Diagnosis of coeliac disease

Abstract

The present disclosure provides methods and assays (for example multiplex assays) for the diagnosis of autoimmune diseases. comprising the (in vitro) concurrent / simultaneous semi-quantitative detection of directed to specific markers as well as qualitative detection of Total IgA.

Description

[0001]M&C PG450988GB 1 Diagnosis of Coeliac Disease FIELD The present disclosure provides assays and methods for use in the diagnosis or detection Coeliac disease. The present disclosure also provides a kit for use in diagnosing or detecting Coeliac disease. BACKGROUND At present, serological testing for Coeliac disease relies on the sequential and progressive detection of IgA antibodies against specific markers, such as tissue transglutaminase (tTg) and total IgA as an initial step in the evaluation of individuals of any age under clinical suspicion of Coeliac disease. However, approximately 2-3% of individuals with celiac disease have selective IgA deficiency. As such, in these cases, IgA-based tests may provide a falsely negative result. Testing of IgG antibodies to deamidated gliadin peptide (DGP) and / or endomysial antibodies (EMA) may be subsequently recommended. However, such an approach to diagnosing Coeliac disease lengthens the process and the first line of IgA dependent serological testing is not suitable for accurate diagnosis of IgA deficient patients. It is recognized in clinical guidelines that no single test for Coeliac disease has a perfect sensitivity or specificity (1). Moreover, there is no automated high-throughput multiplex test currently available on the market that can provide individual results for tTG and DGP for both IgG and IgA antibodies with a single sample in a single testing occasion. This brings additional costing and time constraints and often results in clinicians making decisions on more limit data. Thus, there remains an unmet need in the art for improved methods and assays that enable diagnosis of Coeliac disease with increased sensitivity and / or specificity. SUMMARY The present disclosure provides methods and assays (for example multiplex assays) for the diagnosis of autoimmune diseases, including Coeliac Disease, comprising the (in vitro) concurrent / simultaneous semi-quantitative detection of IgA and IgG antibodies directed to tissue Transglutaminase (tTG) and deamidated gliadin peptide (DGP) as well as qualitative detection of Total IgA. 55650953-1 M&C PG450988GB 2 In a first aspect the disclosure provides a method, assay or multiplex assay for diagnosing an autoimmune disease, wherein said multiplex-assay comprises concurrently probing a sample for: (i) anti-tissue transglutaminase (tTG) IgG and IgA antibodies; (ii) anti-deamidated gliadin peptide (DGP) IgG and IgA antibodies; and (iii) total IgA content; wherein detection of anti-tTG IgA or IgG and / or anti-DGP IgA or IgG in a sample and the absence of IgA, indicates that the sample may have been obtained from a subject suffering from, susceptible or predisposed to an autoimmune disease. In one teaching, the autoimmune disease is Coeliac (or Celiac) disease. Accordingly, the disclosure provides a method, assay or multiplex assay for diagnosing Coeliac disease, wherein said multiplex-assay comprises concurrently probing a sample for: (i) anti-tissue transglutaminase (tTG) IgG and IgA antibodies; (ii) anti-deamidated gliadin peptide (DGP) IgG and IgA antibodies; and (iii) total IgA content; wherein detection of anti-tTG IgA or IgG and / or anti-DGP IgA or IgG in a sample and indicates that the sample may have been obtained from a subject suffering from, susceptible or predisposed to Coeliac disease. The step of probing a sample for total IgA takes account of the fact that some Coeliac sufferers may be IgA deficient and, for those tests that rely on IgA detection (namely the assays for anti-tTG and anti-tDGP IgA), an individual’s IgA status may be important. If someone has an IgA deficiency then any assay looking for either anti-tTG IgA or anti- tDGP IgA will invariably be negative (or at least report a reading lower than it ‘should’). Conducting a total IgA assay puts this result into to context. Specifically – where total IgA is found to be low and the anti-tTG / DGP IgA assays are negative, the user knows to consult the anti-tTG / DGP IgG assays instead – those assays being unaffected by total IgA content. As such, the total IgA level determined by the method of this disclosure is something that places the specific IgA assays (the anti-tTG and anti-tDGP IgA assays) into context. The methods of this disclosure are advantageous as all of the assays needed provide a full coeliac diagnosis are performed together. This is in contrast to prior methods where IgG assays are only conducted upon determination of the IgA status of a sample. This IgA and IgG testing ‘bi-furcation’ leads to delays. In contrast, the present 55650953-1 M&C PG450988GB 3 methods provide all the required information at the same time and there is no need to conduct further tests. One of skill will appreciate that any level of IgA, anti-tTG IgA / IgG or anti-DGP IgA / IgG may be assessed relative to a series of predetermined control values. By way of example, the detection of IgA below a control (or normal) level of IgA, may indicate that the sample has been obtained from a subject with an IgA deficiency. In this regard, an IgA concentration of less than about 7 mg / dL (0.07 g / L) may that indicate that the sample has been obtained from an IgA-deficient subject. A patient of this type may be considered to have selective IgA deficiency. Later in this description is a summary of how a user may determine a control or normal level for each of the targets of the assays described herein (namely IgA, anti-tTG-IgA / IgG and anti-DGP-IgA / IgG). An assay of this disclosure is typically an in vitro assay. The step of probing a sample may comprise a target capture step. The target capture step may comprise contacting the sample with one or more target capture agents. The contacting step may result in the formation of capture agent / target complexes. One of skill will understand that such complexes result from a successful binding event between a capture agent (optionally immobilised to a substrate as described herein) and any capture agent target present in the sample. As such, the contacting step may be performed under conditions which permit binding between any antibodies present in the sample and the capture agents. The capture step may exploit a substrate having one or more target capture agents immobilised thereto. Within the context of this disclosure, the ‘target’ may be an IgA molecule or IgA and / or IgG with specificity or affinity for (i.e. able to bind to) either tTG of DGP. As such, an assay of this disclosure may comprise capture agents which are able to bind (or capture) each of the following targets: (i) IgA; (ii) anti-tTG IgA; (iii) anti-tTG IgG; (iv) anti-DGP IgA; and 55650953-1 M&C PG450988GB 4 (v) anti-DGP IgG. The capture step may comprise contacting (e.g. concurrently contacting) a sample with any (or all) of the following capture agents: (i) an IgA capture agent; and (ii) an anti-tTG IgA capture agent; and (iii) an anti-tTG IgG capture agent; and (iv) an anti-DGP IgA capture agent; and (v) an anti-DGP IgG capture agent. A suitable IgA capture agent may comprise an anti-IgA antibody – such antibodies having an ability to bind IgA. Where the sample is obtained or provided by a human subject, any IgA capture agent should be capable of binding(capturing) human IgA. By way of example, the IgA capture agent may comprise an antibody capable of binding human IgA. In one teaching, the IgA capture agent, may comprise mouse anti-human IgA (for example as supplied by Absolute antibodies (code Ab020441.1)). Where the IgA capture agent comprises an anti-human IgA, that capture agent may be immobilised by printing onto the substrate. The IgA capture agent may be formulated in a print buffer for printing. The print buffer may comprise, sucrose, BSA and PBS. The print buffer may be formulated at about pH 7.2. A suitable anti-tTG IgA and anti-tTG IgG capture agent may comprise open tissue transglutaminase (‘open’ tTG) and / or closed tissue transglutaminase (‘closed’ tTG) or antibody (e.g. IgA / IgG) binding fragments thereof. The term “open conformation tissue transglutaminase” typically refers to tissue transglutaminase that adopts a catalytically active conformation of the protein. An open conformation of the tissue transglutaminase may be typically achieved after the transglutaminase binds a substrate or an analogue thereof which maintains the transglutaminase in an open conformation. The term “closed conformation tissue transglutaminase” typically refers to tissue transglutaminase that adopts a catalytically inactive conformation of the protein. A closed conformation may be achieved if the transglutaminase is not bound to its substrate or bound to an antagonist or inhibitor which maintains its conformation in an inactive state. The tissue transglutaminase of the present disclosure typically refers to tissue transglutaminase 2 (tTG2). A combination of both open tTG and closed tTG may be used as a capture agent in an assay of this disclosure. A combination of open tTG and closed tTG may be referred 55650953-1 M&C PG450988GB 5 to as a tTG ‘blend’. Where the capture agent comprises such a tTG blend (a combination, of the ‘open’ and ‘closed’ tTG forms), the blend may comprise tTG forms in a ratio of (open: closed) 1:1, 2:1, 3:1, 4:1 or 5:1. In one teaching, a tTG blend may comprise may comprise open and closed tTG forms in a ratio of 2: 1 (open:closed). For convenience, these capture agents will be collectively referred to as ‘tTG’. One of skill will appreciate that tTG will be bound by anti-tTG IgA and / or anti-tTG IgG. Where the sample is obtained or provided by a human subject, any anti-tTG IgA / IgG capture agent should be capable of binding(capturing) human anti-tTG IgA or human anti-IgG present in the sample. By way of example, the anti-tTG IgA / anti tTG-IgG capture agent may comprise ‘open’ tTG and / or ‘closed’ tTG. In one teaching the anti-tTG IgA / anti tTG- IgG capture agent comprises a tTG blend as described herein. As stated, IgA and / or IgG binding fragments of any of the tTG capture agents described herein may also be used as (anti-tTG IgA / anti-tTG-IgG) capture agents. Any of the anti-tTG IgA / IgG capture agents may be formulated in a print buffer for printing. The print buffer may comprise, glycerol or sucrose, BSA and a phosphate or carbonate print buffer. The anti-tTG IgA / IgG capture agent may be present at a concentration of about 30 µg / ml (for ‘open’ tTG) and 15 µg / ml (for ‘closed’ tTG) of print buffer. The present inventors have unexpectedly identified the inclusion of both open and closed conformation of tTG increases the specificity and the sensitivity of an assay for the diagnosis of Coeliac disease. Accordingly, the disclosure provides a multiplex assay for diagnosing Coeliac disease, wherein said multiplex-assay comprises concurrently probing a sample for: (i) anti-tissue transglutaminase (tTG) IgG and IgA antibodies; (ii) anti-deamidated gliadin peptide (DGP) IgG and IgA antibodies; and (iii) total IgA content; wherein detection of anti-tTG IgA or IgG and / or anti-DGP IgA or IgG in a sample (, indicates that the sample may have been obtained from a subject suffering from, susceptible or predisposed to Coeliac disease and the sample is probed for anti-tissue transglutaminase (tTG) IgG and IgA antibodies using a tTG blend. 55650953-1 M&C PG450988GB 6 A suitable anti-DGP IgA and anti-DGP IgG capture agent may comprise deamidated gliadin peptide (DGP) or antibody (e.g. IgA / IgG) binding fragments thereof. One of skill will appreciate that the DGP capture agent (in whatever form it takes) will be bound by anti-DGP IgA and / or anti-DGP IgG. Where the sample is obtained or provided by a human subject, any anti-DGP IgA / anti-DGP IgG capture agent should be capable of binding(capturing) human anti-DGP IgA or human anti-DGP IgG. The DGP IgA / IgG capture agent may comprise a blend or mix of at least two types or forms of DGP or DGP from a plurality (e.g. at least two) of different sources. By way of example, the anti-DGP IgA / anti-DGP IgG capture agent may comprise DGP as supplied by BBI and / or Zedira. As stated, IgA and / or IgG binding fragments of these may also be used as anti-DGP IgA / anti-DGP IgG capture agents. The DGP IgA / IgG capture agent may comprise whole DGP or an anti-DGP IgA and / or IgG binding fragment thereof. A DGP IgA / IgG capture agent may comprise a DGP peptide derived from a DGP sequence. A useful DGP peptide is one that may be used to capture anti-DGP IgG and / or anti-DGP IgA in a sample. Whether the capture agent is designed to capture anti-DGP IgG or anti-DGP IgA, the capture agent will be generally referred to as a DGP capture agent. A DGP capture agent (including fusion-type capture agents) may comprise a peptide having a sequence corresponding to (or derived from) SEQ ID NO: 1: SEQ ID NO: 1 NIQVDPSGQVQWLQQQLVPQLQQPLSQQPQQTFPQPQQTFPHQPQQQVPQPQQPQQPFLQPQQP FPQQPQQPFPQTQQPQQPFPQQPQQPFPQTQQPQQPFPQQPQQPFPQTQQPQQPFPQLQQPQQP FPQPQQQLPQPQQPQQSFPQQQRPFIQPSLQQQ A DGP IgA / IgG capture agent may comprise a deaminated form of any of the sequences described herein – including for example SEQ ID NO: 1. A deaminated form of SEQ ID NO: 1 may have the following sequence (SEQ ID NO: 2): SEQ ID NO: 2 55650953-1 M&C PG450988GB 7 NIEVDPSGEVEWLEEELVPELEEPLSEEPEETFPEPEETFPHEPEEEVPEPEEPEEPFLEPEEP FPEEPEEPFPETEEPEEPFPEEPEEPFPETEEPEEPFPEEPEEPFPETEEPEEPFPELEEPEEP FPEPEEELPEPEEPEESFPEEERPFIEPSLEEE Accordingly, a DGP IgA / IgG capture agent may comprise SEQ ID NO: 2 or an anti-DGP IgA and / or anti-DGP IgG binding fragment thereof: A DGP capture agent (including fusion-type capture agents) may comprise a peptide having a sequence selected from the group consisting of: PFPQPQLPY (SEQ ID NO: 3) PYPQPQLPY (SEQ ID NO: 4) PQPQLPYPQ (SEQ ID NO: 5) PQQSFPQQQ (SEQ ID NO: 6) IQPQQPAQL (SEQ ID NO: 7) QQPQQPYPQ (SEQ ID NO: 8) SQPQQQFPQ (SEQ ID NO: 9) QQPFPQQPQ (SEQ ID NO: 10) PQPQQQFPQ (SEQ ID NO: 11) FPQQPEQPYPQQ (SEQ ID NO: 12) FPQQPQQPYPQQ (SEQ ID NO: 13) GIIQPEQPAQL (SEQ ID NO: 14) GIIQPQQPAQL (SEQ ID NO: 15) FSQPEQEFPQPQ (SEQ ID NO: 16) FSQPQQQFPQPQ (SEQ ID NO: 17) LQPQQPFPQQPQQPYPQQPQ (SEQ ID NO: 18) FLQPQQPFPQQPQQPYPQQPQQPFPQ (SEQ ID NO: 19) LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (SEQ ID NO: 20) A DGP capture agent may comprise a deaminated form of any of SEQ ID NOS: 3-20. For example, a DGP capture agent may comprise a peptide having a sequence selected from the group consisting of: PFPEPELPY (SEQ ID NO: 21) PYPEPELPY (SEQ ID NO: 22) PEPELPYPE (SEQ ID NO: 23) PEESFPEEE (SEQ ID NO: 24) 55650953-1 M&C PG450988GB 8 IEPEEPAEL (SEQ ID NO: 25) EEPEEPYPE (SEQ ID NO: 26) SEPEEEFPE (SEQ ID NO: 27) EEPFPEEPE (SEQ ID NO: 28) PEPEEEFPE (SEQ ID NO: 29) FPEEPEEPYPEE (SEQ ID NO: 30) FPEEPEEPYPEE (SEQ ID NO: 31) GIIEPEEPAEL (SEQ ID NO: 32) GIIEPEEPAEL (SEQ ID NO: 33) FSEPEEEFPEPE (SEQ ID NO: 34) FSEPEEEFPEPE (SEQ ID NO: 35) LEPEEPFPEEPEEPYPEEPE (SEQ ID NO: 36) FLEPEEPFPEEPEEPYPEEPEEPFPE (SEQ ID NO: 37) LELEPFPEPELPYPEPELPYPEPELPYPEPEPF (SEQ ID NO: 38) In these sequences (and as compared to SEQ ID NOS: 1-19) the glutamine residues (Q) are replaced with glutamic acid (E). A DGP capture agent may comprise a DGP fusion. A ‘DGP fusion’ may comprise two or more fused (linked or joined) DGP peptides. A ‘DGP’ fusion may comprise any of the deaminated DGP sequences described herein. For example a ‘DGP’ fusion may comprise a deaminated DGP sequence which is fused (linked or joined) to another DGP sequence (for example a sequence comprising SEQ ID NO: 1 or 2 or an anti-DGP IgA / IgG antibody binding fragment thereof). In one teaching, the DGP capture agent may comprise a gliadin fusion, wherein the gliadin fusion comprises deamidated α-gliadin (or an antibody binding fragment thereof) fused to deamidated γ-gliadin (or an antibody-binding fragment thereof). The various components of the DGP fusions described herein may be fused or linked via some form of short peptide linker. Any of the DGP fusions described herein may further comprise a carrier protein. The term ‘carrier protein’ may comprise, for example GST (Glutathione S-transferase), MBP (Maltose Binding Protein), Thioredoxin (Trx), SUMO (Small Ubiquitin-like Modifier). 55650953-1 M&C PG450988GB 9 A DGP capture agent may comprise a mix of any of the specific DGP capture agents described herein. This may be referred to as a DGP ‘blend’. A blend may comprise at least two different DGP capture agents. A DGP ‘blend’ may comprise an equal amount (for example a 50% / 50% mix) of at least two different DGP capture agents. By way of example, a DGP blend may comprise a DGP peptide comprising any of the sequences provided as SEQ ID NOS: 1-2 (or an anti-DGP-antibody binding fragment thereof (such as any of the peptides provided by SEQ ID NOS: 3-38)) and any of the DGP fusions described herein. In one teaching, a DGP capture agent may comprise; (i) a peptide having the sequence of SEQ ID NO: 19; or (ii) a peptide having the sequence of SEQ ID NO: 20; or (iii) a peptide having the sequence of SEQ ID NO: 37; or (iv) a peptide having the sequence of SEQ ID NO: 38; or (iii) a fusion comprising SEQ ID NO: 19 and SEQ ID NO: 20; or. (iv) a fusion comprising SEQ ID NO: 37 and SEQ ID NO: 38. As stated, any of the fusions provided in options (i)-(iv) above may further comprise a carrier protein. In one teaching a DGP capture agent comprises a blend of a fusion comprising SEQ ID NO: 37 and SEQ ID NO: 38 and a peptide sequence comprising SEQ ID NO: 1 or 2. Optionally, the fusion may comprise a carrier protein. In one teaching a DGP capture agent comprises a blend of a fusion comprising SEQ ID NO: 37 and SEQ ID NO: 38 and a peptide sequence comprising SEQ ID NO: 1 (or an anti-DGP IgG / IgA antibody binding fragment thereof). Optionally, the fusion may comprise a carrier protein. In one teaching a DGP capture agent comprises a blend of a fusion comprising SEQ ID NO: 37 and SEQ ID NO: 38 and a peptide sequence comprising SEQ ID NO: 2 (or an anti-DGP IgG / IgA antibody binding fragment thereof). Optionally, the fusion may comprise a carrier protein. Any of the anti-DGP IgA / anti-DGP IgG capture agents may be formulated in a print buffer 55650953-1 M&C PG450988GB 10 for printing. The print buffer may comprise, glycerol or sucrose and a phosphate or carbonate print buffer. The anti-DGP IgA / anti-DGP IgG capture agent may be present at a concentration of about 150 µg / ml of print buffer. Note, where the capture agent comprises a mix of at least two different sources of DGP, an equal amount of each DGP type may be used. For example, where the total amount of DGP in a print formulation is to be 150 µg / ml, and the formulation is to contain a blend of two different DGP sources, then each DGP type may be used at a concentration of 75 µg / ml. As stated, an assay of this disclosure may use a sample. The sample may be provided or obtained from a subject to be tested for an autoimmune disease, such as, for example, Coeliac disease. The subject may be suffering from an autoimmune disease or Coeliac disease and exhibiting one or more associated symptoms. The subject may be healthy or at least asymptomatic, but thought to be predisposed, susceptible or at risk of having or contracting an autoimmune disease or Coeliac disease. The sample may comprise a biological fluid such as, for example blood (e.g. whole blood) or a fraction thereof (e.g. plasma or serum) and / or saliva. The sample may be contacted with the capture agents under conditions which permit binding between any of the relevant targets in the sample (IgA, anti-tTG IgA / anti-tTG IgG or anti-DGP IgA / anti-DGP IgG) and the capture agents. As stated, a single sample may be concurrently, concomitantly or simultaneously contacted with all of the relevant capture agents. A single sample may be contacted with all of the relevant capture agents, in parallel. In other words, the sample may be concurrently, concomitantly or simultaneously (at the same time) contacted with the following capture agents: an IgA capture agent; and an anti-tTG IgA capture agent; and an anti-tTG IgG capture agent; and an anti-DGP IgA capture agent; and an anti-DGP IgG capture agent. As stated, the capture agents may be immobilised to one or more substrates. Moreover, the anti-tTG IgA capture agent; and the anti-tTG IgG capture agent may comprise a tTG 55650953-1 M&C PG450988GB 11 blend as described herein. The anti-DGP IgA capture agent; and the anti-DGP IgG capture agent may comprise a DGP blend. The capture agents may be immobilised in an array, where each capture agent is immobilised (e.g. by printing) to form a discrete dot or spot on a substrate. Other capture agents may be immobilised on the same substrate. An assay of this disclosure may comprise a wash step. The purpose of the wash step (which may take place after the initial contact step as a preliminary wash step) is to remove the sample from the assay (so that it does not contaminate later steps) and therefore any unbound target or ‘off-target’ moieties. Again, this avoids contaminating later steps and reduces the incidence of false positive / negative results. A wash buffer may comprise, for example, PBS, , proclin and tween 20.. The contacting step of any of the assays described herein may result in the formation of capture agent / target complexes. One of skill will understand that such complexes result from a successful binding event between a capture agent (optionally immobilised to a substrate as described herein) and any capture agent target present in the sample. An assay of this disclosure may further comprise a detection step. The detection step may comprise the use of a detection agent which binds to any capture agent / target complexes formed during the contact step and left after any (optional) wash step. The detection agent may comprise an antibody. The antibody may comprise or be conjugated to a detectable (for example optically detectable) moiety. The one or more detection agents may comprise, for example an anti-IgA antibody and / or an anti-IgG antibody conjugated to an optical moiety or an enzyme. For example, the detection agent may be conjugated to a colourimetric or a fluorescent probe. In some examples, the detection agent may be conjugated to an enzyme, such as horseradish peroxidase or alkaline phosphatase. When a suitable substrate is provided, the enzymatic reaction may produce light, fluorescence or colour as a by-product enabling detection of the detection agent. A detection step of this disclosure may exploit absorbance. The detection agent may comprise or be conjugated to HRP. By way of example, where the complex comprises an IgA molecule, the detection agent may comprise an anti-IgA antibody. Where the complex comprises an IgG molecule, the detection agent may comprise an anti-IgG antibody 55650953-1 M&C PG450988GB 12 The detection step may comprise a period of incubation where the detection agent is contacted with the assay for a period of time and under physiological conditions that permit binding between the detection agent and any of the abovementioned complexes. The incubation may be about 20-30 minutes at a temperature of about 30-37 ± 2 º C (for example 35 ± 2 º C). After any detection step incubation period, the assay may be subject to a further wash. The purpose of this wash step (which may take place after the incubation period of the detection step) is to remove the detection agent from the assay (so that it does not contaminate later steps) and therefore any unbound detection agent. Again, this avoids contaminating later steps and reduces the incidence of false positive / negative results. A wash buffer may comprise, for example, PBS, proclin and tween 20. It should be noted that the initial phase of the detection step, where a detection agent is added and incubated with the assay (which may comprise immobilised capture agent / target complexes) may result in the formation of capture agent / target / detection agent complexes. One of skill will appreciate that the presence of these complexes (which only form if the sample contains any of the targets described herein) indicates that the sample has been obtained or provided by a subject having or susceptible, predisposed or at risk of developing, an autoimmune disease, such as, for example, Coeliac disease. Accordingly, the detection step may further comprise a step in which these complexes are revealed to the user. Where the detection agent comprises an optically detectable moiety, the detection step may further comprise the appropriate procedure to render that moiety optically detectable. For example, where the detection agent comprises HRP, the detection step may comprise contacting the assay with an HRP substrate – e.g. 3,3'5,5'- tetramethylbenzidine (TMB); this being a chromogen substrate for horseradish peroxidase. In this way, the presence of capture agent / target / detection agent complexes is revealed through the colourimetric reaction between the HRP moiety of the detection agent and its substrate TMB. One of skill will appreciate that the strength of the detection reaction is proportionate to the amount of target present in the sample. Where the detection step reveals the 55650953-1 M&C PG450988GB 13 presence of capture agent / target / detection agent complexes in the assay, the user may assume that the sample has been provided by, or obtained from, a subject having an autoimmune disease such as Coeliac disease or susceptible, predisposed or at risk of developing the same. In one teaching, the level of a target above / below a control (or threshold / normal) level associated with or indicative of Coeliac disease may be used to diagnose Coeliac disease. A control (normal or threshold) level of IgA may correspond to the level of IgA present in a healthy subject – namely a subject not having, a selective IgA deficiency. Any sample found to have a level of IgA which is lower than the control (or normal) level of IgA may be identified as having selective IgA deficiency. an autoimmune disease or coeliac disease. A control (normal or threshold) level of anti-tTG IgA may correspond to the level of anti- tTG IgA present in a healthy subject – namely a subject not having, or not predisposed or at risk of developing an autoimmune disease (characterised by a presence of anti-tTG IgA) or coeliac disease. Any sample found to have a level of anti-tTG IgA which is higher than the control (or normal) level of anti-tTG IgA may be identified as a sample from a subject suffering from, predisposed, susceptible or at risk of developing, an autoimmune disease or coeliac disease. A control (normal or threshold) level of anti-tTG IgG may correspond to the level of anti- tTG IgG present in a healthy subject – namely a subject not having, or not predisposed or at risk of developing an autoimmune disease (characterised by a presence of anti-tTG IgG) or coeliac disease. Any sample found to have a level of anti-tTG IgG which is higher than the control (or normal) level of anti-tTG IgG may be identified as a sample from a subject suffering from, predisposed, susceptible or at risk of developing, an autoimmune disease or coeliac disease. A control (normal or threshold) level of anti-DGP IgA may correspond to the level of anti- DGP IgA present in a healthy subject – namely a subject not having, or not predisposed or at risk of developing an autoimmune disease (characterised by the presence of anti- DGP IgA) or coeliac disease. Any sample found to have a level of anti-DGP IgA which is higher than the control (or normal) level of anti-DGP IgA may be identified as a sample 55650953-1 M&C PG450988GB 14 from a subject suffering from, predisposed, susceptible or at risk of developing, an autoimmune disease or coeliac disease. A control (normal or threshold) level of anti-tTG IgG may correspond to the level of anti- tTG IgG present in a healthy subject – namely a subject not having, or not predisposed or at risk of developing an autoimmune disease (characterised by the presence of anti- tTG IgG) or coeliac disease. Any sample found to have a level of anti-tTG IgG which is higher than the control (or normal) level of anti-tTG IgG may be identified as a sample from a subject suffering from, predisposed, susceptible or at risk of developing, an autoimmune disease or coeliac disease. The assays described herein may be described as (in vitro) an immunoassay. As it would be appreciated by the skilled person in the art, an in vitro assay provides an advantage of being a non-invasive approach to diagnosis of autoimmune diseases such as Coeliac disease. Moreover, the assay or method disclosed herein are automated and also ‘high- throughput’ in nature. In one teaching, the various assays described herein may be conducted and ‘run’ using the MosaiQ® AiPlex platform. The assays may be further described as ‘multiplex’ assays and in contrast to prior art assays, they are capable of screening samples for the presence of multiple targets (e.g. anti-tTG & DGP IgA and IgG) all together and at the same time (simultaneously and / or concurrently)). In this way, the described assays allow results to be obtained using only one sample and within a very short time frame (e.g. one hour) of being provided with or obtaining a sample. Automated assays or systems of this type allow for continuous random-access testing instead of batch processing, reducing the time-to-multiplexed results. By providing a comprehensive autoantibody panel result swiftly, this enables early, accurate diagnosis of celiac disease and represents a significant improvement over current methods by simplifying complex diagnostic algorithms, improving clinical laboratory workflow, reducing manual intervention and risk of human error and accelerating time to test results. The inclusion of one or more probes which detect tissue transglutaminase IgG antibodies provide the advantage of being able to diagnose autoimmune diseases such as Coeliac disease in individuals with selective IgA deficiency. Approximately 2-3% of individuals with Coeliac disease have selective IgA deficiency. In these cases, IgA-based tests might 55650953-1 M&C PG450988GB 15 be falsely negative. IgG-based tests, such as deamidated gliadin peptide IgG (DGP-IgG) or tTG-IgG, are crucial for accurate diagnosis in these patients. Moreover, detection of IgG antibodies can provide useful diagnostic information, especially when IgA antibodies are negative, but clinical suspicion remains high. Importantly, the complementary (parallel, concomitant, concurrent or simultaneous) testing for a number of targets, as applied in any of the assays described herein, provides a comprehensive screening approach. Specifically, the combined detection of IgA and IgG antibodies provides a more thorough screening approach, especially in complex cases or when initial test results are inconclusive (as they often are with prior art assays). In addition, when IgA and IgG antibodies are probed for and found to be present (positive) in a sample, the diagnostic confidence increases, which supports accurate diagnosis of Coeliac disease and reduces the likelihood of misdiagnosis. Further, the non-invasive approach of the present disclosure allows IgA and IgG antibody levels to be monitored to assess a patient's compliance with a gluten-free diet and their response to treatment over time. As such, the combined IgA and IgG detection tests of the described assays, offers a robust approach for diagnosing and managing Coeliac disease by minimising the risk of false diagnosis due to variations in an individual’s immune response. Moreover, the multiplex assays described herein have been found to be improved in terms of sensitivity and selectivity over prior art assays. In a further aspect of this disclosure, there is provided a substrate having immobilised thereon: an IgA capture agent (for determining the total IgA content of a sample) and an anti-tTG IgA capture agent (for determining the specific anti-tTG IgA content of a sample); and an anti-DGP IgA capture agent (for determining the specific anti-DGP IgA content of a sample). Each capture agent of the substrate may be immobilised as a discrete spot on a surface of the substrate. Each capture agent may be applied by printing. 55650953-1 M&C PG450988GB 16 Each capture agent may be formulated in a print buffer. The print buffer may comprise BSA. In a further aspect, there is provided a substrate having immobilised thereon: an anti-tTG IgG capture agent (for determining the specific anti-tTG IgG content of a sample); and an anti-DGP IgG capture agent (for determining the specific anti-DGP IgG content of a sample). Again, each capture agent of the substrate may be immobilised as a discrete spot on a surface of the substrate. Furthermore, the substrate may be for use in the diagnosis of Coeliac disease. The substrates of this disclosure may further comprise other immobilised capture agents. Each substrate may take the form of a microarray chip. The disclosure further provides a kit comprising any of the substrates described herein. In addition to a substrate, the kit may further comprise: (i) a receptacle for sample collection; and / or (ii) one or more buffers; and / or (iii) an anti-IgA antibody and / or an anti-IgG antibody conjugated to an optically and / or enzymatically detectable probe. The IgA antibody and / or the anti-IgG antibody conjugated to an optically and / or enzymatically detectable probe may be an anti-IgA antibody and / or an anti-IgG antibody conjugated to a fluorescent probe. In other examples, the anti-IgA antibody and / or the anti-IgG antibody may be conjugated to an enzyme for chemiluminescent detection. In one example, the kit may comprise an anti-human IgA antibody and / or an anti-human IgG antibody. 55650953-1 M&C PG450988GB 17 In one aspect, there is provided a method of producing a substrate of this disclosure, said method comprising immobilising one or more of the disclosed capture agents to discrete locations on a surface of the substrate. Each capture agent may be applied using a printing technique. Each capture agent may be formulated in a print buffer, which buffer may comprise BSA. DETAILED DESCRIPTION The present disclosure is described below, by way of example only, with reference to the accompanying Figures, which show: Figure 1: A. Example of MosaiQ array layout for detection of IgA auto-antibodies in Celiac Disease: B. Example of MosaiQ array layout for detection of IgG auto-antibodies in Celiac Disease. The present disclosure will now be described with reference to the following examples. It should be appreciated that these examples are not intended to limit the scope of the claims to the invention but are rather intended to be exemplary of certain embodiments. Any variations in the exemplified methods that occur to the skilled artisan are intended to fall within the scope of the invention. EXAMPLES Materials: Antigens Table 1: Antigens Probe Open tissue transglutaminase (Open tTG) Closed tissue transglutaminase (Closed tTG) Deamidated gliadin peptide (DGP) Deamidated gliadin peptide (DGP) – ZA Substrate Nexterion Epoxysilane coated glass (Schott Minifab) was used for peptide immobilisation and microarray manufacturing. 55650953-1 M&C PG450988GB 18 HM069 (Immuno-trol Celiac G) and HM070 (Immuno-trol Celiac A) are quality control samples (sera) sourced from Theradiag (France), that are positive for tTG, gliadin, and DGP, IgG and IgA respectively. The exact batch-specific concentration of tTG IgA, tTG IgG, DGP IgA, DGP IgG was determined using EliA Celikey IgA, Celikey IgG, GliadinDPIgA and GliadinDPIgG (all from Phadia / Thermo Fisher). Characterised sera samples negative or positive for various celiac autoantibodies were sourced from CerbaXpert (Saint Ouen l'Aumone, France) or Eurofins Biomnis Sample Library (Lyon, France), unless otherwise specified. Methods: Microarray printing and peptide immobilisation 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. The antigen print formulations contained a base buffer (phosphate pH 7.2 or carbonate pH 9.6), a salt (NaCl or KCl) and a viscosity modifier (glycerol or sucrose). Immunoassay methods 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 and human IgA are dispensed concurrently to two side by side microarrays, 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 55650953-1 M&C PG450988GB 19 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) and IgA Conjugate (ref 155052) were supplied by Alive Dx. Results and Discussion: EXAMPLE 1. tTG probe formulation As compared to the closed confirmation of tTG, the open conformation of tTG is considered superior for use in diagnosis purposes to the closed conformation of tTG (see EP2462439). The diagnostic performances of 2 different tTG containing capture probes were compared. Formulation #1 comprised 25 μg / mL of open tTG . Formulation #2 comprised 25 μg / mL of tTG blend in 2:1 open-to-closed ratio. The formulations were printed on the MosaiQ Manufacturing System as described above and the obtained microarrays were assayed using previously characterized sera samples within known sero-status for tTG IgA and tTG IgG. As shown in Table 2 below, ‘blend’ tTG results in higher positive percent agreement (PPA) as well as higher negative percent agreement (NPA) when testing for tTG IgA compared to open tTG. For tTG IgG detection, blend tTG results in higher PPA compared to open tTG as well. Table 2: Performance of tTG IgA detection Capture probe: open tTG Capture probe: blend tTG Reactive Non-reactive Reactive Non-reactive Positive (n=19) 17 2 18 1 Negative (n=19) 1 18 0 19 %Agreement 89.5% 94.7% 94.7 100% PPA NPA PPA NPA 55650953-1 M&C PG450988GB 20 Table 3: Performance of tTG IgG detection Capture probe: open tTG Capture probe: blend tTG Reactive Non-reactive Reactive Non-reactive Positive (n=17) 14 3 15 2 Negative (n=20) 1 19 1 19 %Agreement84.2%95.0% 88.2% 95.0% PPA NPA PPA NPA EXAMPLE 2. Clinical sensitivity and specificity of MosaiQ® AiPlex CD Clinical sensitivity and specificity were evaluated by testing clinically characterized frozen human serum samples with the MosaiQ® AiPlex CD assay and comparing the clinical diagnosis with the result of the associated analyte(s). The clinically characterized samples, using criteria representative of contemporary clinical practice, were provided by Assistance Publique – Hopitaux de Paris (AP-HP, Paris, France) or sourced from external vendors, Serenity Biosciences (Orlando, USA), Sample Smart Ltd (London, UK). They were divided into 2 groups: - Samples from patients diagnosed with Celiac Disease by a certified physician (n=168) - Samples from patients diagnosed with diseases that could be reasonably considered in the differential diagnosis of Celiac Disease. These samples are disease controls (n=467). Table 4: Clinical sensitivity and specificity of MosaiQ Aiplex CD assay IgA IgG tTG IgA DGP IgA tTG IgG DGP IgG True Positive 121 111 41 119 True Negative 462 443 460 444 False Positive 2 22 2 15 False Negative 41 54 103 42 Equivocal 9 5 27 12 Sensitivity% 74.7% 67.3% 28.5% 73.9% 55650953-1 M&C PG450988GB 21 Specificity% 99.6% 95.3% 99.6% 96.7% EXAMPLE 3. Detection of DGP IgA, DGP IgG and tTG IgG in tTG IgA-negative, immune-competent celiac disease patients As expected tTG IgA assay had the highest sensitivity for celiac disease identification with 74.7%, in agreement with data reported in literature. However, when using tTG IgA 5 alone as a first line marker for laboratory testing as recommended in some clinical guidelines, a number of true celiac disease patients are missed and classified as not- diseased (false negatives). In the data presented in Example 2, there are 41 false negatives for tTG IgA and each of these subjects has detectable Total IgA values (Table 5), indicating that they are not IgA-deficient. As such, in routine clinical care, they would 10 not be addressed to tTG or DGP IgG testing for celiac disease. Among these 41 celiac disease patients who were negative for tTG IgA antibodies, 17 had DGP IgA, DGP IgG or tTG IgG antibodies. When these markers are tested simultaneously with tTG IgA, the sensitivity for celiac disease identification increases from 74.7% to 85.2%, an important improvement. Out of the 41 subjects, 15 - 3 were positive for DGP IgA alone and negative for the other markers, - 8 were positive for DGP IgG alone and negative for the other markers, - none were positive for tTG IgG alone and negative for the other markers. These results clearly highlight the utility of testing for DGP IgA, DGP IgG and tTG IgG, concurrently with tTG IgA and Total IgA. 20 Table 5: Results of “non-first line” celiac disease markers in celiac disease patients negative for tTG IgA MosaiQ Aiplex CD index values MosaiQ Aiplex CD interpretation Sample Celiac DGP tTG DGP tTG Total ID Disease IgG IgG IgA IgA IgA DGP tTG DGP tTG (AU / mL) (AU / mL) (AU / mL) (AU / mL) (AU / mL) IgG IgG IgA IgA001 YES0.0 0.0 0.0 0.0 14.6 NEG NEG NEG NEG002 YES2.0 0.0 0.0 0.0 14.5 NEG NEG NEG NEG003 YES16.1 0.0 11.4 6.4 17.1 POS NEG POS NEG004 YES0.0 0.0 0.0 0.0 18.0 NEG NEG NEG NEG005 YES6.4 0.0 3.6 0.0 16.4 NEG NEG NEG NEG006 YES14.7 5.0 0.0 6.9 13.7 POS NEG NEG NEG007 YES2.7 0.0 5.8 0.0 15.3 NEG NEG NEG NEG008 YES1.4 0.0 3.7 0.0 15.9 NEG NEG NEG NEG009 YES15.0 0.0 3.8 0.0 13.9 POS NEG NEG NEG010 YES7.2 0.0 0.0 0.0 14.6 NEG NEG NEG NEG011 YES0.0 0.0 0.0 0.0 15.1 NEG NEG NEG NEG55650953-1 M&C PG450988GB 22 YES1.9 0.0 4.3 0.0 14.6 NEG NEG NEG NEGYES1.0 0.0 0.0 0.0 15.2 NEG NEG NEG NEGYES16.1 0.0 0.0 0.0 13.1 POS NEG NEG NEGYES9.6 0.0 3.8 0.0 13.2 EQV NEG NEG NEGYES1.3 0.0 4.3 0.0 13.5 NEG NEG NEG NEGYES19.1 16.3 7.0 0.0 13.3 POS POS NEG NEGYES19.0 0.0 1.7 0.0 14.2 POS NEG NEG NEGYES4.8 0.0 18.9 0.0 13.7 NEG NEG POS NEGYES18.6 0.0 1.7 0.0 13.4 POS NEG NEG NEGYES0.6 0.0 0.0 5.9 13.5 NEG NEG NEG NEGYES17.4 0.0 11.9 0.0 13.6 POS NEG POS NEGYES15.1 1.8 0.0 4.8 13.7 POS NEG NEG NEGYES0.0 0.0 0.0 6.4 13.7 NEG NEG NEG NEGYES1.0 1.6 19.1 0.0 14.2 NEG NEG POS NEGYES19.3 0.0 10.4 0.0 13.4 POS NEG POS NEGYES6.2 0.0 5.8 0.0 13.9 NEG NEG NEG NEGYES20.2 0.0 13.5 4.0 13.5 POS NEG POS NEGYES15.8 8.9 13.1 0.0 12.9 POS EQV POS NEGYES20.3 1.8 4.0 0.0 12.5 POS NEG NEG NEGYES7.6 0.9 17.0 6.1 13.7 NEG NEG POS NEGYES7.1 0.0 0.0 0.0 13.3 NEG NEG NEG NEG YES0.0 0.0 5.3 5.2 13.2 NEG NEG NEG NEGYES0.5 0.0 4.5 0.0 14.3 NEG NEG NEG NEGYES3.2 0.0 0.0 0.0 13.3 NEG NEG NEG NEGYES0.0 0.0 0.0 0.0 14.1 NEG NEG NEG NEGYES13.5 0.0 4.2 6.7 13.5 POS NEG NEG NEGYES0.4 0.0 5.2 0.0 14.2 NEG NEG NEG NEGYES3.5 0.0 3.4 0.0 14.5 NEG NEG NEG NEGYES5.7 0.0 1.9 0.0 13.9 NEG NEG NEG NEGYES0.0 0.0 0.0 0.0 13.0 NEG NEG NEG NEGEXAMPLE 4. Logistic Regression The clinical data set was divided randomly into 2 subsets. The training subset corresponded to approximately 70% of the whole set (n=454), whereas the test set was the remaining 30% (n=188). Using GraphPad Prism or R, a logistic regression model was fit to the training data subset, estimating the model parameters for the following: Response ~ Intercept + meanGQM_DGP_IgG + meanGQM_tTG_IgG + meanGQM_DGP_IgA + meanGQM_tTG_IgA + meanGQM_Total_IgA, Response is the probability of having celiac disease. For each marker, GQM is the normalized signal metric of the corresponding spot (GQM = raw signal of spot / mean of (normalisation spots signal)*100). The generated logistic regression model was applied to predict the celiac disease probability for each subject in the test subset of the data, and model 55650953-1 M&C PG450988GB 23 performance / value was evaluated by comparing the disease status predicted by the model to the true disease status. As shown in Table 6, the 5-marker model has a validated AUC of 0.91, superior to the AUC of tTG IgA alone which is only 0.86. Taken together with the results of example 3, this analysis exemplifies the benefit of combining tTG IgA, tTG IgG, DGP IgA, DGP IgG and Total IgA altogether for superior celiac disease diagnostic performance. Table 6: Comparison of diagnostic performance of a 5-marker model compared to tTG IgA alone Area Under ROC curve 95% CI 5-marker model train set 0.95 0.92-0.98 5-marker model test set 0.91 0.85-0.97 tTG IgA alone test set 0.86 0.80-0.93 The present disclosure provides an assay for in vitro simultaneous semi-quantitative detection of IgA and IgG antibodies directed to tissue Transglutaminase (tTG) and deamidated gliadin peptides (DGP) as well as qualitative detection of total IgA in serum. Exemplary microarray layout is provided in Figures 1 and 2, and Table 7 provides information related to the probe at each position. Table 7. MosaiQ® AiPlex CD IgA panel probe, spot position and function (with reference to Fig.1 A & B). Position Probe name Function 1, 6, 61, 66 Gridding Reference point for location of probes to enable image analysis (gridding) 16 Positive control Confirmation of addition / presence sample and / or reagents 15 Negative Control Confirmation of absence of nonspecific binding 27, 28 tTG blend Semi-quantitative detection of IgA or IgG antibodies in human serum against human tissue transglutaminase (tTG). 55650953-1 M&C PG450988GB 24 33, 34 Total IgA Qualitative detection of Total IgA antibodies in human serum 39, 40 DGP blend Semi-quantitative detection of IgA or IgG antibodies in human serum against deamidated gliadin peptides 51, 52 CAL IgA or IgG Calibrator IgA or IgG controls for normalisation of results Example 5 Testing a DGP blend The diagnostic performances of 3 different DGP containing capture probes were compared. Antigen 1 (A1) was a recombinant protein comprising SEQ ID NO: 1. Antigen 2 (A2) was a fusion protein comprising SEQ ID NO: 37 and SEQ ID NO: 38 linked to a carrier protein. Antigen 3 (A3) comprised a 50%-%50% blend of A1 and A2. Formulations were printed on the MosaiQ Manufacturing System as described above and the obtained microarrays were assayed using previously characterized sera samples with known sero-status for DGP IgA. As shown in Table 8 below, use of Antigen 3 as capture agent results in higher positive percent agreement (PPA) than when using Antigen 1 or Antigen 2 alone. Table 8 Antigen 1 (A1) Antigen 2 (A2) Antigen 3 (A3) Non- Non- Non- Reactive Reactive Reactive reactive reactive reactive Positive 18 5 20 3 23 1 (n=24) Negative 4 57 5 56 4 57 (n=61) %Agreement 79% 93% 88% 92% 96% 93% PPA NPA PPA NPA PPA NPA References (1) Al-Toma A, Volta U, Auricchio R, Castillejo G, Sanders DS, Cellier C, et al. European Society for the Study of Coeliac Disease (ESsCD) guideline for Coeliac disease and other gluten-related disorders. United European Gastroenterol J.2019;7(5):583-613. 55650953-1

Claims

M&C PG450988GB 25 CLAIMS:

1. A method for diagnosing an autoimmune disease, said method comprising contacting a sample with (i) an anti-tTG IgA / IgG capture agent; and (ii) an anti-DGP IgA / IgG capture agent; wherein the anti-DGP IgA / IgG capture agent comprises a DGP fusion and a DGP peptide.

2. The method of claim 1, wherein the DGP fusion comprises at least two anti-DGP IgA / IgG capture agents.

3. The method of claim 2, wherein the DGP fusion comprises SEQ ID NO: 37 and SEQ ID NO:

38.

4. The method of claims 2-3, wherein the DGP fusion further comprises a carrier protein.

5. The method of claim 1, wherein the anti-DGP IgA / IgG capture agent comprises a DGP fusion and a protein or peptide comprising SEQ ID NO: 1 or 2 or an anti-DGP IgA / IgG binding fragment of either SEQ ID NO: 1 or 2.

6. The method of claim 6, wherein the DGP fusion comprises SEQ ID NO: 37 and SEQ ID NO:

38.

7. The method of any preceding claim, wherein the anti-tTG IgA / IgG capture agent is selected from the group consisting of: (i) open tissue transglutaminase (‘open’ tTG) or an antibody binding fragment thereof; (ii) closed tissue transglutaminase (‘closed’ tTG) or an antibody binding fragment thereof; and (iii) open tTG or an antibody binding fragment thereof and closed tTG or an antibody binding fragment thereof.

8. The method of any preceding claim, wherein the contacting step is performed under conditions which permit binding between any anti-tTG IgA / IgG and / or anti-DGP IgA / IgG in the same and the anti-tTG IgA and anti-DGP IgA / IgG capture agents. 55650953-1M&C PG450988GB 26 9. The method of any preceding claim, wherein a sample found to contain anti-tTG IgA / IgG and / or anti-DGP IgA / IgG may be a sample obtained from or provided by, a subject suffering from or at risk of developing an autoimmune disease.

10. The method of any preceding claim, wherein the autoimmune disease is Coeliac disease.

11. The method of any preceding claim, wherein the method further comprises probing the sample to determine the total IgA content.

12. A multiplex assay for diagnosing Coeliac disease, wherein said multiplex-assay comprises concurrently probing a sample for: (i) anti-tissue transglutaminase (tTG) IgG and IgA antibodies; (ii) anti-deamidated gliadin peptide (DGP) IgG and IgA antibodies; and (iii) total IgA content; wherein detection of anti-tTG IgA or IgG and / or anti-DGP IgA or IgG in a sample indicates that the sample may have been obtained from a subject suffering from, susceptible or predisposed to Coeliac disease, characterised in that the anti-deamidated gliadin peptide (DGP) IgG and IgA antibodies are detected using an anti-DGP IgA / IgG capture agent comprising a DGP fusion and a DGP peptide.

13. The multiplex assay of claim 12, wherein the DGP peptide comprises SEQ ID NO: 1 or 2 and the DGP fusion comprises SEQ ID NOS: 37 and 38.

14. The multiplex assay of claims 12-13, wherein the anti-tissue transglutaminase (tTG) IgG and IgA antibodies are detected using an anti-tTG IgA and anti-tTG IgG capture agent selected from the group consisting of: (i) open tissue transglutaminase (‘open’ tTG) or an antibody binding fragment thereof; (ii) closed tissue transglutaminase (‘closed’ tTG) or an antibody binding fragment thereof; and (iii) open tTG or an antibody binding fragment thereof and closed tTG or an antibody binding fragment thereof. 55650953-1M&C PG450988GB 27 15. A method for diagnosing an autoimmune disease, wherein said method comprises contacting a sample with: (a) an anti-tTG IgG and anti-tTG IgA capture agent selected from the group consisting of: (i) open tissue transglutaminase (‘open’ tTG) or an antibody binding fragment thereof; (ii) closed tissue transglutaminase (‘closed’ tTG) or an antibody binding fragment thereof; and (iii) open tTG or an antibody binding fragment thereof and closed tTG or an antibody binding fragment thereof; and (b) an anti-DGP IgA and anti-DGP IgG capture agent selected from the group consisting of: (i) a peptide having a sequence corresponding to SEQ ID NO: 1 or an anti-DGP IgA and anti-DGP IgG binding fragment thereof; (ii) a peptide having a sequence corresponding to SEQ ID NO: 20 or an anti-DGP IgA and anti-DGP IgG binding fragment thereof; (iii) a peptide having a sequence corresponding to any one of SEQ ID NOS: 2-19 (iv) a peptide having a sequence corresponding to any one of SEQ ID NOS: 21-38 (v) a fusion protein comprising SEQ ID NOS: 36 and 37; (vi) SEQ ID NO: 1 and a fusion protein comprising SEQ ID NO: 36 and 37 wherein detection of anti-tTG IgA or IgG and / or anti-DGP IgA or IgG in a sample and optionally, the absence of IgA, indicates that the sample may have been obtained from a subject suffering from, susceptible or predisposed to an autoimmune disease 16. The method or multiplex assay of any preceding claim wherein the sample is provided or obtained from a subject to be tested for an autoimmune disease or Coeliac disease.

17. The method or multiplex assay of any preceding claim, wherein the sample comprises a biological fluid, blood or a fraction thereof and / or saliva. 55650953-1M&C PG450988GB 28 18. A substrate having immobilised thereon: an IgA capture agent; and an anti-tTG IgA capture agent; and an anti-DGP IgA capture agent; wherein the capture agent of the substrate is immobilised as a discrete spot on a surface of the substrate.

19. A substrate having immobilised thereon: an anti-tTG IgG capture agent; and an anti-DGP IgG capture agent; wherein the capture agent of the substrate is immobilised as a discrete spot on a surface of the substrate.

20. Use of the substrates of claims 18 and 19 in a method of diagnosing an autoimmune disease or Coeliac disease. 55650953-1

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