Misfolded SOD1 assay

Abstract

To solve the problem of the lack of biomarkers that facilitate early diagnosis, demonstrate target binding, monitor disease progression, and / or can serve as surrogate end points for evaluating the efficacy of treatment.SOLUTION: Provided is a novel high-sensitivity method for assaying misfolded SOD1 in a body fluid of a subject, particularly in cerebrospinal fluid. The method is based on a novel high-sensitivity immunoassay utilizing a unique epitope of SOD1 and a corresponding anti-SOD1 antibody. In addition, a kit containing the components of an immunoassay is provided.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates generally to novel, highly sensitive methods for assaying misfolded SOD1 (mSOD1) in the body fluids of a subject, particularly cerebrospinal fluid (CSF), and provides highly sensitive immunoassays for mSOD1 and kits for use in such assays. [Background technology]

[0002] Amyotrophic lateral sclerosis (ALS) is a highly heterogeneous disease with no effective treatment. This is partly because the cause of ALS is largely unknown, with approximately 90% of cases being sporadic (sALS), while only approximately 10% are familial (fALS). Since the 1990s, intensive research has aimed to elucidate the mechanisms involved in motor neuron degeneration. These studies suggest that ALS is a complex disease driven by the combination of several systemic parameters. To date, up to 30 genes have been described as monogenic causes of ALS, the most common of which are C9orf72, SOD1, FUS, and TARDBP / TDP43; for a review, see Vijayakumar et al., Front. Neurol. 10(2019):400. doi:10.3389 / fneur.2019.00400. While genetic linkage and, therefore, genetic markers may be feasible for predicting and correlating susceptibility to fALS, assessment and drug development for sALS are hampered by the lack of biomarkers that can facilitate early diagnosis, demonstrate target engagement, monitor disease progression, and / or serve as surrogate endpoints for evaluating treatment efficacy. Fluid-based biomarkers have the potential to address these issues. An ideal biomarker would demonstrate high specificity and sensitivity for distinguishing ALS from control populations (appropriate disease mimics and other neurological disorders) and monitor disease progression within individual patients. Significant progress has been made in the search for ALS biomarkers using cerebrospinal fluid, serum, and plasma, while urine and saliva biomarkers are still in early stages of development. Some of these candidate biomarkers have demonstrated use in patient stratification, disease course (fast vs. slow progression), and disease severity prediction, or have been used in preclinical and clinical applications. However, although ALS biomarker discovery has shown significant progress over the past decade, validating biomarkers and translating them into the clinic remains more challenging; see review Vu and Bowser, Neurotherapeutics 14 (2017), 119-134. Summary of the Invention [Means for solving the problem]

[0003] The solution to this technical problem is provided by the embodiments characterized in the claims and further disclosed in the following description.

[0004] The present invention generally relates to novel, highly sensitive methods for determining the presence and level, respectively, of misfolded SOD1 (mSOD1) in a body fluid from a subject using an immunoassay comprising an anti-SOD1 antibody as a capture antibody against a specific epitope of SOD1. As illustrated in the accompanying examples and figures, detection of mSOD1 by the method of the present invention or assay of increased levels of mSOD1 compared to control samples is indicative of ALS. More specifically, the assay of the present invention is capable of identifying patients with sALS with a high degree of certainty, which has rarely been possible until now.

[0005] The present invention is based on the unexpected discovery that specific epitopes of SOD1 and therapeutic anti-SOD1 antibodies thereto may also be of particular diagnostic value in detecting mSOD1 in samples of body fluids from subjects suspected of having or at risk of developing ALS and ALS patients, respectively, and may detect and / or distinguish between fALS and sALS.

[0006] Human Cu / Zn superoxide dismutase (SOD1) is a 32 kDa homodimeric metalloenzyme with a genetic locus on chromosome 21. It is primarily localized in the cytosol, nucleus, and peroxisomes of eukaryotic cells, but also in the mitochondrial intermembrane space. It contains an active site that binds a catalytic copper ion and a structural zinc ion. The functional role of SOD1 is to act as an antioxidant enzyme that catalyzes the dismutation of superoxide radicals to dioxygen and hydrogen peroxide, thereby reducing the steady-state concentration of superoxide and oxidative stress on cells (Fridovich, Science 201 (1978), 875-879).

[0007] Mutations in the gene encoding SOD1 account for approximately 20% of familial amyotrophic lateral sclerosis (fALS) cases and a small proportion of sporadic ALS (sALS) cases (Rosen et al., Nature 362 (1993), 59-62; Chio et al., Neurology 70 (2008), 533-537; Kwon et al., Neurobiol. Aging 33 (2012), e1017-1023). ALS is a rapidly progressive and always fatal neurological disease that attacks neurons responsible for controlling voluntary muscles, specifically motor neurons in the spinal cord, brainstem, and motor cortex (Bruijn et al., Annu. Rev. Neurosci. 27 (2004), 723-749). The mechanism by which mutations in SOD1 lead to ALS is not fully understood, but there is evidence for a toxic gain-of-function mechanism in which mutagenic misfolding of SOD1 is associated with toxicity causing degeneration of motor neurons (Julien, Cell 104 (2001), 581-591).

[0008] However, misfolding of wild-type (wt) SOD1 is also thought to be associated with the majority of sALS cases (Bosco et al., Nature Neuroscience 13 (2010), 1396-1403). Wild-type SOD1 is subject to extensive post-translational modifications, including subunit dimerization, formation of an intrasubunit disulfide bond between residues Cys57 and Cys146, and coordination of copper and zinc. Disruption of these processes has all been shown to cause wt SOD1 to aggregate (Durazo et al., J. Biol. Chem. 277 (2009), 15923-15931; Estevez et al., Science 286 (1999), 2498-2500; Rakhit et al., J. Biol. Chem. 279 (2004), 15499-15504; Lindberg et al., Proc. Natl. Acad. Sci. USA 101 (2004), 15893-15898), thus providing a possible pathogenic model for spontaneous forms of ALS. Abnormal alterations of wt SOD1 have also been reported in other neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD).

[0009] SOD1 has been considered as a potential biomarker, but conflicting results have been reported. For example, Jacobsson et al., Brain 124 (2001), 1461-1466, determined the amount, activity, and molecular form of SOD1 in CSF from ALS patients with D90A and other SOD1 mutations and patients without such mutations. They found no differences in the amount of SOD1 protein or enzyme activity among 37 neurological controls, 54 sporadic ALS cases, 12 familial ALS cases, and 10 cases homozygous for the D90A mutation. Similarly, as summarized in Vu and Bowser (2017) above, another study measured CSF SOD1 levels between patients with ALS and neurological controls and found no significant differences between the groups, indicating that SOD1 CSF levels are not a diagnostic biomarker for ALS. In addition, a subsequent study analyzing CSF from ALS patients and controls by ELISA assay using antibodies reacting with different sequence segments of the mSOD1 species showed no significant differences between ALS patients and controls (Zetterstroem et al., J. Neurochem. 117 (2011), 91-99). The authors hypothesized that the estimated concentration of mSOD1 in the CNS interstitium was 1,000-fold lower than the concentration required for significant cytotoxicity in the model system. Thus, Zetterstroem et al. concluded that these results argue against a direct cytotoxic role of extracellular mSOD1 in ALS and therefore that mSOD1 in CSF cannot be used as a biomarker for ALS in patients with and without mutations in the enzyme.

[0010] Recently, Tokuda et al. (Tokuda et al. Molecular Neurodegeneration (2019) 14:42 https: / / doi.org / 10.1186 / s13024-019-0341-5) described an ELISA assay for misfolded wild-type SOD1 in the cerebrospinal fluid of sALS. However, the capture antibody used, C4F6, a monoclonal antibody generated by using recombinant SOD1 with the G93A mutation, showed strong immunoreactivity to the denatured G93A but much lower reactivity to other hSOD1 mutants and very low reactivity to denatured WT hSOD1. Furthermore, the C4F6 antibody has been described to stain spinal cord tissue from A4V fALS cases but not sALS cases; see the characterization of antibody C4F6 by Ayers et al. Acta Neuropathologica Communications 2014, 2:55 Page 2 of 13 http: / / www.actaneurocomms.org / content / 2 / 1 / 55. Therefore, it remains to be shown whether the antibody C4F6 and ELISA assay described by Tokuda et al. (2019) are reliable and suitable for deployment in the clinic.

[0011] In contrast, unbiased experiments performed within the scope of the present invention found that among a subset of different blinded anti-mSOD1 antibodies, all with high but different binding affinities for mSOD1 and covering different epitopes, only two antibodies, designated NI-204.B and NI-204.O, but not the other candidates, reliably detected mSOD1 in tissue, cell, and body fluid samples, whereas some of the other candidates had significantly lower EC2s for denatured, oxidized, and recombinant SOD1 as determined by conventional ELISA assays. 50 The antibody showed excellent activity and was therefore identified as the first choice for use as a capture antibody in immunoassays for mSOD1.

[0012] Decoding of the antibody probes revealed that NI-204.B and NI-204.O share a similar epitope of SOD1 within the amino acid sequence 73-GGPKDEERHVGD-84 shown in SEQ ID NO: 11. When investigating immunoassays for detection according to the present invention, a sandwich ELISA could be established in which antibodies NI-204.B and NI-204.O each served as a capture antibody, and it was found that antibodies NI-204.B and NI-204.O reliably detected mSOD1 in CSF samples from ALS patients as exemplified in the Examples. In principle, the pre-assay to identify a suitable capture antibody and the subsequent immunoassay are based on the assay for mSOD1 described in Gill et al., Sci. Rep. 9 (2019), 6724, https: / / doi.org / 10.1038 / s41598-019-43164-z, see the "Methods" section, with additional modifications for the detection of mSOD1 in body fluids such as CSF, as shown in the Examples.

[0013] Notably, in contrast to antibody C4F6 used in Tokuda et al. (2019), the epitopes recognized by antibodies NI-204.B and NI-204.O are not necessarily associated with mutant SOD1 proteins and are recognized on spinal cord tissue from A4V fALS patients as well as sALS and fALS patients carrying the C9ORF72 hexanucleotide repeat expansion or unknown gene mutations; see Maier et al., Sci. Transl. Med. 10, eaah3924 (2018) 5.

[0014] Therefore, it is reasonable to expect that the assay of the present invention, if it works at all, will be applicable to a wider range of ALS patients than the ELISA assay described in Tokuda et al. (2019).

[0015] Thus, the present invention generally relates to a novel method for assaying mSOD1 in a body fluid of a human subject using an immunoassay, in particular an ELISA assay in which a body fluid, preferably CSF, is contacted with a first anti-SOD1 antibody as a capture antibody and a second anti-SOD1 antibody as a detection antibody.

[0016] This innovative assay is particularly interesting because it allows for the diagnosis of both fALS and sALS by assaying for mSOD1, which serves as a biomarker, in a patient's body fluids. This is important because, while the occurrence of fALS can be largely determined by genetic markers, identifying patients with sALS is much more difficult. Thus, the novel immunoassay can be used to identify and select patients with ALS, particularly sporadic ALS, for treatment with anti-SOD1 antibodies and / or other drugs currently used to treat ALS and its symptoms, respectively.

[0017] The assays of the present invention can also be used to monitor pharmacodynamic changes in levels of mSOD1 in body fluids, preferably CSF, which may facilitate dose optimization of therapeutic agents useful for treating or ameliorating symptoms in patients with ALS. An assay with sufficient sensitivity to allow accurate and precise quantitation of low concentrations of mSOD1 in clinical trials of candidate therapeutics would be beneficial to ALS research efforts. [Brief explanation of the drawings]

[0018] [Figure 1]Detection of mSOD1 in body fluid samples from ALS patients. Quantitative measurement of mSOD1 in CSF was performed using the Ciraplex™ Human Ultrasensitive mSOD1 1-plex Immunoassay Kit (Aushon BioSystems), a single-plex sandwich ELISA. Samples from 10 fALS patients (Figure 1A), 6 sALS patients (Figure 1B), and 10 non-neurological control participants (Figure 1C) were analyzed. The results are shown in bar graphs (Figures 1D and 1E); *p<0.05 (chi-square test for misSOD1-positive / negative cases; or Kruskal-Wallis / Dunn multiple comparison test). DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention relates to a novel highly sensitive immunoassay for detecting mSOD1 in the body fluids of a subject, which uses an antibody that specifically recognizes an epitope located on mSOD1 aggregates, and which can also distinguish subjects suffering from or at risk of developing ALS from healthy volunteers. More specifically, the present invention relates to the embodiments characterized in the claims, disclosed herein, and further illustrated in the following examples and figures.

[0020] Unless otherwise stated, the terms used herein are given the definitions provided in the Oxford Dictionary of Biochemistry and Molecular Biology, Oxford University Press, 1997, ISBN 0 19 850673 2, revised 2000 and reprinted 2003; second edition published 2006, ISBN 0-19-852917-1 978-0-19852917-0.

[0021] As used throughout this specification, the term "assaying" mSOD1 includes determining or measuring the presence / amount / level / concentration of mSOD1 and quantifying mSOD1 and associated expression.

[0022] Furthermore, unless otherwise stated, the terms and expressions used herein to characterize the present invention are given the definitions provided in WO 2012 / 080518 A1, particularly in the subsection "I. Definitions" on pages 10-30, the disclosure of which is expressly incorporated herein by reference. The same applies to the general embodiments disclosed in WO 2012 / 080518 A1 for antibodies, etc.

[0023] As is known in the art, different neurodegenerative diseases, such as ALS, Alzheimer's disease (AD), ALS / Parkinsonism-Dementia Complex (ALS-PDC), Down's syndrome, and Parkinson's disease (PD), exhibit the occurrence of or are associated with mSOD1. Thus, the presence or elevated levels of mSOD1 may indicate the disease. Furthermore, as mentioned above, mutations in the gene encoding SOD1 can cause misfolding, which accounts for approximately 20% of fALS cases and a small proportion of sALS cases, although misfolding of wt SOD1 is also associated with sALS cases. Thus, the presence or elevated levels of mSOD1 indicate ALS.

[0024] Therefore, the method of the present invention can be used as a method for diagnosing ALS, AD, ALS-PDC, Down's syndrome or PD, which method comprises assaying mSOD1 in a sample from a subject to be diagnosed, wherein the presence of mSOD1 in the sample indicates the above-mentioned disease in the subject, and an increased level of mSOD1 in the sample compared to a control indicates the above-mentioned disease in the subject. In a preferred embodiment, the disease diagnosed by the method of the present invention is ALS.

[0025] Since misfolding of SOD1 has been observed in patients with fALS and patients with sALS, and mSOD1 can be detected by the antibodies used in the methods of the present invention, the methods can be used to diagnose patients with fALS and sALS.

[0026] A subject being diagnosed can be asymptomatic or pre-symptomatic for the disease.

[0027] The method of the present invention involves screening for mSOD1 in a sample of a patient's body fluid. The sample analyzed in the assay of the present invention can be any body fluid suspected of pathologically containing mSOD1, such as a blood, CSF, or urine sample. In a preferred embodiment, the sample is whole blood lysate or CSF, preferably CSF.

[0028] The method of the present invention employs an immunoassay comprising contacting a body fluid with a first anti-SOD1 antibody, which is used as a capture antibody. During the course of experiments, two antibodies were identified as suitable for the method of the present invention, both of which bind to an epitope of SOD1 within the amino acid sequence 73-GGPKDEERHVGD-84 set forth in SEQ ID NO:11. These two antibodies are designated NI-204.B and NI-204.O. ​​As described above, NI-204.B was found to be antibody NI-204.12G7 disclosed in WO 2012 / 080518 A1, which binds to an epitope of SOD1 comprising the amino acid sequence 73-GGPKDEERHVG-83 set forth in SEQ ID NO:51 of WO 2012 / 080518 A1. NI-204.O binds to an epitope of SOD1 that includes the amino acid sequence 76-KDEERHVGD-84 (SEQ ID NO: 13).

[0029] In principle, the capture antibody can be any antibody or antibody format that recognizes the epitope 73-GGPKDEERHVGD-84 (SEQ ID NO: 11), preferably an epitope comprising the amino acid sequence 73-GGPKDEERHVG-83 (SEQ ID NO: 12) and / or an epitope comprising the amino acid sequence 76-KDEERHVGD-84 (SEQ ID NO: 13). In principle, such antibodies can be produced against the corresponding antigen in mice, rabbits, goats, or other animals commonly used to produce polyclonal or monoclonal antibodies, or by screening Fv, Fab, or complete IgG libraries. Preferably, the capture antibody is a monoclonal antibody or is derived from a monoclonal antibody.

[0030] In a particular preferred embodiment of the invention, the capture antibody is derived from the human antibody NI-204.12G7 and has, in its variable region, i.e., binding domain, a variable heavy chain (VH) having the amino acid sequence shown in Figure 1B of WO 2012 / 080518 A1. H ) and variable light chain (V L ), or one or more of the CDRs may differ in their amino acid sequence by one, two, three or even more amino acids from those shown in Figure 1B of WO 2012 / 080518 A1, in the case of CDR2 and CDR3, and the capture antibody exhibits substantially the same or identical immunological characteristics as the anti-SOD1 antibody NI-204.12G7 shown in the Examples of WO 2012 / 080518 A1. The locations of the CDRs are shown in Figure 1B and explained in the legend to Figure 1 of WO 2012 / 080518 A1. The corresponding nucleotide sequence is shown in Table II on page 54 of WO 2012 / 080518 A1. Additionally or alternatively, the framework regions or the complete V H and / or V LThe strand is 80% identical to the framework region shown in Figure 1B of WO 2012 / 080518 A1, preferably the framework region shown in Figure 1B of WO 2012 / 080518 A1 as well as the V H and / or V L The NI-204.B antibody is 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the NI-204.B chain, respectively. Furthermore, the cloning and expression of antibody NI-204.B was performed as described in the "Materials and Methods" section on pages 84-88 of WO 2012 / 080518 A1, and therefore, this method is incorporated herein by reference.

[0031] In a particularly preferred embodiment, the capture antibody is V shown in Figure 1B of WO 2012 / 080518 A1. H and / or V L It is characterized by chains.

[0032] Therefore, the capture antibody preferably comprises: (i) a variable heavy chain (VH) comprising VH complementarity-determining regions (CDRs) 1, 2, and 3 and / or a variable light chain (VL) comprising VL CDRs 1, 2, and 3; (a) VH-CDR1 comprises the amino acid sequence of SEQ ID NO: 3 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; (b) VH-CDR2 comprises the amino acid sequence of SEQ ID NO: 4 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; (c) VH-CDR3 comprises the amino acid sequence of SEQ ID NO: 5 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; (d) VL-CDR1 comprises the amino acid sequence of SEQ ID NO: 8 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; (e) VL-CDR2 comprises the amino acid sequence of SEQ ID NO: 9 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; and (f) a variable heavy chain (VH) and / or a variable light chain (VL), wherein VL-CDR3 comprises the amino acid sequence of SEQ ID NO: 10 or a variant thereof, the variant comprising one or two amino acid substitutions; and / or (ii) a VH chain and / or a VL chain, (a) the VH chain comprises the amino acid sequence set forth in SEQ ID NO: 1 or 2 or a variant thereof, wherein the variant comprises one or more amino acid substitutions; and (b) a VH chain and / or a VL chain, wherein the VL chain comprises the amino acid sequence set forth in SEQ ID NO: 6 or 7 or a variant thereof, wherein the variant comprises one or more amino acid substitutions; and preferably the VH and VL chain amino acid sequences are at least 90% identical to SEQ ID NOs: 1 or 2 and 6 or 7, respectively.

[0033] In principle, the capture antibody can be of any form recognizing an epitope, including, for example, a chimeric antibody, a single-chain antibody, a Fab fragment, a bispecific antibody, a fusion antibody, a labeled antibody, or any analog thereof. Corresponding methods for generating such variants are known to those skilled in the art and are described, for example, in Harlow and Lane "Antibodies, A Laboratory Manual," CSH Press, Cold Spring Harbor (1988) First Edition; Second Edition by Edward A. Greenfield, Dana-Farber Cancer Institute (Copyright) 2014, ISBN 978-1-936113-81-1. For example, Fab and F(ab')2 fragments can be produced by proteolytic cleavage of immunoglobulin molecules recombinantly or using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments). F(ab')2 fragments contain the variable region, the light chain constant region, and the CH1 domain of the heavy chain. Such fragments are sufficient for use, for example, in immunodiagnostic procedures involving conjugating immunospecific portions of immunoglobulins to reagents such as radioisotopes. Preferably, the capture antibody has an IgG format, i.e., is a full IgG antibody. Recombinant expression of fully human IgG1 antibodies with human or mouse constant domains can be performed substantially as described in the Examples of WO 2012 / 080518 A1.

[0034] Typically, the method of the present invention further comprises the use of a second antibody as a detection antibody. This antibody can be any anti-SOD1 antibody that binds to mSOD1 at an epitope different from that of the capture antibody 73-GGPKDEERHVGD-84 (SEQ ID NO: 11), such as a commercially available antibody, for example, polyclonal rabbit anti-human SOD1 (Abcam ab52950), rabbit monoclonal anti-human SOD1 (Abcam ab79390) combined with polyclonal biotinylated goat anti-rabbit IgG (Jackson Immuno. 111-065-144), such as those disclosed in Gill et al. (2019), supra, or in WO 2012 / 080518 A1, or listed in Table 3 in Tokuda et al. (2019).

[0035] In one embodiment, the detection antibody is commercially available (Abcam ab185125) and synthetic SOD1 aa 50-150 This rabbit monoclonal antibody [EPR1726] was raised against a peptide and is BSA- and azide-free. Antibody ab185125 is a carrier-free version of ab79390 and is designed for use in antibody labeling with fluorescent dyes, metal isotopes, oligonucleotides, and enzymes. Preliminary epitope analysis suggests that antibody EPR1726 binds to an epitope located in the same loop as the NI-204.12G7 epitope, immediately preceding the NI-204.12G7 epitope, i.e., approximately amino acids (slightly less than 61) 65-75 of human SOD1. Therefore, preferably, the detection antibody for use in the methods of the present invention is an equivalent monoclonal antibody that exhibits binding characteristics similar to those of antibody EPR1726, i.e., an equivalent monoclonal antibody that binds to amino acids 50-150 of human SOD1, particularly amino acids (61) 65-75 of human SOD1. Those skilled in the art are familiar with methods and means for arriving at such equivalent antibodies; see, for example, Harlow and Lane (1988) and Greenfield (2014), Antibodies: A Laboratory Manual, supra.

[0036] Therefore, preferably, the detection antibody binds to a different epitope from the first antibody and from the capture antibody. Therefore, the second antibody is an antibody that does not compete with the first antibody for binding to mSOD1. In principle, the detection antibody can be any type that recognizes mSOD1, and the second antibody is a monoclonal antibody.

[0037] In this regard, during experiments performed according to the present invention, it was found that one candidate from a subset of different blinded anti-mSOD1 antibodies, designated NI-204.G, was not suitable as a capture antibody, but could serve as a suitable second antibody, i.e., a detection antibody, because NI-204.G could bind to mSOD1 in the presence of antibody NI-204.B. Decoding the antibody probe revealed that NI-204.G corresponds to the antibody NI-204.12G3 disclosed in WO 2012 / 080518 A1, which binds to an SOD1 epitope comprising the amino acid sequence 121-HEKADDLGKGGNEES-135 as set forth in SEQ ID NO: 55 of WO 2012 / 080518 A1. Thus, in one embodiment, the detection antibody recognizes the epitope 121-HEKADDLGKGGNEES-135 (SEQ ID NO: 14) and can be of any source and antibody format as described for the capture antibody. Preferably, the detection antibody is derived from the human antibody NI-204.12G3 and has in its variable region, i.e., binding region, the amino acid sequence shown in Figure 1H of WO 2012 / 080518 A1. H and V LThe capture antibody is characterized by comprising the CDRs of the SOD1 chain, or one or more of the CDRs may differ in their amino acid sequence by one, two, three or more amino acids, in the case of CDR2 and CDR3, from those shown in Figure 1H of WO 2012 / 080518 A1, and the capture antibody exhibits substantially the same or identical immunological characteristics as the anti-SOD1 antibody NI-204.12G3 shown in the Examples of WO 2012 / 080518 A1. The locations of the CDRs are shown in Figure 1H of WO 2012 / 080518 A1 and explained in the legend to Figure 1. Additionally or alternatively, the capture antibody may comprise a framework region or a complete V H and / or V L The chains are composed of the framework regions shown in Figure 1H of WO 2012 / 080518 A1 as well as V H and / or V L The strands are 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the respective strands.

[0038] However, as mentioned above, substantially the same epitope and amino acid region recognized by different anti-SOD1 antibodies, in particular the antibodies Abcam ab185125, Abcam ab79390 and NI-204.12G3, respectively, preferably SOD1 aa 100-150 Antibodies that recognize epitopes within SOD1 may also be useful as detection antibodies. Typically, such detection antibodies for use in accordance with the assays of the invention compete with antibodies Abeam ab185125, Abeam ab79390, and / or NI-204.12G3 for binding to SOD1 in a sandwich ELISA format of the invention.

[0039] Preferably, the detection antibody has an IgG format, i.e. is a full IgG antibody. Recombinant expression of a full human IgG1 antibody with human or mouse constant domains can be performed essentially as described in the examples of WO 2012 / 080518 A1.

[0040] In one embodiment of the mSOD1 assay of the present invention, the detection antibody is (i) a variable heavy chain (VH) comprising VH complementarity-determining regions (CDRs) 1, 2, and 3 and / or a variable light chain (VL) comprising VL CDRs 1, 2, and 3; (a) VH-CDR1 comprises the amino acid sequence of SEQ ID NO: 16 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; (b) VH-CDR2 comprises the amino acid sequence of SEQ ID NO: 17 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; (c) VH-CDR3 comprises the amino acid sequence of SEQ ID NO: 18 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; (d) VL-CDR1 comprises the amino acid sequence of SEQ ID NO: 20 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; (e) VL-CDR2 comprises the amino acid sequence of SEQ ID NO: 21 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; and (f) a variable heavy chain (VH) and / or a variable light chain (VL), wherein VL-CDR3 comprises the amino acid sequence of SEQ ID NO: 22 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; and / or (ii) a VH chain and / or a VL chain, (a) the VH chain comprises the amino acid sequence set forth in SEQ ID NO: 15 or a variant thereof, wherein the variant comprises one or more amino acid substitutions; and (b) a VL chain comprising the amino acid sequence set forth in SEQ ID NO: 19 or a variant thereof, wherein the variant comprises one or more amino acid substitutions; and preferably the VH and VL chain amino acid sequences are at least 90% identical to SEQ ID NOs: 15 and 19, respectively.

[0041] In a preferred embodiment of the method of the present invention, the second antibody or a fragment thereof comprises a detectable label (e.g., fluorescent, chemiluminescent, radioactive, enzymatic, nuclear magnetic, heavy metal, tag, flag, etc.); for general techniques, see, for example, Antibodies A Laboratory Manual 2nd edition, 2014 by Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA; for advances in fluorescent labeling strategies for dynamic cell imaging, see Dean and Palmer, Nat. Chem. Biol. 10 (2014), 512-523; and for enzyme-based labeling strategies for antibody-drug conjugates and antibody mimetics, see Falck and Mueller, Antibodies 7 (2018), 4; doi:10.3390 / antib7010004.

[0042] The label can be a directly detectable label, such as a fluorescent label (a physicochemical reporter), or the label can be a ligand, such as biotin, that is bound by a ligand binding partner that contains a directly detectable label.

[0043] In a preferred embodiment of the method of the present invention, the second antibody is conjugated to a ligand binding partner, ie a ligand capable of binding to the ligand binding tag forming a non-covalent protein-ligand interaction.

[0044] According to the present invention, the ligand is a moiety known to those skilled in the art, and includes affinity tags such as His-tags, maltose-binding protein (MBP)-tags, glutathione-S-transferase (GST)-tags, chitin-binding domains or thioredoxins, calmodulin-binding peptides (CBP), FLAG-peptides, Arg-tags, Hat-tags, c-myc-tags, S-tags or streptavidin-binding tags such as Twin-Strep-tags®, and biotin. An overview is provided, for example, in Terpe, Appl. Microbiol. Biotechnol. 60 (2003), 523-533, and exemplary tags, including their amino acid sequences, are listed in Table 2 of Terpe (2003), which is incorporated herein by reference.

[0045] In a preferred embodiment, the ligand is or comprises biotin or a biotin analogue or derivative thereof, i.e., the second antibody used in the method of the invention is biotinylated. Biotinylation of antibodies is generally known in the art, and commercial kits are available, allowing one of skill in the art to generate biotinylated antibodies.

[0046] Therefore, the method of the present invention includes a labeling step using a ligand binding partner that matches the above-mentioned ligand. These ligand binding partners are also known in the art and are summarized, for example, in Terpe, Appl Microbiol Biotechnol 60 (2003), 523-533.

[0047] In a preferred embodiment, the ligand binding partner is streptavidin, avidin, a streptavidin analog, or an avidin analog that binds to the respective biotin or a derivative. The ligand binding partner is included in a conjugate that further comprises a detectable label, which may be a detectable label as specified above. Preferably, the detectable label included in the conjugate is a chromogenic / fluorescent or chemiluminescent label, i.e., an enzyme that can catalyze the conversion of a chromogenic / fluorescent / chemiluminescent substrate. In a preferred embodiment, the detectable label is horseradish peroxidase or β-galactosidase. Thus, the conjugate is a streptavidin-HRP conjugate or a streptavidin-β-galactosidase (SβG) conjugate. These enzymes generate a signal when an appropriate substrate solution is added. For HRP, the chromogenic substrates 3,3',5,5'-tetramethylbenzidine (TMB) or 2,2'-azino-di-[3-ethylbenzothiazoline-6-sulfonic acid] (ABTS) are used, or luminol or luminol containing substrates are used as chemiluminescent substrates, and for β-galactosidase, resorufin β-D-galactopyranoside (RGP) is used.

[0048] The method of the present invention preferably comprises a single-plex assay, which means that only one target is detected. In the method of the present invention, mSOD1 is detected as a single target. However, the assay can also be designed as a multiplex assay.

[0049] Therefore, the method of the present invention comprises at least the following steps: capturing mSOD1 in a subject sample with a first anti-SOD1 antibody attached to a surface, adding a second anti-SOD1 antibody containing a detectable label that enables it to bind to mSOD1, detecting the signal of the label, and comparing the signal of the label with a control.

[0050] In one embodiment, the method comprises the following steps (see also Example 1): First, provide a microplate spotted with the above-mentioned first anti-SOD1 antibody. Such a plate can be manufactured by Aushon BioSystems and is commercially available. Furthermore, the design of microarray immunoassays is generally summarized in Kusnezow et al., Mol. Cell Proteomics 5 (2006), 1681-1696. Preferably, the plate is a 96-well plate.

[0051] A further step involves adding a sample containing a body fluid to the wells of the microplate. The body fluid can be any body fluid as described above, but is preferably CSF. The body fluid can be further modified, for example, purified to remove undesirable components or components that may interfere with the immunoassay. The sample is incubated in the wells under conditions that allow the formation of an antibody-antigen complex, i.e., allow the first anti-SOD1 antibody to bind to mSOD1, if present in the sample. Identification of appropriate conditions can be performed simultaneously with the actual assay or by testing a positive control in advance to adjust the exact parameters. The positive control can be either purified mSOD1 or a sample from a patient with mSOD1-associated ALS. In a preferred embodiment, the incubation is preferably performed for 2 hours at room temperature on a plate shaker set at 600 rpm. In a preferred embodiment, the plate is subsequently washed to remove unbound components.

[0052] The next step involves adding a second anti-SOD1 antibody to the sample, where the second antibody is conjugated to a ligand. The second anti-SOD1 antibody can be the above-mentioned antibody or binding fragment, preferably an antibody that binds to a different binding site of mSOD1 and a different epitope of mSOD1, respectively. As mentioned above, the ligand is a directly detectable label, for example a fluorescent label, or the ligand comprises a label that is bound by a ligand binding partner that comprises a directly detectable label. In a preferred embodiment, the second antibody is biotinylated, i.e., conjugated to biotin or a biotin analog or derivative thereof.

[0053] The mixture is incubated in the well under conditions that allow the formation of additional antibody-antigen complexes, i.e., allow the binding of the second anti-SOD1 antibody to mSOD1 if present in the sample. Conditions must be selected so that both antibodies, i.e., the first and second antibodies, can bind to mSOD1. As mentioned above, appropriate conditions can be tested with a positive control. In a preferred embodiment, the incubation is preferably carried out for 30 minutes at room temperature on a plate shaker set at 600 rpm. In a preferred embodiment, the plate is subsequently washed to remove excess detection antibody.

[0054] The sample and the second antibody can also be added simultaneously to a microplate coated with the first antibody, and incubation can be carried out to allow binding of the first anti-SOD1 antibody to mSOD1 and the second anti-SOD1 antibody to mSOD1.

[0055] If the second antibody is directly labeled with a detectable label, such as a fluorescent label or an enzyme, an appropriate substrate solution is added.

[0056] When indirect labeling is used, a conjugate containing a ligand-binding partner and a detectable label is added. The sample is incubated with the conjugate for another 30 minutes, preferably at room temperature, on a plate shaker set at 600 rpm. The ligand-binding partner contained in the conjugate can be, for example, streptavidin or avidin, or a functional analog or derivative thereof. In a preferred embodiment, the ligand-binding partner is streptavidin or a functional analog or derivative thereof. The detectable label contained in the conjugate can be any detectable label as described above, but is preferably an enzyme capable of catalyzing the conversion of a chromogenic / fluorescent or chemiluminescent substrate. More preferably, the enzyme is horseradish peroxidase (HRP). Thus, the conjugate is a streptavidin-HRP reagent. A washing step is preferably performed, followed by the addition of an appropriate substrate solution, preferably a chromogenic or chemiluminescent substrate solution. In a preferred embodiment, TMB, luminol, or luminol containing a substrate is used.

[0057] The signal from the mixture is imaged. In principle, any commercially available imaging system can be used, particularly one that can detect and measure fluorescent or chemiluminescent signals with high sensitivity. Preferably, imaging is carried out using the Cirascan™ imaging system.

[0058] The signal from each well is detected and measured and compared to a control, preferably assayed in a separate well within the same microplate.

[0059] The control can be a reference standard, i.e., mSOD1. Alternatively or additionally, a sample from a control subject without a neurodegenerative disease is used as a second control, and a difference in the level of mSOD1 in the sample and the control indicates that the subject to be diagnosed has a neurodegenerative disease. In particular, a higher level of mSOD1 in the sample compared to the control sample indicates a disease, particularly ALS. Preferably, the subject to be diagnosed and the control subject are age-matched.

[0060] In one embodiment, the standard comprises serial dilutions of misfolded SOD1 from 200 ng / mL to 3 pg / mL.

[0061] In one embodiment, the microplate is covered with a lid, preferably a lid with a fluid-absorbent matrix filled with fluid to prevent evaporation of the sample during the incubation step. The washing step described above can be performed using any suitable buffer that does not disrupt the binding of the first antibody to the surface, the binding of the first and second antibodies to mSOD1, and the binding of the conjugate to the second antibody. Preferably, washing is performed using the washing buffer from Aushon Biosciences provided in the kit, as described in the Examples. As described above, incubations are performed between different steps of the assay to allow binding of the antibody to mSOD1 and binding of the conjugate to the ligand of the second antibody. Naturally, different incubation times can be selected as long as binding of the antibody and the conjugate is ensured.

[0062] In another embodiment, the method of the invention uses Single Molecule Arrays (Simoa™), also known as digital ELISA. In this approach, target proteins are captured on antibody-coated paramagnetic beads, the captured proteins are labeled with an enzyme label, and single beads are isolated and sealed in an array of femtoliter wells in the presence of the enzyme substrate. The sealing step confines the fluorescent product of the enzyme-substrate reaction to a volume of approximately 40 fL, and within 30 seconds, the fluorescence generated by a single enzyme can be detected on an uncooled CCD camera using a white-light excitation source; see references cited herein, e.g., Rissin et al., Measurement of single protein molecules using digital ELISA. In: Wild, D. (Ed.), The Immunoassay Handbook: Theory and Applications of Ligand Binding, ELISA and Related Techniques, 4th ed. Elsevier, Oxford, UK, and Rivnak et al., A fully-automated, six-plex single molecule immunoassay for measuring cytokines in blood, J. Immunol. Methods, 2015;424:20, Quanterix Whitepaper 6.0 (2015). For example, capture beads, preferably paramagnetic beads with a diameter of about 2.7 μM, having the above-defined first antibody or its binding fragment attached to their surface, can be used in the method of the present invention; see Example 3. The use of such beads allows detection of mSOD1 at the single molecule level. A sample containing the above-defined body fluid is added to the capture beads, and incubation is carried out, allowing capture of mSOD1 by the beads mediated by the first anti-SOD1 antibody, if present in the body fluid. As mentioned above, incubation conditions can be varied, and optimal conditions can be tested with a positive control. Preferably, the incubation time is 30 minutes.Then, a second anti-SOD1 antibody conjugated to the ligand defined above is added, followed by incubation to allow binding of the second anti-SOD1 antibody to the captured misfolded SOD1 on the beads. Preferably, incubation is carried out for 5 minutes. Alternatively, the above two steps can be combined in that the sample and the second antibody are added to the capture beads, followed by incubation to allow capture of misfolded SOD1 present in the body fluid by the beads mediated by the first anti-SOD1 antibody and binding of the second anti-SOD1 antibody to the captured misfolded SOD1 on the beads. When both steps are combined, the incubation time is preferably increased to 35 minutes. If the second antibody is not directly labeled with a detectable label but is labeled with a ligand, a conjugate as defined above containing a ligand-binding partner and a detectable label is added. Preferably, the ligand-binding partner is streptavidin or a functional analog or derivative thereof, and the detectable label is an enzyme capable of generating a colorimetric or fluorescent substrate, preferably β-galactosidase. Incubation is preferably carried out for 5 minutes. The beads are then resuspended as described above, and a fluorescent substrate solution as defined above is added. Preferably, the substrate is resorufin β-D-galactopyranoside (RGP). In the next step, the beads are loaded into femtoliter-sized wells of a microplate configured to hold no more than one bead per well. Preferably, the wells have a width of approximately 4.25 μm and a depth of approximately 3.25 μm. Sealing of the wells with oil and imaging of the fluorescent signal are then performed. In principle, any commercially available imaging system capable of detecting signals with high sensitivity can be used. This assay is based on the commercially available Simoa® assay from Quanterix, and therefore reagents and the Simoa™ optical system are used in the immunoassay. As already mentioned above, washing steps can be performed between the different steps of the assay.

[0063] Thus, in one embodiment of the method of the invention, the second anti-SOD1 antibody is conjugated to a ligand and the method comprises a labeling step with a ligand-binding tag, preferably the method uses a single molecule array (Simoa™) assay and optionally comprises one or more, preferably all of the following steps: (i) attaching a first anti-SOD1 antibody to the surface of capture beads, preferably the beads being paramagnetic beads and / or having a diameter of about 2.7 μM; and (ii)(a) adding a sample containing a body fluid, preferably CSF, and incubating the beads with the sample, thereby allowing capture of misfolded SOD1 present in the body fluid by the beads mediated by the first anti-SOD1 antibody, preferably the incubation time is 30 minutes; and (ii)(b) adding a second anti-SOD1 antibody conjugated to a ligand, preferably biotin or a biotin analogue or derivative thereof, and incubating, thereby allowing the second anti-SOD1 antibody to bind to the captured misfolded SOD1 on the beads, preferably the incubation time is 5 minutes; and (ii)(c) adding a conjugate comprising a ligand binding tag, preferably streptavidin or a functional analogue or derivative thereof, and an enzyme capable of generating a detectable label, preferably a chromogenic or fluorescent molecule, preferably the enzyme is β-galactosidase (streptavidin-β-galactosidase (SβG) conjugate), and incubating, preferably the incubation time is 5 minutes; or (II)(A) adding a sample containing a body fluid, preferably CSF, adding a second anti-SOD1 antibody conjugated to a ligand, preferably biotin or a biotin analog or derivative thereof, and incubating the beads with the sample and the second antibody, thereby allowing capture of misfolded SOD1 present in the body fluid by the beads mediated by the first anti-SOD1 antibody and binding of the second anti-SOD1 antibody to the captured misfolded SOD1 on the beads, preferably for 35 minutes; and (II)(B) adding a conjugate comprising a ligand-binding tag, preferably streptavidin or a functional analogue or derivative thereof, and an enzyme capable of generating a detectable label, preferably a chromogenic or fluorescent molecule, preferably the enzyme is β-galactosidase (streptavidin-β-galactosidase (SβG) conjugate), and incubating, preferably for 5 minutes; and (iii) resuspending the beads in a fluorescent substrate solution, preferably the fluorescent substrate is resorufin β-D-galactopyranoside (RGP); and (iv) loading the beads of step (iii) into an array of femtoliter-sized wells configured to hold no more than one bead per well; and (v) sealing individual beads in femtoliter-sized wells, preferably with oil; and (vi) imaging the fluorescent signal, preferably wherein the imaging is performed by a Simoa™ optical system; optionally, (vii) comparing the assayed level of misfolded SOD1 with a reference standard and / or control.

[0064] Steps (ii)(a), (ii)(b) and (ii)(c) are referred to as the "three-step approach" and steps (II)(a) and (II)(b) are referred to as the "two-step approach."

[0065] As mentioned above, the control can be a reference standard, i.e., mSOD1. Alternatively or additionally, a sample from a control subject without a neurodegenerative disease is used as a second control, and a difference in the level of mSOD1 in the sample and the control indicates that the subject to be diagnosed has a neurodegenerative disease. In particular, a higher level of mSOD1 in the sample compared to the control sample indicates a disease, particularly ALS. Preferably, the subject to be diagnosed and the control subject are age-matched.

[0066] In one embodiment, the standard comprises serial dilutions of misfolded SOD1 from about 1000 ng / mL to 0.244 ng / mL by 4-fold serial dilution to an 8-point calibration curve, from 10 ng / mL to 0.020 ng / mL by 2-fold serial dilution to an 8-point calibration curve, from 50 ng / mL to 0.012 ng / mL by 2-fold serial dilution to a 12-point calibration curve, and / or from about 66.66667 ng / mL to 0.00339 ng / mL by 3-fold serial dilution to a 12-point calibration curve.

[0067] The method of the present invention is highly sensitive, allowing for the detection of minimal amounts of mSOD1 in a subject's CSF. This high sensitivity can be attributed, in particular, to the antibody used in the method of the present invention as the first capture antibody. Depending on the assay, mSOD1 can be detected down to 6-7 pg / mL with acceptable precision. For assays using Aushon BioSystems reagents and instruments, more precise results were obtained within a mSOD1 range of 10.16 pg / mL to 7404.41 pg / mL, resulting in a reportable range of 20.32-14814.82 pg / mL after a 1:2 MRD (minimum required dilution). For assays using Quanterix reagents and instruments, the assay was shown to have an LLOD ranging from 6 pg / mL to 32 pg / mL and an LLOQ ranging from 58 pg / mL to 132 pg / mL, based on a 2× assay background method, with the LLOD ranging from 10.8-13.6 pg / mL, respectively, depending on the capture antibody used, as described in Example 3.

[0068] Thus, the method of the invention preferably has a lower limit of quantitation (LLOQ) for mSOD1 of about ≦20.32 pg / mL and a lower limit of detection (LLOD) of about 7 pg / mL, or an LLOQ of about 58 pg / mL and a LLOD of about 6 or 10 pg / mL.

[0069] Additional single molecule sensing platforms that can be used in accordance with the methods of the present invention are known to those skilled in the art and are under development, such as low background noise fluorescence microscopy and plasmonic and electrical nanotransducers; for reviews, see, e.g., Macchia et al., Analytical and Bioanalytical Chemistry 412 (2020), 5005-5014.

[0070] Given the ability to assay accessible fluids and the desire to have biomarkers confirmed in multiple studies, this would naturally be a useful approach to obtain a complete picture of disease progression in any given patient and to combine the immunoassay of the present invention with the evaluation of other candidate biomarker molecules for ALS. For example, Vijayakumar et al. (2019), supra, suggest combining candidate biomarker molecules from those listed in Table 2 (cystatin C, pNFH, and NFL, all of which reflect neuronal survival; MCP1 as a pro-inflammatory marker; MiR 206 and 133b, which reflect muscle origin and neuromuscular junctions, respectively; and some indicators of metabolic abnormalities, such as homocysteine, glutamate, or cholesterol). Preferably, the immunoassay of the present invention is combined with the evaluation of one or more biomarkers for ALS listed in Tables 1-5 disclosed in Vu and Bowser (2017), supra, fluid-based biomarkers. Thus, the immunoassays of the present invention can facilitate the development of heterogeneous multi-biomarker panels for diagnostic purposes and prognostic or predictive applications.

[0071] The present invention also encompasses therapeutic agents for use in treating or ameliorating symptoms in patients diagnosed according to the methods of the present invention as having ALS or at risk of developing ALS. In a preferred embodiment, the patient has been assayed to have a detectable amount of mSOD1. Preferably, the patient exhibits an increased level of mSOD1 when compared to a control. The control may preferably be a healthy subject age-matched to the patient to be diagnosed.

[0072] In a preferred embodiment, the patient has a level of mSOD1 at least greater than 5 pg / mL, preferably greater than 6 or 7 pg / mL, more preferably greater than 10 pg / mL, more preferably greater than 20 pg / mL, and most preferably greater than 20.32 pg / mL or 58 pg / mL.

[0073] In one embodiment, the therapeutic agent is an anti-SOD1 antibody, preferably an antibody disclosed in WO 2012 / 080518 A1, preferably antibody NI-204.12G7 or an antibody disclosed in WO 2016 / 120810. In another embodiment, the therapeutic agent is an agent for non-specific treatment, such as a drug that reduces SOD1 levels, e.g., pyrimethamine (Lange et al. Ann. Neurol. 81 (2017), 837-848), an agent used in gene silencing, e.g., morpholino oligonucleotides (MOs), or rapamycin. The therapeutic agent may be an agent used in gene therapy approaches. In a preferred embodiment, the therapeutic agent is Rilutek (riluzole) or Radicava (edavarone), both of which are approved by the U.S. Food and Drug Administration for the treatment of ALS. Additionally, the therapeutic agent may be a drug targeted at some of the specific symptoms of ALS, such as a painkiller or muscle relaxant. Therefore, the therapeutic agent is preferably baclofen (Gablofen, Kemstro, Lioresal) or diazepam (Diastat, Valium), which may help relieve spasms. Difficulty swallowing, resulting in saliva accumulation in the mouth, is also a symptom of ALS and can be treated with different drugs that are therapeutic agents according to the present invention. Preferably, the therapeutic agent is Elavil (amitriptyline), trihexyphenidyl, Scopaderm (scopolamine patch) or Robinul (glycopyrrolate).

[0074] The present invention also encompasses kits adapted for carrying out the methods of the invention. Thus, the kits contain the components required to carry out the methods of the invention, preferably the components of preferred embodiments of the methods of the invention.

[0075] In one embodiment, the kit is preferably suitable for use in the method of the invention using a single-plex sandwich ELISA, such as the Ciraplex™ Ultrasensitive immunoassay from Aushon Biosystems, exemplified in Examples 1 and 2, and comprises at least a microplate, preferably including a lid as defined above, with wells pre-spotted with a first anti-SOD1 antibody as defined above. The kit further comprises a detection reagent comprising a second anti-SOD1 antibody as defined above. Additionally or alternatively, the kit may comprise a conjugate comprising a ligand binding partner and a detectable label as defined above, preferably an enzyme capable of catalyzing the conversion of a colorimetric or chemiluminescent substrate, an appropriate substrate solution, calibrated immunoassay standards or controls for mSOD1, buffers, diluents, substrate and / or solution recommendations, and instructions on how to perform the assay of the invention, and / or washing and assay / sample dilution buffers appropriate for immunologically-based diagnostic assays that do not interfere with the method of the invention and allow the antibodies to remain in their active form.

[0076] In another embodiment, the kit is preferably suitable for use in the method of the invention using the Simoa™ assay as exemplified in Example 3 and comprises a capture reagent comprising a first anti-SOD1 antibody as defined above and a detection reagent comprising a second anti-SOD1 antibody as defined above. In an optional embodiment, the kit comprises beads and a suitable femtoliter-sized microplate. Additionally or alternatively, the kit may comprise a conjugate comprising a ligand-binding partner as defined above and a detectable label, preferably an enzyme capable of catalyzing the conversion of a fluorescent substrate, a suitable substrate solution, calibrated immunoassay standards or controls for mSOD1, microplates, buffers, diluents, substrate and / or solution recommendations, and instructions on how to perform the assay of the invention, and / or wash and assay / sample dilution buffers suitable for immuno-based diagnostic assays that do not interfere with the method of the invention and allow the antibodies to remain in their active form.

[0077] Several documents are cited throughout the text of this specification. The contents of all cited references (including literature references, issued patents, published patent applications cited throughout this application, including background sections and manufacturer specifications, instructions, etc.) are expressly incorporated herein by reference; no admission is made that any cited document is actually prior art with respect to this invention.

[0078] A more detailed understanding can be obtained by reference to the following specific examples, which are provided herein for purposes of illustration only and are not intended to limit the scope of the invention. [Example]

[0079] Example 1: Establishment and validation of an immunoassay specific for mSOD1 For quantitative measurement of mSOD1 in CSF, a single-plex sandwich ELISA, Ciraplex™ Human Ultrasensitive mSOD1 1-plex Immunoassay Kit from Aushon BioSystems, was used.

[0080] Test principle: Each well of a 96-well microplate was pre-spotted by Ashon BioSystems with the capture antibody NI-204.B, a monoclonal antibody that specifically recognizes mSOD1 at an epitope within the amino acid sequence 73-GGPKDEERHVGD-84 (SEQ ID NO: 11) of human SOD1, specifically the epitope containing the amino acid sequence 73-GGPKDEERHVG-83 (SEQ ID NO: 12), and captures mSOD1 from the sample of interest. After washing away unbound proteins, biotinylated SOD1 rabbit monoclonal detection antibody EPR1726, available from Abcam as ab185125 or ab79390, was added and allowed to bind to a secondary site on mSOD1 (in this case, ab185125 was used). After removing excess detection antibody, streptavidin-horseradish peroxidase (SA-HRP) was added. HRP is an enzyme that reacts with a substrate to generate a luminescent signal that is detected by the Cirascan™ imaging system. The intensity of the signal generated is directly proportional to the amount of each protein in the standard or sample of interest. The intensity is expressed as an integrated density value (IDV) and exported to SoftMax Pro, where a weighted five-parameter algorithm is used to back-calculate unknown samples using results interpolated from the corresponding standard curve.

[0081] Specifically, the assay was performed as follows: A stock solution of 20 μg / mL mSOD1 (mSOD1 solution diluted with stabilzyme / protease inhibitor cocktail) stored at -70 °C was thawed. Additionally, all assay components were removed from the refrigerator / freezer and stored at room temperature for 30-60 min before use. 1x wash buffer was prepared by adding the entire bottle (50 mL) to 1200 mL of deionized water.

[0082] During the incubation step, a MicroClime® Lid was placed on top of the 96-well microplate. It was filled with deionized (DI) water before use. To do this, the lid was removed from the filling material and placed with the filling trough (the groove around the edge of the lid) and corners facing up. Using a syringe or multichannel pipette, 4 mL of DI water was slowly dispensed into the filling trough at the top of the long edge of the lid. This was repeated for the filling trough at the bottom of the long edge, so that the lid contained a total of 8 mL of DI water. Excess water was removed from the trough using a Kimwipe. When ready for the incubation step, the lid was inverted and placed on top of the 96-well microplate.

[0083] Standards were prepared by thawing the stock and diluting 1:100 with sample diluent. The 1:100 standard was further diluted 1:3 with sample diluent to receive the top standard. The top standard was further diluted 1:3 to give a total of 11 non-zero standards. The zero standard was standard diluent only. Samples and controls were diluted 3-fold with sample diluent.

[0084] The mSOD1-microplate was removed from the pouch and washed six times with ≥300 μL of 1× wash buffer using an Immuno Wash 12 manual washer. The plate was then carefully patted dry on absorbent paper. 50 μL of standards, diluted controls, and diluted samples were added to the appropriate replicate wells, the plate was covered with a MicroClime® lid, and incubated for 2 hours at room temperature on a plate shaker set at 600 rpm. The plate was then washed four times with ≥300 μL of 1× wash buffer using an Immuno Wash 12 manual washer. The plate was then carefully patted dry on absorbent paper. 50 μL of IgG-spiked biotinylated antibody reagent was added to each well, the plate was covered with a MicroClime® lid, and incubated for 30 minutes at room temperature on a plate shaker set at 600 rpm. The plate was then washed four times with ≥300 μL of 1× wash buffer using an Immuno Wash 12 manual washer and patted dry on absorbent paper. 50 μL of streptavidin-HRP reagent was added to each well, the plate was covered with a MicroClime® Lid, and incubated for 30 minutes at room temperature on a plate shaker set at 600 rpm. The plate was then washed four times with ≥300 μL of 1× wash buffer using an Immuno Wash 12 manual washer and patted dry on absorbent paper.

[0085] SuperSignal® substrate solution was prepared no more than 15 min before use, preferably immediately before the final wash step. 50 μL of mixed SuperSignal® substrate solution was added to each well, and the plates were read on an Aushon Cirascan Imaging System within 2–4 min, thereby recording the short and long exposure times for each plate. Integrated density values ​​(IDVs) from Cirasoft were transferred and processed through the SoftMax Pro Protocol, and results were quantified using a weighted 5-parameter logistic curve fit.

[0086] Using this assay, mSOD1 could be quantified to 7.01 pg / mL and 6 pg / mL with acceptable precision. Even more precise results could be obtained within the mSOD1 range of 10.16 pg / mL to 7404.41 pg / mL, resulting in a reportable range of 20.32 to 14814.82 pg / mL after a 1:2 MRD (minimum required dilution) of CSF samples. Thus, the assay has a lower limit of quantitation (LLOQ) of approximately 20.32 pg / mL and a lower limit of detection (LLOD) of approximately 7 pg / mL for mSOD1.

[0087] Example 2: Detection of mSOD1 in CSF samples from fALS and sALS patients CSF samples were analyzed using the assay parameters described above. Specifically, CSF samples from 10 fALS patients with different SOD1 mutations, 6 sALS patients, and 10 non-neurological control (NNC) participants were screened for mSOD1. See Figures 1A, 1B, and 1C. These control participants did not have neurological disorders but may have other diseases. As shown in Figures 1D and 1E, mSOD1 was detected in the CSF of most fALS and sALS patients, i.e., the amount of mSOD1 detected in the CSF of fALS and sALS patients was higher than that in the CSF of control patients.

[0088] Example 3: Establishment and validation of a single molecule array (Simoa™) assay specific for mSOD1 In this experiment, mSOD1 was measured in the subject's body fluids using the Simoa™ assay and antibody NI-204.B as the capture antibody. The Simoa™ assay was performed using a three-step approach (30 min capture, 5 min detection, 5 min enzyme conjugate) and the Quanterix Homebrew Kit, as described above. The sample volume was 100 μl. The capture and detection antibodies, as well as the misfolded SOD antigen stock (667 μg / mL), were stored at 4°C before reagent preparation. The detection antibody was commercially available (Abcam ab185125). CSF samples were kept at -80°C until analysis.

[0089] First, the surface of paramagnetic beads (2.7 μm diameter) was coated with a first anti-mSOD1 antibody (capture antibody). The beads typically contain approximately 250,000 attachment sites. Specifically, the capture antibody was processed according to the standard Quanterix Homebrew assay protocol. The capture antibody was conjugated to the magnetic beads using standard two-step 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) coupling chemistry at an antibody concentration of 0.7 mg / mL and EDC at 0.5 mg / mL.

[0090] A second anti-mSOD1 antibody (detection antibody) was biotinylated at 60-fold molar excess using the standard Quanterix Homebrew biotinylation protocol.

[0091] Capture beads were diluted with the standard bead diluent from the Homebrew Kit to a concentration of 4 x 10 6 1000 beads / mL were added to the sample solution so that there were more beads than target molecules. Incubation was carried out for 30 minutes. Misfolded SOD1 present in the sample was then captured by the capture beads. The CSF sample was diluted 2-fold with Generic Homebrew Sample Diluent. The beads were then washed to remove nonspecifically bound proteins, and 0.1 μg / mL of biotinylated detection antibody was then mixed with the capture beads. Generic Homebrew Detector Diluent was used to dilute the detection antibody. The mixture was incubated for 5 minutes to allow the detection antibody to bind to the captured mSOD1 on the beads.

[0092] Following a second wash step, 300 pM of streptavidin-β-galactosidase (SβG) conjugate (diluted in the SβG diluent provided in the Homebrew Kit) was mixed with the capture beads and incubated for 5 minutes. SβG binds to the biotinylated detection antibody, resulting in enzymatic labeling of the captured misfolded SOD1. In this way, each bead that captured a single protein molecule was labeled with the enzyme. Beads that did not capture a molecule remained unlabeled.

[0093] After the third wash, the captured beads were resuspended in resorufin β-D-galactopyranoside (RGP) substrate solution and transferred to a Simoa Disc, an array of 216,000 femtoliter-sized wells (4.25 μm wide, 3.25 μm deep) sized to hold no more than one bead per well. The wells were then sealed with oil and imaged.

[0094] When misfolded SOD1 is captured and labeled, β-galactosidase hydrolyzes the RGP substrate to a fluorescent product, which provides a signal for measurement. Single-labeled misfolded SOD1 molecules produce sufficient fluorescent signal in 30 seconds to be detected and counted by a Simoa optical system (Simoa HD-1 Analyzer (instrument ID: 2710000020 and 2710000004 STD RUO; software version 1.5)).

[0095] The protein concentration in the test sample is determined by counting the number of wells containing both beads and fluorescent product relative to the total number of wells containing beads. Because the Simoa™ assay allows concentration to be determined digitally, rather than by using a total analog signal, this approach to detecting single immune complexes is called digital ELISA. At low misfolded SOD1 concentrations, the percentage of bead-containing wells in the array with a positive signal is proportional to the amount of misfolded SOD1 present in the sample. At higher target concentrations, when the majority of bead-containing wells have one or more labeled target molecules, the total fluorescent signal is proportional to the amount of misfolded SOD1 present in the sample. The concentration of misfolded SOD1 in unknown samples is interpolated from the standard curve.

[0096] Calibration with mSOD1 was performed by generating a serial dilution standard curve starting at 1 μg / mL (100-fold dilution of 667 μg / mL stock to 6.7 μg / mL) made from an intermediate mSOD1 stock. Dilutions were performed using Generic Homebrew Calibrator Diluent A and a 4-parameter logistic curve-fitting data reduction method (4 PLC, 1 / y). 2 The assay has an LLOD range of 6 pg / mL to 32 pg / mL (mean LLOD: 15.7 pg / mL) and an LLOQ range of 58 pg / mL to 132 pg / mL, based on a 2× assay background method.

[0097] In further tests, mSOD1 was measured in the body fluids of subjects using the Simoa™ assay with another monoclonal antibody, NI-204.O, which specifically recognizes mSOD1 at an epitope within the amino acid sequence 73-GGPKDEERHVGD-84 (SEQ ID NO: 11) of human SOD1, as a capture antibody, namely, antibody NI-204.O, which recognizes an epitope comprising the amino acid sequence 76-KDEERHVGD-84 (SEQ ID NO: 13). The Simoa™ assay was performed essentially as described above, but with different concentrations of reagents. Specifically, conjugation of the capture antibody to paramagnetic beads was performed at an antibody concentration of 0.5 mg / mL; 1,500,000 capture beads / mL were added to the sample solution; the detection antibody was biotinylated at a 60-fold molar excess; 0.75 μg / mL of biotinylated detection antibody was used in the assay; 75 pM SβG was used for labeling; and the CSF sample was diluted 4-fold with Generic Homebrew Sample Diluent. This assay has an LLOD range of 10.8-13.6 pg / mL. [Sequence table] SEQUENCE LISTING <110> AL-S Pharma AG Neurimmune AG <120> Misfolded SOD1 Assay <130> AL57A02 / P-WO <150> EP 20 163 909.3 <151> 2020-03-18 <160> 22 <170> PatentIn version 3.5 <210> 1 <211> 123 <212> PRT <213> Homo sapiens <400> 1 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Thr Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ala Tyr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Arg Glu Gln Gly Leu Glu Trp Met 35 40 45 Gly Val Ile Asn Pro Ser Thr Gly Thr Thr Phe Tyr Ala Gln Asn Phe 50 55 60 Pro Asp Arg Val Ser Val Thr Arg Asp Thr Ser Thr Ser Thr Val Phe 65 70 75 80 Met Glu Leu His Asn Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Ile Ser Glu His Gly Ser Gly Ser Tyr Ser Pro Tyr Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 2 <211> 123 <212> PRT <213> Homo sapiens <400> 2 Glu Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Thr Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ala Tyr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Arg Glu Gln Gly Leu Glu Trp Met 35 40 45 Gly Val Ile Asn Pro Ser Thr Gly Thr Thr Phe Tyr Ala Gln Asn Phe 50 55 60 Pro Asp Arg Val Ser Val Thr Arg Asp Thr Ser Thr Ser Thr Val Phe 65 70 75 80 Met Glu Leu His Asn Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Ile Ser Glu His Gly Ser Gly Ser Tyr Ser Pro Tyr Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 3 <211> 5 <212> PRT <213> Homo sapiens <400> 3 Ala Tyr Tyr Ile His 1 5 <210> 4 <211> 17 <212> PRT <213> Homo sapiens <400> 4 Val Ile Asn Pro Ser Thr Gly Thr Thr Phe Tyr Ala Gln Asn Phe Pro 1 5 10 15 Asp <210> 5 <211> 14 <212> PRT <213> Homo sapiens <400> 5 Ala Ile Ser Glu His Gly Ser Gly Ser Tyr Ser Pro Tyr Tyr 1 5 10 <210> 6 <211> 107 <212> PRT <213> Homo sapiens <400> 6 Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Leu Gly Gln 1 5 10 15 Met Ala Ala Ile Thr Cys Ser Gly Glu Ala Leu Pro Lys Lys Tyr Gly 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Val Pro Val Leu Leu Ile Tyr 35 40 45 Arg Asp Val Glu Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser 50 55 60 Ser Ser Gly Thr Met Val Thr Leu Thr Ile Ser Gly Val Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Leu Ser Ala Asp Ser Ser Gly Thr Trp 85 90 95 Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 7 <211> 107 <212> PRT <213> Homo sapiens <400> 7 Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ser Leu Gly Gln 1 5 10 15 Met Ala Ala Ile Thr Cys Ser Gly Glu Ala Leu Pro Lys Lys Tyr Gly 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Val Pro Val Leu Leu Ile Tyr 35 40 45 Arg Asp Val Glu Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser 50 55 60 Ser Ser Gly Thr Met Val Thr Leu Thr Ile Ser Gly Val Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Leu Ser Ala Asp Ser Ser Gly Thr Trp 85 90 95 Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 8 <211> 11 <212> PRT <213> Homo sapiens <400> 8 Ser Gly Glu Ala Leu Pro Lys Lys Tyr Gly Tyr 1 5 10 <210> 9 <211> 7 <212> PRT <213> Homo sapiens <400> 9 Arg Asp Val Glu Arg Pro Ser 1 5 <210> 10 <211> 10 <212> PRT <213> Homo sapiens <400> 10 Leu Ser Ala Asp Ser Ser Gly Thr Trp Val 1 5 10 <210> 11 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Peptide comprising epitope of NI-204.B and NI-204.O <400> 11 Gly Gly Pro Lys Asp Glu Glu Arg His Val Gly Asp 1 5 10 <210> 12 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Peptide comprising the epitope of NI-204.B <400> 12 Gly Gly Pro Lys Asp Glu Glu Arg His Val Gly 1 5 10 <210> 13 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Peptide comprising the epitope of NI-204.O <400> 13 Lys Asp Glu Glu Arg His Val Gly Asp 1 5 <210> 14 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Peptide comprising the epitope of NI-204.G <400> 14 His Glu Lys Ala Asp Asp Leu Gly Lys Gly Gly Asn Glu Glu Ser 1 5 10 15 <210> 15 <211> 121 <212> PRT <213> Homo sapiens <400> 15 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ile Phe Ser Ser Phe 20 25 30 Gly Met His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Leu Ile Trp Tyr Asp Gly Ser Arg Gln Ser Tyr Ala Asp Ser Val 50 55 60 Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Gly Glu Asp Thr Ala Val Tyr His Cys 85 90 95 Ala Arg Thr Gly Tyr Asp Asp Lys Arg Gly Gly Phe Asp Thr Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 16 <211> 5 <212> PRT <213> Homo sapiens <400> 16 Ser Phe Gly Met His 1 5 <210> 17 <211> 17 <212> PRT <213> Homo sapiens <400> 17 Leu Ile Trp Tyr Asp Gly Ser Arg Gln Ser Tyr Ala Asp Ser Val Arg 1 5 10 15 Gly <210> 18 <211> 12 <212> PRT <213> Homo sapiens <400> 18 Thr Gly Tyr Asp Asp Lys Arg Gly Gly Phe Asp Thr 1 5 10 <210> 19 <211> 107 <212> PRT <213> Homo sapiens <400> 19 Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Ser Gly Asp Ala Leu Ala Lys Gln Tyr Ser 20 25 30 Tyr Trp Tyr Gln His Lys Pro Gly Gln Ala Pro Val Met Val Met Tyr 35 40 45 Lys Asp Arg Glu Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Ser Ser Gly Thr Thr Val Thr Leu Thr Ile Ser Ala Val Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Thr Gly Thr Asp Ser Pro Tyr 85 90 95 Ile Phe Gly Thr Gly Thr Lys Val Thr Val Leu 100 105 <210> 20 <211> 11 <212> PRT <213> Homo sapiens <400> 20 Ser Gly Asp Ala Leu Ala Lys Gln Tyr Ser Tyr 1 5 10 <210> 21 <211> 7 <212> PRT <213> Homo sapiens <400> 21 Lys Asp Arg Glu Arg Pro Ser 1 5 <210> 22 <211> 10 <212> PRT <213> Homo sapiens <400> 22 Gln Ser Thr Gly Thr Asp Ser Pro Tyr Ile 1 5 10

Claims

1. 1. A method for assaying misfolded SOD1 in a sample containing a body fluid of a subject, the body fluid being separated from a sample by a first anti-SOD1 antibody that binds to an epitope of SOD1 within the amino acid sequence 73-GGPKDEERHVGD-84 set forth in SEQ ID NO: 11 as a capture antibody, and a human SOD1 ａａ ５０－１５０ with a second anti-SOD1 antibody that binds to an epitope of

2. The method of claim 1 , wherein the bodily fluid is cerebrospinal fluid (CSF).

3. 3. The method of claim 1 or 2, wherein the presence of misfolded SOD1 is indicative of amyotrophic lateral sclerosis (ALS) in the subject.

4. 4. A method for diagnosing ALS in a subject, comprising the steps of the method of any one of claims 1 to 3, wherein the presence or increased level of misfolded SOD1 in the sample compared to a control is indicative of ALS in the subject.

5. 5. The method of claim 3 or 4, wherein the ALS is familial ALS (fALS).

6. 5. The method of claim 3 or 4, wherein the ALS is sporadic ALS (sALS).

7. The method of any one of claims 1 to 6, wherein the first antibody is a monoclonal antibody.

8. The method of any one of claims 1 to 7, wherein the second antibody is a monoclonal antibody.

9. The first antibody comprises in its variable region, i.e., binding domain: (i) variable heavy chain (V Ｈ ) and variable light chain (V Ｌ ) the six CDRs of (a) VH-CDR1 comprises the amino acid sequence of SEQ ID NO: 3 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; (b) VH-CDR2 comprises the amino acid sequence of SEQ ID NO: 4 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; (c) VH-CDR3 comprises the amino acid sequence of SEQ ID NO: 5 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; (d) VL-CDR1 comprises the amino acid sequence of SEQ ID NO: 8 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; (e) VL-CDR2 comprises the amino acid sequence of SEQ ID NO: 9 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; and (f) VL-CDR3 comprises the amino acid sequence of SEQ ID NO: 10 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; and / or (ii) a VH chain and a VL chain, (a) the VH chain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 1 or 2; and (b) the VL chain comprises a VH chain and a VL chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 6 or 7; The method according to any one of claims 1 to 8, characterized in that it comprises

10. The second antibody binds to an epitope of SOD1 within the amino acid sequence 121-HEKADDLGKGGNEES-135 shown in SEQ ID NO: 14, and in its variable region, i.e., binding domain, (i) V Ｈ and V Ｌ The six CDRs of the chain (a) VH-CDR1 comprises the amino acid sequence of SEQ ID NO: 16 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; (b) VH-CDR2 comprises the amino acid sequence of SEQ ID NO: 17 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; (c) VH-CDR3 comprises the amino acid sequence of SEQ ID NO: 18 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; (d) VL-CDR1 comprises the amino acid sequence of SEQ ID NO: 20 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; (e) VL-CDR2 comprises the amino acid sequence of SEQ ID NO: 21 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; and (f) VL-CDR3 has six CDRs comprising the amino acid sequence of SEQ ID NO: 22 or a variant thereof, wherein the variant comprises one or two amino acid substitutions; and / or (ii) a VH chain and a VL chain, (a) the VH chain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 15; and (b) the VL chain comprises a VH chain and a VL chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 19; 10. The method according to claim 1, wherein the method further comprises:

11. The method of any one of claims 1 to 10, wherein the second anti-SOD1 antibody is conjugated to a detectable label.

12. 12. The method of claim 11, wherein the detectable label is selected from the group consisting of an enzyme, a radioisotope, a fluorescent compound, a chemiluminescent compound, a bioluminescent compound, a tag, a flag, a ligand, and a heavy metal.

13. The second anti-SOD1 antibody is conjugated to a ligand, and the method includes a labeling step with a ligand-binding tag, the method comprising the steps of: (i) providing a microplate having wells spotted with the first anti-SOD1 antibody; and (ii) adding the sample containing the body fluid to the well, followed by incubation, thereby allowing the first anti-SOD1 antibody to capture misfolded SOD1 present in the body fluid, preferably incubation is carried out for 2 hours at room temperature on a plate shaker set at 600 rpm; and (iii) adding the second anti-SOD1 antibody conjugated to a ligand and incubating, thereby allowing the second anti-SOD1 antibody to bind to the captured misfolded SOD1, preferably, incubation is performed for 30 minutes at room temperature on a plate shaker set at 600 rpm; and (iv) adding a conjugate comprising a ligand-binding tag and a detectable label and incubating, preferably for 30 minutes at room temperature on a plate shaker set at 600 rpm; and (v) adding a colorimetric or chemiluminescent substrate solution; and (vi) imaging the signal; optionally, (vii) comparing the assayed level of misfolded SOD1 with a reference standard and / or a control, preferably said reference standard and / or said control being added to the same microplate as said sample.

13. The method of claim 11 or 12, comprising: optionally, the method is a singleplex immunoassay.

14. (i) the microplate is a 96-well plate; (ii) the body fluid is cerebrospinal fluid (CSF); (iii) the ligand is biotin or a biotin analogue or derivative thereof; (iv) the ligand binding tag is streptavidin or a functional analogue or derivative thereof and the detectable label is an enzyme capable of catalysing the conversion of a chromogenic or chemiluminescent substrate, preferably the enzyme is horseradish peroxidase; (v) the substrate is 3,3',5,5'-tetramethylbenzidine (TMB) or luminol substrate; (vi) imaging was performed with a Cirascan™ imaging system; (vii) a washing step is carried out after at least steps (ii), (iii) and / or (iv); and / or (viii) The method of claim 13, wherein the microplate is covered with a lid, preferably a lid having a fluid-absorbing matrix that is filled with fluid during the incubation step.

15. The method of any one of claims 1 to 14, having a lower limit of quantitation (LLOQ) of misfolded SOD1 of ≦20.32 pg / mL.

16. Using single molecule arrays (Simoa™), Preferably, (i) attaching the first anti-SOD1 antibody to the surface of a capture bead; and (ii) (a) adding the sample containing the body fluid and incubating the beads with the sample, thereby allowing the beads to capture misfolded SOD1 present in the body fluid mediated by the first anti-SOD1 antibody; and (ii)(b) adding and incubating the second anti-SOD1 antibody conjugated to a ligand, thereby allowing the second anti-SOD1 antibody to bind to the captured misfolded SOD1 on the beads; and (ii)(c) adding and incubating a conjugate comprising a ligand-binding tag and a detectable label; or (II) (A) adding the sample containing the body fluid, adding the second anti-SOD1 antibody conjugated to a ligand, and incubating the beads with the sample and the second antibody, thereby allowing the capture of misfolded SOD1 present in the body fluid by the beads mediated by the first anti-SOD1 antibody and the binding of the second anti-SOD1 antibody to the captured misfolded SOD1 on the beads; and (II)(B) adding and incubating a conjugate comprising the ligand-binding tag and a detectable label; and (iii) resuspending the beads in a chromogenic / fluorescent substrate solution; and (iv) loading the beads of step (iii) into an array of femtoliter-sized wells configured to hold no more than one bead per well; and (v) sealing the individual beads within the femtoliter-sized wells; and (vi) imaging the signal; optionally, (vii) comparing the assayed level of misfolded SOD1 to a reference standard and / or control The method according to any one of claims 1 to 12, comprising:

17. (i) the beads are paramagnetic beads and / or have a diameter of about 2.7 μM; (ii) (a) the body fluid is CSF and / or the incubation time is 30 minutes; (ii)(b) the ligand is biotin or a biotin analogue or derivative thereof, and / or the incubation time is 5 minutes; (ii)(c) the ligand binding tag is streptavidin or a functional analogue or derivative thereof, the detectable label is an enzyme capable of converting a chromogenic / fluorogenic substrate, preferably the enzyme is β-galactosidase, and / or the incubation time is 5 minutes; (II) (A) the body fluid is CSF, the ligand is biotin or a biotin analog or derivative thereof, and / or the incubation time is 35 minutes; (ii) (B) the ligand-binding tag is streptavidin or a functional analogue or derivative thereof, the detectable label is an enzyme capable of converting a chromogenic / fluorogenic substrate, preferably the enzyme is β-galactosidase, and / or the incubation time is 5 minutes; (iii) the fluorescent substrate is resorufin β-D-galactopyranoside (RGP); (v) the sealing is performed with oil; 17. The method of claim 16, wherein (vi) the imaging is performed by a Simoa™ optical system.

18. The method for assaying misfolded SOD1 according to any one of claims 1 to 17, wherein the anti-SOD1 antibody binding to an epitope of SOD1 within the amino acid sequence 73-GGPKDEERHVGD-84 shown in SEQ ID NO: 11 is used as a capture antibody, and / or human SOD1 is used as a detection antibody. ａａ ５０－１５０ The use of an anti-SOD1 antibody that binds to an epitope of

19. 19. Use according to claim 18, wherein the capture antibody is characterized as defined in claim 9 and / or the detection antibody is characterized as defined in claim 10.

20. 18. A therapeutic agent for use in treating or ameliorating symptoms in a patient diagnosed with or at risk of developing ALS according to the method of any one of claims 1 to 17, wherein preferably said patient has been diagnosed with or at risk of developing sALS, and preferably said patient has been assayed to have a detectable amount of misfolded SOD1 and / or increased levels of misfolded SOD1 when compared to a control.

21. A kit adapted to carry out the method of any one of claims 1 to 17 for assaying misfolded SOD1 in a sample comprising a body fluid of a subject, the kit comprising at least: (i) a first monoclonal anti-SOD1 antibody that binds to an epitope of SOD1 within the amino acid sequence 73-GGPKDEERHVGD-84 set forth in SEQ ID NO: 11; and (ii) Human SOD1 as a detection antibody ａａ ５０－１５０ a detection reagent comprising a second monoclonal anti-SOD1 antibody that binds to an epitope of SOD1, wherein said second anti-SOD antibody is conjugated to a detectable label, preferably said detectable label is selected from the group consisting of an enzyme, a radioisotope, a fluorescent compound, a chemiluminescent compound, a bioluminescent compound, a tag, a flag, a ligand and a heavy metal; and optionally (iii) a conjugate comprising a detectable label-binding tag and a detectable label, preferably wherein the detectable label-binding tag is a ligand-binding tag and the detectable label is an enzyme capable of catalyzing the conversion of a chromogenic, chemiluminescent or fluorescent substrate; (iv) a chromogenic, chemiluminescent, or fluorescent substrate solution; (v) a calibrated immunoassay standard or control for misfolded SOD1; (vi) recommendations for microplates, buffers, diluents, substrates and / or solutions and instructions on how to carry out the assay of any one of claims 1 to 17; and / or (vii) Washing and Assay / Sample Dilution Buffers Kit including:

22. (i) the first antibody is pre-spotted into wells of a microplate, preferably a 96-well microplate including a lid, preferably wherein the first antibody is characterized as described in claim 9; (ii) the detectable label is a ligand, preferably biotin or a biotin analogue or derivative thereof; (iii) the ligand binding tag is streptavidin or a functional analogue or derivative thereof, and the detectable label is an enzyme capable of catalyzing the conversion of a chromogenic or chemiluminescent substrate, preferably, the detectable label is horseradish peroxidase; (v) the substrate solution is a chromogenic or chemiluminescent substrate solution, preferably the substrate is TMB or luminol substrate; and / or (vi) The kit of claim 21, wherein the standard comprises serial dilutions of misfolded SOD1 from 200 ng / mL to 3 pg / mL.

23. (i) a microplate, preferably a 96-well microplate including a lid, in whose wells the first monoclonal anti-SOD1 antibody characterized as claimed in claim 9 is pre-spotted as a capture antibody; (ii) a second biotinylated anti-SOD1 antibody as a detection antibody; (iii) streptavidin-HRP reagent; (v) a substrate solution containing TMB or luminol; (vi) a calibrated immunoassay standard or control for misfolded SOD1; and (vii) Washing and Assay / Sample Dilution Buffers 23. The kit of claim 21 or 22, comprising:

24. (i) the first monoclonal anti-SOD1 antibody is comprised in a capture reagent, preferably the first antibody is characterized as described in claim 9; (ii) the detectable label is a ligand, preferably biotin or a biotin analogue or derivative thereof; (iii) the ligand binding tag is streptavidin or a functional analogue or derivative thereof, and the detectable label is an enzyme capable of converting a chromogenic / fluorogenic substrate, preferably, the detectable label is β-galactosidase; (iv) the substrate solution is a chromogenic or fluorogenic substrate solution, preferably the substrate is resorufin β-D-galactopyranoside (RGP); and / or (v) the standard comprises serial dilutions of misfolded SOD1 from about 1000 ng / mL to 0.244 ng / mL by 4-fold serial dilution to an 8-point calibration curve, from 10 ng / mL to 0.020 ng / mL by 2-fold serial dilution to an 8-point calibration curve, from 50 ng / mL to 0.012 ng / mL by 2-fold serial dilution to a 12-point calibration curve, and / or from about 66.66667 ng / mL to 0.00339 ng / mL by 3-fold serial dilution to a 12-point calibration curve; and optionally 22. The kit of claim 21, further comprising capture beads, preferably paramagnetic beads with a diameter of 2.7 μM.

25. (i) a capture reagent comprising a first monoclonal anti-SOD1 antibody characterized as claimed in claim 9 as a capture antibody; (ii) a second biotinylated anti-SOD1 antibody as a detection antibody; (iii) streptavidin-β-galactosidase (SβG) reagent; (iv) a substrate solution containing RGP; (v) a calibrated immunoassay standard or control for misfolded SOD1; (vi) washing and assay / sample dilution buffers; and (vii) paramagnetic capture beads having a diameter of about 2.7 μM 25. The kit of claim 21 or 24, comprising:

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