Reversible labeling of primary antibodies

Abstract

Antibody conjugates and kits for detecting antigens of interest in cell and tissue samples are described. Target-specific antibodies are reversibly labelled in a site-specific and stoichiometric manner using conjugates that include an Fc binding domain linked to a coiled- coil domain, which in turn is coupled to a labelled peptide nucleic acid (PNA) or a labelled peptide. The labelled conjugate can be released from the target-specific antibody by washing the cells or tissue in a low pH buffer, allowing for cyclic detection of target antigens in the sample. In vitro methods of detecting a target antigen in a cell or tissue sample using the antibody conjugates are also described. Methods of assembling a labelled antibody conjugate are further described.

Description

REVERSIBLE LABELING OF PRIMARY ANTIBODIESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to Indian Provisional Application no. IN202341087634, filed December 21, 2023. The entire contents of the aforementioned application is incorporated by reference herein.SEQUENCE LISTING

[0002] 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 November 19, 2024, is named “TP384994WOl.xml” and is 598,335 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.FIELD OF THE INVENTION

[0003] This disclosure concerns reversibly labelled antibody conjugates and use of the conjugates for detection of target antigens in cell or tissue samples.BACKGROUND OF THE INVENTION

[0004] Studies to examine protein biology involve tracking and imaging of proteins in cells and tissues. To examine protein interactions, spatial protein organization in tissues is interrogated using 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 antibodies have several limitations. The primary methodology for labelling antibodies includes chemical coupling using reactive chemical linkers, which are non-selective and limited in terms of reaction specificity. Furthermore, methods of tagging antibodies with dyes in a specific manner often require antibody engineering. Thus, a need exists for improved methods of detecting proteins of interest in cells and tissues.SUMMARY OF THE INVENTION

[0005] Described herein are antibody conjugates for detecting antigens of interest in cell and tissue samples. Target- specific antibodies are reversibly labelled in a site-specific and stoichiometric manner using conjugates that include an Fc binding domain linked to a coiled- coil domain, which in turn is coupled to a labelled peptide nucleic acid (PNA), or to a labelled peptide. In some aspects, the Fc binding domain binds the CH2-CH3 region of the targetspecific antibody (such as an IgG), making the conjugate both site-specific and stoichiometric as there are two binding sites for the Fc binding domain per antibody molecule. The labelled conjugate is released from the target- specific antibody by washing the cells or tissue in a low pH buffer (e.g., 10 mM glycine, pH 2-4), allowing for cyclic detection of target antigens in the sample.

[0006] Provided herein are conjugates that include an Fc binding domain and a coiled-coil domain, and optionally include a linker positioned between the Fc binding domain and the coiled-coil domain. In some aspects, the Fc binding domain is a bacterially derived immunoglobulin binding domain or a Fc binding protein (FcBP). The Fc binding domain can be synthetically prepared according to known methods. In some aspects, the coiled-coil domain includes a first peptide having a first coiled-coil heptad and a second peptide having a second coiled-coil heptad, and the first coiled-coil heptad and the second coiled-coil heptad non-covalently bind to each other to form the coiled-coil domain.

[0007] In some aspects, the conjugate further includes a first peptide nucleic acid (PNA) conjugated to the second peptide of the coiled-coil domain, and a second PNA, a DNA oligonucleotide, or an RNA oligonucleotide that is complementary to the first PNA (e.g., sufficiently complementary to the first PNA such that the second PNA, the DNA oligonucleotide or the RNA oligonucleotide are capable of hybridizing with the first PNA). In some examples, the second PNA, the DNA oligonucleotide, or the RNA oligonucleotide includes a detectable label (e.g., a fluorophore, a small molecule, a streptavidin, an enzyme, a nanoparticle, allophycocyanin, an oligonucleotide, or a peptide tag). In other aspects, the conjugate further includes a labelled peptide conjugated to the second peptide of the coiled- coil domain.

[0008] In some aspects, the conjugate further includes an immunoglobulin molecule, wherein the Fc region of the immunoglobulin molecule is non-covalently bound to the Fc binding domain, or is genetically fused to the coiled-coil domain. In other aspects, the conjugateincludes an antigen-binding fragment of an antibody, such as a F(ab), a F(ab’)2, a singledomain VHH antibody, or a single-chain variable fragment (scFv). In these aspects, the antigen binding fragment can be genetically fused to the coiled-coil domain.

[0009] 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.

[0010] In specific examples, provided is a conjugate that includes an Fc binding domain; a coiled-coil domain that includes a first peptide having a first coiled-coil heptad and a second peptide having a second coiled-coil heptad, wherein the first coiled-coil heptad and the second coiled-coil heptad non-covalently bind to each other to form the coiled-coil domain; a linker positioned between the Fc binding domain and the coiled-coil domain; a first PNA conjugated to the second peptide of the coiled-coil domain; a second PNA, a DNA oligonucleotide, or an RNA oligonucleotide hybridized to the first PNA, wherein the second PNA, the DNA oligonucleotide, or the RNA oligonucleotide comprises a detectable label; and an immunoglobulin molecule, wherein the Fc region of the immunoglobulin molecule is non- covalently bound to the Fc binding domain. In some instances, the conjugate further includes a purification tag attached to the N-terminus of the Fc binding domain.

[0011] Also provided herein are in vitro methods of detecting a target antigen in cells or tissue, such as cultured cells, formalin-fixed paraformaldehyde-embedded tissue sections, or fresh frozen tumor sections. 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. In some examples, the method includes contacting the cell or tissue with a conjugate that includes an Fc binding domain; a coiled-coil domain including a first peptide having a first coiled-coil heptad and a second peptide having a second coiled-coil heptad, wherein the first coiled-coil heptad and the second coiled-coil heptad non-covalently bind to each other to form the coiled-coil domain; a linker positioned between the Fc binding domain and the coiled-coil domain; a first PNA conjugated to the second peptide of the coiled-coil domain; and an immunoglobulin molecule specific for the target antigen, wherein the Fc region of the immunoglobulin molecule is non-covalently bound to the Fc binding domain; and contacting the cell or tissue with a second PNA, a DNA oligonucleotide, or an RNA oligonucleotide capable of hybridizing to the first PNA, wherein the second PNA, the DNA oligonucleotide, or the RNA oligonucleotide includes a detectable label. In some aspects, the method further includes detecting the antigen by detecting or quantifying the detectable label. In some examples, detecting the antigen includes immunohistochemistry, fluorescencemicroscopy, flow cytometry, Western blot, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (e.g., qPCR or immuno PCR), rolling circle amplification, next generation sequencing (NGS) using NGS-enabled barcoded oligonucleotides with or without biotin for oligonucleotide capture, or nicking enzyme signal amplification. In some examples, the methods further include exposing the cell or tissue to low pH (for example, pH 2 to pH 4) to release the Fc binding domain from the immunoglobulin molecule.

[0012] Further provided herein is a method of assembling a labelled antibody conjugate. In some aspects, the method includes incubating the following components: (i) a fusion protein that includes an Fc binding domain, a linker peptide, and a first coiled-coil heptad repeat; (ii) a second coiled-coil heptad repeat conjugated to a first PNA; (iii) a second PNA, a DNA oligonucleotide, or an RNA oligonucleotide (capable of hybridizing with the first PNA) conjugated to a detectable label, wherein the second PNA, the DNA oligonucleotide, or the RNA oligonucleotide is capable of hybridizing with the first PNA; and (iv) an immunoglobulin molecule. The first coiled-coil heptad repeat and the second coiled-coil heptad repeat non- covalently bind to each other to form a coiled-coil domain; the first PNA hybridizes with the second PNA, the DNA oligonucleotide or the RNA oligonucleotide; and the Fc binding domain non-covalently binds the immunoglobulin molecule, thereby forming an antibody conjugate.

[0013] Kits that include a conjugate disclosed herein are also provided. In some aspects, the kit includes a fusion protein that includes an Fc binding domain, a first coiled-coil heptad repeat, and a linker positioned between the Fc binding domain and the first coiled-coil heptad repeat; a second coiled-coil heptad repeat conjugated to a first PNA; and / or a second PNA, a DNA oligonucleotide, or an RNA oligonucleotide conjugated to a detectable label, wherein the first PNA is capable of hybridizing with the second PNA, the DNA oligonucleotide or the RNA oligonucleotide. In some examples, the kit further includes an immunoglobulin molecule specific for a target antigen.

[0014] 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.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1 is a schematic of an exemplary PNA-labelled antibody conjugate disclosed herein. The exemplary conjugate includes a Z domain fused to a linker peptide (GGGAS; SEQ ID NO: 18) and a first coiled-coil peptide (“Coiled coil I”) containing three repeats of a firstheptad (EIAALEK; SEQ ID NO: 23). A second coiled-coil peptide (“Coiled coil II”) containing four repeats of a second heptad (KIAALKE; SEQ ID NO: 24) non-covalently binds to the first coiled-coil peptide to form a coiled-coil domain. The second coiled-coil peptide is conjugated to a first PNA, which is hybridized to a second PNA (or a DNA or RNA oligonucleotide) labelled with a fluorophore. The Z domain is non-covalently bound to the Fc region of an immunoglobulin molecule (IgG).

[0016] FIG. 2 is a schematic of an exemplary Myc tag-labelled antibody conjugate disclosed herein. This exemplary conjugate includes a Z domain fused to a linker peptide (GGGAS; SEQ ID NO: 18) and a first coiled-coil peptide (“Coiled coil I”) containing three repeats of a first heptad (EIAALEK; SEQ ID NO: 23). A second coiled-coil peptide (“Coiled coil II”) containing four repeats of a second heptad (KIAALKE; SEQ ID NO: 24) non-covalently binds to the first coiled-coil peptide to form a coiled-coil domain. The second coiled-coil peptide is conjugated to a peptide with a Myc tag. A labelled anti-Myc tag antibody is used to detect the conjugate.

[0017] FIG. 3 is a schematic illustrating an exemplary method for assembly of PNA- conjugated antibodies. The following components are combined at a 2:2:2: 1 ratio: (1) a fusion protein comprised of a Z domain, a linker peptide (GGGAS; SEQ ID NO: 18), and a first coiled-coil heptad repeat (EIAALEK; SEQ ID NO: 23); (2) a second coiled-coil heptad repeat (KIAALKE; SEQ ID NO: 2) conjugated to a first PNA; (3) a second PNA labelled with a fluorophore; and (4) an immunoglobulin molecule. The components are incubated at room temperature for approximately one hour to form a PNA-labelled antibody conjugate (bottom).

[0018] FIGS. 4A-4I are images of cyclic immunostaining of mouse duodenum using PNA- labelled antibody conjugates. Mouse duodenum formalin-fixed paraffin-embedded (FFPE) sections were stained with PNA-ALEXA FLUOR™ 647 (“PNA-AF647”) conjugated ACTA2 primary antibody (FIGS. 4B and 4E), a pan-cytokeratin antibody conjugated to eFluor™660 (FIG. 4H), or DAPI to stain for nuclei (FIGS. 4A, 4D and 4G). Composite staining is shown in FIGS. 4C, 4F and 41. In the first cycle of staining, ACTA2 tagged with PNA-AF647 was used to probe mouse duodenum tissue (FIGS. 4A-4C). In the second cycle of staining, a low pH wash was used to release the PNA-AF647 tag conjugated to ACTA2 (FIGS. 4D-4F). In the third cycle of immunostaining, a primary antibody conjugate (pan-cytokeratin-eFluor660) was used to probe the same mouse duodenum section (FIGS. 4G-4I). FIG. 4H shows specific staining of the mucosal layer of mouse duodenum by pan-cytokeratin.

[0019] FIG. 5 is a schematic illustrating the composition of an antibody conjugate that includes a fold-back probe. In this aspect, a single-stranded oligonucleotide is used in place of a second PNA. The fold-back probe includes a single-stranded oligonucleotide (80-150 nucleotides in length) with a 5' end that is complementary to the first PNA sequence and therefore hybridizes to the first PNA. The 3' end of the fold-back probe has a short sequence that is complementary to itself, which self-hybridizes to form a primer that can be used to incorporate fluorophore- labelled dNTPs and / or biotinylated dNTPs using a polymerase such as the Klenow fragment (AGCTCGTGAATTCGGCAGCACTCAATTC; SEQ ID NO: 478, see inset image). The central portion of the oligonucleotide includes a tag- specific barcode sequence that can be amplified using forward and reverse amplification primers that hybridize to sequences flanking the tag-specific barcode.

[0020] FIGS. 6A-6E are schematics illustrating a method of using a fold-back probe for fluorophore and biotin incorporation and NGS-based target quantification. A fold-back oligonucleotide is tethered to Zdomain-E3-K4PNA (FIG. 6A). The fold-back oligonucleotide is labelled with Fluor-dNTPs and Biotin-dNTPs using a polymerase such as the Klenow fragment (FIG. 6B). Target- specific antibodies are tagged with labelled oligonucleotides containing different target- specific barcodes (FIG. 6C). Binding of the labelled antibodies to their respective antigens in immunohistochemistry (IHC) (FIG. 6D). A low-pH wash releases the label into the supernatant which is collected (FIG. 6E). Sample enrichment is performed using streptavidin-coated beads to capture biotinylated oligonucleotide, followed by PCR- based amplification and NGS-based target quantitation.SEQUENCES

[0021] 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.

[0022] SEQ ID NO: 1 is the amino acid sequence of an exemplary Z domain. VDNKFNKEQQNAFYEIEHEPNENEEQRNAFIQSEKDDPSQSANEEAEAKKENDAQAP K

[0023] SEQ ID NO: 2 is the amino acid sequence of an exemplary Z domain variant. VDNKRNKEQQNAFKEIEHEPNENEEQRNGFIQSEKDDPSQSANEEAEAKKENDAQAP K

[0024] SEQ ID NO: 3 is the amino acid sequence of an exemplary Z domain variant.VDNKRNKEQQNAFREILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAP K

[0025] SEQ ID NO: 4 is the amino acid sequence of an exemplary C2 domain. TTYKLVINGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVT E

[0026] SEQ ID NOs: 5-17 are amino acid sequences of exemplary FcBPs.

[0027] SEQ ID NOs: 18-22 are exemplary peptide linkers.

[0028] SEQ ID NO: 23 is the amino acid sequence of an exemplary coiled-coil heptad.EIAALEK

[0029] SEQ ID NO: 24 is the amino acid sequence of an exemplary coiled-coil heptad. KIAALKE

[0030] SEQ ID NOs: 25-477 are nucleic acid sequences of exemplary PNAs (see Table 1).

[0031] SEQ ID NO: 478 is a nucleic acid sequence of an exemplary fold-back oligonucleotide probe, agctcgtgaattcggcagcactcaattcDETAILED DESCRIPTIONI. AbbreviationsACTA2 actin alpha 2, smooth muscleAPC allophycocyaninD API 4 6-diamidino-2-phenylindoleELISA enzyme-linked immunosorbent assayFcBP Fc binding proteinFFPE formalin-fixed paraffin-embeddedIHC immunohistochemistryNGS next generation sequencingPE phycoerythrinPNA peptide nucleic acidII. Summary of Terms

[0032] 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:

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] A “chimeric antibody” has framework residues from one species, such as human, and CDRs (which generally confer antigen binding) from another species.

[0038] 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.

[0039] 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).

[0040] 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.

[0041] 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-V domain; VL domain-linker domain-Vu domain) may be used.

[0042] Coiled-coil domain: A protein domain characterized by a coiled-coil structure having at least 2 alpha-helices coiled together. Coiled-coils are comprised of sequence elements whose hydrophobicity and residue composition are compatible with the structure of amphipathic alpha-helices. Coiled-coils typically contain a repeated pattern of seven hydrophobic and charged amino acid residues referred to as a heptad repeat or coiled-coil heptad. In some aspects herein, the coiled-coil heptad has the amino acid sequence EIAALEK (SEQ ID NO: 23) or KIAALKE (SEQ ID NO: 24). In the context of the present disclosure, the coiled-coil domain includes a first peptide comprising a first coiled-coil heptad and a second peptide comprising a second coiled-coil heptad, which non-covalently bind to each other to form the coiled-coil domain. The first and second peptides include repeats of the coiled-coil heptads, such as three or four repeats (see FIGS. 1-3).

[0043] 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.

[0044] 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:1) Alanine (A), Serine (S), Threonine (T);2) Aspartic acid (D), Glutamic acid (E);3) Asparagine (N), Glutamine (Q);4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0045] Contacting: Placement in direct physical association; includes both in solid and liquid form.

[0046] 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.

[0047] Detectable label: A detectable compound or composition that is conjugated directly or indirectly to another molecule, such as an antibody, peptide, 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).

[0048] 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: 5-17, or a variant thereof having at least 80% identity to any one of SEQ ID NOs: 5-17, 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: 1-3 and an exemplary C2 domain sequence is set forth herein as SEQ ID NO: 4. 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).

[0049] 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.

[0050] 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).

[0051] 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 coiled-coil heptad repeat.

[0052] Heterologous: Originating from a separate genetic source or species.

[0053] 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 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 PNA that is “complementary to a second PNA, a DNAoligonucleotide, or an RNA oligonucleotide” refers to a PNA that is sufficiently complementary to the second PNA, DNA oligonucleotide or RNA oligonucleotide such that the two molecules hybridize. The term “complementary to” does not require 100% complementarity .

[0054] 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).

[0055] 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 coiled-coil domain. Non-limiting examples of peptide linkers include glycine, serine, and glycine-serine linkers. In one example, the linker has the amino acid sequence GGGAS (SEQ ID NO: 4).

[0056] 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.

[0057] Peptide nucleic acid (PNA): Synthetic mimics of DNA in which the phosphodiester backbone is replaced by repetitive units of A-(2-aminocthyl) glycine to which the purine and pyrimidine bases are attached by a methyl carbonyl linker. PNAs hybridize with complementary DNA or RNA oligonucleotides with high affinity and specificity (Pellestor and Paulasova, Eur J Hum Genet 12(9):694-700, 2004). Exemplary PNA sequences for use in the disclosed conjugates are set forth herein as SEQ ID NOs: 25-477.

[0058] 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.

[0059] 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 proteinrepresents 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.

[0060] 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.III. Reversibly-Labelled Antibody Conjugates

[0061] Described herein are antibody conjugates for detecting antigens of interest in cell and tissue samples. Target- specific antibodies are reversibly labelled in a site-specific and stoichiometric manner using conjugates that include an Fc binding domain linked to a coiled- coil domain, which in turn is coupled to a labelled peptide nucleic acid (PNA) or a labelled peptide (see FIG. 1 and FIG. 2). In some aspects, the Fc binding domain binds the CH2-CH3 region of the target- specific antibody (such as an IgG), making the conjugate both site-specific and stoichiometric as there are two binding sites for the Fc binding domain per antibody molecule. In some aspects, the labelled conjugate is released from the target- specific antibody by washing the cells or tissue in a low pH buffer (e.g., in 10 mM glycine, adjusted to pH 2-4), or by treating the cells or tissue with high temperature and / or high salt concentration solutions, allowing for cyclic detection of target antigens in the sample. In other aspects, the labelled conjugate retains the label.

[0062] Provided herein are conjugates that include an Fc binding domain and a coiled-coil domain. In some aspects, the conjugate further includes a linker positioned between the Fc binding domain and the coiled-coil domain.

[0063] 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 or SEQ ID NO: 3. In specific non-limiting examples, the amino acid sequence of the variant Z domain includes or consists of SEQ ID NO: 2 or SEQ ID NO: 3. In other examples, the bacterially derived immunoglobulin binding domain includes a C2 domain (of Streptococcal 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: 4. In a specific non-limiting example, the amino acid sequence of the C2 domain includes or consists of SEQ ID NO: 4.

[0064] 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: 5-17. In a specific non-limiting example, the amino acid sequence of the FcBP includes or consists of any one of SEQ ID NOs: 5-17.

[0065] 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 any one of SEQ ID NOs: 18-22. In specific non-limiting examples, the amino acid sequence of the linker includes or consists of any one of SEQ ID NOs: 18-22.

[0066] In some aspects of the conjugates, the coiled-coil domain includes a first peptide having a first coiled-coil heptad and a second peptide having a second coiled-coil heptad. The first coiled-coil heptad and the second coiled-coil heptad non-covalently bind to each other to form the coiled-coil domain. In some examples, the first peptide of the coiled-coil domain includes two, three, four or five repeats of the first coiled-coil heptad. In particular examples, the first peptide of the coiled-coil domain includes three repeats of the first coiled-coil heptad. In specific non-limiting examples, the amino acid sequence of the first coiled-coil heptad includes EIAALEK (SEQ ID NO: 23). In some examples, the second peptide of the coiled-coil domain includes two, three, four or five repeats of the second coiled-coil heptad. In particular examples, the second peptide of the coiled-coil domain includes four repeats of the secondcoiled-coil heptad. In specific non-limiting examples, the amino acid sequence of the second coiled-coil heptad includes KIAALKE (SEQ ID NO: 24). In one example, the first peptide of the coiled-coil domain has three repeats of EIAALEK (SEQ ID NO: 23) and the second peptide of the coiled-coil domain has four repeats of KIAALKE (SEQ ID NO: 24).

[0067] In some aspects, the conjugates further include a first PNA conjugated to the second peptide of the coiled-coil domain. In some examples, the conjugate further includes a second PNA, a DNA oligonucleotide, or an RNA oligonucleotide that is complementary to (e.g., capable of hybridizing to) the first PNA. In particular examples, the first PNA and the second PNA are each about 8 to about 16 nucleotides in length, such as about 9 to about 15 nucleotides in length, about 10 to about 14 nucleotides in length, or about 11 to about 13 nucleotides in length. In specific non-limiting examples, the first PNA and the second PNA are about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, or about 16 nucleotides in length. In select examples, the first PNA and the second PNA are 12 nucleotides in length. In particular examples, the DNA oligonucleotide or the RNA oligonucleotide is about 10 to about 150, such as about 20 to about 125, about 80 to about 150, or about 50 to about 100 nucleotides in length. In specific non-limiting examples, the DNA oligonucleotide or the RNA 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 some examples, the DNA oligonucleotide is a fold- back probe (see FIGS. 5 and 6A-6E).

[0068] In some examples, the first PNA, the second PNA, the DNA oligonucleotide or the RNA oligonucleotide have a sequence selected from any one of SEQ ID NOs: 25-477. In some examples, the second PNA, the DNA oligonucleotide, or the RNA oligonucleotide includes a detectable label. In particular examples, the detectable label includes a fluorophore, a small molecule (e.g., biotin), a streptavidin, an enzyme (e.g., horseradish peroxidase), a nanoparticle (e.g., a gold nanoparticle), allophycocyanin (APC), an oligonucleotide, or a peptide tag (e.g., a Myc tag, His tag or FLAG tag). In specific examples, the oligonucleotide is labelled, not labelled, dye labelled, or has a self-complementary 3' end for dye incorporation (e.g., a fold- back probe), or the oligonucleotide is an inversion probe.

[0069] In other aspects, the conjugate further includes a labelled peptide conjugated to the second peptide of the coiled-coil domain (FIG. 2). In some examples, the peptide is labelled with a fluorophore, a small molecule (e.g., biotin), a streptavidin, an enzyme (e.g., horseradishperoxidase), a nanoparticle (e.g., a gold nanoparticle), allophycocyanin (APC), an oligonucleotide, or a peptide tag (e.g., a Myc tag, His tag or FLAG tag). In specific examples, the oligonucleotide is labelled, not labelled, dye labelled, or has a self-complementary 3' end for dye incorporation (e.g., a fold-back probe), or the oligonucleotide is an inversion probe.

[0070] 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.

[0071] 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.

[0072] In specific non-limiting examples, the conjugate includes an Fc binding domain; a coiled-coil domain having a first peptide including a first coiled-coil heptad and a second peptide including a second coiled-coil heptad, wherein the first coiled-coil heptad and the second coiled-coil heptad non-covalently bind to each other to form the coiled-coil domain; a linker positioned between the Fc binding domain and the coiled-coil domain; a first PNA conjugated to the second peptide of the coiled-coil domain; a second PNA, a DNA oligonucleotide, or an RNA oligonucleotide hybridized to the first PNA, wherein the second PNA, the DNA oligonucleotide, or the RNA oligonucleotide includes a detectable label; and an immunoglobulin molecule, wherein the Fc region of the immunoglobulin molecule is non- covalently bound to the Fc binding domain (FIG. 1). In some cases, the conjugate further includes a purification tag, such as a purification tag attached to the N-terminus of the Fc binding domain.IV. Kits

[0073] 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 an Fc binding domain, a first coiled-coil heptad repeat, and a linker positioned between the Fc binding domain and the first coiled-coil heptad repeat; a second coiled-coil heptad repeat conjugated to a first PNA; and / or a second PNA, a DNA oligonucleotide, or an RNA oligonucleotide conjugated to adetectable label, wherein the first PNA is capable of hybridizing with the second PNA, the DNA oligonucleotide or the RNA oligonucleotide. In some examples, the kit further includes an immunoglobulin molecule specific for a target antigen, such as an intracellular protein.

[0074] 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 or SEQ ID NO: 3. In specific non-limiting examples, the amino acid sequence of the variant Z domain includes or consists of SEQ ID NO: 2 or SEQ ID NO: 3. In other examples, the bacterially derived immunoglobulin binding domain includes a C2 domain (of Streptococcal 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: 4. In a specific non-limiting example, the amino acid sequence of the C2 domain includes or consists of SEQ ID NO: 4.

[0075] 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: 5-17. In a specific non-limiting example, the amino acid sequence of the FcBP includes or consists of any one of SEQ ID NOs: 5-17.

[0076] 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 any one of SEQ ID NOs: 18-22. In specific non-limiting examples, the amino acid sequence of the linker includes or consists of any one of SEQ ID NOs: 18-22.

[0077] 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. In some examples, the purification tag is attached to the N- terminus of the Fc binding domain.

[0078] In some aspects of the kits, the first coiled-coil heptad repeat includes two, three, four or five repeats. In some examples, the first coiled-coil heptad repeat includes three repeats. In particular examples, the amino acid sequence of the first coiled-coil heptad includes EIAALEK (SEQ ID NO: 23).

[0079] In some aspects of the kits, the second coiled-coil heptad repeat includes two, three, four or five repeats. In some examples, the second coiled-coil heptad repeat includes four repeats. In particular examples, the amino acid sequence of the second coiled-coil heptad includes KIAALKE (SEQ ID NO: 24).

[0080] In some aspects of the kits, the first PNA and the second PNA are each about 8 to about 16 nucleotides in length, such as about 9 to about 15 nucleotides in length, about 10 to about 14 nucleotides in length, or about 11 to about 13 nucleotides in length. In some examples, the first PNA and the second PNA are about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, or about 16 nucleotides in length. In particular examples, the first PNA and the second PNA are 12 nucleotides in length. In particular examples, the DNA oligonucleotide or the RNA oligonucleotide is about 10 to about 150, such as about 20 to about 125, or about 50 to about 100 nucleotides in length. In specific non-limiting examples, the DNA oligonucleotide or the RNA 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 some examples, the DNA oligonucleotide is a fold-back probe (see FIGS. 5 and 6A-6E).

[0081] In some examples, the first PNA, the second PNA, the DNA oligonucleotide or the RNA oligonucleotide have a sequence selected from any one of SEQ ID NOs: 25-477. In some examples, the detectable label conjugated to the second PNA, the DNA oligonucleotide, or the RNA oligonucleotide includes a fluorophore, a small molecule (e.g., biotin), a streptavidin, an enzyme (e.g., horseradish peroxidase), a nanoparticle (e.g., a gold nanoparticle), allophycocyanin (APC), an oligonucleotide, or a peptide tag (e.g., a Myc tag, His tag or FLAG tag). In specific examples, the oligonucleotide is labelled, not labelled, dye labelled, or has aself-complementary 3’ end for dye incorporation (e.g., a fold-back probe), or the oligonucleotide is an inversion probe.

[0082] In some aspects of the kits, 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 nonhuman primate.V. Methods of Detecting Target Antigens

[0083] Also described herein are methods of detecting target antigens in biological samples using the conjugates and kits disclosed herein.

[0084] 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.

[0085] In some aspects, the method of detecting a target antigen in a cell or tissue includes contacting the cell or tissue with a conjugate that includes an Fc binding domain; a coiled-coil domain having a first peptide that includes a first coiled-coil heptad and a second peptide that includes a second coiled-coil heptad, wherein the first coiled-coil heptad and the second coiled- coil heptad non-covalently bind to each other to form the coiled-coil domain; a linker positioned between the Fc binding domain and the coiled-coil domain; a first PNA conjugated to the second peptide of the coiled-coil domain; and an immunoglobulin molecule specific for the target antigen, wherein the Fc region of the immunoglobulin molecule is non-covalently bound to the Fc binding domain; and contacting the cell or tissue with a second PNA, a DNA oligonucleotide, or an RNA oligonucleotide capable of hybridizing to the first PNA, wherein the second PNA, the DNA oligonucleotide, or the RNA oligonucleotide comprises a detectable label; and detecting the detectable label, thereby detecting the target antigen in the cell or tissue. The components of the conjugates are described in detail in sections III and IV.

[0086] In some examples of the disclosed methods, the cell is a cell isolated from a subject. In other examples, the cell is a cultured cell. In some examples, the tissue is a formalin-fixed paraformaldehyde-embedded tissue section.

[0087] In some examples of the methods, detecting the antigen includes immunohistochemistry, fluorescence microscopy, flow cytometry, Western blot, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (e.g., qPCR, immunoPCR), next generation sequencing (NGS) using NGS-enabled barcoded oligonucleotides with or without biotin for oligonucleotide capture (see FIGS. 5 and 6A-6E), rolling circle amplification, or nicking enzyme signal amplification.

[0088] In some examples, the method further includes exposing the cell or tissue to low pH to release the Fc binding domain from the 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.

[0089] 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 cells or tissues 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 cells or tissues with a second labelled antibody, such as a commercially available labelled primary antibody. In yet other examples, the method further includes contact the cells or tissues 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.

[0090] Also provided herein are methods of assembling a labelled antibody conjugate disclosed herein. In some aspects, the method includes incubating the following components: (i) a fusion protein comprising an Fc binding domain, a linker peptide, and a first coiled-coil heptad repeat; (ii) a second coiled-coil heptad repeat conjugated to a first peptide nucleic acid (PNA); (iii) a second PNA, a DNA oligonucleotide, or an RNA oligonucleotide conjugated to a detectable label; and (iv) an immunoglobulin molecule. In this assembly method, the first coiled-coil heptad repeat and the second coiled-coil heptad repeat non-covalently bind to each other to form a coiled-coil domain; the first PNA hybridizes with the second PNA, the DNA oligonucleotide or the RNA oligonucleotide; and the Fc binding domain non-covalently binds the immunoglobulin molecule to form the labelled antibody conjugate (see FIG. 3). In some examples, the components are incubated at room temperature. In some examples, the components (i), (ii), (iii) and (iv) are incubated at a ratio of 2:2:2: 1. The components of the conjugates are described in detail in sections III and IV.VI. PNA, DNA Oligonucleotide and RNA Oligonucleotide Sequences

[0091] Disclosed herein are exemplary PNA (or DNA oligonucleotide or RNA 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 PNA sequences set forth herein as SEQ ID NOs: 25-477. In the sequences below, “T” can be replaced with “U” for RNA sequences.Table 1. PNA SequencesEXAMPLES

[0092] 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: Assembly of Antibody-Z domainE3-PNA-AF647 complex

[0093] This example describes the assembly of a complex that includes a primary antibody, aZ domain, a coiled-coil domain, a first PNA, and a second PNA labelled with ALEXA FLUOR™ 647.

[0094] Materials for complex assembly:1. lOx phosphate buffer saline (PBS) pH 7.4 (Thermo Fisher Scientific Catalogue no. 70011044)2. Autoclaved water3. Primary antibody4. Recombinant Z domain-E3 coiled coil (Z domain - SEQ ID NO: 1; E3 - SEQ ID NO: 5)5. K4 coiled coil-PNA (K4 - SEQ ID NO: 6; PNA - SEQ ID NO: 476)6. Complementary PNA (SEQ ID NO: 477)-ALEXA FLUOR™ 647 (referred to as PNA- AF647).

[0095] Protocol for complex assembly:1. All components were diluted to the desired concentrations. Z domainE3 was diluted in lx PBS, K4 coiled-coil-PNA and PNA-AF647 were diluted in autoclaved water.2. The following reaction was set up such that concentration of primary antibody, Z domainE3, K4 coiled coil-PNA, complementary PNA-ALEXA FLUOR647 were in ratio of 1:2:2:2.NEGATIVETEST CONTROL3. Both the test and negative control tubes were incubated in the dark at room temperature for 1 hour.

[0096] The assembled complex can be used, for example, in immunohistochemical (IHC) staining. The ‘Test’ vial contains the assembled Antibody-Z domainE3-K4 coiled-coil- PNA:PNA-AF647 complex while the ‘Negative Control’ vial contains the K4 coiled coil- PNA:PNA-AF647 to check for background or non-specific signal.Example 2: Testing of the Antibody-Z domainE3-PNA-AF647 complex

[0097] This example describes a study to test the Antibody-Z domainE3-PNA-AF647 complex in IHC using mouse duodenum formalin-fixed paraffin-embedded (FFPE) sections.

[0098] Materials required for IHC staining:1. Slides with FFPE sections of mouse duodenum2. Heating block3. Humidified chamber4. Coplin jars5. HISTOCHOICE® Clearing Agent (Merck Catalogue no. H2779)6. 100%, 95%, 70% and 50% ethanol7. IHC Antigen Retrieval Solution - High pH (10X) (eBioscience™ Catalogue no. 00- 4956-58)8. IX PBS9. IX PBS-TWEEN® (PBST)10. 2% normal goat serum11. Hydrophobic pen12. Primary antibody complexed with PNA (Test and Negative Control)13. DAPI (4',6-diamidino-2-phenylindole)14. Prolong antifade glass mountant15. Cover slips

[0099] Method for IHC staining:1. Slides with FFPE sections of mouse duodenum were placed at 70°C for 5-10 minutes. Once the wax had melted, slides were transferred to HISTOCHOICE® (2x for 10 minutes).2. The deparaffinized slides were serially passaged through 100%, 95%, 70% and 50% ethanol (10 minutes each). Slides were kept in double distilled water for 10 minutes.3. For antigen retrieval (unmasking the antigenic epitope), slides were transferred into IHC antigen retrieval solution and a decloaking chamber at 110°C for 15 minutes.4. Slides were allowed to cool to room temperature (~5 minutes) and then rinsed with lx PBS for 10 minutes. The boundary of the tissue was marked with a hydrophobic pen.5. For blocking, 2% normal goat serum was added onto the sections and incubated in a humidified chamber at room temperature for 1 hour.6. Post incubation, blocking buffer was drained from the slides and the Antibody-Z domainE3-PNA-AF647 complex (‘Test’) was added onto the section, which was incubated for 2 hours at room temperature. A section with only K4 coiled coil- PNA:PNA-AF647 (‘Negative Control’ ) was used as a control.7. Post incubation, the antibody-PNA complex and negative control were washed with lx PBS (3x for 5 minutes).8. Nuclei were stained with DAPI for 10 minutes at room temperature. Excess DAPI was removed by washing the slides with IX PBS for 5 minutes.9. Slides were mounted using prolong antifade glass mountant and imaged using the EVOS™ M7000 Imaging System at 20X magnification.Example 3: Cyclic IHC staining using a low pH wash

[0100] This example describes cyclic IHC staining using a low pH wash to remove the signal from Antibody-Z domainE3-PNA-AF647 and re -probing mouse duodenum FFPE section with a second antibody.

[0101] Materials required for cyclic IHC staining (in addition to those listed in Example 2):1. 0.1% PBS2. 0.01% PBS3. Primary antibody 24. Low pH buffer

[0102] Method for complex assembly:1. Slides with FFPE sections of mouse duodenum were placed at 70°C for 5-10 minutes. Once the wax melted, slides were transferred to HISTOCHOICE® (2x for 10 minutes).2. The deparaffinized slides were serially passaged through 100%, 95%, 70% and 50% ethanol (10 minutes each). Slides were kept in double distilled water for 10 minutes.3. For antigen retrieval (unmasking the antigenic epitope), slides were transferred into IHC antigen retrieval solution and a decloaking chamber at 110°C for 15 minutes.4. Slides were allowed to cool to room temperature (~5 minutes) and then rinsed with lx PBS for 10 minutes. The boundary of the tissue was marked with a hydrophobic pen.5. For blocking, 2% normal goat serum was added onto the sections and incubated in a humidified chamber at room temperature for 1 hour.6. Post incubation, blocking buffer was drained from the slides and the Antibody-Z domainE3-PNA-AF647 complex (‘Test’) was added onto the section, which was incubated for 2 hours at room temperature. A section with only K4 coiled coil- PNA:PNA-AF647 (‘Negative Control’ ) was used as a control.7. Post incubation, the antibody-PNA complex and negative control were washed with lx PBS (3x for 5 minutes).8. Nuclei were stained with DAPI for 10 minutes at room temperature. Excess DAPI was removed by washing the slides with IX PBS for 5 minutes.9. Slides were mounted using prolong antifade glass mountant and imaged using the EVOS™ M7000 Imaging System at 20X magnification.10. Low pH buffer was added to the sections and the sections were incubated for 1 hour at room temperature.11. Sections were imaged using the EVOS™ M7000 Imaging System at 20X magnification to confirm the release of fluorescent signal from Primary Antibody 1.12. pH of the sections was neutralized by incubation with IX PBS for 1 hour, followed by 0.1% PBS and 0.01% PBS for 30 minutes each at room temperature.13. After neutralization, sections were incubated with Primary Antibody 2 for 16 hours at 4 degrees.14. Post incubation, antibody was removed, and sections were washed with lx PBS wash (3x for 5 minutes).

[0103] Slides were mounted using prolong antifade glass mountant and imaged using the EVOS™ M7000 Imaging System at 20X magnification.Example 4: Cyclic immunostaining of mouse duodenum using PNA tagged antibodies

[0104] This example describes the results of cyclic immunostaining of mouse duodenum FFPE sections performed using PNA-ALEXA FLUOR™ 647 conjugated ACTA2 primary antibody and a pan-cytokeratin antibody conjugated to eFluor™660.

[0105] In the first cycle of staining, ACTA2 tagged with PNA-AF647 was used to probe mouse duodenum tissue as described in Example 2. ACTA2 (smooth muscle actin alpha 2) is an actin protein involved in the contractile apparatus of smooth muscle. FIG. 4B shows specific staining of the smooth muscle layer of mouse duodenum by the ACTA2-Z domain-PNA- AF647. Nuclei were stained using DAPI (FIG. 4A) and the composite staining is shown in FIG. 4C.

[0106] In the second cycle of staining, a low pH wash was used to release the PNA-647 tag conjugated to ACTA2. Subsequent loss of signal is shown in FIG. 4E. Nuclei were stained using DAPI (FIG. 4D) and the composite staining is shown in FIG. 4F. Post signal release, pH was neutralized as described in Example 3.

[0107] In the third cycle of immuno staining, a primary antibody conjugate (Pan-Cytokeratin- eFluor660) was used to probe the same mouse duodenum section. Cytokeratin proteins are part of the intermediate filament family. FIG. 4H shows specific staining of the mucosal layer of mouse duodenum by Pan-Cytokeratin. Nuclei were stained using DAPI (FIG. 4G) and the composite staining is shown in FIG. 41.

[0108] These results demonstrate that: (1) primary antibodies tagged with fluorophore labeled PNAs can be used for fluorescent immuno staining in IHC; (2) a low pH based wash (followed by neutralization) effectively detaches the Z domain-PNA-fluorophore signal; and (3) the same tissue can be re -probed with another antibody demonstrating that tissue integrity is intact and not altered by probing with PNA tagged antibodies or low pH treatment. Thus, PNA-tagging can be used as a technology for cyclic immunostaining of FFPE sections.

[0109] 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.INCORPORATION BY REFERENCE

[0110] 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.

Claims

CLAIMS1. A conjugate, comprising: an Fc binding domain; and a coiled-coil domain.

2. The conjugate of claim 1 , further comprising a linker positioned between the Fc binding domain and the coiled-coil domain.

3. The conjugate of claim 1 or claim 2, wherein the Fc binding domain is a bacterially derived or synthetically prepared immunoglobulin binding domain or a Fc binding protein (FcBP).

4. The conjugate of claim 3, 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.

5. The conjugate of claim 4, 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 or SEQ ID NO: 3.

6. The conjugate of claim 3, 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.

7. The conjugate of claim 6, wherein the amino acid sequence of the C2 domain comprises SEQ ID NO: 4.

8. The conjugate of claim 3, wherein the amino acid sequence of the FcBP comprises any one of SEQ ID NOs: 5-17.

9. The conjugate of any one of claims 2-8, wherein the linker is a peptide of about 4 to about 12 amino acids in length.

10. The conjugate of claim 9, wherein the linker is a flexible glycine- serine rich linker.

11. The conjugate of claim 9 or claim 10, wherein the linker comprises or consists of any one of SEQ ID NOs: 18-22.

12. The conjugate of any one of claims 1-11, wherein the coiled-coil domain comprises a first peptide comprising a first coiled-coil heptad.

13. The conjugate of claim 12, wherein the coiled-coil domain further comprises a second peptide comprising a second coiled-coil heptad, wherein the first coiled- coil heptad and the second coiled-coil heptad non-covalently bind to each other to form the coiled-coil domain.

14. The conjugate of claim 12 or claim 13, wherein the first peptide of the coiled-coil domain comprises two, three, four or five repeats of the first coiled-coil heptad.

15. The conjugate of any one of claims 12-14, wherein the first peptide of the coiled-coil domain comprises three repeats of the first coiled-coil heptad.

16. The conjugate of any one of claims 12-15, wherein the first coiled-coil heptad comprises EIAALEK (SEQ ID NO: 23).

17. The conjugate of any one of claims 13-16, wherein the second peptide of the coiled-coil domain comprises two, three, four or five repeats of the second coiled-coil heptad.

18. The conjugate of claim 17, wherein the second peptide of the coiled-coil domain comprises four repeats of the second coiled-coil heptad.

19. The conjugate of claim 17 or claim 18, wherein the second coiled-coil heptad comprises KIAALKE (SEQ ID NO: 24)20. The conjugate of any one of claims 13-19, further comprising a first peptide nucleic acid (PNA) conjugated to the second peptide of the coiled-coil domain.

21. The conjugate of claim 20, further comprising a second PNA, a DNA oligonucleotide, or an RNA oligonucleotide that is complementary to the first PNA.

22. The conjugate of claim 21, wherein the second PNA, the DNA oligonucleotide, or the RNA oligonucleotide is hybridized to the first PNA.

23. The conjugate of claim 21 or claim 22, wherein: the first PNA and the second PNA are about 8 to about 16 nucleotides in length, about 9 to about 15 nucleotides in length, about 10 to about 14 nucleotides in length, or about 11 to about 13 nucleotides in length; or the DNA oligonucleotide or the RNA oligonucleotide is about 10 to about 150, such as about 20 to about 125, or about 50 to about 100 nucleotides in length.

24. The conjugate of any one of claims 21-23, wherein: the first PNA and the second PNA, the DNA oligonucleotide, or the RNA oligonucleotide are about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, or about 16 nucleotides in length; or the DNA oligonucleotide or the RNA 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.

25. The conjugate of claim 24, wherein the first PNA and the second PNA, the DNA oligonucleotide or the RNA oligonucleotide are 12 nucleotides in length.

26. The conjugate of any one of claims 21-25, wherein the first PNA, the second PNA, the DNA oligonucleotide or the RNA oligonucleotide have a sequence selected from any one of SEQ ID NOs: 25-477.

27. The conjugate of any one of claims 21-26, wherein the second PNA, the DNA oligonucleotide, or the RNA oligonucleotide comprises a detectable label.

28. The conjugate of claim 27, wherein the detectable label comprises a fluorophore, a small molecule, a streptavidin, an enzyme, a nanoparticle, allophycocyanin (APC), an oligonucleotide, or a peptide tag.

29. The conjugate of any one of claims 13-19, further comprising a labelled peptide conjugated to the second peptide of the coiled-coil domain.

30. The conjugate of claim 29, wherein the peptide is labelled with a fluorophore, a small molecule, a streptavidin, an enzyme, a nanoparticle, APC, an oligonucleotide, or a peptide tag, optionally wherein the oligonucleotide is labelled, not labelled, dye labelled, or has a self-complementary 3' end for dye incorporation, or the oligonucleotide is an inversion probe or a fold-back probe.

31. The conjugate of any one of claims 27-30, further comprising an immunoglobulin molecule, wherein the Fc region of the immunoglobulin molecule is non- covalently bound to the Fc binding domain.

32. The conjugate of claim 31, 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 single-chain variable fragment (scFv).

33. The conjugate of any one of claims 1-32, further comprising a purification tag.

34. The conjugate of claim 33, wherein the purification tag is attached to the N-terminus of the Fc binding domain.

35. A conjugate, comprising: an Fc binding domain; a coiled-coil domain comprising a first peptide comprising a first coiled-coil heptad and a second peptide comprising a second coiled-coil heptad, wherein the first coiled- coil heptad and the second coiled-coil heptad non-covalently bind to each other to form the coiled-coil domain; a linker positioned between the Fc binding domain and the coiled-coil domain; a first peptide nucleic acid (PNA) conjugated to the second peptide of the coiled- coil domain; a second PNA, a DNA oligonucleotide, or an RNA oligonucleotide hybridized to the first PNA, wherein the second PNA, the DNA oligonucleotide, or the RNA oligonucleotide comprises a detectable label; andan immunoglobulin molecule, wherein the Fc region of the immunoglobulin molecule is non-covalently bound to the Fc binding domain.

36. The conjugate of claim 35, further comprising a purification tag attached to the N-terminus of the Fc binding domain.

37. An in vitro method of detecting a target antigen in a cell or tissue, comprising: contacting the cell or tissue with the conjugate of any one of claims 31-36, wherein the immunoglobulin molecule of the conjugate is specific for the target antigen; and detecting the conjugate, thereby detecting the target antigen.

38. An in vitro method of detecting a target antigen in a cell or tissue, comprising: contacting the cell or tissue with a conjugate comprising: an Fc binding domain; a coiled-coil domain comprising a first peptide comprising a first coiled- coil heptad and a second peptide comprising a second coiled-coil heptad, wherein the first coiled-coil heptad and the second coiled-coil heptad non-covalently bind to each other to form the coiled-coil domain; a linker positioned between the Fc binding domain and the coiled-coil domain; a first peptide nucleic acid (PNA) conjugated to the second peptide of the coiled-coil domain; and an immunoglobulin molecule specific for the target antigen, wherein the Fc region of the immunoglobulin molecule is non-covalently bound to the Fc binding domain; contacting the cell or tissue with a second PNA, a DNA oligonucleotide, or an RNA oligonucleotide capable of hybridizing to the first PNA, wherein the second PNA, the DNA oligonucleotide, or the RNA oligonucleotide comprises a detectable label; and detecting the detectable label, thereby detecting the target antigen.

39. The method of claim 37 or claim 38, wherein the cell is a cell isolated from a subject.

40. The method of claim 37 or claim 38, wherein the cell is a cultured cell.

41. The method of claim 37 or claim 38, wherein the tissue is a formalin- fixed paraformaldehyde-embedded tissue section or a fresh frozen tissue section.

42. The method of any one of claims 37-41, wherein detecting the antigen comprises immunohistochemistry, fluorescence microscopy, flow cytometry, Western blot, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction, rolling circle amplification, next generation sequencing (NGS) using NGS-enabled barcoded oligonucleotides with or without biotin for oligonucleotide capture, or nicking enzyme signal amplification.

43. The method of any one of claims 37 -42, further comprising exposing the cell or tissue to low pH, high temperature and / or high salt concentration to release the Fc binding domain from the immunoglobulin molecule.

44. The method of claim 43, wherein the cell or tissue is exposed to pH 2 to pH 4, optionally wherein the cell or tissue is exposed to a composition comprising 10 mM glycine adjusted to pH 2 to pH 4.

45. The method of claim 43 or claim 44, further comprising contacting the cell with a second conjugate of any one of claims 31-36, wherein the second conjugate has a different detectable label than the first conjugate.

46. A method of assembling a labelled antibody conjugate, comprising contacting the following components:(i) a fusion protein comprising an Fc binding domain, a linker peptide, and a first coiled-coil heptad repeat;(ii) a second coiled-coil heptad repeat conjugated to a first peptide nucleic acid (PNA);(iii) a second PNA, a DNA oligonucleotide, or an RNA oligonucleotide conjugated to a detectable label; and(iv) an immunoglobulin molecule, wherein the first coiled-coil heptad repeat and the second coiled-coil heptad repeat non-covalently bind to each other to form a coiled-coil domain; the first PNA hybridizes with the second PNA, the DNA oligonucleotide or the RNA oligonucleotide; and the Fc binding domain non-covalently binds the immunoglobulin molecule,thereby forming a labelled antibody conjugate.

47. The method of claim 46, wherein the components are incubated at room temperature.

48. The method of claim 46 or claim 47, wherein components (i), (ii), (iii) and (iv) are incubated at a ratio of 2:2:2: 1.

49. The method of any one of claims 46-48, wherein the Fc binding domain is a bacterially derived immunoglobulin binding domain or a Fc binding protein (FcBP).

50. The method of claim 49, 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; or a C2 domain of Streptococcal Protein G or a variant thereof that retains the capacity to bind an antibody Fc region.

51. The method of any one of claims 46-50, wherein the linker is a peptide of about 4 to about 12 amino acids in length.

52. The method of claim 51, wherein the linker is a flexible glycine-serine rich linker.

53. The method of any one of claims 46-52, wherein the first PNA and the second PNA, the DNA oligonucleotide or the RNA oligonucleotide are about 8 to about 16 nucleotides in length, about 9 to about 15 nucleotides in length, about 10 to about 14 nucleotides in length, or about 11 to about 13 nucleotides in length.

54. The method of any one of claims 46-53, wherein the first PNA and the second PNA, the DNA oligonucleotide, or the RNA oligonucleotide are about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, or about 16 nucleotides in length.

55. The method of any one of claims 46-54, wherein the detectable label comprises a fluorophore, a small molecule, a streptavidin, an enzyme, a nanoparticle, allophycocyanin (APC), an oligonucleotide, or a peptide tag.

56. The method of any one of claims 46-55, wherein the immunoglobulin molecule is an IgG.

57. The method of any one of claims 46-56, wherein the conjugate further comprises a purification tag.

58. The method of claim 57, wherein the purification tag is attached to the N-terminus of the Fc binding domain.

59. A kit comprising one or more of the conjugates of any one of claims 1-36.

60. A kit, comprising: a fusion protein comprising an Fc binding domain, a first coiled-coil heptad repeat, and a linker positioned between the Fc binding domain and the first coiled-coil heptad repeat; a second coiled-coil heptad repeat conjugated to a first peptide nucleic acid (PNA); and / or a second PNA, a DNA oligonucleotide, or an RNA oligonucleotide conjugated to a detectable label, wherein the first PNA is capable of hybridizing with the second PNA, the DNA oligonucleotide or the RNA oligonucleotide.

61. The kit of claim 60, further comprising an immunoglobulin molecule specific for a target antigen.

62. The kit of claim 60 or claim 61 wherein the Fc binding domain is a bacterially derived or synthetically prepared immunoglobulin binding domain or a Fc binding protein (FcBP).

63. The kit of claim 62, 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; or a C2 domain of Streptococcal Protein G or a variant thereof that retains the capacity to bind an antibody Fc region.

64. The kit of any one of claims 60-63, wherein the linker is a peptide of about 4 to about 12 amino acids in length.

65. The kit of claim 51, wherein the linker is a flexible glycine-serine rich linker.

66. The kit of any one of claims 60-65, wherein: the first PNA and the second PNA are about 8 to about 16 nucleotides in length, about 9 to about 15 nucleotides in length, about 10 to about 14 nucleotides in length, or about 11 to about 13 nucleotides in length; or the DNA oligonucleotide or the RNA oligonucleotide is about 10 to about 150, about 20 to about 125, or about 50 to about 100 nucleotides in length.

67. The kit of any one of claims 60-66, wherein: the first PNA and the second PNA are about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, or about nucleotides in length; or the DNA oligonucleotide or the RNA 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.

68. The kit of any one of claims 60-67, wherein the detectable label comprises a fluorophore, a small molecule, a streptavidin, an enzyme, a nanoparticle, allophycocyanin (APC), an oligonucleotide, or a peptide tag.

69. The kit of any one of claims 61-68 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 single-chain variable fragment (scFv).

70. The kit of any one of claims 60-69, wherein the conjugate further comprises a purification tag.

71. The kit of claim 70, wherein the purification tag is attached to the N- terminus of the Fc binding domain.

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