Immobilised biomolecule

Abstract

A modified biomolecule for immobilising on a substrate for an assay, comprises a biomolecule, wherein the biomolecule comprises or consists of a cyclic peptide; a linker, wherein the linker is attached to the biomolecule via a N- or C-terminus of the peptide; and a binding group, wherein the binding group is attached to the linker and is capable of covalently binding to the substrate.

Description

[0001] IMMOBILISED BIOMOLECULE

[0002] FIELD

[0003] The present invention relates to a modified biomolecule for immobilising on a substrate for an assay, to a method of immobilising the modified biomolecule on the substrate, to an immobilised biomolecule, and to the use of such an immobilised biomolecule in a method of detecting the presence of a target biomolecule in a sample.

[0004] BACKGROUND

[0005] A microarray is a two-dimensional array of a biological material, or “biomolecules”, such as proteins, DNA, antigens, antibodies, lipids, or peptides, deposited and immobilised on a solid and typically flat substrate, typically made of functionalised glass or plastic. The substrate is generally selected to provide adequate binding with the biomolecule, but also to avoid any degradation of the material or its function, such as denaturation in the case of a protein / peptide. Typically, once the microarray of biological material has been deposited on the substrate, a blocking buffer is applied to the microarray surface to prevent unwanted materials binding to the surface of the substrate. The microarray is then reacted with a sample, and detection is carried out by suitable detection methods, such as the addition of one or more detection reagents, optionally followed by imaging techniques. An example of such microarraybased technology used for blood grouping and donor disease screening is MosaiQ® by AliveDx. Deposition of the biological material is typically carried out by printing, for example using a system such as MosaiQ® Manufacturing System (MMS), Arrayjet’s Inkjet Microarray Technology or Scienion’s sciFLEXARRAYER.

[0006] In order to ensure that the deposited biomolecule is immobilised on the substrate upon printing, it is desirable for the biomolecule to interact with, e.g., bind to, the substrate. Typical substrates have a coating that includes reactive groups which react, e.g., covalently or non-covalently, with the biomolecule, in order to promote binding. An example of such a substrate is a substrate, e.g., glass, coated with a reactive electrophilic material, such as epoxy-containing material, e.g., an epoxysilane material. Such substrates are commonly referred to as “epoxy glass”. An advantage of these substrates is that the epoxy groups in the coating may react with chemical groups that may be present in biomolecules, thereby enabling immobilisation upon printing.

[0007] However, certain small biomolecules, such as peptides, can be challenging to immobilise on substrates without altering or degrading their capacity to interact with other biomolecules. With peptides, and notably cyclic peptides, a key challenge is to ensure that when immobilised they are optimally oriented and spaced away from the substrate surface to maximise their interaction with a sample (often comprising antibodies), and / or detection reagents. This is important to enable a low limit of detection (LoD) and therefore provide a sensitive, reliable, and accurate assay.

[0008] It is known to immobilise peptides to solid surfaces through “biotinylation”, that is, modifying a peptide with biotin, which is then able to bind to a substrate coated with avidin or streptavidin. Biotinylation exploits the protein-substrate complex (that is, a small molecule substrate, not to be confused with a solid surface for immobilisation) that forms upon contacting the biotin-modified biomolecule with the avidin / streptavidin coated substrate. Such a complex is formed through non-covalent intermolecular interactions.

[0009] Biotinylation is a relatively complex immobilisation technique, as it requires at least the printing of streptavidin / avidin onto a substrate prior to binding a peptide comprising biotin. This two-step printing process can result in an increase in process time, cost, and complexity. Additionally, whilst the streptavidin / avidin-biotin interaction is very strong, it is still nonetheless a non-covalent interaction which may have stability issues under particular conditions over extended periods of time. Furthermore, when biotinylation is used for capture phase immobilisation, this prevents use of biotinylated secondary antibodies for the detection step of the immunoassay. As such, it is typically not possible to carry out a 3-step immunoassay (comprising a signal amplification step though streptavidin / avidin-HRP binding, for example), which is often necessary for applications requiring high sensitivity. This is because a secondary biotinylated antibody will bind to the streptavidin / avidin coated substrate non-specifically.

[0010] WO 03 / 044189 A1 describes a nucleotide sequence coding for a modified protein, said sequence comprising a gene coding for said protein, and a nucleotide fragment, ‘polyK’, coding for a succession of at least six lysine residues, for immobilisation on a substrate. It is thought that the positive charge of the lysine residues enables non-covalent interactions with a negatively charged substrate, aiding in immobilisation of the modified protein to the substrate.

[0011] It is an object of the present invention to provide a modified biomolecule for immobilising on a substrate that allows for improved orientation and spacing when immobilised on the substrate and for improved stability through covalent binding, in turn providing an immobilised biomolecule for an assay. It is another object of the present invention to provide for a method of detecting the presence of a target biomolecule in a sample, comprising the use of said immobilised biomolecule. SUMMARY

[0012] The present inventors have found that a biomolecule may be modified so as to improve immobilisation on a substrate. For example, such modification may favourably affect the orientation and spacing of the biomolecule when immobilised for an assay. Specifically, the inventors have identified that a biomolecule that comprises or consists of a peptide can be covalently bound to a substrate, by modifying the biomolecule with a linker and a binding group.

[0013] According to a first aspect of the present invention, there is provided a modified biomolecule for immobilising on a substrate for an assay, the modified biomolecule comprising: a biomolecule, wherein the biomolecule comprises or consists of a peptide; a linker, wherein the linker is attached to the biomolecule; and a binding group, wherein the binding group is attached to the linker and is capable of covalently binding to the substrate.

[0014] The present inventors have identified a method of immobilising a modified biomolecule on a substrate. The modified biomolecule may be a modified biomolecule according to the first aspect. Thus, according to a second aspect of the invention, there is provided a method of immobilising a modified biomolecule on a substrate, comprising: contacting the modified biomolecule with the substrate. The modified biomolecule may comprise: a biomolecule, wherein the biomolecule comprises or consists of a peptide; a linker, wherein the linker is attached to the biomolecule, optionally via a N- or C-terminus of the peptide; and a binding group, wherein the binding group is attached to the linker and is capable of covalently binding to the substrate. The substrate may comprise or may be made of a glass coated with a reactive electrophilic material, such as epoxy glass. The peptide may be a cyclic peptide, which may comprise a ring formed through a disulfide bridge.

[0015] The present inventors have found that immobilised biomolecules with desirable properties for assays are obtainable by the method of the second aspect. Thus, according to a third aspect of the invention, there is provided an immobilised biomolecule obtainable by the method of the second aspect of the invention.

[0016] In a fourth aspect, there is provided an immobilised biomolecule for an assay, immobilised on a substrate. The immobilised biomolecule may be a modified biomolecule according to the first aspect covalently bound to a substrate. The substrate may comprise or may be made of a glass coated with a reactive electrophilic material, e.g. epoxy glass. The present inventors have found that the immobilised biomolecule according to the third or fourth aspect of the invention is notably effective in an assay to detect the presence of an antibody in a sample. Thus, according to a fifth aspect of the invention, there is provided a method of detecting the presence of a target biomolecule in a sample, the method comprising: (a) contacting the sample with an immobilised biomolecule according to the third or fourth aspect of the invention under conditions that allow the formation of a complex between the immobilised biomolecule and the target biomolecule; and (b) contacting the complex with one or more detection reagents.

[0017] DETAILED DESCRIPTION

[0018] Definitions:

[0019] In the discussion that follows, reference is made to a number of terms, which are to be understood to have the meanings provided below, unless a context indicates to the contrary. The nomenclature used herein for defining compounds, in particular the compounds described herein, is intended to be in accordance with the rules of the International Union of Pure and Applied Chemistry (IUPAC) for chemical compounds, specifically the “IUPAC Compendium of Chemical Terminology (Gold Book)” (see A. D. Jenkins et al., Pure & Appl. Chem., 68, 2287-2311 (1996)). For the avoidance of doubt, if an IUPAC rule is contrary to a definition provided herein, the definition herein is to prevail.

[0020] The term “comprising” or variants thereof is to be understood herein to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0021] The term “consisting” or variants thereof is to be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, and the exclusion of any other element, integer or step or group of elements, integers or steps.

[0022] The terms “biological molecule”, “biomolecule”, or “biological material” are well known in the art and relate to any molecule present in organisms that is involved in one or more typically biological processes. Biological molecules may include but are not limited to proteins, peptides, antibodies, antigens, carbohydrates, lipids, nucleic acids, polynucleotides, vitamins, amino acids, and hormones. Biological molecules may be extracted from their natural source. They may be produced by synthetic or biotechnological means. Biological molecules may be of an unnatural origin, have no known biological purpose, or may not be known to be involved in any biological process. They may be engineered or produced in such a way to differ from their natural counterparts.

[0023] The term “complex” used herein is well known in the biochemical arts and relates to a stable association between two or more molecules to form a single unit. Complexes may form as the result of a small-molecule substrate binding to a biological molecule, an antigen binding to an antibody, a protein binding to another protein, or any other inter- biomolecular interaction. When one molecule is bound to one other molecule, this is often referred to as a binary complex. When one molecule is bound to two other molecules, this is often referred to as a ternary complex. As a result of the formation of the complex, the complex may gain or lose particular functions relative to the functions of the constituent molecules. For example, a ligand that binds to a biological molecule to form a complex may result in the biological molecule losing its normal function when part of the complex. On the other hand, a ligand that binds to a biological molecule to form a complex may result in the biological molecule gaining a new or unnatural function when part of the complex. The binding may occur due to any one or more non-covalent intermolecular forces, including but not limited to ionic bonding, hydrogen bonding, Van der Waals forces, London dispersion forces, dipole-dipole interactions, ion-dipole interactions, salt bridges, TT-TT interactions, ion-TT interactions, hydrophobic effects, hydrophilic effects and halogen bonding. Binding to form a complex may also occur due to covalent binding, including but not limited to disulfide bonds, peptide (amide) bonds, ester bonds, glycosidic bonds, and thioester bonds.

[0024] The term “alkyl” is well known in the art and defines univalent groups derived from alkanes by removal of a hydrogen atom from any carbon atom, wherein the term “alkane” is intended to define acyclic branched or unbranched hydrocarbons having the general formula CnH2n+2, wherein n is an integer s 1. Ci-4alkyl refers to any one selected from the group consisting of methyl, ethyl, n-propyl, / so-propyl, n-butyl, sec-butyl, / so- butyl and tert-butyl.

[0025] The term “hydroxy” is well known in the art and defines the univalent group derived from water by the removal of one hydrogen atom. Hydroxy groups are often depicted as -OH.

[0026] The term “thiol” is well known in the art and defines the univalent group derived from hydrogen sulfide by the removal of one hydrogen atom. Thiol groups are often depicted as -SH.

[0027] The term “amino” refers herein to an -NR1R2group, wherein R1and R2are independently H or hydrocarbon-derived substituents, such as alkyl, alkenyl, alkynyl, carbocycle, and their substituted counterparts. R1and R2may be unsubstituted. In some cases, R1and R2are joined together. For example, R1and R2may be joined together in such a way that the nitrogen atom of -NR1R2is part of a ring system. When R1and R2are H, the compound comprising the amino group may be referred to as a primary amine. When one of R1or R2is H, the compound may be referred to as a secondary amine. When neither R1nor R2are H, the compound may be referred to as a tertiary amine. Often, amino refers to the univalent group derived from ammonia by the removal of one hydrogen atom, often depicted as -NH2.

[0028] The term “azide” refers to a compound that comprises a linear monovalent -N3 group, otherwise represented as -N=N+=N".

[0029] The term “epoxy” refers to the univalent group derived from oxirane (a threemembered heterocycle comprising one oxygen atom), by the removal of one hydrogen atom. Epoxy groups are highly electrophilic and are often comprised in coated glass substrates.

[0030] The term “contacting” is used herein to refer to any one or more of the acts of combining, such as reacting, printing, mixing, stirring, slurrying, blending, dissolving, treating, incubating, passing over, flowing over, or otherwise, in any order, and for any length of time.

[0031] The term “about” herein, when qualifying a number or value, is used to refer to values that lie within ± 5% of the value specified. For example, references to spot size at “about 150-200 micrometres” is intended to include sizes of 142.5 and 210 micrometres.

[0032] Modified biomolecules:

[0033] As described above, according to a first aspect of the invention, there is provided a modified biomolecule for immobilising on a substrate for an assay, the modified biomolecule comprising: a biomolecule, wherein the biomolecule comprises or consists of a peptide; a linker, wherein the linker is attached to the biomolecule; and a binding group, wherein the binding group is attached to the linker and is capable of covalently binding to the substrate.

[0034] In some embodiments, the assay is an immunoassay, such as sandwich immunoassay, indirect immunoassay, radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), sandwich enzyme-linked immunosorbent assay, antigen-down immunoassay, fluorescence immunoassay, chemiluminescent immunoassay, competitive exclusion immunoassay, radial diffusion immunoassay, “dipstick” immunoassay, and laminar flow immunoassay. Typically, the assay is a planar immunoassay with capture ligands immobilised on a rigid planar support (microarray).

[0035] As described above, the biomolecule comprises or consists of a peptide.

[0036] The linker may be attached to the biomolecule by any suitable covalent bond.

[0037] In some embodiments, the linker is attached through a group on the biomolecule selected from amino, thio, carboxy, hydroxy, arginine, pyrrolidine, phenol, indole, acetamide, urea, aldehyde, ketone, phosphate, ester, and azide.

[0038] In some embodiments, the linker is attached to the biomolecule through the / V- or C-terminus of the peptide.

[0039] Peptides are understood to comprise an / V-terminus and a C-terminus. An / V- terminus relates to an “end” of the peptide, whereby an amino acid residue of the peptide is attached to the rest of the peptide through only the carboxylic acid, leaving a free amino group, often depicted as -NH2, or-NF . Typically, the linker is be attached to the biomolecule through the / V-terminus.

[0040] In some embodiments where the linker is attached through the / V-terminus, the linker is attached through the amino group of the / V-terminus to form an amide, thioamide, or amidine group with the linker. Typically, the linker is attached through the amino group of the / V-terminus to form an amide group with the linker.

[0041] In some embodiments, the linker is attached to the biomolecule through the C- terminus of the peptide. A C-terminus relates to an “end” of the peptide, whereby an amino acid residue of the peptide is attached to the rest of the peptide through only the amino group, leaving a free carboxy group, often depicted as -CO2H, or -CCh".

[0042] In some embodiments where the linker is attached through the C-terminus, the linker is attached through the carboxy group of the C-terminus to form an amide, ester, or thioester group with the linker.

[0043] In some embodiments, the peptide comprises or consists of a cyclic peptide.

[0044] A cyclic peptide is herein understood as a peptide that comprises a ring, or a cycle. That is to say, a cyclic peptide comprises a circular sequence of bonds. A cyclic peptide may comprise one or more rings. The ring of a cyclic peptide may be formed through the connection of: an / V-terminus and a C-terminus to form a peptide (amide) bond; an / V-terminus and a group on a side chain, for example, the carboxy group on the side chain of a glutamic acid residue; a C-terminus and a group on a side chain, for example, the amino group on the side chain of a lysine residue; or two or more side chains, for example, two cysteine residues to form a disulfide bridge. A ring may also be formed from the modification of an / V-terminus or amino group with a bromoacetyl group, to form a bromoacetamide, which can then react with a thiol group of a cysteine residue to form a thioether, thus forming a ring. In some embodiments, the ring of the cyclic peptide is formed through one or more side chains of the peptide, such as through one or more thiol groups, for example on one or more cysteine residues.

[0045] Typically, the ring is formed through a disulfide bridge. Often where the ring is formed through one or more side chains, the / V- and / or C-terminals of the peptide may be free, for example, to react with a linker. The inventors have surprisingly found that, whilst immobilisation of a cyclic peptide can be improved by modifying the peptide with a linker and binding group, wherein the linker is attached to the biomolecule via a / V- or C-terminus of the peptide, it is surprising that the subsequent analysis and / or detection of the immobilised peptide was not adversely affected despite N-terminus or C-terminus modification of the peptide.

[0046] In some embodiments, the peptide comprises one or more non-proteinogenic amino acids, such as D-amino acids, / V-methyl amino acids, hydroxyproline, betaalanine, citrulline, ornithine, norleucine 3-nitrotyrosine, nitroarginine, and pyroglutamic acid. A peptide that comprises a citrulline amino acid residue may be referred to as a “citrullinated peptide”. The full IIIPAC name for citrulline is 2-amino-5- (carbamoylamino)pentanoic acid. Typically, where the peptide is a citrullinated peptide, the one or more citrullines may have been introduced during peptide synthesis.

[0047] In some embodiments, the peptide comprises or consists of a cyclic peptide. In some embodiments, the peptide comprises or consists of a cyclic citrullinated peptide (CCP). The preparation and sequences of several relevant CCPs can be found in US Patent No. 7,888,133, which is incorporated herein by reference. In some embodiments, the peptide is a CCP, such as any one of SEQ ID NO: 1-12. Typically, the peptide is P2, or SEQ ID NO: 2.

[0048] Peptide sequences may be found in Table 4 below and the enclosed sequence listing.

[0049] Each of sequences 1-13 may be linked / V-terminally and / or C-terminally to 1-4 residues of any amino acids, or synthesised as part of a larger 14 to 22-mer peptide (for SEQ ID NO: 1-12), or synthesised as part of a larger 15 to 23-mer peptide (for SEQ ID NO: 13). That is to say, each of sequences 1-13 may begin and / or end with (X)n, wherein each X is independently any amino acid, and n is 0 to 4. Furthermore, each of sequences 1-14 comprises an intrachain disulfide bond between the two defined cysteine residues. Each defined X is citrulline. For example, in some embodiments, the peptide is SEQ ID NO: 2, or P2, specifically (X)n-CHQFRFXGWSRAAC-(X)n, wherein the / V- and C-terminal X residues are each independently any amino acid; n is an integer from 0 to 4; the defined X (position 7) is citrulline; and there is an intrachain disulfide bond between the cysteines in positions 1 and 14. This applies mutatis mutandis to all of SEQ ID NO: 1-13. SEQ ID NO: 14 may be modified as such at the C-terminus.

[0050] Each of SEQ ID NO: 1-13 may be modified at the / V-terminus (position 1) or C- terminus, typically the / V-terminus, with a linker comprising a binding group, thus forming a modified biomolecule. Where a sequence comprises additional undefined amino acid residues at the C- and / or / V-terminus (described above as linked / V-terminally and / or C- terminally to 1-4 residues of any amino acids, represented as (X)n), the modification is at the respective N- or C-terminus of the variant sequence. That is to say, each of SEQ ID NO: 1-13, and any variants linked / V-terminally and / or C-terminally to 1-4 residues of any amino acids, may be modified at the / V-terminus (position 1) or C-terminus, typically the / V-terminus, with a linker comprising a binding group, thus forming a modified biomolecule. For example, any CCP may be modified with a dPEG4 or dPEGi2 linker. For example, SEQ ID NO: 2 may be modified such that the modified biomolecule comprises the sequence dPEG4-CHQFRFXGWSRAAC, or dPEGi2-CHQFRFXGWSRAAC, respectively. In a further example, SEQ ID NO: 2 may be modified such that the modified biomolecule comprises the sequence dPEG4- (X)n-CHQFRFXGWSRAAC-(X)n, or dPEGi2-(X)n-CHQFRFXGWSRAAC-(X)n, where (X)nis as defined above. This may be written alternatively, for example as dPEG4-(SEQ ID NO: 2), or dPEG4-P2.

[0051] Additionally, P2 may be modified with a polylysine chain at the / V-terminus, typically a four-lysine chain (‘K4-P2’). This sequence, KKKKCHQFRFXGWSRAAC, corresponds to SEQ ID NO: 14, wherein X is citrulline, and there is an intrachain disulfide bond between the cysteines in position 5 and 18.

[0052] In some embodiments, the peptide may be H1 , which is the primary immunodominant domain of the HIV-1 gp41 glycoprotein. H1 , like the CCPs, is a cyclic peptide. H1 corresponds to SEQ ID NO: 13, specifically GLWGCSGKLTCTTAV, wherein there is an intrachain disulfide bond between the cysteines in position 5 and 11. As with the CCPs, SEQ ID NO: 13 may be modified at the / V-terminus (position 1) with a linker comprising a binding group, thus forming a modified biomolecule. For example, H1 may be modified with a dPEG4 linker, such that the modified biomolecule comprises the sequence dPEG4-CHQFRFXGWSRAAC. This may be written alternatively, for example as dPEG4-(SEQ ID NO: 13), or dPEG4-H1 . The peptide may be a non-cyclic peptide. In some embodiments, the peptide may be D1 , which is a 50-mer acyclic peptide encompassing residues 310-359 of the human Zinc Transporter 8 (ZnT8) protein which is found on the surface of pancreatic beta cells. D1 corresponds to SEQ ID NO: 15. SEQ ID NO: 15 may be modified at the / V-terminus (position 1) with a linker comprising a binding group, thus forming a modified biomolecule. For example, D1 may be modified with a dPEG4 or a dPEGi2 linker, such that the modified biomolecule comprises the sequence dPEG4-(SEQ ID NO: 15) or dPEGi2-(SEQ ID NO: 15), which may be written dPEG4-D1 or dPEGi2-D1 .

[0053] The term “immobilising” is well known in the art of assay development, and refers to the attaching or fixing of biomolecules onto a substrate, for use in an assay. Immobilisation is often achieved through one or more intermolecular interactions between the biomolecule and the substrate, for example, covalent and / or non-covalent interactions. Where the intermolecular interaction is a covalent interaction, one or more covalent bonds may be formed between the biomolecule and the substrate, typically through the chemical reaction of a reactive group on the biomolecule with a reactive group on the substrate. Where the biomolecule is a modified biomolecule, the reactive group may be located on the modified portion of the modified biomolecule, for example on a linker or a binding group. Typically, the reactive group may comprise or may consist of a binding group.

[0054] The assay may comprise or may be a microarray. As described above, a microarray is a two-dimensional array of biomolecules, such as peptides, deposited and immobilised on a substrate. In some embodiments, the substrate may be made of glass, silicon, or a polymer such as nitrocellulose. The substrate may be coated with a coating layer which may be selected so as to improve or alter properties of or interaction with the biological material, including adhesion, immobilisation, stabilisation, etc. The coating layer may comprise, may consist essentially of or may consist of a polymer such as hydrophilic polymers or hydrophilic polymer, e.g., polyacrylamide, epoxysilane, or the like. In some embodiments, the substrate may comprise or may be made of a glass coated with a reactive material.

[0055] In some embodiments, a plurality of different biomolecules (such as nucleic acids, proteins, or peptides) are immobilised on the substrate, in which the modified biomolecule of the first aspect may also be immobilised to. That is to say, biomolecules in addition to those disclosed herein may be immobilised to the substrate. A plurality of biomolecules may be 2 or more, such as from 2 to 80, typically from 10-40. The biomolecules may be modified or unmodified. The biomolecules may be immobilised through the methods disclosed herein, or they may be immobilised through methods known to the skilled person, such as those described in Immobilization Techniques for Microarray: Challenges and Applications, Nimse et a / ., Sensors-Basel, 2014, 14, 22208- 22229. The biomolecules may be immobilised prior to, during, or after the modified biomolecules of the first aspect disclosed herein are immobilised. In some embodiments, the substrate comprises a plurality of immobilised biomolecules such that it is capable of use in a microarray, or multiplex assay. That is to say, in some embodiments, the substrate is a substrate for a microarray, or a multiplex assay. The skilled person will recognise that any protein or peptide may be immobilised to a microarray substrate through certain means, as an unmodified or modified biomolecule, and thus the invention is not limited to any particular selection of biomolecules.

[0056] Nonetheless, in some embodiments, the biomolecules are independently selected from a CCP (such as P2), H1 , dsDNA, TRIM21, SS-A 60, SS-B, Sm, Sm / RNP, LI1 RNP, Scl-70, Jo-1 , Chromatin, CENP-B, Ribosomal P Proteins, RNA Polymerase III, and DFS-70. In some embodiments of the first aspect, the substrate comprises a plurality of immobilised biomolecules for use in a microarray, optionally wherein one or more of the plurality of immobilised biomolecules are each independently selected from: dsDNA, TRIM21 ; SS-A 60; SS-B; Sm; Sm / RNP; U1 RNP; Scl-70; Jo-1 ; Chromatin; CENP-B; Ribosomal P proteins; RNA polymerase III; and DFS-70. In some embodiments, one or more of the plurality of immobilised biomolecules each independently comprise a biomolecule selected from: dsDNA, TRIM21 ; SS-A 60; SS-B; Sm; Sm / RNP; LI1 RNP; Scl- 70; Jo-1 ; Chromatin; CENP-B; Ribosomal P proteins; RNA polymerase III; and DFS-70. dsDNA (double stranded DNA) is a nucleic acid antigen for anti-dsDNA antibodies, which are diagnostic of lupus, particularly systemic lupus erythematosus (SLE). Further discussion may be found in the literature, for example: Anti-double stranded DNA antibodies: A rational diagnostic approach in limited-resource settings, Admou et al., Practical Laboratory Medicine, 2022, e00285.

[0057] TRIM21 (tripartite motif-containing protein 21), or TRIM21 / Ro52 / SS-A1 , is an autoantigen implicated in certain autoimmune diseases, such as Sjogren's syndrome and SLE. A related antigen, but not part of the TRIM21 family, is SS-A 60, or R06O / SS- A, also useful for the diagnosis of autoimmune diseases. Further discussion of these antigens may be found in the literature, for example: Autoantigen TRIM21 / Ro52 as a Possible Target for Treatment of Systemic Lupus Erythematosus, Yoshimi et al., International Journal of Rheumatology, 2012, 718237; Diagnostic Utility of Separate Anti- Ro60 and Anti-Ro52 / TRIM21 Antibody Detection in Autoimmune Diseases, Robbins et al., Frontiers in Immunology, 2019, 10, 444; and Clinical associations of anti-SSA / Ro60 and anti-Ro52 / TRIM21 antibodies: Diagnostic utility of their separate detection, Menendez et al., Autoimmunity, 2013, 46(1):32-9.

[0058] SS-B is also an antigen implicated in Sjogren's syndrome and thus antibodies directed against SS-B are useful in the diagnosis of Sjogren's syndrome. Further discussion may be found in the literature, for example: Specificity of anti-SSB as a diagnostic marker for the classification of systemic lupus erythematosus, Rao et al., Experimental and Therapeutic Medicine, 2013, 5, 1710.

[0059] Anti-SM and anti-RNP antibodies are directed against the antigens Sm and Sm / RNP, and implicated in SLE, thus are specific markers for SLE. Further discussion may be found in the literature, for example: Assays for Sm and RNP antibodies: pitfalls and technical considerations, Molden et al., Diagnostic Immunology, 1985, 3(1):24-8; Molecular characterization of the SM and RNP nuclear antigens, Tsay and Chou, Zhonghua Min Guo Wei Sheng Wu Ji Mian Yi Xue Za Zhi, 1989, 22(1):21-30; Anti-Sm and anti-RNP antibodies, Migliorini et al., Autoimmunity, 2005, 38, 47; and Anti-Smith antibody is associated with disease activity in patients with new-onset systemic lupus erythematosus, Ahn et al., Rheumatology International, 2019, 39, 1937.

[0060] LI1 RNP is an antigen that elicits anti-U1 RNP, an autoantibody associated with systemic sclerosis (SSc). These antibodies may be useful in the classification of patients with anti-U1 RNP. Further discussion may be found in the literature, for example: Distribution and antigen specificity of anti-U1 RNP antibodies in patients with systemic sclerosis, Ihn et al., Clinical & Experimental Immunology, 1999, 117, 383.

[0061] Scl-70 is an antigen that elicits anti-Scl-70 antibodies, mainly associated with diffuse systemic scleroderma and thus is a useful marker for diagnosis. Further discussion may be found in the literature, for example: Anti-scl-70, Basu and Reveille, Autoimmunity, 2005, 38, 65.

[0062] Anti-Jo-1 antibodies are implicated in polymyositis (PM) or dermatomyositis (DM), thus the antigen Jo-1 may be useful in the diagnosis of such conditions. Further discussion may be found in the literature, for example: Anti-Jo-1 antibodies in polymyositis or dermatomyositis: evaluation by ELISA using recombinant fusion protein Jo-1 as antigen, Nishikai et al., Rheumatology, 1998, 37, 357.

[0063] Chromatin is a complex of DNA and protein. Anti-chromatin antibodies are a useful marker for SLE, thus detection of such antibodies may be used to help diagnose such conditions. Further discussion may be found in the literature, for example: Anti- chromatin antibodies are a useful marker for lupus nephropathy, Journal of Clinical Pathology, 2003, 56, 820.

[0064] CENP-B, or centromere protein B, is an autoantigen protein that elicits anti- CENP-B antibodies, which are implicated in a variety of conditions, including SSc. Further discussion may be found in the literature, for example: Clinical significance of anti-centromere antibody and anti-CENP-B antibody in sera of patients with primary biliary cirrhosis, Onozuka et al., Rinsho Byori, 1996, 44, 877; Circulating anticentromere CENP-A and CENP-B antibodies in patients with diffuse and limited systemic sclerosis, systemic lupus erythematosus, and rheumatoid arthritis, Russo et al., Journal of Rheumatology, 2000, 27, 142; and Clinical and Molecular Features of Anti-CENP-B Autoantibodies, Prasad et al., Journal of Molecular Pathology, 2021 , 2, 281.

[0065] Anti-ribosomal P protein antibodies are directed towards three specific ribosomal proteins, P0, P1 , and P2, and is a specific serological marker for SLE. Further discussion may be found in the literature, for example: Anti-ribosomal P protein antibodies, Gerli and Caponi, Autoimmunity, 2004, 38, 85; Ribosomal P Autoantibodies are Present Before SLE Onset and are Directed Against non-C Terminal Peptides, Heinlen et al., Journal of Molecular Medicine, 2010, 88, 719; and The diagnostic benefit of antibodies against ribosomal proteins in systemic lupus erythematosus, Shi et al., Advances in Rheumatology, 2020, 60, 45.

[0066] Antibodies directed against RNA polymerase III are a specific marker for SSc. Further discussion may be found in the literature, for example: Anti-RNA polymerase III antibodies: A marker of systemic sclerosis with rapid onset and skin thickening progression, Cavazzana et al., Autoimmunity Reviews, 2009, 8, 580; and Anti-RNA polymerase III antibodies in patients with suspected and definite systemic sclerosis: Why and how to screen, Lazzaroni and Airb, Journal of Scleroderma and Related Disorders, 2018, 3, 214.

[0067] Anti-DFS-70 antibodies (dense fine speckled 70) are considered as a “negative” biomarker for systemic autoimmune rheumatic diseases (SARD) when they are detected as isolated (monospecific) antibodies and other SARD markers are not present. Further discussion may be found in the literature, for example: The significance of autoantibodies to DFS70 / LEDGFp75 in health and disease: integrating basic science with clinical understanding, Ochs et al., Clinical and Experimental Medicine, 2016, 16, 273; and Twenty years of research on the DFS70 / LEDGF autoantibody-autoantigen system: many lessons learned but still many guestions, Ortiz-Hernandez et al., Autoimmunity Highlights, 2020, 11 , 3. In some embodiments, the substrate comprises or is made of a glass coated with a reactive electrophilic material. A reactive electrophilic material is understood to be a material that comprises an electrophilic group, that is, a group that is reactive towards nucleophilic groups. Examples of electrophilic groups include but are not limited to epoxy, aldehyde, and haloalkane. Thus, an epoxysilane material is an example of a reactive electrophilic material comprising an epoxy group.

[0068] Typically, the reactive electrophilic material comprises an epoxy group. Thus, preferably, the substrate may comprise or may be glass coated with an epoxy-containing material, e.g., an epoxysilane material. This type of substrate may be referred to as ‘epoxy glass’.

[0069] As described above, the modified biomolecule comprises a linker, wherein the linker is attached to the biomolecule, and a binding group is attached to the linker.

[0070] A linker is herein understood as any chemical entity capable of joining the biomolecule and the binding group, through a covalent bond at one end of the linker and a covalent bond at the other end of the linker. The skilled person is aware of many different plausible linkers of varying lengths and chemical functionality.

[0071] In some embodiments, the linker is a polyethylene glycol (PEG) chain, such as a discrete polyethylene glycol (dPEG) chain. dPEG is a polyethylene glycol chain wherein the polymer sample comprises a single chain length, rather than a distribution of different chain lengths. That is to say, a dPEG sample comprises a specific single molecular weight polymer. For example, dPEG4 is a PEG with a chain length of 4, wherein the sample consists only of PEG molecules with a chain length of 4. In some embodiments, the linker is dPEG, such as dPEG4 or dPEGi2, typically dPEG4.

[0072] In some embodiments, the modified biomolecule is of the formula (la):

[0073] (la), wherein:

[0074] X1is (CH2)ni (C(O))n2, wherein n1 is an integer selected from 1 to 4; and n2 is 0 or 1 ;

[0075] L is X3CH(R3)CH2, wherein X3is optionally present and is selected from O, S, and NR1; X2is C(X4)X5, wherein X4and X5are independently selected from

[0076] O, S, and NR2;

[0077] R1and R2are independently selected from H, Ci-4alkyl, and C(O)OCi.4alkyl;

[0078] R3is selected from H, Cl, or CH3; n is an integer selected from 1 to 18;

[0079] ‘lBiomoll’ represents the biomolecule; and £ L^GJ ■ represents the binding group.

[0080] In some embodiments, n1 may be selected from 1 to 3, optionally from 1 to 2. Typically, n1 is 2.

[0081] As described above, n2 is 0 or 1. That is to say, the group “C(O)” is optionally present.

[0082] Typically, n2 is 0, i.e., the group is absent.

[0083] In some embodiments, X3may be present, and may be selected from O, S, and NR1. Typically, X3is O.

[0084] R3may be H, Cl, or CH3. Typically, R3is H.

[0085] In some embodiments where X3is NR1, R1is H; Ci-4alkyl, such as methyl or ethyl; or C(O)OCi-4alkyl, such as methyloxycarbonyl, ethyloxycarbonyl, or tert- butyloxycarbonyl.

[0086] Typically, R1is H.

[0087] Typically, X4is O.

[0088] In some embodiments, X5is S or NR2. Typically, X5is NR2.

[0089] In some embodiments where the linker is attached to the biomolecule through the / V-terminus of the peptide, X5is NR2. That is to say, X5may originate from the amino group of the / V-terminus of the peptide, i.e., C(X4)X5is the amide, thioamide, or amidine group formed with the linker, corresponding to X4= O, S, or NR2, respectively.

[0090] In some embodiments where X5is NR2, R2is H; Ci-4alkyl, such as methyl or ethyl; or C(O)OCi-4alkyl, such as methyloxycarbonyl, ethyloxycarbonyl, or tert- butyloxycarbonyl.

[0091] Typically, R2is H.

[0092] In some embodiments, (L)nof formula (la) is joined to X2at the X5group, and X2is joined to the biomolecule at the C(X4) group. That is to say, C(X4)X5is reversed.

[0093] Where C(X4)X5is reversed, the linker may be attached to the biomolecule through the C-terminus of the peptide. In some embodiments where the linker is attached through the C-terminus, C(X4) may originate from the carboxy group of the C-terminus of the peptide, i.e., X4is O, and C(X4)X5is the amide, ester, or thioester group formed with the linker, corresponding to X5= NR2, O, or S, respectively.

[0094] In some embodiments, the linker is derived from or based on a polymer. That is to say, the linker, or ‘L’ portion of formula (la), may substantially comprise an oligomer or polymer, such as polyethylene glycol (PEG), polypropylene glycol (PPG), polyvinyl chloride (PVC), polyethylene (PE), polypropylene (PP), polyethylenimine (PEI). In some embodiments, the linker is derived from PEG, that is to say, of formula (la), X3is O and R3is H.

[0095] In some embodiments, X3and X4is O.

[0096] In some embodiments, X3and X4is O; X5is NR2; and R2is H.

[0097] Typically, n2 is 0; X3and X4is O; X5is NR2; R2is H; and R3is H or CH3.

[0098] Preferably, n1 is 2; n2 is 0; n is selected from 4 to 12, optionally 4 or 12; X3and X4is O; X5is NR2; and R2and R3is H.

[0099] As described above, the modified biomolecule comprises a binding group, wherein the binding group is attached to the linker and is capable of covalently binding to the substrate. That is to say, the binding group may undergo a reaction with the substrate, the substrate comprising a reactive material, to form a covalent bond with the reactive material.

[0100] In some embodiments, the binding group is a nucleophilic group. A nucleophilic group is understood to be a group that is reactive to electrophilic groups, for example, those found on a substrate, particularly epoxy. Examples of nucleophilic groups include but are not limited to amino, azido, hydroxy, and thiol.

[0101] In some embodiments, the binding group is selected from the group consisting of amino, azido, hydroxy, and thiol. Where the binding group is selected from amino, azido, hydroxy, and thiol, the binding group may correspond to the formulae (Illa) to (Hid), respectively:

[0102] (Illa) (lllb) (lllc) (Hid) wherein the wavy line indicates the position of attachment to the linker, i.e., the binding groups depicted correspond to in the formula (la). Typically, the binding group is amino, / .e., the binding group may correspond to formula (Illa).

[0103] In some embodiments, the modified biomolecule is of formula (II):

[0104] Biomol.

[0105] (II), wherein: m is an integer selected from 1 to 18, optionally selected from 2 to 18; and represents the biomolecule.

[0106] That is to say, formula (II) may correspond to formula (la) wherein n1 is 2; n2 is 0; n is selected from 1 to 18; X3and X4are O; X5is NR2; R2and R3are H; and the binding group is amino.

[0107] In some embodiments, m is selected from 2 to 16, 2 to 14, 2 to 12, 3 to 12, or 4 to 12. Typically, m is selected from 4 to 12, more typically m is 4 or 12.

[0108] Formula (II) may correspond to a modified biomolecule that comprises a dPEG linker. For example, where m is 4 or 12, the modified biomolecule comprises dPEG4 or dPEGi2, respectively. That is to say, in some embodiments, the linker comprises dPEGm, wherein m is as defined above.

[0109] The modified biomolecules of the present invention may exist in more than one stereoisomeric forms. For the avoidance of doubt, all stereoisomeric forms of any modified biomolecule of any one previous embodiment of either the first aspect are included within the scope of the invention.

[0110] Stereoisomeric forms include but are not limited to racemic mixtures, scalemic or non-equimolar enantiomeric mixtures, single enantiomers, diastereomeric mixtures, single diastereomers, and mixtures of stereoisomers in any proportion. Whilst some formulae of the abovementioned embodiments may be depicted with a particular stereochemistry, for example, a racemate, a particular enantiomer, or a particular diastereomer, this does not preclude the alternative stereoisomers or stereoisomeric forms from the scope the invention. Also included are solvates and isotopically-labelled modified biomolecules of the invention. Isotopically-labelled compounds are identical to those recited herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number predominantly found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, sulfur, fluorine and chlorine, such as2H ( / .e., deuterium),3H,13C,14C,15N,18O,170,35S,18F, and36CI, respectively.

[0111] A protium atom (H) is a hydrogen atom with zero neutrons. A deuterium atom (D or2H) is a hydrogen atom with one neutron. Naturally occurring hydrogen contains about 0.02 mol% deuterium and 99.98% protium. Physical chemical properties between protium and deuterium are small but measurable. Deuterium is slightly less lipophilic than protium, has a smaller molar volume and carbon-deuterium bonds are shorter than carbon-protium bonds. Deuterium may keep the 3D surface, shape and steric flexibility of a molecule unaltered compared to H. A ratio of deuterium:protium in a compound greater than 1 :99 is considered to be greater than that found naturally in hydrogen. In particular embodiments, where a moiety is specified as being “H”, the ratio of deuterium:protium at this position may be greater than the natural isotopic abundance of deuterium, i.e., the percentage of deuterium found at his position of the compounds of the invention may be greater than its natural isotopic abundance in hydrogen, which is about 0.02 mol%.

[0112] All amorphous and crystalline forms of the modified biomolecules of the invention are included.

[0113] Methods:

[0114] As described above, according to a second aspect of the invention, there is provided a method of immobilising a modified biomolecule on a substrate, comprising: contacting the modified biomolecule with the substrate.

[0115] The modified biomolecule may typically be a modified biomolecule according to the first aspect of the invention.

[0116] The modified biomolecule may comprise: a biomolecule, wherein the biomolecule comprises or consists of a peptide; a linker, wherein the linker is attached to the biomolecule via a N- or C- terminus of the peptide; and a binding group, wherein the binding group is attached to the linker and is capable of covalently binding to the substrate.

[0117] Preferably, the substrate may comprise or may be made of a glass coated with a reactive electrophilic material, such as epoxy glass. The peptide may be a cyclic peptide.

[0118] The peptide, e.g. cyclic peptide, may comprise a ring formed through a disulfide bridge.

[0119] For the avoidance of doubt, the embodiments described in relation to the first aspect of the invention apply mutatis mutandis to the embodiments of the second aspect of the invention.

[0120] As described above, the term “contacting” is used herein to refer to, amongst other things, printing, reacting, and / or treating. The skilled addressee is familiar with relevant and particular contacting conditions, exemplified by the immobilisation techniques discussed in Nimse et al. (supra).

[0121] As described above, according to a third aspect of the invention, there is provided an immobilised biomolecule obtainable by the method according to the second aspect of the invention.

[0122] For the avoidance of doubt, the term “obtainable” includes materials obtained by the method of the second aspect, as well as materials not obtained by the method of the second aspect but that are identical to those obtained by the method of the second aspect.

[0123] As described above, in a fourth aspect, there is provided an immobilised biomolecule for an assay, wherein the immobilised biomolecule is a modified biomolecule according to the first aspect covalently bound to a substrate.

[0124] For the avoidance of doubt, the embodiments described in relation to the first aspect of the invention apply mutatis mutandis to the embodiments of the fourth aspect of the invention. For example, in some embodiments, the modified biomolecule comprises: a peptide, typically a cyclic peptide, such as a CCP or H1 (SEQ ID NO: 1- 13); a linker, typically a PEG linker, such as dPEG4; and a binding group, typically amino; and typically the substrate comprises or is made of a glass coated with a reactive electrophilic material, such as an epoxy group. Furthermore, for example, the substrate may comprise a plurality of immobilised biomolecules for use in a microarray, optionally wherein one or more of the plurality of immobilised biomolecules each independently comprise a biomolecule selected from, or are each independently selected from: dsDNA, TRIM21 ; SS-A 60; SS-B; Sm; Sm / RNP; U1 RNP; Scl-70; Jo-1 ; Chromatin; CENP-B; Ribosomal P proteins; RNA polymerase III; and DFS-70.

[0125] As described above, according to a fifth aspect of the invention, there is provided a method of detecting the presence of a target biomolecule in a sample, the method comprising:

[0126] (a) contacting the sample with an immobilised biomolecule according to the third or fourth aspect of the invention under conditions that allow the formation of a complex between the immobilised biomolecule and the target biomolecule; and

[0127] (b) contacting the complex with one or more detection reagents.

[0128] A “target biomolecule” is understood to be a biomolecule, such as an antibody, antigen, protein, or peptide that is a target analyte of the sample.

[0129] The method of the fifth aspect of the invention may apply to any suitable target biomolecule that may form a complex between the immobilised biomolecule and the target biomolecule.

[0130] In some embodiments, the target biomolecule is an antibody. The antibody may be directed against cyclic citrullinated peptides, or “anti-CCP”, also named anti- citrullinated protein antibodies (ACPA). Antibodies directed against citrullinated proteins or peptides may be useful specific criteria markers, or target biomolecules, for the detection and diagnosis of particular diseases, such as rheumatoid arthritis (RA), and / or connective tissue disorder (CTD). In some embodiments, the target biomolecule is a specific criteria marker for RA and / or CTD.

[0131] In some embodiments, the target biomolecule is directed against H1 , that is to say, it is an “anti-HIV” antibody. The peptide H1 corresponds to the primary immunodominant region on the gp41 protein from HIV-1. Anti-gp41 antibodies can be detected at the earliest stages of HIV-1 infection and persist in those with chronic infection. As such they are very useful for diagnosing individuals who are or have been infected by HIV-1. H1 Anti-HIV-1 gp41 antibody is a useful marker for the detection and diagnosis of individuals who are or have been infected by HIV-1. Further discussion may be found in the literature, for example: Conformational plasticity of the HIV-1 gp41 immunodominant region is recognized non-neutralizing antibodies, Cook et al., Communications Biology, 2022, 5, 291. In some embodiments, the target biomolecule may be an antibody directed against HIV, optionally the target biomolecule is anti-HIV-1 gp41 antibody. A “sample”, as referred to herein, is understood to refer to a sample of a patient, including a tissue, or a tumour, blood, tears, cerebrospinal fluid, urine or faeces sample, in non-limiting embodiments. In some embodiments described herein, the term sample is referred to as a blood sample or biological fluids derived from blood such as serum or plasma, but any other type of sample described herein or known to those skilled in the art may be used as a substitute sample, or in addition to a blood sample. In some embodiments, the sample is anything that may contain the target biomolecule. In some embodiments, the sample may be prepared or treated prior to contacting the sample with the immobilised biomolecule. Typically, the sample is serum or plasma.

[0132] As described above, the term “complex” relates to a stable association between two or more molecules, often biomolecules, to form a single unit. Complexes may form as the result of a small-molecule substrate binding to a biological molecule, an antigen binding to an antibody, a protein binding to another protein, or any other inter- biomolecular interaction. In some embodiments, the complex is between an antigen and an antibody, sometimes referred to as an immune complex, or an antigen-antibody complex, or an antigen-bound antibody. In some embodiments, the immobilised biomolecule comprises an antigen.

[0133] The term “detecting the presence of a target biomolecule” is understood to refer to verifying the presence (or absence) of the target biomolecule in a sample. The term is understood to refer to any further measuring, imaging, and / or quantification or other detecting action relating to the target biomolecule. A detection reagent therefore may be any chemical or biological agent that is added to the complex to facilitate the measuring, imaging, and / or quantification, or any other detecting action, of the target biomolecule in the sample. Detection reagents need not be added simultaneously, but may be added in a sequential manner, with some period of time or process occurring between.

[0134] In some embodiments, the detection reagent is a reagent that is capable of producing a detectable signal, either inherently or in combination with a further detection reagent. Non-limiting examples of detectable signals include fluorescence, chemiluminescence, a radioactive signal, and a chromogenic / colorimetric signal. The skilled addressee will be familiar with many suitable detection reagents and combinations thereof to carry out the invention disclosed herein, and will recognise that the invention is not limited to any particular detection reagent.

[0135] Nonetheless, in some embodiments, one or more detection reagents is or comprises an enzyme, such as a horseradish peroxidase (HRP), an alkaline phosphatase (AP), a p-galactosidase, a glucose oxidase (GOx), an acetylcholinesterase (AChE), a urease, or a dehydrogenase.

[0136] In some embodiments, one or more detection reagents is or comprises a chemiluminescent substrate, such as luminol, isoluminol, an acridinium ester, or a dioxetane derivative.

[0137] In some embodiments, one or more detection reagents is or comprises a fluorescent substrate, such as 4-methylumbelliferyl phosphate (MUP), fluorescein di(|3- D-galactopyranoside) (FDG), Amplex Red, fluorescein isothiocyanate (FITC), and Alexa Fluor.

[0138] In some embodiments, the detection reagent comprises a radioactive isotope, such as iodine-125, iodine-131 , carbon-14, sulfur-35, and tritium (hydrogen-3).

[0139] In some embodiments, the one or more detection reagents is selected from antihuman immunoglobulin antibodies, and chromogenic substrates.

[0140] The term "anti-human immunoglobulin antibodies" refers to a class of antibodies that specifically recognise and bind to human immunoglobulins, i.e., they may form an immune complex with antibodies present in a patient sample. In some embodiments, the anti-human immunoglobulin antibodies are selected from anti-human IgG, IgA, IgM, and IgE. Typically, the anti-human antibody is IgG.

[0141] In some embodiments, the anti-human immunoglobulin antibodies are modified with, or conjugated to, horseradish peroxidase (HRP). That is to say, the anti-human immunoglobulin antibodies are linked to HRP. For example, the anti-human immunoglobulin antibody may be HRP-modified anti-human IgG.

[0142] The term “chromogenic substrates” refers to chemical compounds, typically small-molecules, that upon enzymatic action, and optionally the addition of a further agent (such as an oxidising agent, e.g., hydrogen peroxide (H2O2)), undergo a chemical change that results in a colour change.

[0143] In some embodiments where one or more detection reagents is a chromogenic substrate, the enzymatic action may be carried out by HRP, or the anti-human immunoglobulin antibody-HRP conjugate.

[0144] In some embodiments, the chromogenic substrates are selected from TMB (3,3',5,5'-tetramethylbenzidine), ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), OPD (ort / 70-phenylenediamine), AmplexRed (10-acetyl-3,7- dihydroxyphenoxazine), homovanillic acid, AEC (3-amino-9-ethylcarbazole), and DAB (3,3'-diaminobenzidine), corresponding to the formula (IVa) to (IVg), respectively:

[0145] Typically, the chromogenic substrate is TMB (formula (IVa)). Typically, where the chromogenic substrate is TMB, the anti-human antibody is HRP-modified anti-human IgG.

[0146] BRIEF DESCRIPTION OF THE FIGURES

[0147] Figure 1 : Comparison of indirect immunoassays performed using an unmodified biomolecule (P2 peptide) and a modified biomolecule (d(PEG)4-P2) immobilised onto a substrate as detection antigens (capture phase). Mean signal (SMN) versus the dilutions of L000001S serum sample which contains high titres of anti-CCP antibodies. Plain and dashed lines tagged “Neg” correspond to the mean signal observed when sera which do not contain anti-CCP antibodies are assayed. Data were generated using the automated MosaiQ instrument, with microarrays printed by MMS.

[0148] Figure 2: Comparison of indirect immunoassays performed using 3 different modified biomolecules: (A) d(PEG)4-P2; (B) d(PEG)i2-P2; and (C) K4-P2, immobilised onto a substrate as detection antigens (capture phase). Median signal intensity (AU) versus anti-CCP antibody concentration in n=18 sera containing various levels of anti- CCP antibodies. Dashed lines tagged “LoD” correspond to the limit of detection of each assay. Data were generated by manual immunoassay using microarrays printed on sciFLEXARRAYER S3.

[0149] Figure 3: Performance (method comparison) of an immunoassay using immobilized d(PEG)4-CCP as detection antigen (capture phase). Sera samples with known anti-CCP status (negatives n=29; positives n=30) were assayed using the automated MosaiQ instrument, with microarrays printed by MMS. (A) Box-plots representing positive-negative discrimination. (B) ROC curve (receiver operating characteristic). The area under curve is 1. Figure 4: Comparison of indirect immunoassays performed using an unmodified biomolecule (H1 peptide) and a modified biomolecule (d(PEG)4-H1) immobilised onto a substrate as detection antigens (capture phase). Median signal intensity (AU) anti-HIV positive (n=4) and anti-HIV negative samples (n=3) is represented. Data were generated by manual immunoassay using microarrays printed on sciFLEXARRAYER S3.

[0150] Figure 5: Indirect immunoassays were performed using modified biomolecules (d(PEG)4-D1) (5A) or (d(PEG)i2-D1) (5B) immobilised onto a substrate as detection antigens (capture phase). Mean signal intensity (AU) (n=3) is represented for anti-ZnT8 antibody positive samples (n=11) and for anti-ZnT8 antibody negative samples (n=9). Data were generated by manual immunoassay using microarrays printed on sciFLEXARRAYER S3.

[0151] EXAMPLES

[0152] Materials:

[0153] Peptides

[0154] All peptides were synthesised custom by Biosynth using solid phase methods. Custom peptides were characterised by mass spectral analysis for mass identification (molecular weight) and by ultra-performance liguid chromatography (UPLC) for purity (Table 1).

[0155] Table 1 Substrate

[0156] Nexterion Epoxysilane coated glass (Schott Minifab) was used for peptide immobilisation and microarray manufacturing.

[0157] Samples

[0158] L000001S is a serum sample sourced from Logical Biological (SKU: H007-2). The measured value of anti-CCP antibodies was 2244.16 lll / mL per the information provided by the supplier. The cut-off of the reference method used to determine anti- CCP titres was below 25. Characterised sera samples negative (n=29) or positive (n=30) for anti-CCP antibodies were sourced from CerbaXpert (Saint Ouen I'Aumone, France) or Eurofins Biomnis Sample Library (Lyon, France).

[0159] HIV positive plasma samples (Ref. Q984) were obtained from Etablissement Frangais du Sang, Centre Pays de Loire (Tours, France). All samples were confirmed to be positive for anti-HIV antibodies by a screening technique (Abbott Prism HIV O plus) and a confirmation technique (Inno-LIA HIV l / ll Score Innogenetics). Sample #1 corresponds to batch # 59106470307; sample #2 corresponds to batch # 62091966277; and sample #3 corresponds to batch # 72182466453.

[0160] Samples from individuals with suspected type 1 diabetes were obtained from Eurofins Biomnis Sample Library (Lyon, France). All samples were tested for anti-ZnT8 (Zinc Transporter 8) autoantibodies by ELISA RSR ZnT8 Ab kit (RSR Limited, Cardiff, United Kingdom) according to the instructions of the manufacturer and classified into 2 groups according to the RSR ZnT8 ELISA result: Positive group are positive for anti- ZnT8 antibodies, Negative group are negative for anti-ZnT8 antibodies.

[0161] Methods:

[0162] Microarray printing and peptide immobilisation

[0163] Using sciFLEXARRAYER S3: The sciFLEXARRAYER S3 (Scienion, Berlin, Germany) is an automated piezo driven, non-contact dispensing system of ultra-low volumes specifically designed as an economical entry unit for academia and R&D labs. It is suitable for the production of DNA, protein and glycan arrays for research applications. Printing is performed on glass with dimensions similar to a microscope slide according to the manufacturer’s instructions. The spot size was about 150-200 micrometres in diameter, corresponding to a print volume of about 900-1000 pL. After deposition of spots onto glass, binding is carried out at 2-8 °C, in 50-60% humidity during one day. Using MosaiQ manufacturing system: The MosaiQ instrument (AliveDx, Eysins, Switzerland) is a high-throughput and full traceability system, designed to provide multimodal, multiplexed testing with a single patient sample. The system uses microarrays that can harbour up to 132 spots, has 1 ,000 microarray loading capacity and uses QR codes and RFID tagging for easy management of the ready-to-use reagents. The MosaiQ system microarray was printed and assembled through the MosaiQ manufacturing system using piezo-electric, non-contact printing. The spot size was about 150-200 micrometres in diameter, corresponding to a print volume of about 900-1000 pL.

[0164] The peptide print formulations contained a base buffer (phosphate pH 7.2 or carbonate pH 9.6), a salt (NaCI or KCI) and a viscosity modifier (glycerol or sucrose). The print concentrations are in the 25-300 micromolar range.

[0165] Immunoassay methods

[0166] Manual immunoassay: Slides printed using sciFLEXARRAYER S3 were blocked during 20 minutes with 2% BSA in phosphate buffer. Then, the blocking buffer was discarded and the sample to be tested was added and incubated for 20 min at 37 °C. After several washes in PBS-Tween 20 (0.05%), a secondary antibody conjugated HRP was added (monoclonal anti-human IgG antibody-HRP at 2 microgram / mL in the StabiIZyme HRP Conjugate stabilizer from Surmodics (ref SZ02)). The slides were left for incubation with this detection reagent for 30 min at 37 °C. After several washes in PBS-Tween 20 (0.05%), 3,3’,5,5’-tetramethylbenzidine (TMB) solution was added (SeramunBlau, ref S-710-#-TMB) and incubated 7 min at 37 °C. The colour development reaction was stopped by washing in milli RO water. Image acquisition was carried out using the Microblot Array Reader from Biovendor. Spot images were analysed using the reader software.

[0167] For type 1 diabetes application, slides printed using sciFLEXARRAYER S3 were blocked during 60 minutes with Blockmaster CE510 (JSR Lifesciences). Then, the blocking buffer was discarded and the sample to be tested was added and incubated for 20 min at 37 °C or overnight at 2-8°C. After several washes in PBS-Tween 20 (0.05%), a secondary antibody conjugated to polyHRP80 was added (monoclonal anti-human IgG antibody-polyHRP80 #MAHGP80C (SDT reagents) at 2.5 microgram / mL in the StabiIZyme HRP Conjugate stabilizer from Surmodics (ref SZ02)). The slides were left for incubation with this detection reagent for 30 min at 37 °C. After several washes in PBS-Tween 20 (0.05%), 3,3’,5,5’-tetramethylbenzidine (TMB) solution was added (SeramunBlau, ref S-710-#-TMB) and incubated 7 min at 37 °C. The colour development reaction was stopped by washing in milli RO water. Image acquisition was carried out using the Microblot Array Reader from Biovendor. Spot images were analysed using the reader software.

[0168] MosaiQ system: Single use microarrays were printed and assembled using the MosaiQ manufacturing system (AliveDx). The immunoassays were carried out using the microarrays on the fully automated MosaiQ 125 instrument (AliveDx). The MosaiQ system dilutes the patient sample with sample diluent solution and adds the patient sample to the microarray. An incubation period allows antibodies present in the sample to react with the immobilized peptides. After washing away unbound antibodies, HRP labelled antibodies against human IgG are dispensed to form conjugate complexes during an additional incubation step. Excess conjugate is eliminated during another wash cycle before TMB is added to the array and incubated. Following another wash cycle, images generated by the system for the array are analysed to determine the amount of antibodies bound to each spot. Results are obtained by reading the signal of the MosaiQ for each individual analyte. Wash buffer (ref 155041), MosaiQ Sample Diluent 2 (ref 155002), TMB Substrate (ref 155050) and IgG Conjugate (ref 155051) were supplied by Alive Dx.

[0169] Results and discussion:

[0170] Antibodies directed to CCP (anti-CCP) are highly specific serological markers for rheumatoid arthritis (RA), and are thought to be directly involved in the disease pathogenesis and precede the onset of disease symptoms. Therefore, assays directed towards detecting the presence of anti-CCP would be useful in diagnosing RA and / or CTD. Therefore, it was envisaged that the immobilisation of a CCP could enable such an assay. One of the main challenges was defining a successful strategy to immobilise a small and cyclic peptide, such as a CCP, onto the epoxysilane glass without decreasing its antigenic reactivity. The present inventors have identified and compared 3 different / V-terminal modifications to enable this assay: amino(PEG)4_P2, amino(PEG)i2_P2 and K4_P2 (Scheme 1). amino(PEG)4_P2 amino(PEG)i2_P2 K4_P2

[0171] Scheme 1

[0172] The amino(PEG) modifications may be referred to as “dPEGm”, wherein m is the PEG chain length. Initial testing showed that each modification tested could bind to the substrate, thus prototype assays were developed. Comparison of the modified biomolecule versus the unmodified biomolecule demonstrated a clear advantage to using the modified version (Fig. 1). Table 2 shows the signal-to-noise ratio (SNR) for the modified biomolecule and unmodified biomolecule.

[0173] Table 2: Signal-to-noise (positive-to-negative) ratio of data presented in Fig. 1.

[0174] The prototype based on amino(PEG)4_P2 overall had the best performance, thus was selected for further evaluation and optimisation (Fig. 2). Table 3 shows the SNR for each modification tested.

[0175] Table 3: Signal-to-noise (positive-to-negative) ratio for 6 sera among those included in

[0176] Fig. 2. The performance of the modified biomolecule in an immunoassay was assessed in several tests. Fig. 3A shows box-plots demonstrating the positive-negative discrimination of sera samples with known anti-CCP status. Fig. 3B shows the corresponding receiver operator characteristic (ROC); the area under the curve (AUC) is 1 , demonstrating perfect identification of positive and negative samples.

[0177] Furthermore, the inventors have demonstrated that they are able to immobilise a further type of peptide for an assay, H1 , through the preparation of a modified biomolecule comprising H1 , a linker, and a binding group. H1 is a peptide encompassing the primary immunodominant region for HIV-1 gp41 , and most HIV-infected individuals raise antibodies directed against the gp41 protein and its immunodominant region (antiHIV). Therefore, assays directed towards detecting the presence of anti-HIV would be useful in diagnosing HIV. As with a CCP, H1 is a cyclic peptide, thus shares the challenges associated with immobilisation for assays. The inventors prepared a modified biomolecule comprising H1 , a dPEG4 linker, and an amino binding group, and immobilised to epoxysilane glass substrate. Fig. 4 shows the median signal intensity from an assay comparing unmodified H1 to modified H1 on positive and negative samples. The results clearly show the improved sensitivity and reliability in positivenegative discrimination when using the modified peptide.

[0178] Furthermore, the inventors have demonstrated that they are able to immobilise a further type of peptide for an assay, D1 , through the preparation of modified biomolecules comprising D1 , a linker, and a binding group. D1 is a 50-mer acyclic peptide encompassing residues 310-359 of the human Zinc Transporter 8 (ZnT8) protein which is found on the surface of pancreatic beta cells (See SEQ ID 15 in Table 4). Autoantibodies directed against ZnT8 can be used to help diagnose type 1 diabetes and assess the risk of developing the disease. Fig. 5 shows the mean signal intensity from an assay using modified D1 for positive and negative samples. The results indicate that the modified peptide is able to provide positive-negative discrimination.

[0179] The inventors have therefore shown unequivocally that the modified biomolecules, capable of immobilisation on a substrate, are highly effective in a method of detecting the presence of a target biomolecule. Table 4: Amino acid sequences, / V-terminal to C-terminal. Each of sequences 1-13 may be linked / V-terminally and / or C-terminally to 1-4 residues of any amino acids. That is to say, each of sequences 1-13 may begin and / or end with (X)n, wherein each X is independently any amino acid, and n is 0 to 4. Sequence 14 may be linked C-terminally to 1-4 residues of any amino acids. Each of sequences 1-14 comprises an intrachain disulfide bond between the two defined cysteine residues. Each defined X is citrulline.

Claims

CLAIMS1 . A modified biomolecule for immobilising on a substrate for an assay, the modified biomolecule comprising: a biomolecule, wherein the biomolecule comprises or consists of a cyclic peptide; a linker, wherein the linker is attached to the biomolecule via a / V- or C-terminus of the peptide; and a binding group, wherein the binding group is attached to the linker and is capable of covalently binding to the substrate.

2. The modified biomolecule of claim 1 , wherein the modified biomolecule is of formula (la):(la), wherein:X1is (CH2)ni (C(O))n2, wherein n1 is an integer selected from 1 to 4; and n2 is 0 or 1 ;L is X3CH(R3)CH2, wherein X3is optionally present and is selected from O, S, and NR1;X2is C(X4)X5, wherein X4and X5are independently selected from O, S, and NR2;R1and R2are independently selected from H, Ci-4alkyl, and C(O)OCi- 4alkyl;R3is selected from H, Cl, or CH3; n is an integer selected from 1 to 18;‘lBiomoll’ represents the biomolecule; and£L^GJ ■ represents the binding group.

3. The modified biomolecule of claim 2, wherein:X3and X4are O; orX3and X4are O; X5is NR2; and R2is H; or n2 is 0; X3and X4are O; X5is NR2; R2is H; and R3is H or CH3; orn1 is 2; n2 is 0; n is selected from 4 to 12; X3and X4are O; X5is NR2; and R2and R3are H.

4. The modified biomolecule of any one preceding claim, wherein the binding group is a nucleophilic group, such as amino, azido, hydroxy, and thiol.

5. The modified biomolecule of any one preceding claim, wherein the binding group is amino.

6. The modified biomolecule of any one preceding claim, wherein the modified biomolecule is of formula (II):Biomol.(II), wherein: m is an integer selected from 1 to 18, optionally selected from 2 to 18; andrepresents the biomolecule.

7. The modified biomolecule of claim 6, wherein m is selected from 4 to 12, optionally m is 4 or 12.

8. The modified biomolecule of any one preceding claim, wherein the substrate comprises or is made of a glass coated with a reactive electrophilic material, optionally an epoxy group.

9. The modified biomolecule of any one preceding claim, wherein the substrate comprises a plurality of immobilised biomolecules for use in a microarray.

10. The modified biomolecule of claim 9, wherein one or more of the plurality of immobilised biomolecules each independently comprise a biomolecule selected from, or are each independently selected from: dsDNA, TRIM21 ; SS-A 60; SS-B; Sm; Sm / RNP; LI1 RNP; Scl-70; Jo-1 ; Chromatin; CENP-B; Ribosomal P proteins; RNA polymerase III; and DFS-70.

11. The modified biomolecule of any one preceding claim, wherein the linker is attached to the biomolecule through an / V-terminus of the peptide.

12. The modified biomolecule of any one preceding claim, wherein the cyclic peptide comprises a ring formed through a disulfide bridge.

13. The modified biomolecule of any one preceding claim, wherein the biomolecule comprises any one of SEQ ID NO: 1-13.

14. The modified biomolecule of any one of claims 1 to 13, wherein the biomolecule is a cyclic citrullinated peptide, optionally wherein the biomolecule comprises any one of SEQ ID NO: 1-12.

15. The modified biomolecule of any one of claims 1 to 13, wherein the biomolecule comprises SEQ ID NO: 2 or SEQ ID NO: 13.

16. A method of immobilising a modified biomolecule on a substrate, comprising: contacting the modified biomolecule with the substrate, wherein the substrate comprises or is made of a glass coated with a reactive electrophilic material, wherein the modified biomolecule comprises: a biomolecule, wherein the biomolecule comprises or consists of a peptide; a linker, wherein the linker is attached to the biomolecule via a / V- or C- terminus of the peptide; and a binding group, wherein the binding group is attached to the linker and is capable of covalently binding to the substrate.

17. The method of claim 16, wherein the contacting is under conditions that allow the formation of a covalent bond between the biomolecule and the substrate.

18. The method of claim 16 or claim 17, wherein the substrate comprises or consists of epoxy glass.

19. An immobilised biomolecule obtainable by the method of any one of claims 16 to18.

20. An immobilised biomolecule for an assay, wherein the immobilised biomolecule is a modified biomolecule covalently bound to a substrate, wherein the substrate comprises or is made of a glass coated with a reactive electrophilic material, wherein the modified biomolecule comprises: a biomolecule, wherein the biomolecule comprises or consists of a peptide; a linker, wherein the linker is attached to the biomolecule via a / V- or C- terminus of the peptide; and a binding group, wherein the binding group is attached to the linker and is capable of covalently binding to the substrate.21 . The immobilised biomolecule of claim 19, wherein the substrate comprises or is made of epoxy glass.

22. A method of detecting the presence of a target biomolecule in a sample, the method comprising:(a) contacting the sample with an immobilised biomolecule according to any one of claims 19 to 21 under conditions that allow the formation of a complex between the immobilised biomolecule and the target biomolecule; and(b) contacting the complex with one or more detection reagents.

23. The method of claim 22, wherein the target biomolecule is selected from: an anti- CCP antibody; and anti-HIV-1 gp41 antibody.

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