How to diagnose multiple sclerosis

Abstract

The present invention provides immunoassay-based methods for diagnosing and monitoring multiple sclerosis (SEP) using MSBI1.176 or MSBI2.176 Rep protein or fragments thereof as antigens that bind to antibodies against MSBI1.176 or MSBI2.176 Rep protein from a subject sample.

Description

[Technical field]

[0001] The present invention relates to a method for the diagnosis of multiple sclerosis, which is based on a bridging ELISA immunoassay for detecting antibodies against the BMMF1 Rep protein in a sample from a subject. [Background technology]

[0002] Neurodegenerative diseases of the central nervous system (CNS), which cause progressive loss of neuronal structure and function, are particularly devastating to affected patients and their families. Among these neurodegenerative diseases are, for example, multiple sclerosis (MS), Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), and stroke. Due to the complexity of the CNS, many of these diseases remain poorly understood to date.

[0003] One of the most common progressive neurodegenerative diseases is multiple sclerosis (MS). MS is a chronic inflammatory demyelinating disease of the CNS and is the leading cause of neurological disability in young or middle-aged adults. Multiple sclerosis (MS) is an autoimmune disease with autoimmune activity against central nervous system (CNS) antigens. It affects approximately 2.3 million individuals worldwide (Non-Patent Document 1), and there is currently no available therapeutic treatment. It is primarily a disease of young adults, with 70%-80% of patients presenting to the physician between the ages of 20-40 years (Non-Patent Document 2), and onset occurs earlier but is often not recognized by the patient as a symptom of the disease. MS has a gender bias affected by the phenotype, with approximately 64%-70% of diagnosed patients being female (Non-Patent Document 2).

[0004] Symptoms associated with MS include sensory changes (hypoesthesia and dysesthesias), muscle weakness, muscle spasms, movement difficulties, coordination and balance difficulties (ataxia), speech problems (dysarthria) or swallowing problems (dysphagia), vision problems (nystagmus, optic neuritis and reduced vision, or diplopia), fatigue, acute or chronic pain, bladder and bowel difficulties. Varying degrees of cognitive impairment and affective symptoms of depression or unstable mood are also common symptoms. The main clinical measure of disability progression and symptom severity is the Expanded Disability Status Scale (EDSS). Further symptoms of MS are well known in the art and are described in standard medical and neurological textbooks.

[0005] The pathogenesis of MS is due to the breakdown of T lymphocyte tolerance to CNS self-antigens, leading to chronic inflammation followed by demyelination and neuroaxonal degeneration, mainly in periventricular and juxtacortical white matter (Non-Patent Document 3). Axonal damage occurs already early in the disease, independent of demyelination, and is best correlated with clinical disability during the progressive course of MS (Non-Patent Document 4). Similarly, neuronal damage and atrophy in gray matter, with a predilection for the cingulate gyrus, frontotemporal cortex, and hippocampus, have also been shown to occur from the earliest stages and likely play an important role in clinical progression (Non-Patent Document 5).

[0006] There are four major clinical types of MS: 1) relapsing-remitting MS (RR-MS), characterized by clearly defined relapses, with complete recovery or residual deficits at recovery; periods between relapses of the disease characterized by a lack of disease progression; 2) secondary progressive MS (SP-MS), characterized by an initial relapsing-remitting course followed by progression with or without occasional relapses, minor remissions, and plateaus; 3) primary progressive MS (PP-MS), characterized by disease progression from the very beginning of onset, with occasional plateaus and temporary minor improvements; 4) progressive relapsing MS (PR-MS), characterized by progressive disease onset, with apparent acute relapses, with or without complete recovery; periods between relapses characterized by continued progression.

[0007] Clinically, the disease most frequently manifests as a relapsing-remitting disease and, to a lesser extent, as a steady progression of neurological disability. Relapsing-remitting MS (RR-MS) is characterized by recurrent attacks of focal or multifocal neurological dysfunction. Attacks may occur, remit, and relapse seemingly randomly for many years. Remissions are often incomplete, and as one attack follows another, a stepwise downward progression occurs, with increasing permanent neurological deficits. The usual course of RR-MS is characterized by repeated relapses, with the eventual development of disease progression for the majority of patients. The subsequent course of the disease is unpredictable, but most patients with relapsing-remitting disease (RR-MS) eventually transition to secondary progressive disease. In the relapsing-remitting phase, relapses alternate with periods of clinical inactivity, which may or may not result in sequelae, depending on the presence or absence of neurological deficits between episodes. The periods between relapses in the relapsing-remitting phase are clinically stable. On the other hand, patients with progressive MS, as defined above, show a steady increase in deficits, either from the time of onset or during the post-episode period, although this designation does not exclude the further occurrence of new relapses.

[0008] This disease is characterized by inflammation in parts of the CNS, leading to loss of the myelin sheath around neuronal axons (demyelination), axon loss, and eventual death of nerve cells (neurons), oligodendrocytes, and glial cells. For a comprehensive review of MS and current treatments, see, for example, Non-Patent Document 6.

[0009] More recently, it has been shown that, in addition to T cells, B lymphocytes (expressing the CD20 molecule) play a central role in MS and may influence the underlying pathophysiology through at least four specific functions: 1. Antigen presentation: B cells can present self-neuroantigens to T cells and activate them. 2. Cytokine production: B cells in patients with MS produce abnormal proinflammatory cytokines that can activate T cells and other immune cells. 3. Production of autoantibodies: B cells produce autoantibodies that can cause tissue damage and activate macrophages and natural killer (NK) cells. 4. Formation of follicle-like aggregates: B cells reside in ectopic lymphoid follicle-like aggregates that are associated with microglial activation, local inflammation, and neuronal loss in the nearby cortex.

[0010] Despite much research into the disease and multidisciplinary approaches to the problem, some pathogenic mechanisms of multiple sclerosis remain unclear and the etiology is unknown.

[0011] A recent paper by K. Bjornevik et al. (Non-Patent Document 7) reports on the analysis of 10 million blood samples from young adults on active duty in the US military; 955 of them were diagnosed with MS during their time on the job. The paper notes that the risk of MS increased 32-fold after infection with the Epstein-Barr virus (EBV), but not after infection with other viruses, such as the similarly transmitted cytomegalovirus (CMV). In an accompanying commentary, epidemiologist Alberto Ascherio, senior author of the study, concludes that, in essence, pretty much: "If you're not infected with EBV, you're not going to get MS." It's rare to get such black-and-white results. However, others, in their comments on this paper, have suggested that because EBV is ubiquitous and approximately 95% of adults have been infected with EBV, most of whom were infected during childhood or young adulthood, infection alone is insufficient for diagnosis, even if EBV was the inciting event in MS - a situation also discussed by the authors of the Bjornevik et al. paper itself.

[0012] For relapsing-remitting multiple sclerosis, several treatments that modify the course of the disease have been approved by regulatory agencies. These treatments include interferon beta, glatiramer acetate, mitoxantrone, natalizumab, fingolimod, teriflunomide, dimethyl fumarate, alemtuzumab, rituximab, and daclizumab. Interferon and glatiramer acetate are first-line treatments that reduce relapses by about 30% (see, e.g., J. Med. Soc. 1999, 141: 111-112). Natalizumab reduces relapse rates more than interferon, but has adverse effects and is therefore reserved as a second-line drug for patients who do not respond to other treatments or who have severe disease (see, e.g., J. Med. Soc. 1999, 141: 111-112). Treatment of clinically isolated syndrome (CIS) (the first clinically neurological symptom in multiple sclerosis) with interferon reduces the likelihood of progression to clinically definite MS (see, e.g., J. Med. Soc. 1999, 141: 111-112). Recently, new monoclonal antibodies such as ocrelizumab, alemtuzumab and daclizumab have shown potential as treatments for MS. Ocrelizumab, an anti-CD20 B cell-targeting monoclonal antibody, has shown beneficial effects in both relapsing and primary progressive MS in phase II and III clinical trials (NCT00676715, NCT01247324, NCT01412333, NCT01194570).

[0013] The diagnosis of MS is currently based on clinical examination by a medical professional. Such examinations include testing the patient's ability to perform certain physical activities. Several tests have been developed and are routinely performed by medical professionals. These tests aim to evaluate walking, balance, and other motor skills. Examples of tests currently performed include the Expanded Disability Status Scale (EDSS) and Multiple Sclerosis Functional Composite (MSFC). These tests require the presence of a medical professional for evaluation and assessment, and are currently performed on an outpatient basis in a doctor's office or hospital. Recently, there have been efforts to monitor MS patients using smartphone devices to collect data on MS patients in their natural environment (Non-Patent Document 12). In addition, diagnostic tools are being used to diagnose MS. Such tools include neuroimaging, analysis of cerebrospinal fluid, evoked potentials, etc. Magnetic resonance imaging (MRI) of the brain and spinal cord can visualize demyelination (lesions or plaques). A contrast agent containing gadolinium can be administered intravenously to mark active plaques, allowing the differentiation of acute inflammation from the presence of older lesions not associated with symptoms at the time of evaluation. Analysis of cerebrospinal fluid obtained by lumbar puncture can provide evidence of chronic inflammation of the central nervous system. The cerebrospinal fluid can be analyzed for oligoclonal immunoglobulin bands, an inflammatory marker present in 75-85% of MS patients (Non-Patent Document 13). However, none of the aforementioned techniques are specific for MS. Therefore, confirmation of the diagnosis of MS requires repeated clinical and / or MRI examinations to demonstrate the dissemination in space and time of the disease, which is a prerequisite for MS diagnosis. Moreover, none of the aforementioned methods are suitable for the diagnosis of early stages of MS, when patients already show some (non-specific) symptoms but also do not show signs of demyelination visible on brain MRI. The current diagnostic procedure for multiple sclerosis is rather long and complex. Early diagnosis of MS is needed, since even with currently available treatments, early therapeutic intervention as early as possible is most effective in the long term.Furthermore, there are currently no easy, cost-effective methods for monitoring disease progression or treatment success. [Prior art documents] [Non-patent literature]

[0014] [Non-Patent Document 1] GBD 2016 Multiple Sclerosis Collaborators, Lancet Neurol. 2019, vol. 18, pp. 269-85 [Non-Patent Document 2] Noonan et al., Neurology 2002, 58, pp. 136-138 [Non-Patent Document 3] Compston, A. and Coles A. (2008) Lancet. 372:1502-1517 [Non-Patent Document 4] Kornek et al. (2000), Am J Pathol. 157:267-276 [Non-Patent Document 5] Fisher et al. (2008), Ann Neurol. 64:255-265 [Non-Patent Document 6] McAlpine's Multiple Sclerosis, by Alastair Compston et al., 4th edition, Churchill Livingstone Elsevier, 2006 [Non-Patent Document 7] K. Bjornevik et al., Science 10, 1126 / science.abj8222 (2022) [Non-Patent Document 8] zur Hausen et al., 2017, Curr. Top. Microbiol. Immunol., Volume 407, 83-116 [Non-Patent Document 9] Borkosky et al., 2012, PLoS One 7(2):e32160, 2012 [Non-Patent Document 10] Tsang 2011, The Australian family physician 40 (12): 948-55 [Non-Patent Document 11] Compston 2008, Lancet 372(9648): 1502-17 [Non-Patent Document 12] Bove 2015, Neurol Neuroimmunol Neuroinflamm 2 (6): 62 [Non-Patent Document 13] Link 2006, J Neuroimmunol. 180 (1-2): 17-28 Summary of the Invention [Problem to be solved by the invention]

[0015] Therefore, the technical problem underlying the present invention can be said to be the provision of means and methods that meet the aforementioned needs. [Means for solving the problem]

[0016] The technical problem is solved by the embodiments characterized in the claims and described herein below.

[0017] Bjornevik et al., 2022 conclude that their experiments demonstrate that EBV is the cause of MS, but they do not rule out the contribution of other factors.

[0018] The presence of additional factors has already been discussed by the inventors in previous patent applications and in the literature (WO 2016 / 005054 (=US 2020 / 0002723); zur Hausen et al., Nature Reviews Clinical Oncology, 2015, zur Hausen et al., Curr. Top.Microbiol. Immunol. 2017, Volume 407, 83-116). Thus, according to the inventors, MS is due to: First, the disease was caused by two different infectious agents. same Latent infection of cells, one of which is most likely EBV, the other acquired through consumption of milk or beef (beef and milk factor - BMMF), each of which latently infects individual cells, but occasionally the genomes of both infectious agents occur within the same cell; · The second prerequisite is the reactivation of EBV into the lytic cycle. In the case of EBV, this is probably related to increased levels of transforming growth factor β (TGFβ), which is negatively regulated by the activated vitamin D receptor; · the third event, the amplification of BMMF, leading to partial inhibition of EBV DNA synthesis and the formation of BMMF proteins or particles or the spread of its nucleic acid to neighboring cells via neuronal interconnections; · This is followed by infection of neighboring cells with expression of BMMF proteins; Finally, the T cell response against BMMF leads to the destruction of diseased cells and, in the case of MS, to plaque formation. This supports the clinical observations of the localized appearance of lesions, usually originating from central veins, and of a focal local immune response in early lesions.

[0019] A spectrum of 19 different but partially related DNA molecules of BMMF subgroup 1 was isolated from different test materials (multiple sclerosis (MS) brain tissue, bovine serum, milk) (Funk et al., 2014, Genome Announc 2(4):e00846-14; Gunst et al. 2014, Genome Announc 2(4): e00847-14; Lamberto et al. 2014, Genome Announc 2(4):e00848-14; Whitley et al. 2014, Genome Announc 2(4); e00849-14; Falida et al. 2017, Genome Announc 5(17):e00266-17, zur Hausen, H., et al. 2017, Curr. Top .Microbiol. Immunol., Volume 10, 2017). 407, 83-116).

[0020] Of these isolates, two DNA molecules were isolated from brain tissue of one MS patient (Whitley et al. 2014, Genome Announc 2(4); e00849-14). We called these isolates MSBI1.176 (MSBI, multiple sclerosis brain isolate) (1,766 bp) and MSBI2.176 (1,766 bp) and named them "MSBI1 genome" and "MSBI2 genome", respectively. Both belong to the BMMF family. The large open reading frames (ORFs) of the isolates encode putative DNA replication proteins that share high similarity between them (de Villiers et al. Emerg. Microbes Infect. 2019, 8:1205-1218). Another common feature is the presence of iteron-like tandem repeats. Alignment of this repeat region shows core single nucleotide variations (zur Hausen et al., Curr. Top.Microbiol. Immunol. 2017, Volume 407, 83-116). These iteron-like repeats may constitute binding sites for Rep proteins. The sequences of the isolates have been deposited in the EMBL Databank under accession numbers LK931491 (MSBI1.176) and LK931492 (MSBI2.176) (Whitley C. et al., 2014), aligned and described in WO 2016 / 005054 (=US Patent Application Publication No. 2020 / 0002723).

[0021] The isolation of two types of BMMF from autopsy lesions of MS (Whitley et al. 2014, Genome Announc 2(4); e00849-14) seems to suggest their possible involvement in the pathogenesis of MS. A property of herpes-type viruses is that they amplify small single-stranded to double-stranded DNA of other DNA-containing infectious agents with which they are co-infected (or superinfected) (Schlehofer at al., 1986, Heilbronn et al., 1993). More recently, amplification of small single-stranded TT virus DNA by EBV has been reported (Borkosky et al. 2012). So far, TT virus has not been associated with MS. However, the similarity of the genome structure of BMMF, in addition to the finding of BMFF in MS lesions and the elevated BMMF antibody titers in MS patients compared to non-MS controls, led to the inclusion of BMMF amplification induced by EBV reactivation as a second factor. Therefore, EBV-induced amplification was included in the above model of MS pathogenesis (zur Hausen et al. 2017).

[0022] The inventors found that the MSBI1 or MSBI2 genome showed significant production of transcribed RNA and that the Rep protein encoded by the MSBI1 or MSBI2 genome was expressed in human cells (Eilebrecht et al., Scientific Reports 8:2851, 2018). Furthermore, serological tests in human samples identified antibodies reactive against the MSBI1 protein. As already mentioned above, according to our hypothesis, MS results from a latent infection of the same cells by two different infectious agents, one of which is EBV and the other acquired by the consumption of milk (zur Hausen et al., Nature Reviews Clinical Oncology, 2015, zur Hausen et al., Curr. Top.Microbiol. Immunol. 2017, Volume 407, 83-116). The latter one is called beef and milk factor (BMMF). Finally, the T cell response against BMMFs, and especially against MSBI1 Rep, leads to the destruction of diseased cells and, in the case of MS, to plaque formation (zur Hausen et al., Nature Reviews Clinical Oncology, 2015, zur Hausen et al., Curr. Top.Microbiol. Immunol. 2017, Volume 407, 83-116). This supports the clinical observations of the localized appearance of the lesions, usually starting from the central veins, and of the focal local immune response in (early) lesions (Moreno MA et al., Neurol Neuroimmunol Neuroinflamm. 2018;5(4):e466). The detection of this immune response should therefore be a suitable means to diagnose and monitor the progression of MS.

[0023] Thus, the present invention provides methods for diagnosing and monitoring MS based on immunoassays using MSBI1.176 or MSBI2.176 Rep protein or fragments thereof as antigens for binding antibodies against MSBI1.176 or MSBI2.176 Rep protein from a sample from a subject.

[0024] The present invention provides a method for the immunological determination of MS in a sample using a bridging immunoassay comprising a capture antigen and a tracer antigen (also called a detection antigen).

[0025] In a preferred embodiment of the present invention, the capture antigen is immobilized on the solid phase via a specific binding pair. Such binding pairs (first component / second component) are, for example, streptavidin or avidin / biotin, antibody / antigen (see, for example, Hermanson, GT, et al., Bioconjugate Techniques, Academic Press, 1996), lectin / polysaccharide, steroid / steroid binding protein, hormone / hormone receptor, enzyme / substrate, IgG / protein A and / or G, etc. Preferably, the capture antigen is conjugated to biotin and immobilized via immobilized avidin or streptavidin (most preferably streptavidin).

[0026] In a preferred embodiment, the tracer antigen can be replaced with anti-human antibodies, preferably anti-human IgG or IgM antibodies. These anti-human IgG or IgM antibodies are mainly produced in goats, mice or rabbits, and can be monoclonal or polyclonal. Numerous examples of suitable anti-human IgG or IgM antibodies are commercially available, for example from Thermo Fisher, Promega, Abcam, or Jackson Immuno Research.

[0027] Preferably, the tracer antigen / antibody is labeled with a detectable label or a detectable tag.

[0028] Examples of detectable labels / tags are chromogens (fluorescent or luminescent groups and dyes), enzymes, NMR-active groups or metal particles. Detectable labels / tags may also be photoactivatable crosslinking groups, such as azide or azirine groups. Particularly preferred is labeling with horseradish peroxidase (HRP).

[0029] Both binding of antigen to solid phase via biotin / streptavidin and HRP labeling of antigen are standard procedures and well known to those skilled in the art. Briefly, the coupling of biotin (N-hydroxysuccinimide ester (NHS)-biotin) is carried out through the primary amine of lysine residues at pH 4-10 in at least 20-fold excess over antigen (see, for example, Thermo Scientific™ EZ-Link™ NHS-Biotin biotinylation documentation, or Diner, I. et al., J Biol Chem. 2014 Dec 19; 289(51): 35296-35313). Alternatively, carboxyl group coupling is performed using EDC and Thermo Scientific™ EZ-Link™ Hydrazid-Biotin at acidic pH (based on the Thermo Scientific™ EZ-Link™ Hydrazid-Biotin paper used in Roncato F et al. Nat Commun 2018, 9: 4070). HRP coupling is based on a two-step process, first adding N-succinimidyl-S-acetylthioacetate (SATA) to the target antigen at a primary amine, such as lysine, to introduce a protected sulfhydryl group at pH 4-10 (see, for example, Thermo Scientific™ Pierce™ SATA (N-succinimidyl-S-acetylthioacetate) paper used in Duncan, RJS, et al. Anal Biochem (1983) 132:68-73). In the second step, maleimide-activated HRP is conjugated to the antigen via the previously introduced sulfhydryl groups (see, for example, Thermo Scientific™ EZ-Link™ Maleimide Activated Horseradish Peroxidase conjugation article, Pleiner, T., Bates, M., Gorlich, D, J Cell Biol 2018; (217):3 1143-1154).In an alternative technical setup, HRP is coupled to primary amines using Thermo Scientific™ EZ-Link™ Plus Activated Peroxidase in a single-step process, preferably at basic pH (see, for example, the text on Thermo Scientific™ EZ-Link™ Plus Activated Peroxidase coupling in Chen H et al. Journal: J Immunol Res 2018, 2018:4894705).

[0030] In an alternative embodiment, in order to increase the stability of the biotin Rep antigen over a longer period (months to years, long-term storage) and to simplify and shorten the operation (SOP) for the end user, a technical option was implemented to immobilize, stabilize and store the biotin Rep oligomer on a microtiter plate. In this way, the immobilization of the biotin Rep on the streptavidin microtiter plate is already prepared and is not performed by the user. Thus, a specific sugar preparation (e.g. containing 10-30% (e.g. 20%) saccharose (or sucrose), 0.01-0.1% (e.g. 0.06%) 5-bromo-5-nitro-1,3-dioxane, and optionally 1% (porcine) gelatin) was tested to allow the immobilization of the biotin Rep oligomer on the microtiter plate, the subsequent drying and storage of the material, as well as the confirmation of the proper detection of BMMF antibodies after reactivation of the material when used in a diagnostic test setting. In this regard, see Example 4. [Brief description of the drawings]

[0031] [Figure 1] Overview of the principle of BMMF-Rep specific serological testing based on the generation of BMMF Rep antigens and the setup of a bridging ELISA assay [Figure 2A]Quantification of antibody concentrations against oligomeric Rep (oligomeric assay). Individuals (male and female) aged 53 years or less at the time of sample collection were subgrouped into 26 healthy control serum samples (HC) and 70 MS serum samples (MS). [Figure 2B] Receiver operating characteristic curves (ROC) including the area under the ROC curve (ROC A), specificity and sensitivity (%), and P value for ROC analysis. [Figure 2C] Subtype classification of MS based on the clinical course. [Figure 3A] Quantification of antibody concentrations against aggregate Rep (aggregate assay). Women aged 40 years or younger at the time of sample collection are subgrouped into 11 healthy control serum samples (HC) and 27 MS serum samples (MS). [Figure 3B] Receiver operating characteristic curves (ROC) including the area under the ROC curve (ROC A), specificity and sensitivity (%), and P value for ROC analysis. [Figure 4] Incidence and sex ratios of multiple sclerosis in Scotland, 2010-2017 (Reprinted from Kearns et al., J. of Neurology, 266, pp. 2376-2386 (2019)). [Diagram 5] Epidemiology of multiple sclerosis in Austria (Reprinted from Salhofer-Polanyi et. Al., Neuroepidemiology, 49, pp. 40-44 (2017)). [Figure 6] Clinical course of MS (Reprinted from Filippi, M., et al. Nat Rev Dis Primers 4, 43 (2018)). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] The term "sample" includes, but is not limited to, whole blood, serum, or plasma from an individual suspected of having MS or an individual in whom a diagnosis of MS has already been confirmed. A sample may be, for example, a "post-treatment" sample, where the sample is obtained after one or more treatments, or may be a "baseline sample," e.g., used for initial diagnosis or used as a baseline for assessing disease progression.

[0033] The term "subject" or "patient" as used herein relates to a human individual suffering from or suspected of suffering from MS, i.e. a human individual who may already exhibit some or all of the symptoms commonly associated with said disease.

[0034] The term "solid phase" refers to non-fluid materials, including particles (including microparticles and beads) made from materials such as polymers, metals (paramagnetic, ferromagnetic particles), glass, and ceramics; gel materials such as silica, alumina, polymer gels; capillaries, zeolites, and other porous materials that can be made from polymers, metals, glass, and / or ceramics; electrodes; microtiter plates; solid strips; cuvettes, tubes, or other spectrometer sample containers. The solid phase component of the assay is distinguished from an inert solid surface that the assay may contact in that the "solid phase" comprises at least one moiety on its surface that is intended to interact with the capture antigen. The solid phase may be a fixed component such as a tube, strip, cuvette, or microtiter plate, or a non-fixed component such as beads and microparticles. According to the present invention, the solid phase is most preferably a microtiter plate.

[0035] Immunoassays are well known to those skilled in the art. Methods for carrying out such assays, as well as practical applications and procedures, are summarized in relevant textbooks (e.g., Colowick, SP and Caplan, NO (eds.), "Methods in Enzymology", Academic Press, dealing with immunological detection methods). In one embodiment, the immunoassay is an enzyme-linked immunosorbent assay (ELISA). In a preferred embodiment, the immunoassay is a bridging ELISA, which is believed to function similarly to the "anti-drug antibody assay" (ADA test).

[0036] The term "bridging ELISA" as used herein refers to an ELISA that includes a solid support and a capture antigen or fragment thereof (specific for the antibody in the sample of interest) immobilized on the solid support. In such an ELISA, a certain amount of target antibody (e.g., anti-MSBI1 or 2 Rep antibody contained in the sample) binds to the capture antigen. The bound antibody forms an antigen-antibody-antigen complex through binding to a second labeled antigen or fragment thereof (i.e., a tracer antigen or fragment thereof), and this complex is detected through a substrate reaction, such as a dye reaction generated based on the labeled antigen, which may alternatively constitute a chemiluminescent or fluorescent label for the detection of the antigen-antibody-antigen complex.

[0037] As used herein, the term "epitope" refers to the site on an antigen recognized by an antibody or binding fragment.

[0038] As used herein, the term "control" refers to a sample from a subject or group of subjects that does not have a disease or any condition. The terms "healthy control" or "control" are used interchangeably.

[0039] The term "non-denatured" as used herein refers to a protein in its native conformation (eg, 3D conformation) or in a conformation sufficient to confer functionality.

[0040] The term "reference material" or "calibrator" as used herein refers to a material that can be used in an assay for calibration purposes. It can be, for example, a known antibody against MSBI1 Rep. Examples are the Rep-specific mouse monoclonal antibodies AB8 or AB11 (Bund et al., PNAS 2021 Vol. 118 No. 12 e2025830118), or those mentioned in WO 2018 / 069296 (=US Patent Application Publication No. 2019 / 0270795), in particular AB3-6 (deposited under the accession number DSM ACC 3329 at DSMZ Braunschweig as "MSBI1 381-6-2"). These reference materials are also useful as quality controls for the purity of antigen preparations. For example, AB3-6 shows significantly increased reactivity to oligomeric Rep antigen in bridging ELISA; AB11 shows high reactivity with aggregate antigen in bridging ELISA, and AB8 reacts with both oligomers and aggregates.

[0041] The term "vector" encompasses any vehicle suitable for recombinant expression of a protein and / or fragment thereof. Potential expression vectors include, but are not limited to, cosmids, plasmids, or modified viruses (e.g., replication-defective retroviruses, adenoviruses, adeno-associated viruses), etc.

[0042] The term "host cell" includes a wide variety of prokaryotic and eukaryotic host cells. For example, the proteins of the invention can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus), yeast cells, or mammalian cells.

[0043] As used herein, "Rep protein" refers to DNA replication-related protein (RepB). Rep protein contains DNA binding activity and may be essential for initiating replication of episomal / viral DNA molecules. In general, Rep protein refers to Rep protein from the group of Small Sphinx Genome (Whitley et al., 2014). In particular, Rep protein is wild-type MSBI1 or MSBI2 Rep protein, or mutant MSBI1 or MSBI2 Rep protein with two point mutations (MSBI1 Rep 27 / 154E or MSBI2 Rep 27 / 155E) compared to the wild-type sequence. Preferably, the wild-type MSBI1 Rep protein is derived from MSBI1.176, which has the amino acid sequence shown in SEQ ID NO: 1 and is deposited in the EMBL data bank under accession number LK931491. Preferably, the wild type MSBI2 Rep protein has the amino acid sequence shown in SEQ ID NO: 4 and is derived from MSBI2.176 deposited in the EMBL data bank under accession number LK931492. Preferably, the mutant MSBI1 Rep protein has the amino acid sequence shown in SEQ ID NO: 3 and the MSBI2 Rep protein has the amino acid sequence shown in SEQ ID NO: 5. "Rep protein" also encompasses fragments and variants of the protein capable of binding to an anti-Rep antibody specific for a Rep protein having an amino acid sequence of SEQ ID NO: 1, 3, 4 or 5. Preferably, such fragments are immunogenic fragments of a protein having an amino acid sequence of SEQ ID NO: 1, 3, 4 or 5, encompassing at least one epitope of an anti-Rep protein antibody against a Rep protein of SEQ ID NO: 1, 3, 4 or 5, and preferably comprising at least 7, 8, 9, 10, 15, 20, 25 or 50 consecutive amino acids. In certain embodiments, the fragment comprises or consists essentially of a domain of a Rep protein, for example the N-terminal conserved region, the C-terminal variable region, the first or second DNA binding domain.A variant of a protein includes one or more amino acid deletions, substitutions or additions compared to SEQ ID NO: 1, 3, 4 or 5, has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the amino acid sequence of SEQ ID NO: 1, 3, 4 or 5, and is capable of binding to an anti-Rep antibody specific for a Rep protein having the amino acid sequence of SEQ ID NO: 1, 3, 4 or 5. The definition of variant includes, for example, polypeptides that include one or more analogs of an amino acid (including, for example, unnatural amino acids, peptide nucleic acids (PNAs), etc.), polypeptides with substituted linkages, and polypeptides with other modifications known in the art, both naturally occurring and non-naturally occurring. The term "Rep protein" includes fusion proteins with heterologous amino acid sequences, leader sequences or Tag sequences, etc.

[0044] The synthesized wild-type or mutant MSBI1 or MSBI2 Rep proteins (including Rep fragments and Rep variants as defined above) can be prepared by classical chemical synthesis. The synthesis can be performed in homogeneous solution or in solid phase. The polypeptides according to the invention can also be prepared by recombinant DNA techniques. For example, the mutant proteins (MSBI1 Rep27 / 154E or MSBI2 Rep27 / 155E) were engineered such that the possibility of aggregation of the two amino acid sequences between residues 25-31 (RLILLAII) of MSBI1 and 2 Rep and residues 151-155 of MSBI1 Rep and residues 152-156 (LLICW) of MSBI2 Rep was minimized by two single point mutations. The basis for cloning was the MSBI1 or MSBI2 Rep DNA sequence codon-optimized for Rep expression in the human system encoding the original wild-type MSBI1 or MSBI2 Rep primary amino acid sequence. The nucleotides encoding amino acid 27 (L, leucine, DNA codon CTA) as well as the nucleotides encoding amino acid 154 (C, cysteine, DNA codon TGT) were replaced with the nucleotides encoding the amino acid glutamic acid in MSBI1.176 (E, DNA codon GAG). The nucleotides encoding amino acid 27 (L, leucine, DNA codon CTA) as well as the nucleotides encoding amino acid 155 (C, cysteine, DNA codon TGT) were replaced with the nucleotides encoding the amino acid glutamic acid in MSBI2.176 (E, DNA codon GAG). For further details, see WO 2019 / 008052.

[0045] Unless otherwise specified in this application, "MSBI Rep" refers to both the MSBI1 Rep and MSBI2 Rep proteins, in particular those derived from MSBI1.176 and MSBI2.176.

[0046] As mentioned above, the expression of both wild-type and mutant Rep proteins can be carried out by recombinant DNA techniques. These are well known to those skilled in the art. Suitable vectors and host cells are well known and commercially available. See further Example 1, where the expression and purification of the proteins are described. In a particularly preferred embodiment, the wild-type and mutant Rep proteins are expressed separately in a host cell, preferably E. coli, where each nucleic acid is codon-optimized for expression in E. coli (SEQ ID NOs: 7 and 8). Recombinant production provides a mixture of Rep oligomers (1-4 Rep subunits) and aggregates (5-9 Rep subunits) for both the wild-type and mutant Rep proteins. However, due to two point mutations in the mutant sequence, the mutants tend to form fewer aggregates than the wild type. Non-denaturing purification of the mutant Rep oligomer and aggregate mixture is performed at pH 5.0-9.8, most preferably pH 7.0-9.5, preferably in a buffer containing 30 ml of 1xPBS (pH 7.4), 300 mM NaCl, 1% TritonX-100, 5 mM imidazole, 5 mM β-mercaptoethanol, containing benzonase and proteinase inhibitors, and loaded onto an affinity chromatography column. Elution and storage of the mutant Rep oligomer and aggregate mixture is performed in the pH range of pH 4-11, preferably pH 4.8-9.8, most preferably pH 5.0-9.5. Non-denaturing purification of wild-type Rep oligomer and aggregate mixtures is performed at pH 5.0-9.8, most preferably pH 7.0-9.5, preferably in a buffer containing 30 ml of 1x PBS (pH 7.4), 300 mM NaCl, 1% TritonX-100, 5 mM imidazole, 5 mM β-mercaptoethanol, containing benzonase and proteinase inhibitors, and loaded onto an affinity chromatography column. Elution and storage of wild-type Rep oligomer and aggregate mixtures is performed at a pH range of pH 4-11, preferably pH 4.8-9.8, most preferably pH 5.0-9.5 or pH 4.0-6.0.The entire preparation from the mutant and half of the preparation from the wild type are each separately subjected to biotinylation according to the standard procedure described above. The remaining half of the wild type preparation is coupled to HRP according to the standard procedure described above. The biotinylated mutant preparation and the biotinylated wild type preparation are each separately subjected to size exclusion chromatography (SEC) to separate the biotinylated oligomers and aggregates from each other. This results in mutant biotinylated oligomers (MBO), mutant biotinylated aggregates (MBA), wild type biotinylated oligomers (WBO), and wild type biotinylated aggregates (WBA). Furthermore, the HRP-bound wild type preparation is subjected to size exclusion chromatography to separate the HRP-bound oligomers (HRP-O) and HRP-bound aggregates (HRP-A) from each other. These different molecules are used to establish two immunoassays: -One is an oligomer assay in which mutant biotinylated oligomer (MBO) is bound to a 96-well plate coated with streptavidin. Then, the sample of interest is added to the plate simultaneously or in rapid succession with HRP-conjugated oligomer (HRP-O). After incubation and washing, a substrate reaction is initiated to detect oligomer-reactive antibodies, and the antibody level against MSBI1 Rep or MSBI2 Rep in serum is quantified. - The other is an aggregate assay in which wild-type biotinylated aggregates (WBA) are bound to a 96-well plate coated with streptavidin. Then, the sample of interest is added to the plate simultaneously or in rapid succession with HRP-conjugated aggregates (HRP-A). After incubation and washing, a substrate reaction is initiated for detection of aggregate-reactive antibodies to quantify the antibody levels against MSBI1 Rep or MSBI2 Rep in serum.

[0047] The assay can be calibrated using known antibodies against MSBI1 Rep. Examples are Rep-specific mouse monoclonal antibodies AB8 or AB11 (Bund et al., PNAS 2021 Vol. 118 No. 12 e2025830118), or those mentioned in WO 2018 / 069296 (=US Patent Application Publication No. 2019 / 0270795) or WO 2019 / 008052 (=US Patent Application Publication No. 2020 / 0123520), in particular AB3 (deposited under the accession number DSM ACC 3329 at DSMZ Braunschweig as "MSBI1 381-6-2").

[0048] Serum containing increased amounts of anti-Rep antibodies indicates that the corresponding subject has been reliably exposed to Rep-related proteins for a period of time long enough to mount a Rep-specific immune response or that the subject itself expresses Rep.

[0049] In the course of the present invention, the assay has been validated using a large number of blood samples from healthy individuals (=controls) and patients with a confirmed history of MS. The results are shown in Figures 2 and 3. In Figure 2, the age of patients and controls is restricted to 53 years or less, since it is generally known that the incidence of MS declines rapidly after the age of 55 (see Figures 4 and 5). Furthermore, since MS is a disease that is more prevalent in young women (see Figures 4 and 5), in Figure 3, a restriction to women aged 40 years or less is applied. It can therefore be concluded that the aggregate assay is particularly suitable for young women, while the oligomer assay is useful for both men and women up to about the age of 55.

[0050] The assay of the present invention is suitable for diagnosing or monitoring all possible active or inactive forms of MS, i.e. relapsing-remitting MS (RR-MS), secondary progressive MS (SP-MS), primary progressive MS (PP-MS) and / or progressive relapsing MS (PR-MS), see Figure 2(c).

[0051] Moreover, the assay is particularly suitable for the initial diagnosis of multiple sclerosis (MS) in the pre-symptomatic phase (early diagnosis) and for monitoring the progression of the disease and / or the success of treatment, i.e., treatment monitoring. The suitability of the assay of the present invention for early diagnosis is important, since many patients exhibit clinically isolated syndrome (CIS) but are not considered to have multiple sclerosis (MS) because they do not meet the general diagnostic criteria (see FIG. 6). Such pre-symptomatic patients lose valuable time before treatment can be initiated, since the diagnosis is delayed and symptoms may progress. In this regard, see also Compston et al. (2008, Lancet 372(9648): 1502-17), in which it was confirmed that the treatment of clinically isolated syndrome (CIS) with interferon reduces the chance of progression to clinically defined multiple sclerosis.

[0052] Furthermore, the assays are suitable for monitoring disease progression and therapeutic success during treatment.

[0053] The present invention also provides an ELISA kit comprising (a) an MSBI1 Rep or MSBI2 Rep protein or a fragment thereof as a capture antigen immobilized on a solid phase via a specific binding pair, (b) an MSBI1 or MSBI2 Rep protein or a fragment thereof conjugated to a detectable label as a tracer antigen, (c) a dye, chemiluminescent or fluorescent substrate, and (d) instructions for using the kit in a method for diagnosing or monitoring multiple sclerosis.

[0054] In this disclosure, the term "a" should be understood to mean "at least one". As used herein, terms of degree such as "substantially", "about", and "approximately" refer to a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be interpreted as including at least ±5% deviation of the modified term, if the deviation does not negate the meaning of the word it modifies.

[0055] As used herein, the terms "include(s)" and "including" are meant to be open-ended and are understood to mean "comprise(s)" and "comprising," respectively.

[0056] The sequential labeling of the method steps provided herein by numbers and / or letters is not meant to limit the method or any embodiment thereof to the particular sequence indicated. Various publications, including patents, patent applications, published patent applications, accession numbers, technical papers, and academic papers, are cited throughout this specification. Each of these cited references is incorporated herein by reference in its entirety and for all purposes.

[0057] The following figures are illustrative of the present disclosure:

[0058] Figure 1: Overview of the principle of the BMMF-Rep specific serological test based on the generation of BMMF Rep antigens and the setup of a bridging ELISA assay For the mutant, Rep oligomers (1-4 Rep subunits) and aggregates (5-9 Rep subunits) are generated by non-denaturing purification followed by buffering to pH 4-11. For the wild type, Rep oligomers (1-4 Rep subunits) and aggregates (5-9 Rep subunits) are generated by non-denaturing purification followed by buffering to pH 4-11. The entire preparation from the mutant and half of the preparation from the wild type are subjected to biotinylation separately. The other half of the wild type preparation is coupled to HRP. The biotinylated mutant preparation and the biotinylated wild type preparation are subjected to size exclusion chromatography (SEC) separately to separate the biotinylated oligomers and aggregates from each other. This results in the mutant biotinylated oligomers (MBO), mutant biotinylated aggregates (MBA), wild type biotinylated oligomers (WBO) and wild type biotinylated aggregates (WBA). Furthermore, the HRP-conjugated wild-type preparation is subjected to size-exclusion chromatography to separate HRP-conjugated oligomers (HRP-O) and aggregates (HRP-A) from each other.

[0059] Oligomer Testing : The mutant biotinylated oligomer (MBO) is bound to a 96-well plate coated with streptavidin. The sample of interest is then added to the plate simultaneously or in rapid succession with the HRP-conjugated oligomer (HRP-O). After incubation and washing, a substrate reaction is initiated for the detection of antibodies reactive to the oligomer to quantify the antibody levels in the serum. Calibration of the test is performed using a test sample containing a known antibody, namely the Rep-specific mouse monoclonal antibody AB8 (Bund et al., PNAS 2021 Vol. 118 No. 12 e2025830118).

[0060] Aggregate Testing: Wild-type biotinylated aggregates (WBA) are bound to a 96-well plate coated with streptavidin. The sample of interest is then added to the plate simultaneously or in rapid succession with the HRP-conjugated aggregates (HRP-A). After incubation and washing, a substrate reaction is initiated for the detection of antibodies reactive to the aggregates to quantify the antibody levels in the serum. Calibration of the test is performed using test samples containing a known antibody, namely the Rep-specific mouse monoclonal antibody AB8 (Bund et al., PNAS 2021 Vol. 118 No. 12 e2025830118).

[0061] Figure 2: Quantification of antibody concentration against oligomeric Rep (oligomeric assay) (A) Individuals (males and females) aged 53 years or less at the time of sample collection are subgrouped into 26 healthy control serum samples (HC) and 70 MS serum samples (MS). Two tests are performed for each serum, using 10 μl of serum per test. Measured antibody concentrations are significantly increased in MS patients compared to healthy controls (tested by Mann Whitney U test, scatter plot showing medians with 95% CI). (B) Receiver operator curve (ROC) including area under the ROC curve (ROC A), specificity and sensitivity (%), and P value of the ROC analysis. (C) Subtype classification based on the clinical course of MS.

[0062] Figure 3: Quantification of antibody concentration against aggregate Rep (aggregation assay) (A) Women aged 40 years or younger at the time of sample collection are subgrouped into 11 healthy control serum samples (HC) and 27 MS serum samples (MS). Two tests are performed for each serum, using 10 μl of serum per test. Measured antibody concentrations in MS patients are significantly increased compared to healthy controls (tested by Mann-Whitney U test, scatter plot showing medians with 95% CI). (B) Receiver operating characteristic curve (ROC) including the area under the ROC curve (ROC A), specificity and sensitivity (%), and P value of the ROC analysis.

[0063] Figure 4: Incidence and sex ratio of multiple sclerosis in Scotland 2010-2017 (Reprinted from Kearns et al., J. of Neurology, 266, pp. 2376-2386 (2019)).

[0064] Figure 5: Epidemiology of multiple sclerosis in Austria (reprinted from Salhofer-Polanyi et. Al., Neuroepidemiology, 49, pp. 40-44 (2017)).

[0065] Figure 6: Clinical course of MS (Reprinted from Filippi, M., et al. Nat Rev Dis Primers 4, 43 (2018)) The National Multiple Sclerosis Society's Advisory Committee for Clinical Trials in Multiple Sclerosis has classified the clinical course of multiple sclerosis into four: relapsing-remitting multiple sclerosis (RRMS), secondary progressive multiple sclerosis (SPMS), primary progressive multiple sclerosis (PPMS), and progressive relapsing multiple sclerosis (PRMS). Revisions of these phenotypes have been proposed to include clinically isolated syndrome (CIS) to represent patients whose first clinical symptoms show features of inflammatory demyelination and may be multiple sclerosis but do not meet the diagnostic criteria for it (pre-symptomatic phase). Within each subtype, the disease can be classified as active or inactive, defined by the occurrence of relapses or lesions detected using MRI.

[0066] The above disclosure generally describes the present application. A more complete understanding can be obtained by reference to the following specific examples. These examples are set forth for illustrative purposes only and are not intended to limit the scope of the present application. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient. Although specific terms have been employed herein, these terms are used in a descriptive sense and not for purposes of limitation.

[0067] The following non-limiting examples are illustrative of the present disclosure. EXAMPLES

[0068] Example 1: Purification of non-denatured H1MSBI Rep protein antigen Nucleotide acid molecules encoding the full-length Rep open reading frame (ORF) or its mutant form (mut) identified in the H1MSB.1 genome (isolated from the brain of a patient with multiple sclerosis, WT), respectively, were cloned into an expression plasmid (pEXP5-CT, Invitrogen, C-terminal 6xHis tag) to allow protein expression in Escherichia coli (SoluBL21, Genlantis) by IPTG induction (0.66 mM IPTG) for 18 h at 16 °C (LB medium, ampicillin selection). The gene expression parts were selected to express either the wild-type Rep amino acid sequence found in the H1MSB.1 genome to allow specific generation of HRP-linked oligomeric test antigen later, or a variant with two point mutations (L27E and C154E) to generate appropriate amounts of biotin-linked oligomeric test antigen. After induction of gene expression, cells expressing Rep are washed with 1×PBS (pH 7.4), aliquoted, pelleted, and stored at −80° C. for further use.

[0069] For separate purification of the two Rep variants (WT and mut) based on IMAC / His affinity purification, cells are lysed by sonication on ice in lysis buffer (30 ml 1x PBS, pH 7.4, 300 mM NaCl, 1% TritonX-100, 5 mM imidazole, 5 mM β-mercaptoethanol (containing benzonase and proteinase inhibitors)). After 30 min incubation at 4°C and centrifugation, the cleared lysate is loaded onto an IMAC column (His60 Ni Superflow Resin, Clontech) and washed with 50 ml washing buffer (lysis buffer containing 35 mM imidazole, all purification steps were performed at 4°C). Wild-type (wt) Rep protein is eluted with 5 ml of PBS lysis buffer (pH 5) containing 300 mM imidazole, and the Rep double mutant is eluted with 5 ml of carbonate buffer (pH 9.5) containing 300 mM imidazole.

[0070] The quality of purification is determined by Coomassie protein staining and Western blotting with anti-His and anti-Rep protein antibodies. Rep-containing protein eluates are pooled to a final concentration of approximately 1.2 mg / ml. The purity of the purified Rep target protein is calculated by densitometry to be greater than 98%.

[0071] Example 2: Biotin / HRP conjugation of Rep for bridging ELISA serology testing Solutions of purified wild-type (wt) and mutant Rep proteins consist of a mixture of excess Rep monomers, dimers and trimers (called oligomers) and, to a lesser extent, aggregates with more than four Rep subunits (called aggregates). The wt protein purification (pH 5) is subjected to biotinylation or HRP coupling after rebuffering to pH 9.5. The preparation containing mutant Rep (pH 9.5) is subjected to conjugation to HPR without buffer exchange.

[0072] Coupling of biotin (N-hydroxysuccinimide ester (NHS)-biotin) is carried out at pH 9.5 through the primary amines of lysine residues in at least 20-fold excess compared to the antigen (see, for example, Thermo Scientific™ EZ-Link™ NHS-Biotin biotinylation documentation, or Diner, I. et al., J Biol Chem. 2014 Dec 19; 289(51): 35296-35313). HRP coupling is based on a two-step process, firstly by addition of N-succinimidyl-S-acetylthioacetate (SATA) to the target antigen at the primary amine, e.g., lysine, to introduce protected sulfhydryl groups at pH 9.5. In the second step, after rebuffering to neutral pH (PBS pH 7.5) by gel filtration, maleimide-activated HRP is coupled to the antigen via the previously introduced sulfhydryl groups.

[0073] After conjugation, in order to analyze the generated antigen preparations, to remove excess biotin and HRP label, and to rebuffer the antigens for long-term and intermediate storage and for final serum testing in assay buffer, both the generated biotin-conjugated Rep antigen preparations and the HRP-conjugated Rep antigen preparations, including oligomers and aggregates, are subjected to size exclusion chromatography (SEC) based on a 30 cm Sephadex column, respectively. During SEC, the elution of the conjugated Rep proteins is monitored by UV / visible spectroscopy, allowing size separation and the collection of fractions containing oligomeric and aggregated biotin and HRP conjugated preparations.

[0074] After SEC separation, portions of the HRP-conjugated Rep antigen preparation are either used immediately or stored at -20°C in PBS (8.1 mM disodium hydrogen phosphate, 1.5 mM potassium dihydrogen phosphate, 136 mM sodium chloride, 2.7 mM potassium chloride, pH 7.3) supplemented with an equal volume of glycerol.

[0075] After SEC separation, the biotin-conjugated Rep antigen preparation portion is either used immediately or stored at -20°C in carbonate buffer (0.1 M sodium bicarbonate, 0.5 M sodium chloride, pH 9.4) with the addition of an equal volume of glycerol.

[0076] To allow detection of antibodies reactive against oligomers, biotin-conjugated Rep oligomers and HRP-conjugated Rep oligomers are utilized together in an oligomer-bridging ELISA test setup (oligomer test). Detection of antibodies reactive against aggregates is performed by combining biotin-conjugated Rep aggregates and HRP-conjugated Rep aggregates in an aggregate-bridging ELISA test setup (aggregate test).

[0077] Example 3: Quantification of anti-Rep antibodies in human serum by bridging ELISA assay Biotin-Rep oligomers or biotin-Rep aggregates are separately immobilized on streptavidin-coated assay plates by incubation for 60 min at 20-25 °C. After washing away unbound material, serum samples (or calibration / control antibodies) are pipetted onto the plate, followed by the addition of HRP-Rep oligomers. After 2 h of coincubation at 20-25 °C, unbound material is washed away. After this washing step, 3,3',5,5'-tetramethylbenzidine (TMB) substrate is added to the wells, which reacts with HRP to produce color (substrate reaction). After 30 min of incubation, the reaction is stopped with HCl and the plate is read at 450 nm using a plate reader. The amount of color produced is directly proportional to the amount of antibody reacting with the Rep antigen in the sample. The concentration of antibodies reactive to aggregates and / or oligomers in samples of interest is calculated based on a standard curve generated with a standard antibody (anti-Rep mouse monoclonal antibody AB8) run in parallel on each oligomer or aggregate test plate.

[0078] A calibration curve can be established by plotting standard concentrations on the x-axis (linear scale) against standard absorbance on the y-axis (linear scale). Antibody concentrations of patient samples can then be read off the calibration curve. A four-parameter (4-PL, 4-parameter logistic) curve fit or equivalent method is used for automated data reduction. Standards and samples were measured in duplicate or triplicate. If samples were pre-diluted differently from the standards, concentrations are obtained by multiplying the values ​​read off the calibration curve by the difference in dilution factor.

[0079] The results are shown in Figure 2 (oligomer assay) and Figure 3 (aggregate assay). Both studies show that the method of the invention is suitable for providing a reliable distinction between MS and healthy controls.

[0080] Example 4: Protocol for stabilization of Biotin-Rep on polystreptavidin-coated wells for long-term storage and reactivation of test plates In an alternative test setup, an appropriate amount of biotin-Rep oligomers is immobilized on the wells of a polystreptavidin-coated microtiter plate in 100 μl of assay buffer (1-16 hours on a shaker at 21° C.), followed by four washing steps and stabilization in stabilization solution (200 μl / well; 20% saccharose, 0.06% 5-bromo-5-nitro-1,3-dioxane, and optionally 1% (porcine) gelatin) for 1 hour at 21° C. After stabilization of the Rep antigen on the microtiter plate, the solution is removed and the microtiter plate is dried at room temperature for at least 48 hours, sealed in a flat bag containing desiccant, and stored long-term at 2-8° C. (until reactivation with assay buffer for diagnostic use).

[0081] Table 1 below shows a comparison of the detection optical density (OD) of the oligomeric Rep assay with and without stabilization of the biotinylated Rep oligomers before testing the samples. The biotinylated Rep oligomers were immobilized on streptavidin-coated microtiter plates, then stabilized, the plates were dried, stored at 21° C. for 7 days, and reactivated for testing ("stabilized"), or immobilized on the microtiter plates immediately before testing according to the standard protocol ("reference"). Comparable OD values ​​were observed for the standard antibody AB8 (standard curve) and the reference antibodies AB3 and AB11, as well as for the control serum with and without 50 ng / ml of AB3. [Table 1]

[0082] Preferred embodiments of the present invention will now be described. 1. An immunoassay for the detection and / or measurement of antibodies to MSBI Rep protein in a sample of a subject, the method comprising: (a) coating a solid support with a capture antigen, which is a mutant MSBI Rep oligomer, via a specific binding pair; (b) contacting the capture antigen with the sample under conditions to form a capture antigen:anti-MSBI Rep antibody complex; (c) removing unbound sample; (d) contacting the capture antigen:anti-MSBI Rep antibody complex with a tracer compound, which is a wild-type MSBI Rep oligomer conjugated to a detectable label or an anti-human antibody conjugated to a detectable label, to form a capture antigen:anti-MSBI Rep antibody:tracer compound complex; (e) removing unbound tracer compound; (f) adding a dye, chemiluminescent or fluorescent substrate; (g) detecting and / or measuring the capture antigen:anti-MSBI Rep antibody:tracer compound complex via a detectable reaction of the substrate. An immunoassay comprising:

[0083] 2. An immunoassay for the detection and / or measurement of antibodies to MSBI Rep protein in a sample of a subject, the method comprising: (a) coating a solid support with a capture antigen, which is a wild-type MSBI Rep aggregate, via a specific binding pair; (b) contacting the capture antigen with the sample under conditions to form a capture antigen:anti-MSBI Rep antibody complex; (c) removing unbound sample; (d) contacting the capture antigen:anti-MSBI Rep antibody complex with a tracer compound, which is a wild-type MSBI Rep aggregate conjugated to a detectable label or an anti-human antibody conjugated to a detectable label, to form a capture antigen:anti-MSBI Rep antibody:tracer compound complex; (e) removing unbound tracer compound; (f) adding a dye, chemiluminescent or fluorescent substrate; (g) detecting and / or measuring the capture antigen:anti-MSBI Rep antibody:tracer compound complex via a detectable reaction of the substrate. An immunoassay comprising:

[0084] 3. The immunoassay of embodiment 1 or 2, wherein the specific binding pair is streptavidin / biotin, wherein streptavidin is immobilized on a solid support and biotin is conjugated to the capture antigen.

[0085] 4. The immunoassay of any of embodiments 1-3, wherein the detectable label is horseradish peroxidase.

[0086] 5. The immunoassay of any of embodiments 1 to 4, wherein the substrate is 3,3',5,5'-tetramethylbenzidine.

[0087] 6. The immunoassay of any of embodiments 1 to 5, wherein the anti-human antibody is an anti-human IgG or IgM antibody.

[0088] 7. A kit comprising: (a) the MSBI Rep protein or a fragment thereof as a capture antigen immobilized on a solid phase via a specific binding pair; (b) as a tracer compound, an MSBI Rep protein or a fragment thereof conjugated to a detectable label, or an anti-human antibody conjugated to a detectable label; (c) a dye, chemiluminescent or fluorescent substrate, and (d) instructions for using the kit in a method for diagnosing or monitoring multiple sclerosis. Including the kit.

[0089] 8. The kit of embodiment 7, wherein the specific binding pair is streptavidin / biotin, wherein streptavidin is immobilized on a solid support and biotin is conjugated to the capture antigen.

[0090] 9. The kit of embodiment 7 or 8, wherein the detectable label is horseradish peroxidase.

[0091] 10. The kit according to any one of embodiments 7 to 9, wherein the substrate is 3,3',5,5'-tetramethylbenzidine.

[0092] 11. The kit according to any of embodiments 7 to 10, wherein the anti-human antibody is an anti-human IgG or IgM antibody.

[0093] 12. Use of an immunoassay according to any one of embodiments 1 to 6 or a kit according to any one of embodiments 7 to 10 for diagnosing or monitoring multiple sclerosis.

[0094] Overview of Arrays TIFF2025507383000003.tif244170 TIFF2025507383000004.tif255170 TIFF2025507383000005.tif250170 TIFF2025507383000006.tif200170

Claims

1. 1. An immunoassay for the detection and / or measurement of antibodies against MSBI Rep protein in a sample of a subject, comprising: (a) coating a solid support with a capture antigen, which is a mutant MSBI Rep oligomer, via a specific binding pair; (b) contacting the capture antigen with the sample under conditions to form a capture antigen:anti-MSBI Rep antibody complex; (c) removing unbound sample; (d) contacting the capture antigen:anti-MSBI Rep antibody complex with a tracer compound, which is a wild-type MSBI Rep oligomer conjugated to a detectable label or an anti-human antibody conjugated to a detectable label, to form a capture antigen:anti-MSBI Rep antibody:tracer compound complex; (e) removing unbound tracer compound; (f) adding a dye, chemiluminescent or fluorescent substrate; (g) detecting and / or measuring the capture antigen:anti-MSBI Rep antibody:tracer compound complex via a detectable reaction of the substrate; An immunoassay comprising:

2. 2. The immunoassay of claim 1, wherein the mutant MSBI Rep oligomer comprises 1 to 4 Rep subunits and the wild-type MSBI Rep oligomer comprises 5 to 9 Rep subunits.

3. 1. An immunoassay for the detection and / or measurement of antibodies against MSBI Rep protein in a sample of a subject, comprising: (a) coating a solid support with a capture antigen, which is a wild-type MSBI Rep aggregate, via a specific binding pair; (b) contacting the capture antigen with the sample under conditions to form a capture antigen:anti-MSBI Rep antibody complex; (c) removing unbound sample; (d) contacting the capture antigen:anti-MSBI Rep antibody complex with a tracer compound that is a wild-type MSBI Rep aggregate conjugated to a detectable label or an anti-human antibody conjugated to a detectable label to form a capture antigen:anti-MSBI Rep antibody:tracer compound complex; (e) removing unbound tracer compound; (f) adding a dye, chemiluminescent or fluorescent substrate; (g) detecting and / or measuring the capture antigen:anti-MSBI Rep antibody:tracer compound complex via a detectable reaction of the substrate; An immunoassay comprising:

4. The immunoassay of claim 3, wherein the wild-type MSBI Rep aggregates contain 5 to 9 Rep subunits.

5. The immunoassay of any one of claims 1 to 4, wherein the specific binding pair is streptavidin / biotin, wherein streptavidin is immobilized on a solid support and biotin is conjugated to a capture antigen.

6. The immunoassay of any one of claims 1 to 5, wherein the detectable label is horseradish peroxidase.

7. The immunoassay of any one of claims 1 to 6, wherein the substrate is 3,3',5,5'-tetramethylbenzidine.

8. The immunoassay of any one of claims 1 to 7, wherein the anti-human antibody is an anti-human IgG or IgM antibody.

9. A kit comprising: (a) the MSBI Rep protein or a fragment thereof as a capture antigen immobilized on a solid phase via a specific binding pair; (b) as a tracer compound, the MSBI Rep protein or a fragment thereof conjugated to a detectable label, or an anti-human antibody conjugated to a detectable label; (c) a dye, chemiluminescent or fluorescent substrate, and (d) instructions for using the kit in a method for diagnosing or monitoring multiple sclerosis. Including the kit.

10. 10. The kit of claim 9, wherein the specific binding pair is streptavidin / biotin, wherein streptavidin is immobilized on a solid support and biotin is conjugated to a capture antigen.

11. The kit of claim 9 or 10, wherein the detectable label is horseradish peroxidase.

12. The kit according to any one of claims 9 to 11, wherein the substrate is 3,3',5,5'-tetramethylbenzidine.

13. The kit according to any one of claims 9 to 12, wherein the anti-human antibody is an anti-human IgG or IgM antibody.

14. Use of an immunoassay according to any one of claims 1 to 8 or a kit according to any one of claims 9 to 13 for diagnosing or monitoring idiopathic sclerosis.

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