region. In particular examples, the amino acid sequence of the C2 domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 3. In a specific non-limiting example, the amino acid sequence of the C2 domain includes or consists of SEQ ID NO: 3.

[0112] In other aspects of the kits, the Fc binding domain of the fusion protein is a Fc binding protein (FcBP). In some examples, the amino acid sequence of the FcBP is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 4-16. In a specific non-limiting example, the amino acid sequence of the FcBP includes or consists of any one of SEQ ID NOs: 4-16.

[0113] In some aspects of the kits, the relaxase domain of the fusion protein is an endonuclease protein, or a portion of a relaxase endonuclease protein, obtained from Porcine circovirus 2 (PCV2), Wheat dwarf virus (WDV) or Agrobacterium tumefaciens. In some aspects of the conjugates, the relaxase domain is a VirD2 protein or a portion of VirD2 protein. In some examples, the amino acid sequence of the relaxase domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 17-19. In a specific non-limiting example, the amino acid sequence of the relaxase domain includes or consists of any one of SEQ ID NOs: 17-19.

[0114] In some aspects of the kits, the linker component of the fusion protein is a peptide of about 4 to about 12 amino acids in length, such as about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11 or about 12 amino acids in length. In some examples, the linker is a flexible glycine-serine rich linker. In particular examples, the amino acid sequence of the linker is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 37-41. In specific non-limiting examples, the amino acid sequence of the linker includes or consists of any one of SEQ ID NO: 37-41.

[0115] In some aspects of the kits, the relaxase domain of the fusion protein is covalently attached to an oligonucleotide. In particular examples, the oligonucleotide is about 10 to about 150 nucleotides in length, or about 20 to about 125 nucleotides in length, or about 50 to about 100 nucleotides in length. In select examples, the oligonucleotide is about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, or about 150 nucleotides in length. In particular examples, the nucleic acid sequence of the oligonucleotide is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 20-36. In specific non-limiting examples, the nucleic acid sequence of the oligonucleotide includes or consists of any one of SEQ ID NOs: 20-36.

[0116] In some examples, the oligonucleotide includes a qPCR handle which is designed synthetically. In some examples, each qPCR handle has unique 5’ and 3’ priming sites for applications such as PCR and PLA. In some examples, the qPCR handle length is about 80 base pairs in length, about 90 base pairs in length, about 100 base pairs in length, about 110 base pairs in length, about 120 base pairs in length, about 130 base pairs in length, about 140 base pairs in length, or about 150 base pairs in length. In some examples, the qPCR handle can be used in NGS profiling when used with a single priming site and is about 8 base pairs in length, about 10 base pairs in length, about 12 base pairs in length, about 14 base pairs in length, about 16 base pairs in length, about 18 base pairs in length, or about 20 base pairs in length. In some particular examples, the qPCR handle further includes a label, a barcode, or an RNA polymerase binding site. In some examples, the oligonucleotide includes a detectable label. In particular examples, the label is directly or indirectly detectable. In particular examples, the label includes a fluorescent agent, a hapten, biotin, a nucleic acid analog, or a digoxygenin label. In particular examples, the label is a fold-back probe. In particular examples, the nucleic acid sequence of the fold-back probe is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 24-36. In specific non-limiting examples, the nucleic acid sequence of the fold-back probe includes or consists of any one of SEQ ID NOs: 24-36. In particular examples, the fold-back probe includes one or more fluorescent agent. In particular examples, the fluorescent agent is a xanthene, a fluorescein, a rhodamine, a rhodol, a roseamine, a carbopyranone, an indole, an indacene, a borapolyazaindacene, a furan, a benzofuran, a cyanine, a benzocyanine, a benzopyrilium, a pyrene, a coumarin, a styryl, a squarine, a resorufin, an anthraquinone, an acridine or a benzophenoxazine. In some aspects, the kit further includes an immunoglobulin molecule non-covalently bound to the Fc binding domain. In some examples, the immunoglobulin molecule is an IgG, such as a human IgG of any isotype (e.g., IgGl, IgG2, IgG3 or IgG4). In other aspects, the immunoglobulin molecule is an IgA, IgD, IgE or IgM, such as a human IgA, IgD, IgE or IgM. In other aspects, the immunoglobulin molecule is from a non-human species, such as rabbit, mouse, rat or non-human primate.

[0117] In some aspects of the kits, the fusion protein further includes a purification tag. In some examples, the purification tag is a His tag, a Myc tag or a FLAG tag, or another purification tag known in the art. A skilled person is capable of selecting an appropriate purification tag. In some examples, the purification tag is attached to the N-terminus of the Fc binding domain.

[0118] In specific non-limiting examples, the kit includes a fusion protein which includes an Fc binding domain, a relaxase domain, and a linker positioned between the Fc binding domain and the relaxase domain; an oligonucleotide covalently attached to the relaxase domain; and an immunoglobulin molecule, wherein the Fc region of the immunoglobulin molecule is non- covalently bound to the Fc binding domain. In some aspects, the fusion protein further includes a purification tag, such as a purification tag attached to the N-terminus of the Fc binding domain.

[0119] Various ancillary materials will frequently be employed in a method in accordance with the present disclosure. In some aspects, buffers and / or stabilizers are present in the kit components. In another exemplary embodiment, the kits include indicator solutions or indicator "dipsticks", blotters, culture media, cuvettes, and the like. In some aspects, the kits include indicator cartridges (where a kit component is bound to a solid support) for use in an automated detector. In some aspects, the kit further comprises molecular weight markers, wherein said markers are selected from phosphorylated and non-phosphorylated polypeptides, calcium-binding and non-calcium binding polypeptides, sulfonated and non- sulfonated polypeptides, and sialylated and non-sialylated polypeptides. In some aspects, the kit further comprises a member selected from a fixing solution, a detection reagent, a standard, a wash solution, and combinations thereof. In some aspects, the kit further includes at least one of the following: a buffering agent, a purification medium, a vial comprising the sample, and an organic solvent. V. Methods of Detecting Target Antigens

[0120] Also described herein are methods of detecting target antigens in biological samples using the conjugates and kits disclosed herein.

[0121] The compositions and methods of the present disclosure provide for on-demand sitespecific release of the oligonucleotide tag for qPCR and / or NGS quantification or release of labelled oligonucleotide for spectral channel re-use in multiplex imaging. In some aspects, the release of the oligonucleotide tag is achieved using low-pH to release the PRO-tag from the antibody. In other aspects, the oligonucleotide tag is detached from the antibody-PRO-tag for qPCR and NGS profiling using a site specific nickase / endonuclease

[0122] Provided are in vitro methods of detecting a target antigen in a cell or tissue. In some aspects, the method includes contacting the cell or tissue with a conjugate disclosed herein, wherein the immunoglobulin molecule of the conjugate is specific for the target antigen; and detecting the conjugate, thereby detecting the target antigen.

[0123] In some aspects, the method includes contacting a sample with a conjugate that includes any one of an antigen binding protein, an antibody, an antibody fragment, or a protein comprising an Fc binding domain; a relaxase domain; and an oligonucleotide covalently attached to the relaxase domain; incubating the sample with the conjugate for an amount of time sufficient to allow the conjugate to bind to a target within the sample; and detecting the conjugate. In some aspects, the method includes contacting a sample with a conjugate that includes an Fc binding domain; a relaxase domain; a linker positioned between the Fc binding protein and the relaxase domain; and an oligonucleotide comprising a label, wherein the oligonucleotide is covalently attached to the relaxase domain; incubating the sample with the conjugate for an amount of time sufficient to allow the conjugate to bind to a target within the sample; and detecting the conjugate. The components of the conjugates are described in detail in sections III and IV.

[0124] In some aspects, the method includes contacting a sample with the any one of an antigen binding protein, an antibody, an antibody fragment, or a protein comprising an Fc binding domain; a relaxase domain; and an oligonucleotide covalently attached to the relaxase domain, wherein the antigen binding protein, antibody, antibody fragment, or immunoglobulin molecule of the conjugate is specific for an antigen present in the sample; and detecting the conjugate. In some aspects, the method includes contacting a sample with an antibody, antibody fragment, or immunoglobulin molecule specific for an antigen present in the sample; incubating the sample with the antibody, antibody fragment, or immunoglobulin molecule for an amount of time sufficient to allow the antibody, antibody fragment, or immunoglobulin molecule to bind to the antigen forming an antigen complex; adding a conjugate that includes an Fc binding domain; a relaxase domain; a linker positioned between the Fc binding protein and the relaxase domain; and an oligonucleotide comprising a label, wherein the oligonucleotide is covalently attached to the relaxase domain, and incubating for an amount of time sufficient to allow the Fc binding domain to non-covalently bind to the Fc region of the antibody, antibody fragment, or immunoglobulin molecule; and detecting the conjugate.

[0125] In some examples of the disclosed methods, the sample is live cells, intracellular fluids, extracellular fluids, sera, biological fluids, biological fermentation media, environmental samples, industrial samples, viruses, proteins, peptides, buffer solutions, blood cells, immune cells, cultured cells, cellular extracts, tissue, muscle tissue, neurons, extracellular vesicles, vascular tissue, blood fluids, saliva, urine, water, soil, wastewater, sea water, pharmaceuticals, foodstuffs, or beverages. In some examples of the disclosed methods, the sample is immobilized on a polymeric membrane, within a polymeric gel, on a microparticle, on a microarray, on a silicon chip, on a glass slide, on a microwell plate, and on a microfluidic chip. In some examples of the methods disclosed herein the sample is a tissue, wherein the tissue is tumor tissue, epidermal tissue, muscle tissue, bone marrow tissue, neural tissue, brain tissue, organ tissue, or human biopsy tissue. In some examples, the tissue is a formalin-fixed paraformaldehyde-embedded tissue section or a fresh frozen tissue section.

[0126] In some examples of the methods, detecting the sample or the antigen in the sample includes immunohistochemistry, fluorescence microscopy, flow cytometry, immunoprecipitation (IP) assays, western blot, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (e.g., qPCR, immunoPCR), rolling circle amplification, next generation sequencing (NGS) using NGS-enabled barcoded oligonucleotides, or nicking enzyme signal amplification.

[0127] In some examples, a recombinant fusion protein comprising a ligand relaxase domain chimera can be covalently attached to an oligonucleotide (a “ligand-PRO-tag”). In some examples, the fold-back probe can be labelled with a dye suitable for sorting in a flow cytometry based assay. In some examples of this method, multiple ligand-PRO-tags are labelled each with specific dyes that can be assayed in flow cytometry. (See FIG. 12A). In some examples, the fold-back oligonucleotide probe, is designed with an 8, 10, or 12 base barcode, and a site specific cleavage site. This can be used to profile the transcript for the receptor, the total cell transcriptome and the ligand copy number (barcode tags) in either qPCR, single cell droplet qPCR, NGS or single cell NGS assays. (FIG. 12B). The copies of tag barcode for ligand-PRO-tags bound to cell can be profiled in relation to the cell receptors or cell transcriptome to derive an assessment of ligands occupied per cell. In some examples, a PRO- tag comprising a fusion protein is an antigen relaxase domain fusion, covalently bound to a detectably labeled oligonucleotide (an “antigen-PRO-tag). The antigen-PRO-tag can be used for rapid labelling of B -cells, and for B-cell receptor (BCR) selection using flow-cytometry (FIG. 12C) or capturing B-cells when the oligonucleotide is labeled with, for example, biotin, or digoxygenin as a selective pull-down tag or to a specific complementary oligonucleotide (FIG. 12D), where an antigen-PRO-tag when bound to its specific B-cell receptor (BCR), can be detected, quantified and used to enrich antigen positive cells from a pool of a B-cell in a cell population.

[0128] In some examples, the method further includes exposing the sample to low pH, high temperature and / or high salt concentration to release the Fc binding domain from the antibody, antibody fragment, or immunoglobulin molecule. In specific examples, the cell or tissue is exposed to pH 2 to pH 4, such as pH 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9 or 4.0.

[0129] In some aspects, the method includes cyclic immunostaining involving exposing the cells or tissues to low pH to release the Fc binding domain of the first antibody conjugate, followed by additional rounds of immunostaining, such as two, three, four, five, six, seven, eight, nine or ten rounds of immuno staining (such as to detect two, three, four, five, six, seven, eight, nine or ten different antigens). In some examples, the method includes contacting the sample with a second conjugate disclosed herein, wherein the second conjugate has a different detectable label than the first conjugate. In other examples, the method further includes contacting the sample with a second labelled antibody, such as a commercially available labelled primary antibody. In yet other examples, the method further includes contacting the sample with a second antibody that is not labelled. In the latter example, the unlabeled antibody can be detected, for example, using a secondary antibody that is labelled. In some examples, the sample is exposed to a composition comprising 10 mM glycine adjusted to pH 2-pH 4. VI. DNA Oligonucleotide Sequences

[0130] Disclosed herein are exemplary DNA oligonucleotide sequences that can be used in the conjugates disclosed herein. The exemplary sequences have been computationally designed to: (i) have a specific melting temperature (>55°C); (ii) not form self-complementarity structures or dimers; and (iii) have minimal sequence similarity to human and mouse genomes. Table 1 lists the sequences set forth herein as SEQ ID NOs: 20-36 and 42-45.

[0131] Table 1. DNA oligonucleotide Sequences

[0132] EXAMPLES

[0133] The following examples are provided to illustrate particular features of certain aspects of the disclosure, but the scope of the claims should not be limited to those features exemplified. Example 1: Synthesis of a PRO-tag comprising an antibody binding domain, a relaxase domain and an oligonucleotide This example describes the synthesis of a PRO-tag, starting with the expression and purification of a fusion protein comprising a Z domain (SEQ ID NO: 1) and a VirD2 relaxase domain (SEQ ID NO: 19) (Zdom-VirD2 fusion protein), followed by conjugation of the recombinant Zdom-VirD2 fusion protein with a VirD2 oligonucleotide.

[0134] A. Expression and purification ofZdom-VirD2 protein:

[0135] Materials for protein expression:

[0136] • 10X Phosphate Buffer Saline pH 7.4 (Gibco™-Catalogue#: 70011044)

[0137] • Autoclaved water

[0138] • Imidazole (Sigma Aidrich-Catalog#: I202-500G)

[0139] • Glycerol (Thermo Scientific'™-Catalogue#: 038988. AP)

[0140] • 5M NaOH to adjust pH

[0141] • Zdom-VirD2 gene (SEQ ID NOs: 1 and 19) synthesized and cloned in pET28a vector; sequence reference: Pansegrau, etal. Proc. Natl. Acad. Sci. USA 90: 11538- 11542 (1993)

[0142] • BL21(DE3) competent cells (Invitrogen™-Catalogue#: C600003)

[0143] • IPTG (Sigma-Catalogue#: I6758-10G)

[0144] • Luria Broth Base (Miller's LB Broth Base), powder (Invitrogen™-Catalogue#: 12795-084)

[0145] • Kanamycin sulfate (Gibco™-Catalogue#: 119815-024-5g)

[0146] • Halt protease and phosphatase inhibitor cocktail (100X) (Thermo Scientific™- Catalogue#: 1861284)

[0147] • HisPur™ Ni-NTA Resin (Thermo Scientific™-Catalogue#: 88222)

[0148] • 2-Mercaptoethanol, 1000X (Gibco™-Catalogue#: 21985-023 50 mL)

[0149] • HiLoad™ 16 / 600 Superdex™ 75 pg column (Cytiva-Catalogue#: 28989333) Protein expression and purification:

[0150] E. coli BL21(DE3) competent cells were transformed with the Zdom-VirD2 synthetic gene construct by heat shock at 42°C for 30 seconds and the transformation mix was spread on LB Agar (kanamycin, 50 pg / ml) plates. A single colony was inoculated from the plate in 50 ml LB broth containing 50 pg / ml kanamycin and the primary culture was incubated at 37°C, 200 RPM, overnight. A secondary culture was setup in IL LB broth (containing 50 pg / ml kanamycin) using 1% of the primary culture as the inoculum and was incubated at 37°C, 200 RPM for 2.5 hours. On reaching an optical density at 600 nm (ODeoo) of 0.8, the culture was induced with 0.5 mM IPTG and grown at 16°C, 200 RPM, overnight. Post- induction, the bacterial culture pellet was prepared by centrifugation at 5000g, 4°C for 10 minutes and the pellet was stored at -80 °C.

[0151] Buffers for protein purification:

[0152] A protease inhibitor cocktail and 2 mM 2-Mercaptoethanol was added to 50 mL lysis buffer and the stored pellet was resuspended in the buffer and kept on ice for 30 minutes. The pellet was lysed by sonication at Amplitude 60, for 5 minutes (10 seconds on and 25 seconds off). Supernatant and pellet fractions were separated by high-speed centrifugation at 14,000g for 45 minutes at 4°C. The resulting supernatant was collected and loaded on to 1 ml packed HisPur™ Ni-NTA Resin (Thermo Fisher Scientific). The column was washed with 15 mL wash buffer containing 50 mM imidazole (wash buffer was prepared by diluting elution buffer with lysis buffer such that the buffer contained the required concentration of imidazole). Finally, the protein was eluted using an imidazole gradient from 100 mM-500 mM imidazole. Protein that was eluted from the Ni-NTA purification (200 mM-400 mM) was pooled and concentrated using a 3 kDa Centricon™ centrifugal filter. The concentrated protein was filtered using 2 pm syringe filters. The concentrated, filtered protein was injected into a size exclusion chromatography (SEC) Superdex™ 75 column and the purified protein was collected.

[0153] The size exclusion chromatography eluates were run on a 4-12% Bis Tris gel under reducing and non-reducing conditions. In FIG. IB, lane PL represents concentrated protein injected into the column and lanes 12-16 represent pure protein eluates in fractions no. 12-16.

[0154] Results and Conclusions:

[0155] A recombinant fusion protein of bacterial VirD2 protein was designed with an N- terminal 6X-His tag and a Z-domain and was cloned in a bacterial expression vector pET28a. The Zdom-VirD2 fusion protein was successfully expressed in E. coli BL21(DE3) strain at 37°C. The soluble fraction of bacterial transformants were affinity purified on a Ni-NTA column using a gradient of imidazole concentration. The Ni-NTA eluates were pooled, concentrated, and further purified on a size exclusion chromatography (SEC) column. Eluted fraction numbers 12-16 were verified for containing the Zdom-VirD2 fusion protein on an SDS-PAGE gel stained with Coomassie G-250 under reducing and non-reducing conditions (FIG. IB). The pure fraction(s) of a 35 kDa Zdom-VirD2 fusion protein was purified and confirmed on an SDS-PAGE gel after size exclusion chromatography.

[0156] B. Oligo conjugation assay with recombinant Zdom-VirD2

[0157] Material for oligo complex assembly:

[0158] • Nuclease free water; DEPC-treated water (Invitrogen™-Catalogue# AM9922)

[0159] ® VirD2 oligo (SEQ ID NO. 23); sequence reference: Pansegrau, et al. Proc. Natl. Acad. Sci. USA 90: 11538-11542 (1993)

[0160] • Purified Zdom-VirD2 fusion protein (see above part A)

[0161] • iBlot™ 2 Transfer Stacks, nitrocellulose, mini (Invitrogen™-Catalogue# IB23002)

[0162] • NuPAGE™ 4-12% Bis-Tris Protein Gel, 10 well (Invitrogen™' Catalogue# NP0321BOX)

[0163] • 5% skimmed milk prepared in 1X-PBS-T (0.1% Tween-20)

[0164] • NuPAGE™ LDS Sample Buffer (4X) (Invitrogen™-Catalogue# NP0007) • 6X-His Tag Monoclonal Antibody (HIS.H8), HRP (Invitrogen™-Catalogue# MA1- 21315-HRP)

[0165] • SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Scientific™, Catalogue# 34580)

[0166] PRO -tag assembly:

[0167] A 10X Buffer was prepared as follows: 200 mM Tris pH 8.8, 500 mM NaCl in nuclease free water. Five reactions were setup using different volumes of purified Zdom-VirD2 fusion protein as described in the table below.

[0168] Two controls were setup: one that did not contain the oligonucleotide (VirD2 only) and another that did not contain protein (Reaction 1). The reactions were incubated at 37°C for 3 hours and the reactions were stopped by adding 2X LDS sample buffer (supplemented with DTT) to the reaction vials. The samples were heated at 70°C for 10 minutes and 30 pL sample were loaded on a 4-12% gel. The gel was transferred onto a nitrocellulose membrane using an iBlot2 dry blotting system. The nitrocellulose membrane was blocked with 5% skimmed milk and then incubated with a 6X-Histidine mouse antibody (1:3000 dilution, MA1-21315-HRP) for 3 hours at room temperature and washed three times with 1X-PBS-T for 5 min. The blots were developed using West Pico Plus and viewed on iBright FL1500.

[0169] Results and Conclusions:

[0170] To confirm relaxase activity of the purified Zdom-VirD2 fusion protein, an oligo conjugation assay was performed in vitro using a VirD2 specific oligonucleotide (SEQ ID NO: 23). Five different reaction conditions were set up for this experiment as mentioned in the table above. There were 2 control reactions: 1) “VirD2 only” that lacked the VirD2 recognition oligo but contained the Zdom-VirD2 fusion protein; and 2) “Reaction 1” which lacked the Zdom- VirD2 fusion protein but contained the VirD2 recognition oligo. “Reaction 2”, “Reaction 3”, and “Reaction 4” contained various concentrations of the Zdom-VirD2 fusion protein (2pg, 4pg, 6pg, respectively) along with the remaining components of the oligo conjugation reaction (see table). The Reaction mix was incubated at 37°C for 3 hours (VirD2 reaction conditions and recognition oligo were designed as per Pansegrau, et al. Proc. Natl. Acad. Sci. USA 90: 11538-11542 (1993)). After reaction termination, VirD2 relaxase activity was checked by testing reaction mixes on a western blot probed with a 6X-His tag antibody.

[0171] Upon probing with the 6X-His tag antibody, a significant shift in mass was observed in Reactions 2-5 due to Zdom-VirD2-oligo adduct formation (FIG. 1C). The Zdom-VirD2 fusion protein was also observed at around expected mass ~35 kDa in “VirD2 only” control and in Reactions 2-4 due to unreacted Zdom-VirD2 fusion protein. These results confirmed that the Zdom-VirD2 fusion protein has an active relaxase VirD2 domain as demonstrated by the formation of a covalently linked oligo adduct of higher mass.

[0172] Example 2: Expression and purification of protein-relaxase domain fusion (Z domain-PCV2 fusion)

[0173] This example describes the expression and purification of a fusion protein comprising a Z domain (SEQ ID NO: 1) and a Porcine circovirus 2 (PCV2) relaxase domain (SEQ ID NO: 17) (Zdom-PCV2 fusion protein).

[0174] Materials for protein expression, Ni-NTA purification and size exclusion chromatography:

[0175] • 10X Phosphate Buffer Saline pH 7.4 (Gibco™-Catalogue#: 70011044)

[0176] • Autoclaved water

[0177] • NaH2PO4(Sigma-Catalogue#: S8282-1KG)

[0178] • 5 M NaCl (Invitrogen™-Catalogue#: AM9759) Imidazole (Sigma Aidrich-Catalogue#: I202-500G)

[0179] Glycerol (Thermo Scientific™-Catalogue#: 038988. AP)

[0180] • 5M NaOH to adjust pH

[0181] • Zdom-PCV2 relaxase fusion (SEQ ID NOs: 1 and 17) synthesized and cloned in pET28a vector

[0182] • BL21(DE3) competent cells (Invitrogen™-Catalogue#: C600003)

[0183] • IPTG (Sigma-Catalogue#: I6758-10G)

[0184] • Luria Broth Base (Miller's LB Broth Base), powder (Invitrogen™-Catalogue#: 12795-084)

[0185] • Kanamycin sulfate (Gibco™-Catalogue#: 119815-024-5g)

[0186] • Halt protease and phosphatase inhibitor cocktail (100X) (Thermo Scientific™- Catalogue#: 1861284)

[0187] • SimplySafe™ SafeStain (Invitrogen™-Catalogue#: LC60665)

[0188] • HisPur™ Ni-NTA Resin (Thermo Scientific™-Catalogue#: 88222)

[0189] • 2-Mercaptoethanol, 1000X (Gibco™-Catalogue#: 21985-023, 50 mL)

[0190] • iBlot™ 2 Transfer Stacks, nitrocellulose, mini (Invitrogen™-Catalogue# IB23002)

[0191] • NuPAGE™ 4-12% Bis-Tris Protein Gel, 10 well (Invitrogen™- Catalogue# NP0321BOX)

[0192] • 5% skimmed milk prepared in 1X-PBS-T (0.1% Tween-20)

[0193] • NuPAGE™ LDS Sample Buffer (4X) (Invitrogen™-Catalogue# NP0007)

[0194] • 6X-His Tag Monoclonal Antibody (HIS.H8), HRP (Invitrogen™-Catalogue# MA1- 21315-HRP) • SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Scientific™, Catalogue# 34580)

[0195] • HiLoad™ 16 / 600 Superdex™ 75 pg column (Cytiva-Catalogue#: 28989333)

[0196] Protein expression and Ni-NTA purification (FIG. 2A):

[0197] E. coli BL21(DE3) competent cells were transformed with a Zdom-PCV2 relaxase fusion gene construct by heat shock (42°C, 30 seconds) and the transformation mix was spread on an LB Agar (kanamycin, 50 pg / ml) plate. A single colony from the plate was inoculated in 30 ml LB broth containing 50 pg / ml kanamycin and the primary culture was incubated at 37°C, 200 RPM, overnight. A secondary culture was setup in IL LB broth (containing 50 pg / ml kanamycin) using 1% of primary culture as the inoculum and was grown at 37°C, 200 RPM for 2.5 hours. On reaching an optical density at 600 nm (ODeoo) of 0.8, the culture was induced with 1 mM IPTG and grown at 37°C, 200 RPM for 3-4 hours or at 18°C, 200 RPM for 16 hours. Post-induction, the bacterial culture pellet was prepared by centrifugation at 5000g, 4°C for 10 minutes and the pellet was stored at -80 °C.

[0198] A protease inhibitor cocktail and 2 mM 2-Mercaptoethanol were added to 50 mL lysis buffer and the stored pellet was resuspended in the buffer and kept on ice for 30 minutes. The pellet was lysed by sonication at Amplitude 60, for 5 minutes (10 seconds on and 25 seconds off) and supernatant and pellet fractions were separated by high-speed centrifugation at 14,000g for 45 minutes at 4°C. The supernatant and pellet fractions of the uninduced sample and induced samples at 37°C and 18°C were run on SDS-PAGE and stained with Coomassie G-250 stain (SimplySafe™ SafeStain, FIG. 2B).

[0199] Western blot for expression confirmation (FIG. 2C):

[0200] To confirm expression of the Zdom-PCV2 fusion protein, a western blot was performed on supernatant and pellet fractions of the uninduced sample and induced samples at 37°C and 18°C. The blot was incubated with a 6X-Histidine mouse antibody (1:3000 dilution, MA1- 21315-HRP) for 3 hours at room temperature and washed three times with 1X-PBS-T for 5 minutes. The blots were developed using West Pico Plus and viewed on an iBright™ FL1500.

[0201] Ni-NTA purification and size exclusion chromatography (FIGs. 2D and 2E):

[0202] The supernatant fraction of the sonicated lysate was collected and loaded onto a 1 ml packed HisPur™ Ni-NTA Resin. The column was washed with 10 mL wash buffer containing 30 mM imidazole (wash buffer was prepared by diluting elution buffer with lysis buffer such that the buffer contained the required concentration of imidazole). Finally, the protein was eluted using an imidazole gradient from 50 mM-250 mM imidazole.

[0203] Protein eluted from the Ni-NTA purification (50 mM -250 mM) was pooled and concentrated using a 3 kDa Centricon™ centrifugal filter. The concentrated fusion protein was filtered using 2 pm syringe filters and the fusion protein was injected into a size exclusion chromatography Superdex 75 column and the purified fusion protein was collected. To confirm purity of the SEC eluates, fractions 11-19 were run on SDS-PAGE and stained with Coomassie G-250 stain (SimplySafe™ SafeStain, FIG. 2E).

[0204] Buffers for protein purification:

[0205] Results and Conclusions:

[0206] A recombinant fusion protein of viral relaxase domain PCV2 (SEQ ID NO: 17) was designed with an N-terminal 6X-His tag and a Z-domain (SEQ ID NO: 1) and cloned in a bacterial expression vector pET28a. The Zdom-PCV2 fusion protein was successfully expressed in E. coli BL21(DE3) strain at 37°C and 18°C. A 21 kDa band corresponding to the Zdom-PCV2 fusion protein was observed in a Coomassie stained gel in both supernatant and pellet fractions of samples induced with IPTG, compared to uninduced sample (FIG. 2B).

[0207] Expression of the Zdom-PCV2 fusion protein in E. coli was confirmed by western blot using a 6X-His tag antibody. Expression of the Zdom-PCV2 fusion protein was observed at the expected mass of 21 kDa in samples induced with IPTG in both supernatant and pellet fractions at 37°C and 18°C (FIG. 2C).

[0208] These results demonstrated that the recombinant fusion protein of the Z-domain and viral relaxase domain PCV2 were expressed in E. coli in a soluble form. The supernatant fraction of the Zdom-PCV2 fusion protein expressed in E. coli was loaded on to a Ni-NTA resin for affinity purification. The His-tagged Zdom-PCV2 fusion protein was eluted with a gradient of imidazole concentration ranging from 50 mM-250 mM. Pooled Ni-NTA eluates were concentrated and loaded onto a size exclusion chromatography (SEC) column to get pure fractions of the fusion protein. FIG. 2D shows a chromatograph of the eluted fractions from the SEC column. SEC eluted fractions 11 to 19 were run on an SDS-PAGE to check for purity and integrity of the Zdom-PCV2 fusion protein. FIG. 2E represents a Coomassie stained gel of the SEC eluted fractions of the Zdom-PCV2 fusion protein. Pure protein was observed in fractions 14-19 at expected mass of 21 kDa for the Zdom-PCV2 fusion.

[0209] Example 3: Assembly of a PRO-tag with a biotinylated oligonucleotide

[0210] This example describes the assembly of a biotinylated PRO-tag by combining the Zdom-PCV2 fusion protein from Example 2 with a biotin-conjugated PCV2 oligonucleotide (SEQ ID NO: 20) as shown schematically in FIG. 3A.

[0211] Material for covalent assembly:

[0212] • Nuclease free water; DEPC-treated water (Invitrogen™-Catalogue# AM9922)

[0213] • Biotin conjugated oligo for PCV2 (SEQ ID NO. 20)

[0214] • Purified Z-Domain-PCV2-relaxase fusion protein

[0215] • 10X reaction buffer (500 mM HEPES, 500 mM NaCl, 10 mM MgCb, 10 mM MnCh)

[0216] • Pierce™ Silver Stain kit (Thermo Scientific™, Catalogue# 24612)

[0217] • 1X-PBS-T (1X-PBS with 0.1% Tween-20)

[0218] • iBlot™ 2 Transfer Stacks, nitrocellulose, mini (Invitrogen™-Catalogue# IB23002)

[0219] • NuPAGE™ 4-12% Bis-Tris Protein Gel, 10 well (Invitrogen™- Catalogue# NP0321BOX)

[0220] • 5% skimmed milk prepared in 1X-PBS-T (0.1% Tween-20) NuPAGE™ LDS Sample Buffer (4X) (Invitrogen™-Catalogue# NP0007)

[0221] • 6X-His Tag Monoclonal Antibody (HIS.H8), HRP (Invitrogen™-Catalogue# MA1- 21315-HRP)

[0222] • SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Scientific™, Catalogue# 34580)

[0223] Covalent assembly of the biotinylated PRO -tag:

[0224] Four reaction conditions were setup as described in the table below:

[0225] The reaction was incubated at 37°C for 30 minutes and reaction was stopped by adding 2X LDS sample buffer (supplemented with DTT) to the reaction vials. The samples were heated at 95°C for 10 minutes and 30 pl of each sample was loaded on a 4-12% gel in duplicates. The first gel was transferred onto a nitrocellulose membrane for western blot analysis. The nitrocellulose membrane was blocked with 5% milk for 1 hour, incubated with a 6X histidine mouse antibody (1:3000 dilution, MA1-21315-HRP) for 3 hours at room temperature and washed three times with 1X-PBS-T for 5 minutes. The blot was developed using West Pico Plus and viewed on iBright FL1500 (FIG. 3B, left). The second gel was stained using a silver stain kit as per kit’s protocol (FIG. 3B, right).

[0226] Results and Conclusions:

[0227] To confirm endonuclease / relaxase activity of the purified Zdom-PCV2 fusion protein, an oligo conjugation assay was performed in vitro. Four different reaction conditions were set up for this experiment as mentioned in the table above. There were two control reactions: 1) “No Zdom-PCV2” that lacked the Zdom-PCV2 fusion protein but contained the PCV2 recognition oligo (SEQ ID NO: 20); and 2) “No Oligo” that lacked the PCV2 recognition oligo but contained the Zdom-PCV2 protein. The “No Buffer” reaction lacked the reaction buffer while the “Full Reaction” contained all of the reaction components for the relaxase domain reaction (see table above). The reaction mix was incubated at 37°C for 30 minutes (PCV2 relaxase domain reaction conditions and recognition oligo were designed as per Lovendahl, et al. J. Am. Chem. Soc. 139:7030-7035 (2017)). After reaction termination, PCV2 relaxase / endonuclease activity was checked by testing the reaction mixes on a western blot probed with a 6X-His tag antibody. A mass shift at ~30 kDa was observed in the “Full Reaction” due to Zdom-PCV2-oligo adduct formation. In the absence of reaction buffer in “No Buffer”, unreacted Zdom-VirD2 fusion protein (~21 kDa) was observed along with the Zdom- PCV2-oligo adduct. Unreacted Zdom-PCV2 fusion protein was observed in the “No Oligo” reaction.

[0228] These results confirmed that the Zdom-PCV2 fusion protein has an active relaxase domain as demonstrated by the formation of a covalently linked oligo adduct of higher mass. Furthermore, in the absence of reaction buffer oligo conjugation is incomplete.

[0229] Example 4: Assembly of a PRO-tag with a fluorescently-labeled oligonucleotide

[0230] This example describes the assembly of a fluorescently-labeled PRO-tag by combining the Zdom-PCV2 fusion protein from Example 2 with an Alexa Fluor™ 647-conjugated PCV2 oligonucleotide (SEQ ID NO: 21) as depicted schematically in FIG. 4A.

[0231] Material for covalent assembly:

[0232] • Nuclease free water; DEPC-treated water (Invitrogen™-Catalogue# AM9922)

[0233] • Alexa Fluor™ 647 conjugated oligo for PCV2-relaxase (SEQ ID NO. 21)

[0234] • Purified Z-Domain-PCV2-relaxase fusion protein

[0235] • 10X reaction buffer (500 mM HEPES, 500 mM NaCl, 10 mM MgCb, 10 mM MnCh)

[0236] • Pierce™ Silver Stain kit (Thermo Scientific™, Catalogue# 24612)

[0237] • 1X-PBS-T (1X-PBS with 0.1% Tween-20)

[0238] • iBlot™ 2 Transfer Stacks, nitrocellulose, mini (Invitrogen™-Catalogue# IB23002) • NuPAGE™ 4-12% Bis-Tris Protein Gel, 10 well (Invitrogen™- Catalogue# NP0321BOX)

[0239] • 5% skimmed milk prepared in 1X-PBS-T (0.1% Tween-20)

[0240] • NuPAGE™ LDS Sample Buffer (4X) (Invitrogen™-Catalogue# NP0007) • 6X-His Tag Monoclonal Antibody (HIS.H8) (Invitrogen™-Catalogue# MA1-

[0241] 21315)

[0242] • Donkey anti -Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 800 (Invitrogen™-Catalogue# A32789)

[0243] Covalent assembly of the fluorescently-labeled. PRO-tag: Six reaction conditions were setup with various oligo concentrations as mentioned below:

[0244] The reaction was incubated at 37°C for 30 minutes and was stopped by adding 2X LDS sample buffer (supplemented with DTT) to the reaction vials. The samples were heated at 95°C for 10 minutes and 30 pl of each sample was loaded on a 4-12% gel in duplicates. The first gel was transferred on to a nitrocellulose membrane for western blot analysis. The nitrocellulose membrane was blocked with 5% milk for 1 hour. The blot was viewed in the AF647 channel of an iBright FL1500 (FIG. 4B, left) before incubating with a 6X histidine mouse antibody (1: 1000 dilution, MAI-21315) for 16 hours at 4°C. The next day, the blot was washed three times with 1X-PBS-T for 5 minutes. The blot was subsequently incubated with Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 800 (A32789, 1: 10,000 dilution) for 1 hour at room temperature. After 1 hour, the blot was washed three times with 1X-PBS-T for 5 minutes. The blot was viewed in the AF800 channel (FIG. 4B, right) of an iBright FL1500 and then multiplexed with AF647 (FIG. 4C, left) and images were captured. The second gel was stained using silver stain kit as per kit’ s protocol (FIG. 4C, right).

[0245] Results and Conclusions:

[0246] Endonuclease / relaxase activity of the purified Zdom-PCV2 fusion protein was tested with a fluorophore conjugated oligo. The PCV2 relaxase domain recognition oligo was conjugated with Alexa Fluor™ 647 (SEQ ID NO: 21). The conjugation assay was performed in vitro with four different oligo concentrations: 50 pmol, 100 pmol, 200 pmol and 300 pmol with relevant controls as mentioned in the table above.

[0247] There were two control reactions: 1) “No Zdom-PCV2” that lacked the Zdom-PCV2 fusion protein but contained the PCV2 recognition oligo; and 2) “No Oligo” that lacked the PCV2 recognition oligo but contained the Zdom-PCV2 fusion protein. The remaining four reactions comprised all of the reaction components with varying concentrations of Alexa Fluor™ 647-conjugated oligo (as mentioned in table above). The reaction mix was incubated at 37°C for 30 minutes. After reaction termination, PCV2 relaxase / endonuclease activity was checked by:

[0248] 1) capturing fluorescent signal of the Alexa Fluor™ 647-conjugated oligo and whether there was a mass shift due to covalent adduct formation between Zdom-PCV2 relaxase and oligo; and

[0249] 2) checking for a mass shift on a western blot upon probing with a mouse anti-6X-His tag antibody followed by an anti-mouse secondary antibody conjugated with Alexa Fluor™ 800.

[0250] A mass shift due to covalent adduct formation at ~30 kDa was observed in reactions containing all components. With increased oligo concentration, an increase in Alexa Fluor™ 647 signal from the labeled oligo was observed (red channel, FIG 4B, left panel). The same mass shift was observed when probed with an anti-His antibody and an Alexa Fluor™ 800 secondary antibody (green channel, FIG 4B, right panel), which binds to the his-tagged Zdom- PCV2 relaxase fusion protein. Upon capture of the signal in dual channel / multiplex mode, a mass shift due to covalent adduct formation showed an upper band with a dual signal. A lower band with only the green signal from the His tag antibody was also observed indicating there was a subset of oligo in the pool that formed a covalent adduct with the fusion protein but lacked the Alexa Fluor™ 647 conjugate (FIG 4C, left panel). This was also confirmed by a silver-stained gel (FIG. 4C, right panel). In the absence of either the Zdom-PCV2 fusion protein or the oligo in the reaction, no signal or unreacted Zdom-PCV2 fusion protein (~21 kDa) was observed respectively.

[0251] These results confirm that relaxase domain in the Zdom-PCV2 fusion protein covalently linked an oligonucleotide (containing a PCV2 recognition sequence) conjugated with either biotin or a fluorophore.

[0252] Example 5: Antibody labeling with a PRO-tag and Detection by ELISA

[0253] This example describes antibody labeled with a biotinylated PRO-tag followed by detection of the labeled antibody by ELISA as depicted schematically in FIG. 5A.

[0254] Material for ELISA:

[0255] • Recombinant ATG4 protein (purified in-house using an expression construct)

[0256] • IL-6 (Interleukin-6), Recombinant human protein (Gibco™ Catalogue# CTP0061)

[0257] • 1X-PBS-T (1X-PBS with 0.1% Tween-20)

[0258] • Purified ZDomain-PCV2 relaxase fusion protein

[0259] • 1 OX reaction buffer

[0260] • Biotin conjugated oligo for PCV2 (SEQ ID NO. 20)

[0261] • Coating Buffer A (Thermo Scientific™ Catalogue# CB07100)

[0262] • Coating Buffer B (Thermo Scientific™ Catalogue# CB01100)

[0263] • 5% BSA prepared in 1X-PBS-T • Nunc Maxisorp ELISA Plate (Thermo Scientific™ Catalogue# 442404)

[0264] • ELISA TMB Stabilized Chromogen (Invitrogen™ Catalogue# SB02)

[0265] • Stop solution (Thermo Scientific™ Catalogue# N600)

[0266] • ATG4B Recombinant Rabbit Monoclonal Antibody (1HC6LC10), 0.5 mg / ml (Invitrogen™ Catalogue# 701882)

[0267] • IL-6 Recombinant Rabbit Monoclonal Antibody (4H16L21), 0.5 mg / ml (Invitrogen™ Catalogue# 701028)

[0268] • Pierce™ Streptavidin Poly-HRP (Thermo Scientific™ Catalogue# 21140)

[0269] Assembly of PRO-tag and conjugation to primary antibody: A PRO-tag was prepared by assembling a biotinylated PCV2 oligo (SEQ ID NO: 20) with the Zdomain-PCV2 fusion protein of Example 2 as follows at 37°C for 30 minutes:

[0270] A scaled up reaction volume was prepared as per requirement in downstream steps.

[0271] The primary antibody and the Zdom-PCV2-biotin oligo complex (biotinylated PRO-tag) were combined to allow for conjugation as follows in a multi- well plate: 1 pmol (150 ng) of antibody per well and 2 pmol of Zdom-PCV2-oligo complex (biotinylated PRO-tag) per well and incubated at 37°C for 1.5-2 hrs.

[0272] Enzyme-linked immunosorbent assay (ELISA): Coating buffers A and B were mixed in a 1: 1 ratio. A diluted 200 ng / pl stock of recombinant IL-6 antigen or ATG4B antigen were prepared to 200 ng / 100 pl in coating buffer. Serial dilutions of each antigen in coating buffer mix were made as follows: 100 ng / 100 pl, 50 ng / 100 pl, 25ng / 100 pl, 12.5 ng / 100 pl and 6.25 ng / 100 pl. A Maxisorp ELISA plate was coated with 100 pl of diluted antigen in duplicates and kept at 4°C overnight. The next day, the antigen was discarded and wells were blocked with 250 pl of 5% BSA (prepared in IX- PBS-T) at 37°C for 2-3 hrs. The blocking solution was discarded. The PRO-tag-labeled antibody conjugate was prepared in 5% BSA (in 1X-PBS-T) in a 1:2 molar ratio (Ab:PRO-tag) for each well, i.e. 1 pmol of Ab: 2 pmol of oligo tag in 100 pl per well. 100 pl of Ab:PRO-tag per well was added and incubated for 1.5 hours at 37°C. After 1.5 hours, the Ab:PRO-tag was discarded and wells were washed thoroughly three times with 1X-PBS-T and the plate was tapped to remove excess liquid. A 1:7500 dilution of Streptavidin-poly HRP in 5% BSA was prepared and 100 pl was added per well of either of the two antigens and incubated for 30 minutes. After 30 minutes, the wells were washed three times with 1X-PBS-T and excess liquid was drained. 100 pl of pre-warmed TMB substrate was added to each well and incubated at room temperature for 5 min in the dark. After incubation, the reaction was stopped with 100 pl stop solution. Absorbance at 450 nm was measured in an ELISA plate reader.

[0273] Results and Conclusions:

[0274] The Z-domain is known to bind to the Fc region of rabbit and human IgGs. To confirm the functionality of the PRO-tag in an immunoassay, a PRO-tag bound antibody was tested in an ELISA. Either IL-6 or ATG4 antigens were coated in different concentrations: 200 ng, 100 ng, 50 ng, 25 ng, 12.5 ng and 6.25 ng per well. A biotinylated oligo (SEQ ID NO: 20) was covalently attached to the Zdom-PCV2 fusion protein. This PRO-tag was allowed to bind to a rabbit primary antibody (anti-IL-6 or anti-ATG4). After blocking the wells with 5% BSA, the PRO-tag bound antibody was added to antigen coated wells. As a control, only PRO-tag (without antibody) was also added to a row of coated antigen. Any non-specific binding was washed off with 1X-PBS-T. Streptavidin-HRP was diluted to 1:7,500 dilution, added to all wells and incubated. After incubation, excess / non- specifically bound streptavidin-HRP was washed off. TMB substrate was added to wells and incubated for 5 minutes. Reaction was stopped by adding stop solution and absorbance was read at 450 nm in an ELISA plate reader. As a function of antigen concentration, the PRO-tag bound antibody was confirmed to be functionally active in an ELISA application as seen by titration curves in FIG. 5B for both antigens IL-6 and ATG4.

[0275] Example 6: Stability of antibody-PRO-tag complex in an immunoprecipitation assay

[0276] This example describes stability studies of an antibody labeled with a biotinylated PRO- tag as analyzed by an immunoprecipitation assay as depicted schematically in FIG. 6A.

[0277] Material for immunoprecipitation:

[0278] • HisPur™ Ni-NTA Magnetic Beads (Thermo Scientific™- Catalog# 88831)

[0279] • 1X-PBS-T (1X-PBS with 0.2% Tween-20)

[0280] 1X-PBS

[0281] • Biotin conjugated oligo for PCV2 (SEQ ID NO. 20)

[0282] • Purified ZDomain-PCV2 relaxase fusion protein

[0283] • IL-6 (Interleukin-6), Recombinant human protein (Gibco™ Catalogue# CTP0061) 10X reaction buffer

[0284] • Nuclease free water; DEPC-treated water (Invitrogen™-Catalogue# AM9922)

[0285] • IL-6 Recombinant Rabbit Monoclonal Antibody (4H16L21), 0.5 mg / ml (Invitrogen™-Catalogue# 701028)

[0286] • IL-6 Monoclonal Antibody (6708) (Invitrogen™-Catalogue# MA5-23698)

[0287] • Rabbit IgG Isotype control (Invitrogen™-Catalogue# 02-6102)

[0288] • Pierce™ Streptavidin Poly-HRP (Thermo Scientific™ Catalogue# 21140)

[0289] • iBlot™ 2 Transfer Stacks, nitrocellulose, mini (Invitrogen™-Catalogue# IB23002) NuPAGE™ 4-12% Bis-Tris Protein Gel, 10 well (Invitrogen™- Catalogue#

[0290] NP0321BOX)

[0291] 5% skimmed milk prepared in 1X-PBS-T (0.1% Tween-20)

[0292] • NuPAGE™ LDS Sample Buffer (4X) (Invitrogen™-Catalogue# NP0007)

[0293] • Goat anti-Mouse IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Invitrogen™-Catalogue# A28177)

[0294] • Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Invitrogen™-Catalogue# A27036)

[0295] • SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Scientific™, Catalogue# 34580)

[0296] Protocol for immunoprecipitation:

[0297] A biotinylated PRO-tag comprising a Zdomain-PCV2 fusion protein from Example 2 covalently attached to a biotinylated PCV2 oligo (SEQ ID NO: 20) was prepared as described below. Reaction volumes can be scaled up as required.

[0298] The reaction was incubated at 37°C for 30 minutes. Binding of anti-IL-6 antibody to the biotinylated PRO-tag was done in a 1:2 (antibody:PRO-tag) ratio. 6 pg of antibody (40 pmol) was added to 48 pl of oligo tag (80 pmol) and incubated for 2 hours at 37°C. 800 ng of recombinant IL6 protein was added per reaction and incubated with PRO-tag bound IL-6 antibody for 2-3 hours at 37°C at 350 RPM. Six reaction conditions were set up and the volume was made up with IX reaction buffer, as described in the table below:

[0299] 10 pl (125pg) of HisPur™ Ni-NTA Magnetic Beads were added to a 1.5 ml microcentrifuge tube. 190 pl of Equilibration Buffer (IX Buffer) were added to the beads, then vortexed for 10 seconds to mix. The tube was placed onto a magnetic stand to collect the beads against the side of the tube and supernatant was discarded. 200 pl of equilibration buffer were added to the tube, then the beads were vortexed for 10 seconds, the beads were collected by placing the tube on a magnetic stand and the supernatant was discarded (performed twice). The loading sample was prepared (reaction mix prepared earlier) by diluting the reaction mix with an equal volume of equilibration buffer (100 pl). 200 pl of diluted reaction mix was added to the tube containing the Ni-NTA beads, the beads were vortexed for 10 seconds, then mixed on an end-over-end rotator for 30 minutes to Ihour at room temperature. The beads were collected by placing the tube on a magnetic stand and the supernatant was saved for downstream analysis.

[0300] 100 pl of Wash Buffer (1X-PBS containing 25 mM Imidazole) was added to the sample, then vortexed for 10 seconds to mix. The beads were collected by placing the tube on a magnetic stand, then removed and the supernatant was discarded. The wash and collecting steps were repeated. 20 pl of Elution Buffer (1X-PBS containing 250 mM Imidazole) were added to the tube, then vortexed for 15 seconds. Subsequently, the tube was vortexed for 15 seconds, every 5 minutes for 15 minutes. The beads were collected by placing the tube on a magnetic stand, the supernatant containing the His-tagged protein was carefully removed and the elution steps were repeated twice.

[0301] Detection on Western Blot: The wash and eluents of every reaction were collected and equal amounts of 2X loading dye were added to them and heated at 70°C for 10 minutes. The samples were run on a 4-12% Bis-Tris gel for 1 hour at 150 V (Constant voltage). Two sets of gels were run. The gels were transferred onto nitrocellulose membranes using an iBlot2 apparatus. The blots were blocked with 5% milk prepared in 1X-PBS-T for 1 hour at room temperature. The first blot was probed with an anti-IL6 antibody (MA5-23698, 1:500 dilution), the second blot was probed with an anti-His HRP antibody (MA1-21315-HRP, 1:2000 dilution), and both blots were incubated overnight at 4°C. The next day, the blots were washed three times with 1X-PBS-T.

[0302] The first blot (anti-IL6 blot) was probed with a Goat anti-Mouse secondary antibody (A28177, 1:5000 dilution) for 1 hour at room temperature. After 3 washes with 1X-PBS-T, the blot was developed with West Pico Plus and imaged using iBright FL1500 (FIG. 6B, top panel).

[0303] The second blot that was incubated with anti-His HRP was developed with West Pico Plus and imaged using an iBright FL1500 (FIG. 6B, middle panel). After capturing the signal for the His tag antibody, this blot was probed with a Goat anti-Rabbit secondary antibody (A27036, 1 : 10,000 dilution) for 3 hours at room temperature. After 1X-PBS-T washes, the blot was developed with West Pico Plus and imaged using iBright FL1500 (FIG. 6B, bottom panel).

[0304] Results and Conclusions:

[0305] Immunoprecipitation was performed with recombinant IL-6 protein and a biotinylated PRO-tag (Zdom-PCV2-oligo) conjugated anti-IL-6 antibody. All components of immunoprecipitation IL-6 antigen and PRO-tag conjugated IL-6 antibody were allowed to interact with each other. The immunocomplex was allowed to bind to a Ni-NTA column via an N-terminal 6X-His tag of the Zdom-PCV2 fusion protein, washed to remove non-specific binding and eluted using 250 mM imidazole. Wash and eluted fractions were analyzed on a western blot using antibodies against IL-6, 6X-His tag and anti-rabbit secondary antibody.

[0306] Combining the results from three western blot conditions, all components of the immunoprecipitation: IL-6 antigen, anti-IL-6 rabbit antibody and His-tagged Zdom-PCV2- oligo tag were detected (FIG. 6B top, bottom, and middle, respectively). These results suggest the entire immunocomplex will be pulled down on a Ni-NTA column only if IL-6 antigen is bound by IL-6 antibody that is conjugated with a PRO-tag having a His-tagged Zdom-PCV2 fusion protein. Therefore, the PRO-tag conjugated antibody was stable and functional in an immunoprecipitation assay. Example 7: Antibody-PRO-tag complex in an immuno-PCR assay (single antigen)

[0307] This example describes a single antigen immuno-PCR assay of an antibody labeled with a PRO-tag comprising a qPCR handle as depicted schematically in FIG. 7 A.

[0308] Materials for Immuno-PCR:

[0309] • Recombinant ATG4 protein (purified in-house using an expression construct)

[0310] • IL-6 (Interleukin-6), Recombinant human protein (Gibco™-Catalogue# CTP0061)

[0311] • 1X-PBS-T (1X-PBS with 0.1% Tween-20)

[0312] • Purified ZDomain-PCV2-relaxase fusion protein

[0313] • 1 OX reaction buffer

[0314] • Coating Buffer A (Thermo Scientific™-Catalogue# CB07100)

[0315] • Coating Buffer B (Thermo Scientific™-Catalogue# CB01100)

[0316] • 5% BSAprepared in 1X-PBS-T

[0317] • Nunc Maxisorp ELISA Plate (Thermo Scientific™-Catalogue# 442404)

[0318] • qPCR handle #1 for PCV (SEQ ID NO: 35)

[0319] • qPCR handle #2 for PCV (SEQ ID NO: 36)

[0320] • Nuclease free water; DEPC-treated water (Invitrogen™-Catalogue# AM9922)

[0321] • ATG4B Recombinant Rabbit Monoclonal Antibody (1HC6LC10), 0.5 mg / ml (InvitrogenlM-Catalogue# 701882)

[0322] • IL6 Recombinant Rabbit Monoclonal Antibody (4H16L21), 0.5 mg / ml (Invitrogen1!V:-Catalogue# 701028)

[0323] • Forward primer for qPCR handle #1 (SEQ ID NO: 42); T7 promoter primer

[0324] Reverse primer for qPCR handle #1 (SEQ ID NO: 43); M13 Reverse primer Forward primer for qPCR handle #2 (SEQ ID NO: 44); SP6 promoter primer

[0325] Reverse primer for qPCR handle #2 (SEQ ID NO: 45); T3 promoter primer

[0326] • Proteinase K, Recombinant, PCR Grade (Thermo Scientific™-Catalogue# EO0491)

[0327] • 1X-TE Buffer (lOmM Tris-Cl, pH 8, ImM EDTA)

[0328] • PowerUP™ SYBR Green Master Mix for qPCR (Applied Biosystems™- Catalogue# A25742)

[0329] • MicroAmp™ Fast Optical 96-Well Reaction Plate, 0.1 ml (Applied Biosystems™- Catalogue# 4346907)

[0330] • MicroAmp™ Optical Adhesive Film (Applied Biosystems™-Catalogue# 4311971)

[0331] Immuno-PCR Analysis:

[0332] Coating buffer A and B were mixed in a 1: 1 ratio. A diluted 200 ng / pl stock of recombinant IL-6 and recombinant ATG4 were prepared to 200ng / 100pl in coating buffer and serial dilutions of IL-6 and ATG4 were made in coating buffer mix: lOOng / lOOpl, 50ng / 100pl, 25ng / 100pl, 12.5ng / 100pl and 6.25ng / 100pl. A Maxisorp ELISA plate was coated with 100 pl antigen of each concentration in duplicates in rows A and B for IL-6 and rows C and D for ATG4 and kept at 4°C overnight. The next day, the antigen was discarded and the wells were blocked with 250 pl of 5% BSA in 1X-PBS-T at 37°C for 2 hrs. The Zdomain-PCV2 relaxase tag with qPCR handle #1 (for IL-6) or qPCR handle #2 (for ATG4) were prepared separately as described in the table below at 37°C for 1 hour.

[0333] The anti-IL-6 rabbit antibody complex with Zdom-PCV2-qPCR#l tag and anti-ATG4B rabbit antibody complex with Zdom-PCV2-qPCR#2 tag were prepared as follows: 4 pmol (600 ng) of Ab per well and 2 pmol of Zdom-PCV2-oligo tag per well were combined and incubated at 37°C for 2 hrs. After complex formation, Ab-PRO-tag complexes were diluted in 5% BSA such that each 100 pl consists of Ab:PRO-tag in 2: 1 ratio (4pmol:2pmol). 100 pl of this mix was added to row A (containing IL-6 Ab complex) and row C (containing ATG4 Ab complex). To rows B and D, equivalents amounts were added of Zdom-PCV2-qPCR#l and Zdom-PCV2- qPCR#2 diluted in 100 pl of 5% BSA as controls. The plate was incubated at 37°C for 1.5-2 hrs. After incubation, the plate contents were discarded, the plate was washed thrice vigorously with IX-PBS-T and excess liquid was drained by tapping. To each well, 100 pl of 1X-TE buffer and 4 pl of Proteinase K were added and the plate was incubated at 55°C for 20 minutes to degrade all protein components. The contents were transferred to a PCR plate and Proteinase K enzyme was inactivated at 95°C for 15 min. The sample in each well was diluted to 1: 100. qPCR reactions were set up with 2 pl of 1:100 diluted fraction as DNA template per reaction as follows:

[0334] The fold difference was calculated in immuno-PCR wells (rows A and C) for each antigen concentration by normalizing to the PRO-tag control (rows B and D for IL-6 and ATG4 respectively).

[0335] Results and Conclusions: PRO-tag bound antibody was tested in an immuno-PCR assay. Either IL-6 or ATG4 antigens were coated in an ELISA plate in different concentrations: 200 ng, 100 ng, 50 ng, 25 ng, 12.5 ng and 6.25 ng per well. A long oligo that can be used a qPCR handle was covalently attached to a Zdom-PCV2 fusion protein. This PRO-tag was allowed to bind to a rabbit primary antibody (anti-IL-6 or anti-ATG4). After blocking the wells with 5% BSA, PRO-tag bound antibody was added to antigen coated wells. qPCR handle# 1 was used for IL-6 antibody and qPCR handle# 2 was used for ATG4 antibody. As a control, only PRO-tag (without antibody) was also added to a row of coated antigen. Any non-specific binding was washed off with IX- PBS-T. All protein components in the well were digested by Proteinase K at 55°C followed by heat inactivation at 95°C. Essentially, oligonucleotide should be intact in the mix, which can be detected by molecular amplification of the PCR handle. qPCR was done with primers specific to each qPCR handle to quantify antigen. Background signal was captured from antigen coated wells incubated with only PRO-tag. As a function of antigen concentration, PRO-tag bound antibody was able to quantify antigen in an immuno-PCR assay application as seen by titration curves in FIG. 7B for both antigens IL- 6 and ATG4.

[0336] Example 8: Antibody-PRO-tag complex in a dual antigen immuno-PCR assay

[0337] This example describes a dual antigen immuno-PCR assay of an antibody labeled with a PRO-tag comprising a qPCR handle as depicted schematically in FIG. 8A.

[0338] Materials for Immuno-PCR:

[0339] • Recombinant ATG4 protein (purified in-house using an expression construct)

[0340] • IL-6 (Interleukin-6), Recombinant human protein (Gibco™-Catalogue# CTP0061)

[0341] • 1X-PBS-T (1X-PBS with 0.1% Tween-20)

[0342] • Purified ZDomain-PCV2-relaxase fusion protein

[0343] • 1 OX reaction buffer

[0344] • Coating Buffer A (Thermo Scientific™-Catalogue# CB07100)

[0345] • Coating Buffer B (Thermo Scientific™-Catalogue# CB01100) 5% BSA prepared in 1X-PBS-T

[0346] • Nunc Maxisorp ELISA Plate (Thermo Scientific™-Catalogue# 442404)

[0347] • qPCR handle #1 for PCV (SEQ ID NO: 35)

[0348] • qPCR handle #2 for PCV (SEQ ID NO: 36)

[0349] • Nuclease free water; DEPC-treated water (Invitrogen™-Catalogue# AM9922)

[0350] • ATG4B Recombinant Rabbit Monoclonal Antibody (1HC6LC10), 0.5 mg / ml (Invitrogen^-Catalogue# 701882)

[0351] • IL6 Recombinant Rabbit Monoclonal Antibody (4H16L21), 0.5 mg / ml (InvitrogenlM-Catalogue# 701028)

[0352] • Forward primer for qPCR handle #1 (SEQ ID NO: 42); T7 promoter primer

[0353] • Reverse primer for qPCR handle #1 (SEQ ID NO: 43); M13 Reverse primer

[0354] • Forward primer for qPCR handle #2 (SEQ ID NO: 44); SP6 promoter primer

[0355] • Reverse primer for qPCR handle #2 (SEQ ID NO: 45); T3 promoter primer

[0356] • Proteinase K, Recombinant, PCR Grade (Thermo Scientific™-Catalogue# EO0491)

[0357] • 1X-TE Buffer (lOmM Tris-Cl, pH 8, ImM EDTA)

[0358] • PowerUP™ SYBR Green Master Mix for qPCR (Applied Biosystems™- Catalogue# A25742)

[0359] • MicroAmp™ Fast Optical 96-Well Reaction Plate, 0. 1 ml (Applied Biosystems™- Catalogue# 4346907)

[0360] • MicroAmp™ Optical Adhesive Film ( Applied Biosystems™-Catalogue# 4311971 )

[0361] Immuno-PCR Analysis: Coating buffers A and B were mixed in a 1: 1 ratio. A diluted 200 ng / pl stock of recombinant IL-6 and recombinant ATG4 was prepared in such a way that each well contains both antigens at the same time. A Maxisorp ELISA plate was coated with IL-6 and ATG4 in duplicate wells as follows: a) 50ng of IL-6 and 150 ng of ATG4 in total lOOpl coating buffer, b) lOOng of IL-6 and 100 ng of ATG4 in total lOOpl coating buffer, c) 150ng of IL-6 and 50 ng of ATG4 in total lOOpl coating buffer. Coated antigen was kept at 4°C overnight. The next day, antigen was discarded and the wells were blocked with 250 pl of 5% BSA in 1X-PBS-T at 37°C for 2 hrs. The Zdomain-PCV2 relaxase tag (PRO-tag) with qPCR handle #1 (for IL-6) or qPCR handle #2 (for ATG4) were prepared separately as described below at 37°C for 1 hour.

[0362] The anti-IL-6 rabbit antibody conjugate with the Zdom-PCV2-qPCR#l tag and the anti- ATG4B rabbit antibody conjugate with Zdom-PCV2-qPCR#2 tag were prepared as follows: 4 pmol (600 ng) of Ab per well and 2 pmol of PRO-tag per well were combined and incubated at 37 deg for 2 hrs. After conjugate formation, both the PRO-tag bound anti-IL6 antibody and the PRO-tag bound anti-ATG4B antibody were mixed in 5% BSA such that each 100 pl consists of Ab:PRO-tag in 2: 1 ratio (4pmol:2pmol) of both the antibody conjugates. 100 pl of this mix was added to all wells coated with antigens. To rows of coated antigens, equivalent amounts were added (2pmol per well) of mixed Zdom-PCV2-qPCR#l and Zdom-PCV2- qPCR#2 tags diluted in 100 pl of 5% BSA as controls. The plate was incubated at 37°C for 1.5-2 hrs. After incubation, the plate contents were discarded, washed thrice vigorously with IX-PBS-T and drained of excess liquid by tapping. To each well, 100 pl of 1X-TE buffer and 4 pl of Proteinase K were added and incubated at 55°C for 20 minutes to degrade all protein components. The contents were transferred to a PCR plate and Proteinase K enzyme was inactivated at 95°C for 15 min. Sample in each well was diluted to 1: 100. qPCR reactions were set up with 2 pl of 1:100 diluted fraction as DNA template per reaction as follows:

[0363] The fold difference was calculated in immuno-PCR wells for each antigen concentration by normalizing to the PRO-tag control for IL-6 and ATG4 separately.

[0364] Results and Conclusions:

[0365] PRO-tag bound antibodies were tested in a dual antigen immuno-PCR assay. IL-6 and ATG4 antigens were mixed in different concentrations and coated in duplicates in an ELISA plate. A different qPCR handle was used for each antigen and the PRO-tags were prepared with handle#l and handle#2. Each PRO-tag was allowed to bind to its respective rabbit primary antibody (anti-IL-6 or anti-ATG4). qPCR handle#l was used for IL-6 antibody and qPCR handle#2 was used for ATG4 antibody. After blocking the wells with 5% BSA, PRO-tag bound antibodies were mixed and added to antigen coated wells. As a control, only PRO-tags mixed (without antibody) were also added to a row of coated antigen. Any non-specific binding was washed off with 1X-PBS-T. All protein components in the well were digested by Proteinase K at 55°C followed by heat inactivation at 95°C. qPCR was done with primers specific to each qPCR handle to quantify antigen. Background signal was captured from antigen coated wells incubated with only PRO-tag.

[0366] In this experiment, each PRO-tag conjugated antibody was titrated as a function of antigen concentration, IL-6 (FIG. 8B, grey bars) and ATG4B (FIG. 8B, black bars) in a dual antigen immuno-PCR assay as represented by fold difference in FIG. 8B.

[0367] Example 9: On-demand antibody labeling using a detectably-labeled PRO-tag

[0368] This example describes an antibody labeled with a PRO-tag comprising a fold-back probe as depicted schematically in FIG. 9A. Materials for on-demand labelling and immunoblotting:

[0369] Recombinant ATG4 protein (purified in-house using an expression construct)

[0370] • IL-6 (Interleukin-6), Recombinant human protein (Gibco™-Catalogue# CTP0061)

[0371] • 1X-PBS-T (1X-PBS with 0.1% Tween-20)

[0372] • Purified ZDomain-PCV2-relaxase fusion protein

[0373] • 1 OX reaction buffer

[0374] • Fold back oligo for relaxase tagging and self-labeling 2A-P4 (SEQ ID NO: 32)

[0375] • Nuclease free water; DEPC-treated water (Invitrogen™-Catalogue# AM9922)

[0376] • iBlot™ 2 Transfer Stacks, nitrocellulose, mini (Invitrogen™-Catalogue# IB23002)

[0377] • NuPAGE™ 4-12% Bis-Tris Protein Gel, 10 well (Invitrogen™' Catalogue# NP0321BOX)

[0378] • 5% skimmed milk prepared in 1X-PBS-T (0.1% Tween-20)

[0379] • NuPAGE™ LDS Sample Buffer (4X) (Invitrogen™-Catalogue# NP0007)

[0380] • Aminoallyl-dUTP-XX-AF647, (Jena Biosciences Catalogue# NU-803-XX-AF647- L)

[0381] • Deoxynucleotide (dNTP) Solution Set (NEB, Catalogue# N0446S)

[0382] • Klenow Fragment (3’ -> 5’ exo-) (NEB, Catalogue# M0212S)

[0383] • 10X NEBuffer 2

[0384] • 500 mM EDTA solution

[0385] • ATG4B Recombinant Rabbit Monoclonal Antibody (1HC6LC10), 0.5 mg / ml (Invitrogen1 M-C ata I ogue# 701882) • IL6 Recombinant Rabbit Monoclonal Antibody (4H16L21), 0.5 mg / ml (Invitrogen1^-Catalogue# 701028)

[0386] • Pierce™ Protein-Free Blocking Buffer (Thermo Scientific™'Catalogue# 37572)

[0387] • Donkey anti -Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 800 (Invitrogen™-Catalogue# A32808)

[0388] Immunobloting assay:

[0389] 500 ng and 250 ng of recombinant ATG4B and recombinant IL-6 antigens were run on an SDS-PAGE (4-12% Bis-Tris gel). Separate gels were run for ATG4B and IL-6. The gels were transferred onto nitrocellulose membranes using iBlot2 system. Post-transfer, the membranes were blocked with 5% milk prepared in 1X-PBS-T for 1 hour.

[0390] Labeled PRO-tag assembly and antibody conjugation:

[0391] Fold-back probe 2A-P4 oligo (SEQ ID NO: 32) was used for the relaxase reaction as follows:

[0392] The reaction was incubated at 37°C for 30-45 minutes. After the relaxase reaction, self- labeling Klenow reaction was set up as follows:

[0393] Reaction was incubated at 37°C for 20 minutes and stopped by adding 500 mM EDTA to a final concentration of 10 mM. The Alexa Fluor 647 labeled tag was assembled with primary antibodies at 37°C for 1.5 hours as follows: For IL-6 and ATG4B antibodies individually,

[0394] Fluorescent western blot analysis:

[0395] To 1 ml of Protein-Free Blocking buffer, 100 pl of labeled antibody (prepared in previous step) was added and incubated the western blot membrane at 4°C overnight on a rocker. The next day, the contents were discarded and the membranes were washed with IX- PBS-T for 10-15 minutes. The Alexa Fluor™ 647 signal was captured in iBrightl500 FL (red channel). After signal capture, the blot was incubated in a 1 :20,000 dilution of Goat anti-Rabbit Alexa Fluor™ Plus 800 for 45 minutes. The contents were discarded and blot was washed with 1X-PBS-T. The Alexa Fluor™ Plus 800 signal was captured and compared to the signal intensity of the labeled tag (green channel).

[0396] Results and Conclusions: A fold-back probe is a synthetic single- stranded sequence designed in such a way that the oligonucleotide sequence folds back on to itself to bind to complementary sequence to form a hairpin with a free 3 ’ -OH end. The 3 ’ -end is available for extension using a DNA polymerase. To test the concept of on-demand labeling of antibodies with fluorophores and its application in immunoassays, a fold-back probe was covalently attached to a Zdom-PCV2 fusion protein followed by 3 ’-extension using fluorescently labeled dNTPs using the Klenow fragment (FIG. 9B, top panel). After fluorescent labeling of the PRO-tag, it was allowed to bind to a primary antibody of choice for downstream testing in an immunoassay (FIG. 9A).

[0397] A primary antibody conjugated with fluorescently labeled fold-back PRO-tag was tested on a western blot (schematically shown in FIG. 9A). Recombinant ATG4 and IL-6 protein were run on an SDS-PAGE and probed with respective labeled antibodies. The bottom panel in FIG. 9B shows western blots of anti-ATG4 antibodies (bottom left) or anti-IL-6 antibodies (bottom right) labeled with an Alexa Fluor™ 647-labeled fold-back PRO-tag and detected by direct visualization of the Alexa Fluor™ 647 label (1stand 3rdpanels). Fluorescent signal on primary antibody from the PRO-tag was compared to a conventional approach of using a secondary antibody labeled with an Alexa Fluor™ 800 labeled (2ndand 4thpanels).

[0398] These results suggest:

[0399] 1) a PRO-tag can be labeled with a fluorophore / label of choice using a fold-back design of oligonucleotide;

[0400] 2) the labeled PRO-tag was able to bind to a rabbit primary antibody with aid of Z-domain; and

[0401] 3) the labeled PRO-tag was functional in a western blot and was able to detect recombinant antigen successfully.

[0402] The signal captured directly from the PRO-tag was equivalent to signal captured from a labeled secondary antibody.

[0403] Example 10: Stability of the detectably-labeled PRO-tag as an antibody label This example analyzes the stability an antibody labeled with a PRO-tag comprising a fold-back probe as depicted schematically in FIG. 10A.

[0404] Materials required:

[0405] • Transferrin human (Sigma Aidrich-Catalogue# T3309-100MG)

[0406] • IL-6 (Interleukin-6), Recombinant human protein (Gibco™-Catalogue# CTP0061)

[0407] • 1X-PBS-T (1X-PBS with 0.1% Tween-20)

[0408] • Purified ZDomain-PCV2-relaxase fusion protein

[0409] • 1 OX reaction buffer

[0410] • Fold back oligo for relaxase tagging and self-labeling 2A-P4 (SEQ ID NO: 32)

[0411] • Nuclease free water; DEPC-treated water (Invitrogen™-Catalogue# AM9922)

[0412] • iBlot™ 2 Transfer Stacks, nitrocellulose, mini (Invitrogen™-Catalogue# IB23002)

[0413] • NuPAGE™ 4-12% Bis-Tris Protein Gel, 10 well (Invitrogen™' Catalogue# NP0321BOX)

[0414] • 5% skimmed milk prepared in 1X-PBS-T (0.1% Tween-20)

[0415] • NuPAGE™ LDS Sample Buffer (4X) (Invitrogen™-Catalogue# NP0007)

[0416] • Aminoallyl-dUTP-XX-AF647, (Jena Biosciences Catalogue# NU-803-XX-AF647- L)

[0417] • Deoxynucleotide (dNTP) Solution Set (NEB, Catalogue# N0446S)

[0418] • Klenow Fragment (3’ -> 5’ exo-) (NEB, Catalogue# M0212S)

[0419] • 10X NEBuffer 2

[0420] • 500 mM EDTA solution • Transferrin Recombinant Rabbit Monoclonal Antibody (101), 1 mg / ml ( Invi trogen1 M-C at al ogue# #M A5 -30565)

[0421] • IL6 Recombinant Rabbit Monoclonal Antibody (4H16L21), 0.5 mg / ml ( Inv itrogen1M-Catalogue# 701028) • ATG4B Recombinant Rabbit Monoclonal Antibody (1HC6LC10), 0.5 mg / ml

[0422] (Invitrogen™ Catalogue# 701882)

[0423] • EZH2 Recombinant Rabbit Monoclonal Antibody (8H32L43), 0.5 mg / ml (Invitrogen™ Catalogue# 702492)

[0424] • Pierce™ Protein-Free Blocking Buffer (Thermo Scientific™'Catalogue# 37572) Immunobloting analysis:

[0425] 500 ng of recombinant IL-6 and human transferrin were run on an SDS-PAGE (4-12% Bis-Tris gel). The gel was transferred onto a nitrocellulose membrane using iBlot2 system. Post-transfer, the membrane was blocked with 5% milk prepared in 1X-PBS-T for 1 hour.

[0426] Labeled PRO-tag assembly and antibody conjugation: Fold-back probe 2A-P4 oligo (SEQ ID NO: 32) was used for the relaxase reaction as follows:

[0427] The reaction was incubated at 37°C for 30-45 minutes. After the relaxase reaction, self-labeling Klenow reaction was set up as follows:

[0428] The reaction was incubated at 37°C for 20 minutes and stopped by adding 500 mM EDTA to a final concentration of 10 mM. The Alexa Fluor 647 labeled PRO-tag was assembled with the Transferrin antibody at 37°C for 1.5 hours as follows:

[0429] Dark channel western blot analysis:

[0430] To 1 ml of Protein-Free Blocking buffer, 100 pl of labeled Transferrin antibody (prepared in previous step) was added along with 4 pg of IL-6 antibody (unlabeled) and incubated the western blot membrane at 4°C overnight on a rocker. The next day, the contents were discarded and the membrane was washed with 1X-PBS-T for 10-15 minutes. The Alexa Fluor™ 647 signal was captured in iBrightl500 FL (see red channel FIG. 10C, left). The Alexa Fluor™ 647 signal was observed for transferrin protein only and not for IL-6.

[0431] After single channel signal capture, the blot was incubated in a 1:20,000 dilution of Goat anti-Rabbit Alexa Fluor™ Plus 800 for 45 minutes. The contents were discarded and the blot was washed with 1X-PBS-T. The Alexa Fluor 647 signal (red channel) and Alexa Fluor™ Plus 800 (blue channel) signal were captured together to show whether the PRO-tag is stable in a mixed antibody solution (see FIG. IOC, right). Dual channel signal was observed in the case of Transferrin only; therefore, the PRO-tag appears to be stable in the presence of multiple analytes along with mixed antibody solution.

[0432] Results and Conclusions:

[0433] To test whether the PRO-tag dissociates from one antibody binds to a different antibody in a mixed antibody solution, a labeled antibody (conjugated with PRO-tag) and an unlabeled second antibody were mixed together to test in a western blot assay. Recombinant transferrin and IL-6 were run on an SDS-PAGE and probed with a mix of labeled (anti-TF) and unlabeled antibodies (anti-IL-6) (FIG. 10A). FIG. 10B shows there is no dissociation of the PRO-tag from one antibody to another.

[0434] The Alexa Fluor™ 647 signal was observed for only the anti-transferrin antibody and not the anti-IL-6 antibody (red channel, left panel, FIG. 10B). To confirm that the IL-6 antibody was indeed bound to IL-6 antigen and there was no dissociation of the Alexa Fluor™ 647 signal from the transferrin antibody, the blot was probed using an Alexa Fluor™ Plus 800-labeled secondary antibody (GAR-AF800) that binds to both the anti-transferrin antibody (TF) and the anti-IL-6 antibody (IL). The band in the TF lane was labeled with both the Alexa Fluor™ 647 and the Alexa Fluor™ 800-labeled secondary antibody (multiplex, dual channel-red, blue) demonstrating that there is no dissociation of the PRO-tag between antibodies. On the other hand, IL-6 lane was labeled with only Alexa Fluor™ 800-labeled secondary antibody (single channel, blue channel) (FIG. 10B, right panel).

[0435] This experiment demonstrates the PRO-tag stability under assay conditions and an absence of dissociation in a mixed antibody solution with multiple analytes.

[0436] A few more antigen and antibody combinations were tested to demonstrate the stability of the PRO-tag and absence of dissociation from antibody. ATG4 and IL-6 were tested where an anti-ATG4 antibody was conjugated with the Alexa Fluor™ 647 labeled PRO-tag and unlabeled IL-6 antibody was added. When the signal was captured in the Alexa Fluor™ 647 channel, only ATG4 protein was observed and no signal was observed for IL-6 protein. As a control, upon probing this blot with an anti-rabbit Alexa Fluor 800 conjugated secondary antibody, both proteins ATG4 and IL-6 were captured (FIG. 10C, left panel). Similarly, when an anti-IL-6 conjugated to the PRO-tag labeled with AF647 and mixed with unlabeled anti- EZH2 antibody, only the IL-6 protein was observed (FIG. 10C, right panel). Example 11: PRO-tags for labeling of antibody fragments (e.g., Fab) and / or singlechain antibodies (VHH)

[0437] This example represents the design of a PRO-tag that is a recombinant fusion protein of a VHH antibody fragment with a relaxase domain which is then covalently linked to a labeled oligonucleotide sequence via the relaxase domain (a VHH-PRO-tag). This VHH-PRO- tag has multiple advantages:

[0438] • All components of this design can be covalently linked to each other i.e. Antibody fragment-Relaxase-Oligo (VHH-PRO-tag) making these interactions more robust.

[0439] • This design is modular, easier to synthesize, can be engineered and is customizable.

[0440] • VHH-PRO-tag complex is much smaller in size (~40 kDa) as compared to a full-length antibody-PRO-tag complex (-180 kDa).

[0441] • Labeled VHH-PRO-tag complex can be directly used in an immunoassay.

[0442] A recombinant fusion of VHH and relaxase domain is created with a 6x-His tag for purification and expressed in a bacterial host. Purified VHH-relaxase fusion is covalently linked to an oligonucleotide designed for desired application. This chimera in itself is capable of binding to the VHH’s cognate antigen and is available for use an immunoassay. FIG. 11 shows a schematic of a labeled VHH-PRO-tag complex in which the VHH domain is bound to its cognate antigen in an immunoassay. A single modular fusion like this enables covalent oligo conjugation with an antibody fragment directly. Such an assembly eliminates the need of incubation of PRO-tag with a full-length antibody required for non-covalent binding.

[0443] Example 12: Ligand-relaxase chimeras covalently linked to an oligonucleotide

[0444] This example depicts additional embodiments of the PRO-tag where the protein is a ligand (a “ligand-PRO-tag”) or where the protein is an antigen (an “antigen-PRO-tag”).

[0445] A) A recombinant fusion of a ligand and a relaxase domain that is covalently linked to a labeled oligonucleotide (a “ligand-PRO-tag”) can be used in a flow cytometry-based assay. This ligand-PRO-tag when bound to its cognate receptor on a particular cell can be assayed using flow cytometry. Multiple ligand-PRO-tags each labeled with a different dye can be assayed for their respective receptor binding on a single cell or a cell population (FIG. 12A). In FIG. 12A, Ligand 1 is a ligand-PRO-tag comprising a fusion of Ligand 1, a relaxase domain, and an oligo labeled with Dye 1, and Ligand 2 is a ligand-PRO-tag comprising a fusion of Ligand 2, a relaxase domain, and an oligo labeled with Dye 2. Each ligand-PRO-tag when bound to their respective receptor can be assayed by the fluorescence signal of their respective dyes. In this example, the labeled oligo is a fold-back probe.

[0446] B) In another example depicted in FIG. 12B, a recombinant fusion of a ligand and a relaxase domain is covalently linked to a dye-labeled fold back probe which comprises a sitespecific cleavage sequence and a unique barcode. Each ligand-PRO-tag when bound to its respective receptor, is assayed by measuring the fluorescent signal that is captured from the bound ligand-PRO-tag. The barcode on the fold-back probe can be released using a nicking enzyme or site- specific endonuclease, in a sequence- specific manner. The released barcode can be used to quantify the copy number of the ligand bound to the receptor by either qPCR or NGS-based assays. The copies of tag barcode for ligands bound to cell can be profiled in relation to the cell receptors or cell transcriptome to derive an assessment of ligands occupied per cell.

[0447] C) In another example, a recombinant fusion of an antigen and a relaxase domain is covalently linked to a dye-labeled fold-back oligo. Labeled antigen-PRO-tag when bound to its respective B-cell receptor (BCR) can be used for B-cell selection in a cell population. In FIG. 12C, labeled antigen-PRO-tag- 1 binds to BCR-1 on B-cell- 1 and labeled antigen-PRO- tag-2 binds to BCR-2 on B-cell-2. Each B-cell can be selected based on the specific fluorescent signal of its bound antigen.

[0448] D. In another example, an antigen-relaxase fusion is covalently linked with a biotinylated oligo. Specific B-cells can be pulled down using streptavidin-beads that bind biotinylated oligonucleotide when the labeled antigen-PRO-tag is bound to its specific BCR (FIG. 12D). The capture of specific antigen binding pools or specific ligand binding cells can be performed using complementary oligonucleotides in selective pull-down enrichment . This design can be for B-cell selection and enrichment in a mixed pool of cell population.

[0449] E. In another example, FIG. 12E describes the use of domain- specific PRO-tags for use in domain- specific enrichment of B-cells. The left diagram of FIG. 12E depicts a receptor / protein-1 that has 4 domains: I, II, III and IV (FIG. 12E, left). Fusion proteins of one of the receptor / protein-1 domains with a relaxase domain and covalently attached to unique oligo tags are designed and allowed to bind to a B-cell population (domain fusions depicted in FIG. 12E, right). In this example, B-cells expressing BCRs against each of these domains can be selected or enriched using epitope / domain-enriched tags. Total signal from all 4 labeled tags (I, II, III and IV) can be used to quantify / select B-cells expressing antibodies against receptor-

[0450] 1. Signal from each domain-specific tag can be selected and used to enrich B-cell population for each domain.

[0451] It will be apparent that the precise details of the methods or compositions described may be varied or modified without departing from the spirit of the described aspects of the disclosure. We claim all such modifications and variations that fall within the scope and spirit of the claims below.

Claims

CLAIMS1. A conjugate comprising: a protein chosen from a ligand, a protein domain, an antigen, an antigen binding protein, an antibody, an antibody fragment, a single-domain antibody, a VHH antibody or a protein comprising an Fc binding domain; a relaxase domain; and an oligonucleotide covalently attached to the relaxase domain.

2. The conjugate of claim 1, further comprising a linker positioned between the protein and the relaxase domain.

3. The conjugate of claim 2, wherein the linker comprises any one of SEQ ID NO: 37-41.

4. The conjugate of any one of claims 1-3, further comprising a purification tag.

5. The conjugate of claim 4, wherein the purification tag is attached to the N- terminus of the protein.

6. The conjugate of any one of claims 1-5, wherein the Fc binding domain is a bacterially derived or a synthetically prepared immunoglobulin binding domain or a Fc binding protein (FcBP).

7. The conjugate of claim 6, wherein the bacterially derived immunoglobulin binding domain comprises a Z domain or a variant thereof that retains the capacity to bind an antibody Fc region.

8. The conjugate of claim 7, wherein:the amino acid sequence of the Z domain comprises SEQ ID NO: 1; or the amino acid sequence of the variant Z domain comprises SEQ ID NO: 2.

9. The conjugate of claim 7, wherein the bacterially derived immunoglobulin binding domain comprises the C2 domain of Streptococcal Protein G or a variant thereof that retains the capacity to bind an antibody Fc region.

10. The conjugate of claim 9, wherein the amino acid sequence of the C2 domain comprises SEQ ID NO: 3.

11. The conjugate of any one of claims 1-10, wherein the amino acid sequence of FcBP comprises any one of SEQ ID NO: 4-16.

12. The conjugate of any one of claims 1-11, wherein the relaxase domain is obtained from Porcine circovirus 2 (PCV2), Wheat dwarf virus (WDV) or Agrobacterium tumefaciens.

13. The conjugate of any one of claims 1-12, wherein the relaxase domain is obtained from VirD2 protein.

14. The conjugate of claim 12, wherein the relaxase domain comprises SEQ ID NO: 17 or 18.

15. The conjugate of claim 13, wherein the relaxase domain obtained from VirD2 protein comprises SEQ ID NO: 19.

16. The conjugate of any one of claims 1-15, wherein the nucleic acid sequence of the oligonucleotide comprises any one of SEQ ID NO: 20-36.

17. The conjugate of any one of claims 1-16, wherein the oligonucleotide is about 10 to about 150 nucleotides in length, about 20 to about 125 nucleotides in length, or about 50 to about 100 nucleotides in length.

18. The conjugate of any one of claims 1-17, wherein the oligonucleotide comprises a qPCR handle.

19. The conjugate of claim 18, wherein the qPCR handle further comprises a PCR priming site, a label, a barcode, or an RNA polymerase binding site.

20. The conjugate of any one of claims 1-19, wherein the oligonucleotide comprises a label.

21. The conjugate of claim 20, wherein the label is directly or indirectly detectable.

22. The conjugate of claim 20 or claim 21 , wherein the label comprises a fluorescent agent, a hapten, biotin, a nucleic acid analog, or a digoxygenin label.

23. The conjugate of any one of claims 1-22, wherein the oligonucleotide is a fold- back probe.

24. The conjugate of claim 23, wherein the fold -back probe comprises any one of SEQ ID NO: 24-36.

25. The conjugate of claim 23 or claim 24, wherein the fold-back probe comprises one or more fluorescent agent.

26. The conjugate of any one of claims 22-25, wherein the fluorescent agent is chosen from a xanthene, a fluorescein, a rhodamine, a rhodol, a roseamine, a carbopyranone, an indole, an indacene, a borapolyazaindacene, a furan, a benzofuran, a cyanine, abenzocyanine, a benzopyrilium, a pyrene, a coumarin, a styryl, a squarine, a resorufin, an anthraquinone, an acridine and a benzophenoxazine.

27. The conjugate of any one of claims 1-26, further comprising an immunoglobulin molecule, wherein the Fc region of the immunoglobulin molecule is non-covalently bound to the Fc binding domain.

28. The conjugate of claim 27, wherein the immunoglobulin molecule is an IgG, or the immunoglobulin molecule is a F(ab), a F(ab’)2, a single-domain VHH antibody, or a singlechain variable fragment (scFv).

29. A conjugate, comprising: an Fc binding domain; a relaxase domain; a linker positioned between the Fc binding protein and the relaxase domain; and an oligonucleotide comprising a label, wherein the oligonucleotide is covalently attached to the relaxase domain.

30. The conjugate of claim 29, further comprising: an immunoglobulin molecule, wherein the Fc region of the immunoglobulin molecule is non-covalently bound to the Fc binding domain.

31. The conjugate of claim 30, further comprising a purification tag attached to the N-terminus of the Fc binding domain.

32. A method comprising: contacting a sample with the conjugate of any one of claims 1-31; incubating the sample with the conjugate for an amount of time sufficient to allow the conjugate to bind to a target within the sample; and detecting the conjugate.

33. A method, comprising: contacting a sample with the conjugate of any one of claims 1-28 or 30-31, wherein the antigen binding protein, antibody, antibody fragment, or immunoglobulin molecule of the conjugate is specific for an antigen present in the sample; and detecting the conjugate.

34. A method, comprising: contacting a sample with an antibody, antibody fragment, or immunoglobulin molecule specific for an antigen present in the sample; incubating the sample with the antibody, antibody fragment, or immunoglobulin molecule for an amount of time sufficient to allow the antibody, antibody fragment, or immunoglobulin molecule to bind to the antigen forming an antigen complex; adding the conjugate of claim 29 and incubating for an amount of time sufficient to allow the Fc binding domain to non-covalently bind to the Fc region of the antibody, antibody fragment, or immunoglobulin molecule; and detecting the conjugate.

35. The method of any one of claims 32-34, wherein the sample is chosen from live cells, intracellular fluids, extracellular fluids, sera, biological fluids, biological fermentation media, environmental samples, industrial samples, viruses, proteins, peptides, buffer solutions, blood cells, immune cells, cultured cells, cellular extracts, tissue, muscle tissue, neurons, extracellular vesicles, vascular tissue, blood fluids, saliva, urine, water, soil, wastewater, sea water, pharmaceuticals, foodstuffs, and beverages.

36. The method of any one of claims 32-25, wherein the sample is immobilized on a polymeric membrane, within a polymeric gel, on a microparticle, on a microarray, on a silicon chip, on a glass slide, on a microwell plate, and on a microfluidic chip.

37. The method of claim 35, wherein the tissue is chosen from tumor tissue, epidermal tissue, muscle tissue, bone marrow tissue, neural tissue, brain tissue, organ tissue, and human biopsy tissue.

38. The method of any one of claims 35-37, wherein the tissue is a formalin-fixed paraformaldehyde-embedded tissue section or a fresh frozen tissue section.

39. The method of any one of claims 32-38, wherein the detecting step comprises immunohistochemistry, fluorescence microscopy, flow cytometry, immunoprecipitation (IP) assay, western blot, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction, qPCR, immunoPCR, rolling circle amplification, next generation sequencing (NGS) using NGS -enabled barcoded oligonucleotides, or nicking enzyme signal amplification.

40. The method of any one of claims 32-38, further comprising exposing the sample to low pH, high temperature and / or high salt concentration to release the Fc binding domain from the antibody, antibody fragment, or immunoglobulin molecule.

41. The method of claim 40, wherein the sample is exposed to pH 2 to pH 4, optionally wherein the sample is exposed to a composition comprising 10 mM glycine adjusted to pH 2 -pH 4.

42. The method of claim 40 or claim 41, further comprising contacting the sample with a second conjugate of any one of claims 1-28 or claim 30-31, wherein the second conjugate has a different detectable label than the first conjugate.

43. A kit, comprising: one or more of the conjugates of any one of claims 1-31; and instructions for use according to any of the methods of claims 32-42.

44. The kit according to claim 43, further comprising at least one of the following: a buffering agent, a purification medium, a vial comprising the sample, and an organic solvent.

45. A kit comprising: a fusion protein comprising an antigen binding protein and a relaxase domain; and an oligonucleotide comprising a label.

46. A kit, comprising: a fusion protein comprising an Fc binding protein and a relaxase domain; and an oligonucleotide comprising a label.

47. The kit of claim 46, further comprising an antibody, antibody fragment, or immunoglobulin molecule.

48. The kit of any one of claims 45-47, wherein the fusion protein further comprises a purification tag.

49. The kit of claim 48, wherein the purification tag is attached to the N-terminus of the fusion protein.

50. The kit of any one of claims 46-49, wherein the Fc binding domain is a bacterially derived or a synthetically prepared immunoglobulin binding domain or a Fc binding protein (FcBP).

51. The kit of claim 50, wherein the bacterially derived immunoglobulin binding domain comprises a Z domain or a variant thereof that retains the capacity to bind an antibody Fc region.

52. The kit of claim 51, wherein: the amino acid sequence of the Z domain comprises SEQ ID NO: 1; or the amino acid sequence of the variant Z domain comprises SEQ ID NO: 2.

53. The kit of claim 51, wherein the bacterially derived immunoglobulin binding domain comprises the C2 domain of Streptococcal Protein G or a variant thereof that retains the capacity to bind an antibody Fc region.

54. The kit of claim 53, wherein the amino acid sequence of the C2 domain comprises SEQ ID NO: 3.

55. The kit of any one of claims 46-54, wherein the amino acid sequence of FcBP comprises any one of SEQ ID NO: 4-16.

56. The kit of any one of claims 44-55, wherein the relaxase domain is obtained from Porcine circovirus 2 (PCV2), Wheat dwarf virus (WDV) ox Agrobacterium tumefaciens.

57. The kit of any one of claims 44-55, wherein the relaxase domain is obtained from VirD2 protein.

58. The kit of any one of claims 44-55, wherein the relaxase domain comprises SEQ ID NO: 17 or 18.

59. The kit of claim 57, wherein the relaxase domain obtained from VirD2 protein comprises SEQ ID NO: 19.

60. The kit of any one of claims 44-59, wherein the nucleic acid sequence of the oligonucleotide comprises any one of SEQ ID NO: 20-36.

61. The kit of any one of claims 44-59, wherein the oligonucleotide is about 10 to about 150 nucleotides in length, about 20 to about 125 nucleotides in length, or about 50 to about 100 nucleotides in length.

62. The kit of any one of claims 44-61, wherein the oligonucleotide comprises a qPCR handle.

63. The kit of claim 62, wherein the qPCR handle further comprises a PCR priming site, a label, a barcode, or an RNA polymerase binding site.

64. The kit of any one of claims 44-63, wherein the oligonucleotide comprises a label.

65. The kit of claim 64, wherein the label is directly or indirectly detectable.

66. The kit of claim 64 or claim 65, wherein the label comprises a fluorescent agent, a hapten, biotin, a nucleic acid analog, or a digoxygenin label.

67. The kit of any one of claims 44-66, wherein the oligonucleotide is a fold-back probe.

68. The kit of claim 67, wherein the fold-back probe comprises any one of SEQ ID NO: 24-36.

69. The kit of claim 67 or 68, wherein the fold-back probe comprises one or more fluorescent agent.

70. The kit of any one of claims 45-69, further comprising instructions for use according to any of the methods of claims 32-42.

71. A method comprising: contacting a sample with the conjugate of any one of claims 1-4 or 12-26; incubating the sample with the conjugate for an amount of time sufficient to allow the conjugate to bind to a target within the sample; anddetecting the conjugate.

72. The method of claim 71, wherein the sample comprises a cell population.

73. The method of claim 72, wherein the cell population comprises B cells.

74. The method of any one of claims 71-73, wherein the conjugate comprises a ligand, an antigen, or a protein domain.

75. The method of any one of claims 71-74, wherein the conjugate comprises a fold- back probe.

76. The method of any one of claims 71-75, further comprising selecting or enriching for B-cells that are bound to the conjugate.

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