Horseradish peroxidase copolymers, conjugates, and uses thereof

Abstract

Provided herein are copolymers comprising a lysine-rich polypeptide and horseradish peroxidase, conjugates of such copolymers with proteins (e.g., antibodies, streptavidin, and the like), methods of making copolymer conjugates, and methods of using such conjugates, e.g., in assays for measuring a target in a sample.

Description

[0001] HORSERADISH PEROXIDASE COPOLYMERS, CONJUGATES, AND USES THEREOF

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 565,989, filed on March 15, 2024, the disclosure of which is incorporated herein by reference in its entirety.

[0004] FIELD

[0005] Provided herein are copolymers comprising a lysine-rich polypeptide and horseradish peroxidase, conjugates of such copolymers with proteins (e.g., antibodies, streptavidin, and the like), methods of making copolymer conjugates, and methods of using such conjugates, e.g., in assays for measuring a target in a sample.

[0006] BACKGROUND

[0007] Horseradish peroxidase (HRP) is a widely used enzyme in many different assay applications, as it allows for chromogenic or chemiluminescent detection of substrates in a variety of assays and detection techniques such as enzyme-linked immunosorbent assay (ELISA), Western blots (e.g. capillary electrophoresis western blots), immunohistochemistry (IHC), and immunocytochemistry (ICC). HRP can also be used for luminescent detection of substrates, e.g., via tyramide signal amplification (TSA)). For example, HRP is conjugated to antibodies for use in IHC.

[0008] HRP is conjugated to targeting molecules for use in these types of assays. As HRP is a glycoprotein, modification of HRP via oxidation of its sugar backbone is well-known. In particular, oxidation using sodium periodate gives the corresponding sugar-derived aldehyde, and reductive amination of the aldehydes and an amino group on a targeting molecule (e.g., an antibody or a protein) gives the HRP-conjugate. These conjugates typically consist of monomers or short oligomers of HRP conjugated to the targeting molecule, but these typically generate only weak output signals.

[0009] There is a need for HRP conjugates capable of providing enhanced signal to noise (S / N) in these assays.

[0010] SUMMARY

[0011] In one aspect, disclosed herein is a copolymer comprising: a lysine-rich polypeptide and horseradish peroxidase.

[0012] In some embodiments, the lysine-rich polypeptide is a poly-lysine. In some embodiments, the lysine-rich polypeptide is poly-L-lysine. In some embodiments, the poly-lysine has a molecular weight of about 1 kDa to about 50 kDa. In some embodiments, the poly-lysine has a molecular weight of about 1 kDa to about 15 kDa. In some embodiments, the poly-lysine has a molecular weight of about 1 kDa to about 5 kDa. In some embodiments, the poly-lysine has a molecular weight of about 4 kDa to about 15 kDa.

[0013] In some embodiments, the copolymer has a molecular weight of greater than 7000 kDa. In some embodiments, the copolymer has a molecular weight of about 20 MDa to about 50 MDa.

[0014] In another aspect, disclosed herein is a method of preparing copolymer disclosed herein (e.g., a copolymer comprising a lysine-rich polypeptide and horseradish peroxidase), the method comprising:

[0015] (a) reacting horseradish peroxidase with sodium periodate to generate an aldehyde- functionalized horseradish peroxidase; and

[0016] (b) reacting the aldehyde-functionalized horseradish peroxidase with the lysine-rich polypeptide to generate the copolymer.

[0017] In another aspect, disclosed herein is a conjugate comprising: (i) a copolymer disclosed herein (e.g., a copolymer comprising a lysine-rich polypeptide and horseradish peroxidase); and (ii) a protein.

[0018] In some embodiments, the copolymer is directly bound to the protein via a covalent bond. In some embodiments, the copolymer is bound to the protein via a linker that comprises a moiety resulting from a reaction between two complementary reactive groups. In some embodiments, the two complementary reactive groups are selected from: an azide and an alkyne; a thiol and a maleimide; and a trans-cyclooctene and a tetrazine. In some embodiments, the linker comprises a moiety selected from:

[0019] In some embodiments, the protein is an antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an antibody fragment. In some embodiments, the protein is avidin or streptavidin, or a derivative thereof.

[0020] In another aspect, disclosed herein is a conjugate described herein (e.g., a conjugate comprising: (i) a copolymer comprising a lysine-rich polypeptide and horseradish peroxidase; and (ii) a protein), for use in a method of measuring a target in a sample.

[0021] In another aspect, disclosed herein is a method of preparing a conjugate, the conjugate comprising: (i) a copolymer comprising a lysine-rich polypeptide and horseradish peroxidase; and (ii) a protein, wherein the method comprises:

[0022] (a) reacting horseradish peroxidase with sodium periodate to generate an aldehyde- functionalized horseradish peroxidase; and

[0023] (b) reacting the aldehyde-functionalized horseradish peroxidase with the lysine-rich polypeptide to generate a copolymer; and

[0024] (c) reacting the copolymer with the protein (e.g., an antibody) to generate the conjugate.

[0025] In some embodiments, the protein is an antibody. In some embodiments, the protein is avidin or streptavidin, or a derivative thereof.

[0026] In another aspect, disclosed herein is a method of preparing a conjugate, the conjugate comprising: (i) a copolymer comprising a lysine-rich polypeptide and horseradish peroxidase; and (ii) a protein, wherein the method comprises:

[0027] (a) reacting horseradish peroxidase with sodium periodate to generate an aldehyde- functionalized horseradish peroxidase; and

[0028] (b) reacting the aldehyde-functionalized horseradish peroxidase with the lysine-rich polypeptide and the protein to generate the conjugate.

[0029] In some embodiments, the protein is an antibody. In some embodiments, the protein is avidin or streptavidin, or a derivative thereof.

[0030] In another aspect, disclosed herein is a method of measuring a target in a sample, the method comprising contacting the sample with a conjugate comprising: (i) a copolymer comprising a lysine-rich polypeptide and horseradish peroxidase; and (ii) a protein (e.g., an antibody).

[0031] In another aspect, disclosed herein is a method of measuring a target in a sample, the method comprising:

[0032] (a) contacting the sample with a primary antibody that binds to the target, if present in the sample;

[0033] (b) contacting the sample with a conjugate comprising: (i) a copolymer comprising lysine-rich polypeptide and horseradish peroxidase; and (ii) a protein, wherein the conjugate binds to the primary antibody; and

[0034] (c) detecting a signal from the conjugate.

[0035] In another aspect, disclosed herein is a method of measuring a target in a sample, the method comprising:

[0036] (a) contacting the sample with a biotinylated primary antibody that binds to the target, if present in the sample;

[0037] (b) contacting the sample with a conjugate comprising: (i) a copolymer comprising lysine-rich polypeptide and horseradish peroxidase; and (ii) a protein selected from avidin and streptavidin, or a derivative thereof, wherein the avidin or streptavidin or derivative thereof of the conjugate binds to the biotinylated primary antibody; and

[0038] (c) detecting a signal from the conjugate.

[0039] In another aspect, disclosed herein is a method of measuring a target in a sample, the method comprising:

[0040] (a) contacting the sample with a primary antibody that binds to the target, if present in the sample;

[0041] (b) contacting the sample with a conjugate comprising: (i) a copolymer comprising a lysine-rich polypeptide and horseradish peroxidase; and (ii) an antibody, wherein the antibody of the conjugate binds to the primary antibody; and

[0042] (c) detecting a signal from the conjugate.

[0043] BRIEF DESCRIPTION OF THE DRAWINGS

[0044] FIG. 1 is a scheme showing a representative synthesis of a copolymer disclosed herein.

[0045] FIG. 2 is a scheme showing a representative synthesis of a conjugate disclosed herein.

[0046] FIG. 3 shows Simple Western data using an HRP-poly-lysine copolymer conjugate of IgG disclosed herein. FIG. 4 shows Simple Western data using an HRP-poly-lysine copolymer conjugate of IgG disclosed herein.

[0047] DETAILED DESCRIPTION

[0048] Polymers of HRP (polyHRP) conjugated to a targeting module (e.g., an antibody or streptavidin) have the potential to provide enhanced signal and signal / noise (S / N) in a variety of assays and detection techniques including ELISA (e.g. microfluidic ELISA), Western blots (e.g. capillary electrophoresis western blots), IHC, ICC, and TSA. However, in practice, such polymers are not trivial to develop. Disclosed herein are copolymers comprising a lysine-rich polypeptide and HRP. These large copolymers have high activities in assays such as immunoassays.

[0049] Definitions

[0050] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those that are well known and commonly used in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0051] As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise.

[0052] As used herein, the term “and / or” includes any and all combinations of listed items, including any of the listed items individually. For example, “A, B, and / or C” encompasses A, B, C, AB, AC, BC, and ABC, each of which is to be considered separately described by the statement “A, B, and / or C.”

[0053] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6- 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0054] Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75thEd., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Sorrell, Organic Chemistry, 2ndedition, University Science Books, Sausalito, 2006; Smith, March’s Advanced Organic Chemistry: Reactions, Mechanism, and Structure, 7thEdition, John Wiley & Sons, Inc., New York, 2013; Larock, Comprehensive Organic Transformations, 3rdEdition, John Wiley & Sons, Inc., New York, 2018; and Carruthers, Some Modern Methods of Organic Synthesis, 3rdEdition, Cambridge University Press, Cambridge, 1987; the entire contents of each of which are incorporated herein by reference.

[0055] As used herein, the term “alkyne” refers to a moiety containing a carbon-carbon triple bond. An alkyne can be a straight-chain alkyne such as an ethynyl group (-C=CH), or a cyclic moiety containing a carbon-carbon triple bond, such as a cyclooctynyl group.

[0056] As used herein, the term “aryl” refers to a radical of a monocyclic, bicyclic, or tricyclic 4n+2 aromatic ring system (e.g., having 6, 10, or 14 n electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms (“Ce-Ci4 aryl”). In some embodiments, an aryl group has six ring carbon atoms (“Ce aryl,” i.e., phenyl). In some embodiments, an aryl group has ten ring carbon atoms (“Cio aryl,” e.g., naphthyl such as 1- naphthyl and 2-naphthyl). In some embodiments, an aryl group has fourteen ring carbon atoms (“Ci4 aryl,” e.g., anthracenyl and phenanthrenyl).

[0057] As used herein, the term “arylene” refers to a divalent aryl radical.

[0058] As used herein, the term “azide” refers to a moiety -N3.

[0059] As used herein, the term “cycloalkyl” refers to a radical of a saturated carbocyclic ring system containing three to ten carbon atoms and zero heteroatoms. The cycloalkyl may be monocyclic, bicyclic, bridged, fused, or spirocyclic. Representative examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, adamantyl, bicyclo[2.2.1]heptanyl, bicyclo[3.2.1]octanyl, and bicyclo[5.2.0]nonanyl.

[0060] As used herein, the term “cycloalkylene” refers to a divalent cycloalkyl radical.

[0061] As used herein, the term “heteroaryl” refers to a radical of a 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6 or 1071 electrons shared in a cyclic array) having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur (“5-10 membered heteroaryl”). In heteroaryl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. Heteroaryl bicyclic ring systems can include one or more heteroatoms in one or both rings. “Heteroaryl” also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused (aryl / heteroaryl) ring system. Bicyclic heteroaryl groups wherein one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, and the like) the point of attachment can be on either ring, i.e., either the ring bearing a heteroatom (e.g., 2-indolyl) or the ring that does not contain a heteroatom (e.g., 5-indolyl). Exemplary 5-membered heteroaryl groups containing one heteroatom include, without limitation, pyrrolyl, furanyl and thiophenyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, without limitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, without limitation, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, without limitation, tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, without limitation, pyridinyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, without limitation, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include, without limitation, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, without limitation, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzo thiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6- bicyclic heteroaryl groups include, without limitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl.

[0062] As used herein, the term “heteroarylene” refers to a divalent heteroaryl radical.

[0063] As used herein, the term “heterocyclyl” refers to a radical of a 3- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon (“3-10 membered heterocyclyl”). In heterocyclyl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. A heterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”) or a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic heterocyclyl”), and can be saturated or can be partially unsaturated. Heterocyclyl bicyclic ring systems can include one or more heteroatoms in one or both rings. “Heterocyclyl” also includes ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more cycloalkyl groups wherein the point of attachment is either on the cycloalkyl or heterocyclyl ring, or ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclyl ring system. A heterocyclyl group may be described as, e.g., a 3-7-membered heterocyclyl, wherein the term “membered” refers to the non-hydrogen ring atoms, i.e., carbon, nitrogen, oxygen, sulfur, boron, phosphorus, and silicon, within the moiety. Exemplary 3 -membered heterocyclyl groups containing one heteroatom include, without limitation, azirdinyl, oxiranyl, and thiorenyl. Exemplary 4-membered heterocyclyl groups containing one heteroatom include, without limitation, azetidinyl, oxetanyl, and thietanyl. Exemplary 5 -membered heterocyclyl groups containing one heteroatom include, without limitation, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2, 5-dione. Exemplary 5-membered heterocyclyl groups containing two heteroatoms include, without limitation, dioxolanyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclyl groups containing three heteroatoms include, without limitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing one heteroatom include, without limitation, piperidinyl (e.g., 2,2,6,6-tetramethylpiperidinyl), tetrahydropyranyl, dihydropyridinyl, pyridinonyl (e.g., l-methylpyridin-2-onyl), and thianyl. Exemplary 6- membered heterocyclyl groups containing two heteroatoms include, without limitation, piperazinyl, morpholinyl, pyridazinonyl (2-methylpyridazin-3-onyl), pyrimidinonyl (e.g., 1- methylpyrimidin-2-onyl, 3-methylpyrimidin-4-onyl), dithianyl, dioxanyl. Exemplary 6- membered heterocyclyl groups containing two heteroatoms include, without limitation, triazinanyl. Exemplary 7-membered heterocyclyl groups containing one heteroatom include, without limitation, azepanyl, oxepanyl and thiepanyl. Exemplary 8-membered heterocyclyl groups containing one heteroatom include, without limitation, azocanyl, oxecanyl and thiocanyl. Exemplary 5-membered heterocyclyl groups fused to a Ce aryl ring (also referred to herein as a 5,6-bicyclic heterocyclyl ring) include, without limitation, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, benzoxazolinonyl, and the like. Exemplary 5 -membered heterocyclyl groups fused to a heterocyclyl ring (also referred to herein as a 5, 5 -bicyclic heterocyclyl ring) include, without limitation, octahydropyrrolopyrrolyl (e.g., octahydropyrrolo[3,4-c]pyrrolyl), and the like. Exemplary 6- membered heterocyclyl groups fused to a heterocyclyl ring (also referred to as a 4,6- membered heterocyclyl ring) include, without limitation, diazaspirononanyl (e.g., 2,7- diazaspiro[3.5]nonanyl). Exemplary 6-membered heterocyclyl groups fiised to an aryl ring (also referred to herein as a 6,6-bicyclic heterocyclyl ring) include, without limitation, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like. Exemplary 6-membered heterocyclyl groups fused to a cycloalkyl ring (also referred to herein as a 6,7-bicyclic heterocyclyl ring) include, without limitation, azabicyclooctanyl (e.g., ( 1 ,5)-8- azabicyclo[3.2.1]octanyl). Exemplary 6-membered heterocyclyl groups fused to a cycloalkyl ring (also referred to herein as a 6,8-bicyclic heterocyclyl ring) include, without limitation, azabicyclononanyl (e.g., 9-azabicyclo[3.3.1]nonanyl).

[0064] As used herein, the term “heterocyclylene” refers to a divalent heterocyclyl radical. As used herein, the term “conjugate” refers to a molecule in which two elements are linked together through covalent bonds, either directly or via a linker.

[0065] As used herein, the term “polypeptide” refers to a polymeric compound in which two or more amino acids form a single chain in which the amino acids are connected by amide bonds (-C(O)NH-). The amino acids in a polypeptide may comprise natural amino acids, nonnatural amino acids, amino acid analogs, and / or modified amino acids.

[0066] As used herein, the term “tetrazine” refers to a 1,2,4,5-tetrazinyl moiety. As used herein, the term “thiol” refers to a group -SH.

[0067] When a group or moiety can be substituted, the term “substituted” indicates that one or more (e.g., 1, 2, 3, 4, 5, or 6; in some embodiments 1, 2, or 3; and in other embodiments 1 or 2) hydrogens on the group indicated in the expression using “substituted” can be replaced with a selection of recited indicated groups or with a suitable substituent group known to those of skill in the art (e.g., one or more of the groups recited below), provided that the designated atom’ s normal valence is not exceeded. Substituent groups include, but are not limited to, alkyl, alkenyl, alkynyl, alkoxy, acyl, amino, amido, amidino, aryl, azido, carbamoyl, carboxyl, carboxyl ester, cyano, cycloalkyl, cycloalkenyl, guanidino, halo, haloalkyl, haloalkoxy, heteroaryl, heterocyclyl, hydroxy, hydrazino, imino, oxo, nitro, phosphate, phosphonate, sulfonic acid, thiol, thione, or combinations thereof.

[0068] As used herein, in chemical structures the indication: represents a point of attachment of one moiety to another moiety (e.g., a substituent group to the rest of the compound). For compounds described herein, groups and substituents thereof may be selected in accordance with permitted valence of the atoms and the substituents, such that the selections and substitutions result in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.

[0069] When substituent groups are specified by their conventional chemical formulae, written from left to right, such indication also encompass substituent groups resulting from writing the structure from right to left. For example, if a bivalent group is shown as -CH2O-, such indication also encompasses -OCH2-; similarly, -OC(O)NH- also encompasses - NHC(O)O-. When linker moieties are shown, the linkers can be attached to other moieties of the compound in either direction.

[0070] Copolymers

[0071] Disclosed herein is a copolymer, in which the copolymer comprises a lysine-rich polypeptide and horseradish peroxidase.

[0072] As used herein, the term “lysine-rich polypeptide” refers to a polypeptide in which at least 25% of the amino acids are lysine. In some embodiments, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the amino acids in the polypeptide are lysine. In some embodiments, 100% of the amino acids are lysine, i.e., the lysine-rich polypeptide is poly-lysine. In some embodiments, the lysine -rich polypeptide is poly-L-lysine. In some embodiments, the lysine-rich polypeptide is poly-D-lysine. In some embodiments, the lysine- rich polypeptide is poly-DL-lysine. In some embodiments, the lysine-rich polypeptide comprises one or more lysine residues that are in a salt form (i.e., wherein the terminal -NH2 group of the lysine side change is positively charged and associated with an appropriate counteranion). For example, in some embodiments, the lysine-rich polypeptide is poly-lysine hydrobromide.

[0073] When the lysine-rich polypeptide includes amino acids other than lysine, the remaining amino acids may be any combination of naturally occurring and non-naturally occurring amino acids. The amino acids may be the L-isomers or the D-isomers. Exemplary non-naturally occurring amino acids include beta-alanine ( -Ala), N-alpha-methyl-alanine (Me-Ala), aminobutyric acid (Abu), gamma-aminobutyric acid (y-Abu), aminocaproic acid (s-Ahx), aminoisobutyric acid (Aib), aminomethylpyrrolecarboxylic acid, aminopiperidinecarboxylic acid, aminoserine (Ams), aminotetrahydropyran-4-carboxylic acid, arginine N-methoxy-N-methylamide, beta-aspartic acid ( -Asp), azetidinecarboxylic acid, 3- (2-benzothiazolyl) alanine, alpha-tert-butylglycine, 2-amino-5-ureido-N-pentanoic acid (citrulline, Cit), beta-cyclohexylalanine (Cha), acetamidomethyl-cysteine, alphaaminobutylglycine, N-aminoethyl, diaminobutyric acid (Dab), diaminopropionic acid (Dpr), dihydroxyphenylalanine (DOPA), dimethylthiazolidine (DMTA), gamma-glutamic acid (y- Glu), homoserine (Hse), hydroxyproline (Hyp), isoleucine N-methoxy-N-methylamide, methyl-isoleucine (Melle), 4-piperidinecarboxylic acid (isonipecotic acid) (Isn), methylleucine (MeLeu), methyl-lysine, dimethyl-lysine, trimethyl-lysine, methane proline, methionine sulfoxide (Met(O)), methionine sulfone (Met(O2)), norleucine (Nle), methylnorleucine (Me-Nle), norvaline (Nva), ornithine (Om), p- aminobenzoic acid (PABA), penicillamine (Pen), Methyl phenylalanine (MePhe), 4-chlorophenylalanine (Phe(4-Cl)), 4- fluorophenylalanine (Phe(4-F)), 4-nitrophenylalanine (Phe(4-NO2)), 4-cyanophenylalanine ((Phe(4-CN)), phenylglycine (Phg), piperidinylalanine, piperidinylglycine, 3,4- dehydroproline, pyrrolidinylalanine, sarcosine (Sar), selenocysteine (Sec), U-benzyl- phosphoserine, 4-amino-3-hydroxy-6-methylheptanoic acid (Sta), 4-amino-5-cyclohexyl-3- hydroxypentanoic acid (ACHPA), 4-amino-3-hydroxy-5-phenylpentanoic acid (AHPPA), l,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), tetrahydropyranylglycine, thienylalanine (Thi), U-benzyl-phosphotyrosine, O-phosphotyrosine, methoxy tyrosine, ethoxytyrosine, O-(bis-dimethylamino-phosphono)tyrosine, tyrosine tetrabutylamine sulfate, methyl-valine (MeV al), 1 -amino- 1 -cyclohexanecarboxylic acid (Acx), aminopentanoic acid, P-cyclopropyl-alanine (Cpa), propargylglycine (Prg), allylglycine (Alg), 2-amino-2- cyclohexyl -propionic acid (2-Cha), tert-butylglycine (Tbg), vinylglycine (Vg), 1 -amino- 1- cyclopropanecarboxylic acid (Acp), 1 -amino- 1 -cyclopropanecarboxylic acid (Acpe), alkylated 3-mercaptopropionic acid, and 1-amino-l-cyclobutanecarboxylic acid (Acb).

[0074] In some embodiments, the lysine-rich polypeptide (e.g., poly-lysine) has a molecular weight of about 1 kDa to about 50 kDa, about 1 kDa to about 15 kDa, about 1 kDa to about 5 kDa, or about 4 kDa to about 15 kDa. In some embodiments, the lysine-rich polypeptide (e.g., poly-lysine, such as poly-L-lysine) has a molecular weight of about 1 kDa to about 50 kDa, or about 1 kDa to about 15 kDa. For example, in some embodiments, the lysine-rich polypetide has a molecular weight of about 1 kDa, about 2 kDa, about 3 kDa, about 4 kDa, about 5 kDa, about 6 kDa, about 7 kDa, about 8 kDa, about 9 kDa, about 10 kDa, about 11 kDa, about 12 kDa, about 13 kDa, about 14 kDa, about 15 kDa, about 16 kDa, about 17 kDa, about 18 kDa, about 19 kDa, about 20 kDa, about 25 kDa, about 30 kDa, about 35 kDa, about 40 kDa, about 45 kDa, or about 50 kDa. In some embodiments, the lysine-rich polypeptide (e.g., poly-lysine, such as poly-L- lysine) has a molecular weight of about 1 kDa to about 5 kDa. In some embodiments, the lysine-rich polypeptide (e.g., poly-lysine, such as poly-L- lysine) has a molecular weight of about 4 kDa to about 15 kDa.

[0075] Some lysine-rich polypeptides, such as poly-lysines (including poly-L-lysine, poly-D- lysine, and poly-DL-lysine) are commercially available at a variety of molecular weights. If it is not purchased commercially, a lysine-rich polypeptide can be synthesized using known techniques for preparing polypeptides (e.g., solid-phase peptide synthesis).

[0076] One skilled in the art will appreciate that the molecular weight of lysine -rich polypeptides such as poly-lysine can be determined by measuring their viscosities. Commercial suppliers of poly-lysine, for example, provide the product at a range of molecular weights, such as about 1 kDa to about 5 kDa, or about 4 kDa to about 15 kDa. See, e.g., poly-L-lysine hydrobromide sold by Sigma- Aldrich, product nos. P0879 and P6516, respectively. This molecular weight range is determined by viscosity, as stated by the supplier. Methods for determining molecular weight by viscosity are disclosed in, e.g., Yaron et al. Biochmi. Biophs. Acta 69: 397-399 (1963); and U.S. Patent No. 8,699,600.

[0077] HRP is readily available from a variety of commercial suppliers, the enzyme (MW ~40 kDa) being derived from the roots of the horseradish plant. HRP catalyzes the reaction of hydrogen peroxide with organic, electron-donating substrates (e.g. luminol or 3, 3’, 5,5’- tetramethylbenzidine TMB) to furnish highly colored products, allowing for chromogenic, chemiluminescent, or luminescent detection of substrates as discussed above. HRP suitable for use in the copolymers disclosed herein can be purchased commercially from a variety of suppliers, such as Roche and Worthington Biochemical Co.

[0078] To generate the copolymers, the lysine-rich polypeptide can be conjugated to HRP via known methods for preparing HRP conjugates. For example, HRP is a glycoprotein, and oxidation of the glycan moieties using sodium periodate generates reactive aldehyde groups, which can be used for conjugation to primary amines such as those in the lysine-rich polypeptide. See, e.g., Hermanson, Greg T. (2013) Bioconjugate Techniques, 3rd edition. Thus, in some embodiments, disclosed herein is a method of preparing a copolymer, comprising: (a) reacting horseradish peroxidase with sodium periodate to generate an aldehyde-functionalized horseradish peroxidase; and (b) reacting the aldehyde-functionalized horseradish peroxidase with the lysine-rich polypeptide to generate the copolymer.

[0079] In some embodiments, the resultant copolymer has a weight average molecular weight of greater than about 7000 kDa. For example, in some embodiments, when purified by size exclusion chromatography (SEC), the copolymers may elute in the void volume and thus may be characterized as having a molecular weight of greater than the upper working range limit of the column. In some embodiments, the copolymer has a weight average molecular weight of about 20 MDa to about 100 MDa, or about 20 MDa to about 50 MDa. For example, in some embodiments, the copolymer has a weight average molecular weight of about 20 MDa, about 20 MDa, about 21 MDa, about 22 MDa, about 23 MDa, about 24 MDa, about 25 MDa, about 26 MDa, about 27 MDa, about 28 MDa, about 29 MDa, about 30 MDa, about 31 MDa, about 32 MDa, about 33 MDa, about 34 MDa, about 35 MDa, about 36 MDa, about 37 MDa, about 38 MDa, about 39 MDa, about 40 MDa, about 41 MDa, about 42 MDa, about 43 MDa, about 44 MDa, about 45 MDa, about 46 MDa, about 47 MDa, about 48 MDa, about 49 MDa, or about 50 MDa.

[0080] Conjugates

[0081] In some aspects, provided herein are conjugates. In some embodiments, provided herein are conjugates comprising a copolymer described herein and a protein.

[0082] In some embodiments, the copolymer is directly bound to the protein via a direct covalent bond. In some embodiments, the copolymer is bound to a protein via a linker that comprises a moiety resulting from a reaction between two complementary reactive groups, such as reactive groups known in various bioconjugation reactions. Such moieties would result, for example, from reaction of an azide with an alkyne, a thiol and a maleimide, a transcyclooctene and a tetrazine, or other complementary reactive groups. For example, reactions of azides with terminal alkynes or with strained alkynes (e.g., a difluorinated cyclooctyne or dibenzoazacyclooctyne) results in formation of a 1,2,3-tetrazine ring. For example, in some embodiments, the linker includes a moiety selected from: In some embodiments, the linker may include additional atoms or groups. For example, some embodiments, the linker comprises additional groups derived from reactive compounds that are used to generate conjugates. For example, in some embodiments the protein (e.g., antibody) can be reacted with 2-iminothiolane (Traut’s reagent) to install a thiol moiety. Reaction of sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-l-carboxylate (sulfo-SMCC) with an -NH2 group on the copolymer installs a reactive maleimido group, which in turn can react with the protein thiol to yield the conjugate. In such embodiments, the linker comprises the following moiety:

[0083] In some embodiments, the linker separates the copolymer and the protein by about 5 A, about 10 A, about 20 A, about 50 A, about 100 A, about 150 A, about 200 A, about 300 A, about 400 A, about 500 A, about 600 A, about 700 A, about 800 A, about 900 A, about 1000 A, or any suitable range therebetween (e.g., about 5-100 A, about 50-500 A, about 150-700 A, etc.). In some embodiments, the linker separates the copolymer and the protein by about 1- 200 atoms (e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 60, about 70, about 80, about 90, about 100, about 120, about 140, about 160, about 180, about 200, or any suitable ranges therebetween (e.g., about 2-20, about 10-50, etc.)).

[0084] The linker can include one or more groups independently selected from methylene (- CH2-), ethylene (-CH=CH-), ethynylene -C=C-), ether (-O-), amine (-NH-), thioether (-S-), carbonyl (-C(O)-), thiocarbonyl (-C(S)-), sulfonyl (-S(O)2-), arylene, cycloalkylene, heteroarylene, and heterocyclylene moieties, each of which may be optionally substituted with one or more substituents, or any combination thereof. For example, the above moieties can be combined to form additional groups that may be included in the linker, e.g., a carbonyl group and an ether group can together provide an ester moiety (-C(O)O-); a carbonyl group and two ether groups can together provide a carbonate moiety (-OC(O)O-); a carbonyl group and an amine group can together provide an amide moiety (-C(O)NH-); a carbonyl group and two amine groups can together provide a urea moiety (-NHC(O)NH-); a carbonyl group together with an amine group and an ester group can provide a carbamate moiety (- OC(O)NH-); a carbonyl group together with a thioether and an amine group can provide an S-thiocarbamate moiety; a thiocarbonyl group together with an ether and an amine group can provide an O-thiocarbamate moiety; multiple methylene groups can together form an alkylene chain; etc.

[0085] In some embodiments, the linker comprises one or more alkylene groups (e.g., - (CH2)n-, wherein n is 1-12, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or any suitable range therebetween). In some embodiments, the linker comprises one or more branched alkylene groups. In some embodiments, the linker comprises one or more ethylene glycol units (- CH2CH2O-).

[0086] In some embodiments, the protein is an antibody. The term “antibody” is used in the broadest sense and refers to antibodies and antibody fragments. Accordingly, the term “antibody” refers to monoclonal antibodies, monospecific antibodies (e.g., which can either be monoclonal, or may also be produced by other means than producing them from a common germ cell), polyclonal antibodies, multi- specific antibodies, human antibodies, humanized antibodies (fully or partially humanized), animal antibodies such as, but not limited to, a bird (for example, a duck or a goose), a shark, a whale, and a mammal, including a non-primate (for example, a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, etc.) or a non-human primate (for example, a monkey, a chimpanzee, etc.), recombinant antibodies, chimeric antibodies, single-chain Fvs (“scFv”), single chain antibodies, single domain antibodies, Fab fragments, F(ab’) fragments, F(ab’)2 fragments, disulfide-linked Fvs (“sdFv”), and anti-idiotypic (“anti-Id”) antibodies, dual-domain antibodies, dual variable domain (DVD) or triple variable domain (TVD) antibodies (dual- variable domain immunoglobulins and methods for making them are described in Wu, C., et al., Nature Biotechnology, 25(ll):1290-1297 (2007) and PCT International Application WO 2001 / 058956, the contents of each of which are herein incorporated by reference), or domain antibodies (dAbs) (e.g., such as described in Holt et al., Trends in Biotechnology 21:484-490 (2014)), and including single domain antibodies sdAbs that are naturally occurring, e.g., as in cartilaginous fishes and camelid, or which are synthetic, e.g., nanobodies, VHH, or other domain structure), and functionally active epitopebinding fragments of any of the above. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, namely, molecules that contain an analyte-binding site. Immunoglobulin molecules can be of any type (for example, IgG, IgE, IgM, IgD, IgA, and IgY), class (for example, IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), or subclass. An antibody fragment, which is expressly included in the term “antibody”, refers a portion of an intact antibody that retain the ability to specifically bind to an antigen (see, generally, Holliger et al., Nat. Biotech., 23(9): 1126-1129 (2005)) (e.g., comprises the antigen-binding site or variable region). Any antigen-binding fragment of the antibody described herein is within the scope of the present disclosure. The antibody may not include the constant heavy chain domains (e.g., CH2, CH3, or CH4, depending on the antibody isotype) of the Fc region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab’ fragments, Fab’-SH fragments, F(ab’)2 fragments, Fd fragments, Fv fragments, diabodies, single-chain Fv (scFv) molecules, single-chain polypeptides containing only one light chain variable domain, single-chain polypeptides containing the three CDRs of the light-chain variable domain, single-chain polypeptides containing only one heavy chain variable region, and single-chain polypeptides containing the three CDRs of the heavy chain variable region.

[0087] In some embodiments, the conjugate serves as a primary antibody in a method of measuring a target. In some embodiments, the conjugate serves as a secondary antibody in a method of measuring a target.

[0088] In some embodiments, the conjugate comprises a protein that binds to biotin. In some embodiments, the protein is avidin, streptavidin, or a derivative thereof.

[0089] The conjugates can be prepared by a number of methods. For example, in some embodiments, a conjugate is prepared by:

[0090] (a) reacting horseradish peroxidase with sodium periodate to generate an aldehyde- functionalized horseradish peroxidase;

[0091] (b) reacting the aldehyde-functionalized horseradish peroxidase with the lysine-rich polypeptide to generate a copolymer; and

[0092] (c) reacting the copolymer with the protein to generate the conjugate.

[0093] In other words, in these embodiments, the copolymer is formed first, followed by conjugation to the protein to generate the conjugate.

[0094] In other embodiments, a conjugate is prepared by: (a) reacting horseradish peroxidase with sodium periodate to generate an aldehyde-functionalized horseradish peroxidase; and (b) reacting the aldehyde-functionalized horseradish peroxidase with the lysine-rich polypeptide and the protein to generate the conjugate. In other words, in these embodiments, the generation of the copolymer and the conjugation to the protein are conducted in one step. Methods of Use

[0095] The conjugates provided herein find use in methods of measuring a target in a sample. “Measuring a target in a sample’ does not necessarily indicate that the target is present in the sample, but rather refers to methods of determining whether a target is present in the sample. In some embodiments, “measuring a target in a sample” involves determining that the target is present in the sample. In some embodiments, “measuring a target in a sample” involves measuring an amount, quantity, or level of a target in a sample. In some embodiments, “measuring a target in a sample” involves determining that the target is not present in the sample. The term “target” is used in the broadest sense and is inclusive of any suitable moiety to be detected in a sample, including proteins (e.g. antibodies), metabolites, carbohydrates, glycopeptides, lipids, nucleic acid (e.g. DNA, RNA), cells, etc. In some embodiments, the target is directly immobilized on a substrate (e.g. an ELISA plate, such as for a direct format ELISA). In some embodiments, the target is not directly immobilized on a substrate. In some embodiments, a primary antibody is immobilized on a substrate (e.g. an ELISA plate, such as for an indirect or a sandwich format ELISA) and the target, if present in the sample, binds to the immobilized primary antibody. The methods provided herein may be performed on any suitable sample type. The sample may be a biological sample. The term “biological sample” is used in the broadest sense and is inclusive of any sample comprising a biological material obtained from a subject, such as blood, serum, plasma, saliva, sputum, urine, tissue, etc. The sample may be subjected to one or more pretreatment or processing steps prior to use in the methods (e.g. detection with the conjugates) provided herein.

[0096] In some embodiments, provided herein is a method of measuring a target in a sample, comprising contacting the sample with a conjugate provided herein. The conjugates provided herein find use in numerous methods of measuring a target in a sample, including immunoassays such as ELISA (e.g. microfluidic ELISA) and western blot, and microscopy techniques such as immunohistochemistry, immunocytochemistry, and the like. For example, in some embodiments the method of measuring a target in a sample is an ELISA format immunoassay (e.g. sandwich ELISA, direct ELISA, indirect ELISA, competitive ELISA). In some embodiments, the conjugate is used as a primary antibody in a method of measuring a target in a sample. For example, in some embodiments the conjugate comprises a copolymer described herein and an antibody, and is used as a primary antibody in an ELISA format assay (e.g. microfluidic ELISA), such as a direct ELISA. As another example, in some embodiments the method of measuring a target in a sample is a western blot. In some embodiments, the western blot is a capillary electrophoresis western blot. For example, the conjugates provided herein can be used as a secondary antibody (e.g. detection antibody) in a western blot, including in a capillary electrophoresis western blot.

[0097] In some embodiments, provided herein is a method of measuring a target in a sample, comprising contacting the sample with a conjugate provided herein and detecting a signal from the conjugate. The conjugates provided herein may function as the primary antibody and / or the secondary antibody in methods of measuring a target in a sample. In some embodiments, the conjugate is the primary antibody in a method of measuring a target in a sample. For example, in some embodiments a method of measuring a target comprises contacting a sample with a conjugate provided herein, wherein the conjugate binds to the target, if present in the sample, and measuring a signal from the conjugate. As another example, in some embodiments, the method of measuring a target comprises contacting a sample with a conjugate comprising a copolymer provided herein and an antibody, wherein the antibody binds to the target, if present in a sample, and measuring a signal from the conjugate. In some embodiments, the target is immobilized on a substrate (e.g. coated on a plate). For example, in some embodiments provided herein is a direct ELISA format immunoassay comprising contacting a sample with a conjugate provided herein and detecting a signal from the conjugate. In some embodiments, the conjugate comprises an antibody which binds to the target, if present in the sample, and a signal from the conjugate is measured to directly detect the target in the sample. In such embodiments, the conjugate is the primary antibody for detection of target in the sample. In some embodiments, the target is biotinylated and the conjugate comprises a protein that binds to biotin (e.g. avidin, streptavidin, or a derivative thereof). In some embodiments, the conjugate binds to the biotinylated target, if present in the sample, and a signal from the conjugate is measured to directly detect the biotinylated target in the sample. In some embodiments, the biotinylated target is bound to a primary antibody (e.g. immobilized on a surface of a substrate), and the conjugate comprising the protein that binds to biotin is the secondary antibody for detection of the biotinylated target. One or more wash steps may be performed, such as prior to detection, to remove unbound conjugate.

[0098] In some embodiments, provided herein is a method contacting the sample with a primary antibody that binds to the target, if present in the sample, contacting the sample with a conjugate provided herein, and detecting a signal from the conjugate. In such embodiments, the conjugate serves as the secondary antibody for detection of the target. For example, in some embodiments provided herein is an indirect or a sandwich ELISA format immunoassay (e.g. microfluidic ELISA) comprising contacting the sample with a primary antibody that binds to the target, if present in the sample, contacting the sample with a conjugate provided herein, and detecting a signal from the conjugate. In some embodiments, such as in the indirect ELISA format immunoassay, the target is immobilized on a substrate (e.g. coated on an ELISA plate). In some embodiments, such as in the sandwich ELISA format, the primary antibody is immobilized on the substrate, and the target binds to the immobilized primary antibodies. As another example, in some embodiments provided herein is a western blot format immunoassay (e.g. a capillary electrophoresis western blot assay) comprising contacting the sample with a primary antibody that binds to the target, if present in the sample, contacting the sample with a conjugate provided herein, and detecting a signal from the conjugate. In some embodiments, (including the indirect ELISA, sandwich ELISA, and western blot methods such as capillary electrophoresis western blots) the conjugate binds to the primary antibody, and a signal from the conjugate is detected to measure the target in the sample. In some embodiments, the method comprises performing one or more wash steps. For example, one or more wash steps may be performed after contacting the sample with the primary antibody, such that unbound primary antibody is removed from the sample prior to contacting the sample with the conjugate. Alternatively or in combination, one or more wash steps may be performed after contacting the sample with the conjugate to remove unbound conjugate prior to detecting the signal.

[0099] In some embodiments, the primary antibody is biotinylated. In some embodiments, a biotinylated antibody binds to a target, if present in the sample, and a conjugate provided herein comprising avidin, streptavidin, or a derivative thereof binds to the biotinylated primary antibody, thereby producing a detectable signal when the target is present in the sample. In some embodiments, the primary antibody comprises an antibody that binds to the target, if present in the sample, and the conjugate comprises a secondary antibody raised against the host species used to generate the primary antibody. For example, if the primary antibody is raised in rabbit, the secondary antibody should be an anti-rabbit secondary antibody raised in a host species other than rabbit.

[0100] In some embodiments, provided herein is a method of measuring a target in a sample comprising contacting the sample with a biotinylated primary antibody that binds to the target, if present in the sample, contacting the sample with a conjugate provided herein comprising avidin, streptavidin, or a derivative thereof, and detecting a signal from the conjugate. The conjugate binds to the biotinylated primary antibody due to the interaction between biotin present in the primary antibody and the avidin, streptavidin, or derivative thereof present in the conjugate. In some embodiments, provided herein is a method of measuring a target in a sample comprising contacting the sample with a primary antibody that binds to the target, if present in the sample, contacting the sample with a conjugate provided herein comprising an antibody (e.g. a secondary antibody raised against the host species used to generate the primary antibody) and detecting a signal from the conjugate.

[0101] For any of the methods provided herein, suitable blocking and / or washing steps may be performed as known in the art. For example, the method may involve blocking steps to reduce non-specific binding, and / or one or more wash steps to remove blocking buffer, remove unbound primary antibody, and / or remove unbound conjugate prior to signal detection. The steps of the methods provided herein (e.g. blocking, incubating with primary antibody, washing, incubating with the conjugate) may be performed at any suitable temperature for any suitable duration of time to achieve the desired effect. For example, contacting the sample with a conjugate provided herein may involve incubating the sample and the conjugate at a suitable temperature (e.g. room temperature, or a temperature chilled below room temperature such as 4°C) for a suitable incubation time (e.g. 10 minutes to 24 hours).

[0102] The signal from the conjugate (e.g. from the HRP) may be measured by any suitable method. Generally, detection of a signal from HRP involves contacting the sample with the conjugate, as described herein, optionally washing the sample to remove unbound conjugate, and subsequently contacting the sample with an HRP substrate. The HRP substrate is selected based upon the mode of detection / immunoassay performed. In some embodiments, the substrate is a chromogenic substrate. Chromogenic substrates are particularly useful for ELISAs or other colorimetric assays. Suitable chromogenic substrates include, but are not limited to 3,3',5,5'-tetramethylbenzidine (TMB) and 2,2' -azino-di-[3-ethylbenzthiazoline-6- sulfonic acid] (ABTS).

[0103] In some embodiments, the substrate is a chemiluminescent substrate. Suitable chemiluminescent substrates include luminol-based and acridan-based reagents. In some embodiments, one or more enhancers are added to enhance the signal from the HRP. In some embodiments, the signal is normalized to a control value, and the normalized signal is indicative of the level, amount, or presence of the target in the sample.

[0104] Examples

[0105] Materials

[0106] HRP: Roche (product no. 10814407001)

[0107] NaICU: Millipore Sigma (product no. S1878) Poly-L-lysine HBr (mol wt 1-5 kDa): Millipore Sigma (product no. P0879)

[0108] Poly-L-lysine HBr (mol wt 4-15 kDa): Millipore Sigma (product no. P6516)

[0109] Streptavidin: Agilent (product no. SA10-100)

[0110] Sodium cyanoborohydride (NaBH CN) solution (5M in 1 M NaOH): Millipore Sigma, Prod 296945

[0111] Lysine: Millipore Sigma (product no. L5501)

[0112] L-Cysteine: Millipore Sigma (product no.30089) lodoacetamide: Millipore Sigma (product no. 11149)

[0113] Sulfo-SMCC: Thermo Scientific (product no. 22322).

[0114] Traut’s Reagent (2-Iminothiolane.HCl): Thermo Scientific (product number 26101).

[0115] Anti-Mouse IgG: Bio-Techne (product no. D-201-C-ABS2).

[0116] Anti-Rabbit IgG: Bio-Techne (product no. D-301-C-ABS2).

[0117] Column: TOSOH Bioscience TSKGel G3000SW 7.5mm x 30 cm column (product no 0005789).

[0118] PBS: Gibco PBS pH 7.2 (10X) (product no. 70013-016). Diluted to IX using deionized water and pH adjusted (7.2) using 2 M HC1 (aq).

[0119] 20x Borate buffer: Thermo Scientific (product no. 28341).

[0120] 0.5M EDTA, pH 8.0: Invitrogen (product no. 15575-038).

[0121] Dialysis tubing: Snakeskin™ dialysis tubing, Thermo Scientific, product no. 68700

[0122] 10 kDa Slide- A-Lyzer Mini Dialysis Devices: Thermo Scientific (product no. 88404).

[0123] 7K MWCO zeba-spin de-salting column: Thermo Scientific (product no. 89890).

[0124] 5K MWCO PD SpinTrap G25 column: Cytiva (product no. 28918004).

[0125] 5K MWCO PD MiniTrap G25 columns: Cytiva (product no. 28918007).

[0126] Spectra Por Float- A-Lyzer G2 / 1000KDa: Spectrum Labs Inc. (product no. G235037).

[0127] BCA kit: Pierce™ BCA Protein Assay Kit: Thermo Scientific (product no. 23225).

[0128] 12-230 kDa Separation Module: Bio-Techne (product no. SM-W004).

[0129] Biotinylated Ladder EZ 2: Bio-Techne (product no. 042-484).

[0130] Antibody Diluent 2: Bio-Techne (product no. 042-203)

[0131] Milk-Free Antibody Diluent: Bio-Techne (product no. 043-524).

[0132] Biotin Antibody: Fitzgerald (product no. 10-3155).

[0133] Anti-mouse Secondary Antibody: Bio-Techne (product no. 042-205).

[0134] LuminoLS: Bio-Techne (product no. 043-311.

[0135] Peroxide: Bio-Techne (product no. 043-379).

[0136] Quantikine HS ELISA human IL-6 kit: Bio-Techne (product no. HS600C). Experimental Methods

[0137] Example la One-Step Polymerization-Conjugation Method

[0138] HRP Aldehyde: HRP (18.3 mg, 0.42 pmol) was dissolved in deionized water (1.83 mL) and treated with a solution of NaIO4 (3.0 mg, 14.2 pmol) in water (178 |iL). The mixture was end-over-end rotated at 25 °C in the dark for 90 min. The solution was then dialyzed using a 10 kDa Slide-A-Lyzer in the dark for 60 min. Water was replaced and the dialysis repeated for a further 60 min to give HRP-aldehyde (-2 mL)

[0139] 1-5 kDa Poly-L-Lys Sample Preparation: 1-5 kDa Poly-L-Lys.HBr (4.8 mg) was dissolved in DI water (280 pL) and adjusted to pH 9.0 using IM LiOH (aq) (11 pL). Polymerization with Anti-Mouse IgG: The mixture was protected from light throughout the protocol. HRP aldehyde (0.25 mL / 2.5 mg in water) was added to a freshly prepared mixture of 1 M Na2CC>3 (25 pL, pH 9.5), 1-5 kDa poly-L-Lys solution (17 pL, see above for preparation) and anti-mouse IgG (32 pL, 0.25 mg; 7.7 mg / mL in PBS). The mixture was end- over-end rotated at 25 °C in the dark overnight to give a brown solution. NaBH CN (12.5 pL; 5.0 M in IM NaOH (aq)) was added and the mixture was rotated at 25 °C for 60 min. L- Lysine solution (25 pL; 40 mg / mL in 100 mM Na2CO , adjusted to pH 9.5 using 1 M HC1 (aq)) was added and the mixture was rotated at 25 °C for a further 60 min.

[0140] The solution was dialyzed against PBS using a 10 kDa Slide-A-Lyzer in the dark for 60 min. Buffer was replaced and the dialysis repeated for a further 60 min to give IgG-HRP- poly-Lys conjugate (~0.3 mL).

[0141] Polymerization with Anti-Rabbit IgG: The mixture was protected from light throughout the protocol. HRP aldehyde (0.25 mL / 2.5 mg in water) was added to a freshly prepared mixture of 1 M Na2CO3 (25 pL, pH 9.5), 1-5 kDa poly-L-Lys solution (17 pL, see above for preparation) and anti-rabbit IgG (41 pL, 0.25 mg; 6.0 mg / mL in PBS). The mixture was end-over-end rotated at 25 °C in the dark overnight to give a brown solution. NaBlLCN (12.5 pL; 5.0 M in IM NaOH (aq)) was added and the mixture was rotated at 25 °C for 60 min. L-Lysine solution (25 pL; 40 mg / mL in 100 mM Na2CO3, adjusted to pH 9.5 using 1 M HC1 (aq)) was added and the mixture was rotated at 25 °C for a further 60 min.

[0142] The solution was dialyzed against PBS using a 10 kDa Slide-A-Lyzer in the dark for 60 min. Buffer was replaced and the dialysis repeated for a further 60 min to give IgG-HRP- poly-Lys conjugate (-0.3 mL). SEC Purification of IgG Conjugates: The mixtures were further purified by SEC chromatography using a TOSOH Bioscience TSKGel G3000SW 7.5mm x 30 cm column, eluting with 0.4 M NaCl (aq) (pre-filtered through a 0.2 uM filter) at 0.8 mL / min. The IgG- HRP-poly-Lys conjugate eluted at the void volume as a brown solution and was collected accordingly (~1.2 mL @ -0.9 mg / mL by BCA analysis).

[0143] Polymerization with Streptavidin: HRP aldehyde (250 pL / 2.5 mg in water) was added to a freshly prepared mixture of 1 M Na2COs buffer (25 pL, pH 9.5), 1-5 kDa poly-L- Lys solution (17 pL, see above for preparation) and Streptavidin (25 pL, 0.25 mg; 10 mg / mL in water). The mixture was end-over-end rotated at 25 °C in the dark overnight to give a brown solution. NaBH CN (12.5 pL; 5.0 M in IM NaOH (aq)) was added and the mixture was rotated at 25 °C for 60 min. L-Lysine solution (25 pL; 40 mg / mL in 100 mM Na2CO , adjusted to pH 9.5 using 1 M HC1 (aq)) was added and the mixture was rotated at 25 °C for a further 60 min.

[0144] The solution was dialyzed against PBS using a 10 kDa Slide- A-Lyzer for 60 min. The buffer was replaced and dialysis continued for a further 60 min to give Streptavidin-HRP- poly-Lys conjugate.

[0145] 4-15 kDa Poly-L-Lys Sample Preparation: 4-15 kDa Poly-L-Lys.HBr (21.2 mg) was dissolved in DI water (3.15 mL) and adjusted to pH 9.0 using IM LiOH (aq) (45 pL). Polymerization with Anti-Mouse IgG: The mixture was protected from light throughout the protocol. HRP aldehyde (250 pL / 2.5 mg in water, prepared as described above) was treated with 1 M Na2COs (25 pL, pH 9.5), 4-15 kDa poly-L-Lys solution (20 pL, see above for preparation) and anti-mouse IgG (34 pL, 0.27 mg; 8.0 mg / mL in PBS). The mixture was end- over-end rotated at 25 °C in the dark overnight to give a brown solution. NaBH CN (25 pL; 5.0 M in IM NaOH (aq)) was added and the mixture was rotated at 25 °C for 60 min. L- Lysine solution (100 pL; 40 mg / mL in 100 mM Na2CO , adjusted to pH 9.5 using 1 M HC1 (aq)) was added and the mixture was rotated at 25 °C for a further 60 min.

[0146] The solution was dialyzed against PBS using a 10 kDa Slide- A-Lyzer in the dark for 60 min. Buffer was replaced and the dialysis repeated for a further 60 min to give IgG / Poly- HRP conjugate (-0.5 mL). SEC purification (as described above) afforded polymer (0.8 mL @ 0.19 mg / mL by BCA analysis). Example lb

[0147] Co-polymerization of HRP and Poly-L-Lys Followed by Conjugation to a Protein 1-5 kDa Poly-L-Lys Sample Preparation: 1-5 kDa Poly-L-Lys.HBr (8.54 mg) was dissolved in DI water (500 pL) and adjusted to pH 9.0 using IM LiOH (aq) (20 pL).

[0148] Polymerization: HRP aldehyde in water (1000 pL, prepared as described in example la) was treated with 1 M Na2COs (100 pL, pH 9.5) and 1-5 kDa poly-L-Lys solution (68 pL, see above for preparation). The mixture was end-over-end rotated at room temperature in the dark overnight to give a brown solution. NaBH CN (12.5 pL; 5.0 M in IM NaOH (aq)) was added and the mixture was rotated at room temperature for 60 min. L-Lysine solution (12.5 pL; 80 mg / mL in 100 mM Na2CO , adjusted to pH 9.5 using 1 M HC1 (aq)) was added and the mixture was rotated at room temperature for a further 60 min. The solution was then dialyzed using PBS in a 10 kDa Slide- A-Lyzer in the dark for 120 min. PBS was replaced and the dialysis repeated for a further 60 min to give HRP-poly-Lys copolymer (~1.0 mL @ 4.77 mg / mL)

[0149] Conjugation of IgG:

[0150] Activation of poly-HRP: HRP-poly-Lys copolymer (377 pL; 4.77 mg / mL in PBS) was treated with PBS (276 pL) and sulfo-SMCC (100 pL; 1 mg / mL in PBS), the mixture being end-over-end rotated at room temperature for 45 min to give poly-HRP maleimide.

[0151] Activation of IgG: Dissolved Anti-mouse IgG (0.5 mg) in PBS containing 5mM EDTA (100 pL, adjusted to pH 7.2 using 2M HC1 (aq)). Added a freshly prepared solution of Traut's reagent (11 pL; 1 mg / mL in PBS,) to the IgG solution. End-over-end rotated at RT for 60 min and immediately de-salted using a 5K MWCO PD SpinTrap G25 columns to give thiolated-IgG solution (0.1 mL).

[0152] Conjugation of IgG to HRP-Polymer: Poly-HRP maleimide (0.21 mL; 2.4 mg / mL, prepared as described above) was treated with thiolated-IgG (100 pL, freshly prepared as above) and the mixture was end-over-end rotated at room temperature overnight. Cysteine (25 pL; 50 mg / mL cysteine.HCl in PBS adjusted to pH 7.2 using 2M NaOH) and the mixture was rotated for 15 min. lodoacetamide (30 pL; 100 mg / mL in 20x borate buffer) was added and the mixture was rotated at room temperature for a further 15 min. The solution was then dialyzed against PBS in a 10 kDa Slide- A-Lyzer in the dark for 60 min. PBS was replaced and the dialysis repeated for a further 60 min to give IgG-HRP-poly-Lys conjugate (~0.3 mL) SEC Purification of IgG Conjugates: IgG-HRP-poly-Lys conjugate was further purified by SEC chromatography using a TOSOH Bioscience TSKGel G3000SW 7.5mm x 30 cm column, eluting with 0.4 M NaCl (aq) (pre-filtered through a 0.2 uM filter) at 0.8 mL / min. The IgG-HRP-poly-Lys co-polymer eluted at the void volume as a brown solution and was collected accordingly (~0.8 mL @ 0.29 mg / mL by BCA analysis).

[0153] 4-15 kDa Poly-L-Lys Sample Preparation: 4-15 kDa Poly-L-Lys.HBr (21.4 mg) was dissolved in DI water (2000 pL) and adjusted to pH 9.5 using IM LiOH (aq) (50 pL).

[0154] Polymerization: HRP aldehyde in water (9 mL, prepared as described in example la) was treated with 1 M Na2COs (750 pL, pH 9.5) and 4-15 kDa poly-L-Lys solution (485 pL, see above for preparation). The mixture was end-over-end rotated at room temperature in the dark overnight to give a brown solution. NaBH CN (450 pL; 5.0 M in IM NaOH (aq)) was added and the mixture was rotated at room temperature for 60 min. L- Lysine solution (1800 pL; 20 mg / mL in 100 mM Na2CO , adjusted to pH 9.5 using 1 M HC1 (aq)) was added and the mixture was rotated at room temperature for a further 120 min.

[0155] The resultant mixture was dialyzed against PBS (2 L, pH 7.2) using a 7 kDa dialysis membrane in the dark for 90 min. Buffer was replaced and the dialysis repeated for a further 360 min to give HRP-poly-Lys co-polymer (-11 mL @ 2.16 mg / mL by BCA analysis).

[0156] Conjugation of Streptavidin.

[0157] Activation of poly-HRP: HRP-poly-Lys co-polymer (0.5 mL @ 2.16 mg / mL) was treated with sulfo-SMCC (0.5 mg, 1.16 pmol), the mixture being end-over-end rotated at room temperature. After 30 min, a further portion of sulfo-SMCC (0.5 mg, 1.16 pmol) was added and rotation continued for a further 30 min. The mixture was then dialyzed against PBS using a 10 kDa Slide- A-Lyzer in the dark for 60 min. The buffer was replaced and dialysis continued for a further 60 min to give poly-HRP maleimide.

[0158] Activation of Streptavidin: Dissolved Streptavidin (10.5 mg) in PBS containing 5mM EDTA (1.05 mL, adjusted to pH 7.2 using 2M HC1 (aq)). Added a freshly prepared solution of Traut's reagent (288 pL; 2 mg / mL in PBS) to the Streptavidin solution. End-over- end rotated at RT for 60 min and immediately de-salted using a 7K MWCO zeba-spin desalting column to give thiolated-Streptavidin solution (1.5 mL).

[0159] Conjugation of Streptavidin to HRP-Polymer: Poly-HRP maleimide (0.5 mL) was treated with thiolated-Streptavidin (158 pL, prepared as described above) and the mixture was end-over-end rotated at room temperature overnight. Cysteine (25 pL; 50 mg / mL cysteine.HCl in PBS adjusted to pH 7.2 using 2M NaOH) and the mixture was rotated for 15 min. lodoacetamide (40 pL; 100 mg / mL in PBS) was then added followed immediately by 20x borate buffer (20 pL). The mixture was rotated at room temperature for a further 15 min. Purification by Dialysis: The conjugate was dialyzed against PBS using a Spectra Por Float- A-Lyzer G2 / 1000KDa in the dark at room temperature overnight. The buffer was replaced and dialysis continued overnight again. This was repeated until analysis by SDS- PAGE showed removal of unreacted Streptavidin.

[0160] Conjugation of IgG:

[0161] Activation of IgG: Dissolved Anti- mouse IgG (3.0 mg) (or Anti-Rabbit IgG (3.0 mg)) in PBS containing 5mM EDTA (0.60 mL, adjusted to pH 7.2 using 2M HC1 (aq)). Added a freshly prepared solution of Traut's reagent (23 pL; 3 mg / mL in PBS) to the IgG solution. End-over-end rotated at RT for 60 min and immediately de-salted using 2 x 5K MWCO PD MiniTrap G25 columns to give thiolated IgG solution (0.6 mL).

[0162] Conjugation of IgG to HRP-Polymer: IgG-HRP-poly-Lys co-polymer conjugate was prepared as described above for Streptavidin conjugation except Anti-mouse IgG (1 mg I 1 mg HRP maleimide) or Anti-rabbit IgG (1 mg / 1 mg HRP maleimide) was used in place of Streptavidin.

[0163] Example 2 Use of Conjugates - Western Blot

[0164] Anti-mouse IgG and anti-rabbit IgG derived poly-HRP conjugates were prepared as described in Example 1 and tested using the Jess Simple Western platform. The optimized copolymers gave a consistently higher signal than the signal generated using commercially available material (cf + / -40% variability seen for commercially available material).

[0165] In a typical test assay, the biotinylated ladder EZ 2 was used as the sample of interest. A block is performed using AD2, followed by treatment with mouse (or rabbit) anti-biotin primary IgG. This is then detected using the matching species (anti-mouse or anti-rabbit) poly-HRP conjugate with signal generated by treatment with luminol / H2O2.

[0166] HRP-IgG conjugates prepared by co-polymerization of 4-15 kDa (or 1-5 kDa) poly-L- Lys and HRP followed by subsequent IgG conjugation gave material of high activity, data comparable to the control lot (FIG. 3).

[0167] Similarly, poly-HRP-IgG conjugates prepared by co-polymerization of 1-5 kDa poly- L-Lys, HRP and IgG were shown to give high signal, in excess of, or comparable, to those generated using commercially available material (FIG. 4).

[0168] Streptavidin conjugates prepared via either route gave similarly positive results when tested using a similar assay (no primary antibody required), particularly those prepared by copolymerization of 4-15 kDa (or 1-5 kDa) poly-L-Lys and HRP followed by subsequent streptavidin conjugation. Example 3

[0169] Use of Conjugates - ELISA

[0170] Streptavidin-poly-HRP conjugates were prepared as described above and tested using the Bio-Techne IL-6 Quantikine HS ELISA kit. The optimized copolymers gave high signal, comparable to those generated using commercially available material (Table 1).

[0171] Table 1. IL-6 Quantikine HS ELISA data using commercially available Streptavidin-poly-HRP conjugates Poly-HRP-Streptavidin conjugates prepared by co-polymerization of 4-15 kDa (or 1-5 kDa) poly-L-Lys and HRP followed by subsequent Streptavidin conjugation gave material of high activity, data comparable to the control lot. Poly-HRP-Streptavidin conjugates prepared by co-polymerization of 1-5 kDa poly-L-Lys, HRP and Streptavidin were shown to give moderate signal compared to those generated using commercially available material (Table 2).

[0172] Table 2. IL-6 Quantikine HS ELISA data using Streptavidin-poly-HRP conjugates prepared as described herein

Claims

CLAIMS1. A copolymer comprising: a lysine-rich polypeptide and horseradish peroxidase.

2. The copolymer of claim 1 , wherein the lysine-rich polypeptide is a poly-lysine.

3. The copolymer of claim 2, wherein the lysine-rich polypeptide is poly-L-lysine.

4. The copolymer of claim 2 or claim 3, wherein the poly-lysine has a molecular weight of about 1 kDa to about 50 kDa.

5. The copolymer of claim 4, wherein the poly-lysine has a molecular weight of about 1 kDa to about 15 kDa.

6. The copolymer of claim 4, wherein the poly-lysine has a molecular weight of about 1 kDa to about 5 kDa.

7. The copolymer of claim 4, wherein the poly-lysine has a molecular weight of about 4 kDa to about 15 kDa.

8. The copolymer of any one of claims 1-7, wherein the copolymer has a molecular weight of greater than 7000 kDa.

9. The copolymer of any one of claims 1-8, wherein the copolymer has a molecular weight of about 20 MDa to about 50 MDa.

10. A method of preparing the copolymer of any one of claims 1-9, comprising:(a) reacting horseradish peroxidase with sodium periodate to generate an aldehyde- functionalized horseradish peroxidase; and(b) reacting the aldehyde-functionalized horseradish peroxidase with the lysine-rich polypeptide to generate the copolymer.

11. A conjugate comprising the copolymer of any one of claims 1-9 and a protein.

12. The conjugate of claim 11, wherein the copolymer is directly bound to the protein via a covalent bond.

13. The conjugate of claim 11, wherein the copolymer is bound to the protein via a linker that comprises a moiety resulting from a reaction between two complementary reactive groups.

14. The conjugate of claim 13, wherein the two complementary reactive groups are selected from: an azide and an alkyne; a thiol and a maleimide; and a trans-cyclooctene and a tetrazine.

15. The conjugate of claim 13, wherein the linker comprises a moiety selected from:

16. The conjugate of any one of claims 11-15, wherein the protein is an antibody.

17. The conjugate of claim 16, wherein the antibody is an IgG antibody.

18. The conjugate of claim 16, wherein the antibody is an antibody fragment.

19. The conjugate of any one of claims 11-15, wherein the protein is avidin or streptavidin, or a derivative thereof.

20. The conjugate of any one of claims 11-19, for use in a method of measuring a target in a sample.

21. A method of preparing the conjugate of any one of claims 11-20, comprising:(a) reacting horseradish peroxidase with sodium periodate to generate an aldehyde- functionalized horseradish peroxidase; and(b) reacting the aldehyde-functionalized horseradish peroxidase with the lysine-rich polypeptide to generate a copolymer; and(c) reacting the copolymer with the protein to generate the conjugate.

22. The method of claim 21, wherein the protein is an antibody.

23. The method of claim 21, wherein the protein is avidin or streptavidin, or a derivative thereof.

24. A method of preparing a conjugate of any one of claims 11-20, comprising:(a) reacting horseradish peroxidase with sodium periodate to generate an aldehyde- functionalized horseradish peroxidase; and(b) reacting the aldehyde-functionalized horseradish peroxidase with the lysine-rich polypeptide and the protein to generate the conjugate.

25. The method of claim 24, wherein the protein is an antibody.

26. The method of claim 24, wherein the protein is avidin or streptavidin, or a derivative thereof.

27. A method of measuring a target in a sample, the method comprising contacting the sample with the conjugate of any one of claims 11-20.

28. A method of measuring a target in a sample, the method comprising: a) contacting the sample with the conjugate of any one of claims 11-20, wherein the conjugate binds to the target, if present in the sample; and b) detecting a signal from the conjugate.

29. A method of measuring a target in a sample, the method comprising: a) contacting the sample with a primary antibody that binds to the target, if present in the sample; b) contacting the sample with the conjugate of any one of claims 11-20; wherein the conjugate binds to the primary antibody; and c) detecting a signal from the conjugate.

30. A method of measuring a target in a sample, the method comprising: a) contacting the sample with a biotinylated primary antibody that binds to the target, if present in the sample; b) contacting the sample with the conjugate of claim 19, wherein the avidin or streptavidin or derivative thereof of the conjugate binds to the biotinylated primary antibody; and c) detecting a signal from the conjugate.

31. A method of measuring a target in a sample, the method comprising: a) contacting the sample with a primary antibody that binds to the target, if present in the sample; b) contacting the sample with the conjugate of any one of claims 16-18, wherein the antibody of the conjugate binds to the primary antibody; and c) detecting a signal from the conjugate.

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