Antibody biomarker to diagnose idiopathic inflammatory myopathies

Abstract

The present disclosure provides methods of detecting and treating idiopathic inflammatory myopathies (IIM). It was surprisingly discovered that autoantibodies against dysferlin are common in subjects having IIM patients and that the levels of dysferlin antibodies are linked with the severity and the progression of the disease. In some embodiments, it includes a method of treating idiopathic IIM in a subject, comprising, obtaining a sample from the subject, measuring the level of one or more dysferlin autoantibodies in the subject, and administering a therapeutically effective dose of an agent that inhibits one or more dysferlin autoantibodies, when an increase of IIM was measured in a subject.

Description

Docket No. 103362-068WO1 ANTIBODY BIOMARKER TO DIAGNOSE IDIOPATHIC INFLAMMATORY MYOPATHIESGOVERNMENT SUPPORT CLAUSEThis invention was made with government support under Grant No. R01AR084519 awarded by the National Institutes of Health. The Government has certain rights in the invention.CROSS REFERENCE TO RELATED APPLICATIONSThis application claims the benefit of U.S. Provisional Patent Application 63 / 719,884, filed November 13, 2024, which is hereby incorporated herein by reference in its entirety.FIELD OF THE INVENTIONThe present disclosure relates to methods of detecting and treating idiopathic inflammatory myopathies (IIM).BACKGROUND OF THE INVENTIONIdiopathic inflammatory myopathies (IIM) are a group of disorders in which autoimmune responses produce a chronic state of inflammation resulting in degeneration of skeletal muscle structure and function. These IIM include five major conditions: polymyositis (PM), dermatomyositis (DM), anti synthetase syndrome (ASyS), juvenile dermatomyositis (JDM), inclusion body myositis (IBM), and immune-mediated necrotizing myopathy (IMNM). While the pathogenesis of myositis remains largely unknown, T-cell mediated autoimmune processes and antibody-mediated mechanisms are known to play a significant role, possibly driven by genetic and / or environmental factors. Efforts to better understand the pathogenesis of IIM is complicated by the highly heterogeneous nature of these diseases. Treatment options involve systemic immunosuppression that provide a degree of efficacy for the acute management; however, immune suppression often leads to significant complications.What is urgently needed are novel and effective methods of detecting and treating IIM to address the unmet needs associated with these diseases.Docket No. 103362-068WO1 SUMMARY OF THE INVENTIONDisclosed herein are methods of detecting and treating idiopathic inflammatory myopathies (IIM).Accordingly, in one aspect, disclosed herein is a method of treating idiopathic inflammatory myopathies (IIM) in a subject, comprising, obtaining a sample from the subject, measuring the level of one or more dysferlin autoantibodies in the subject, and administering a therapeutically effective dose of an agent that inhibits one or more dysferlin autoantibodies, when an increase of IIM was measured in a subject.In some embodiments, an increase in the level of one or more dysferlin autoantibodies compared to a control indicates the presence of IIM or risk thereof in the subject. In some embodiments, the control comprises a standardized level of one or more dysferlin autoantibodies from subjects without IIM. In some embodiments, the control comprises a sample from the subject at an earlier time period.In some embodiments, the agent that inhibits one or more dysferlin autoantibodies comprises a small molecule, a peptide, a protein, an antibody, an antibody fragment, or a combination thereof.In some embodiments, the agent that inhibits one or more dysferlin autoantibodies is formulated in a delivery vehicle.In some embodiments, the delivery vehicle comprises a liposome, a lipid nanoparticle, an exosome, an extracellular vesicle, a viral particle, or a combination thereof.In some embodiments, the sample comprises blood or any blood component. In some embodiments, the blood comprises whole blood. In some embodiments, the blood component comprises serum or plasma.In some embodiments, the method produces at least a 50% decrease in disease severity of a subject treated with an agent that inhibits one or more dysferlin autoantibodies compared to an untreated subject.In some embodiments, the method further comprises measuring the level of dysferlin in the sample, wherein a decrease in the level of dysferlin compared to a control indicates the presence of IIM or risk thereof in the subject.In some embodiments, the agent that inhibits one or more IIM-causing anti-dysferlin autoantibodies, when administered to a subject with IIM, increases membrane repair even in the presence of a dysferlin antibody, compared to a control not administered with the agent. In some embodiments, the agent that inhibits one or more IIM-causing anti-dysferlinDocket No. 103362-068WO1autoantibodies, when administered to a subject with IIM, decreases the severity of myositis when compared to a control not administered with the agent.In some embodiments, the agent that inhibits one or more anti-dysferlin autoantibodies is formulated in an oil-in-water emulsion, a nano-emulsion, a nanoparticle, an excipient, a diluent, a salt, a buffer, a stabilizer, a lipid, or combinations thereof.In one aspect, disclosed herein is a method of diagnosing IIM in a subject, comprising, obtaining a sample from the subject and measuring level of one or more dysferlin autoantibodies. In some embodiments, an increase in the level of one or more dysferlin autoantibodies compared to a control indicates the presence of IIM or risk thereof in the subject.In some embodiments, at least one additional biomarker is also measured in the subject. In some embodiments, the method further comprises measuring the level of dysferlin in the sample, wherein a decrease in the level of dysferlin compared to a control indicates the presence of IIM or risk thereof in the subject.In some embodiments, a threshold level is established, and wherein, when the level of one or more dysferlin autoantibodies increases above that level, it is indicative of the presence of IIM or the progression of IIM in a subject who has already been diagnosed with IIM.In some embodiments, the IIM is selected from polymyositis (PM), dermatomyositis (DM), anti synthetase syndrome (ASyS), juvenile dermatomyositis (JDM), inclusion body myositis (IBM), and immune-mediated necrotizing myopathy (IMNM).In some embodiments, the subject is a human. In some embodiments, the human subject is a child or an adult.A summary of embodiments of the invention is described in further detail below.BRIEF DESCRIPTION OF FIGURESThe accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects described below.FIG. 1 shows that Idiopathic immune myopathies (IIM) consist of chronic inflammation leading to myofiber death within unknown cause. Reproduced from Myositis, in Photos, Myositis Support and Understanding Association (MSU).FIGs. 2A-2B show that Foxp3- / Y Syt7- / ~ adoptive transfer mouse model revealed a defect in membrane integrity as shown in FIG. 2 A and repair as shown in FIG. 2B. Reproduced from McElhanon KE et al. JCI. 2020 Aug 3; 130(8):4440-4455.Docket No. 103362-068WO1FIGs. 3A-3C show that normal amounts of physiological eccentric contraction generates disruptions in the muscle sarcolemma.FIG. 3A shows muscle contraction.FIG. 3B shows a high-resolution microscopy image of muscle contraction. Reproduced from R.L. Lieber, T.M. Woodburn, and J. Friden, “Muscle Damage Induced by Eccentric Contractions of 25% Strain, ’’Journal of Applied Physiology 70, no. 6 (1991): 2498-2507 and Schoenfeld, B. Science and Development of Muscle Hypertrophy. Second Edition. Champaign, IL: Human Kinetics

[2021] . ISBN: 978-1-4925-9767-4 (print). ISBN 9781492597704 (pdf).FIG. 3C shows that repair of the membrane is essential for preventing the loss of intracellular components into the extracellular milieu.FIG. 4 shows that plasma membrane repair is a mechanism involved in maintaining membrane integrity and viability.FIGs. 5A-5G show that TRIM72 autoantibodies are elevated in IIM patient sera and compromises sarcolemmal repair. Images of sarcolemma when treated with basal-media (basal) or IIM patient derived serum-treated (serum) in healthy (FIG. 5A), PM (FIG. 5C), and DM (FIG. 5E) patients are shown. The AF / F0in healthy (FIG. 5B), PM (FIG. 5D), and DM (FIG. 5F) patients are shown. Reproduced from McElhanon KE et al. JCI. 2020 Aug 3; 130(8) : 4440-4455.FIG. 5G shows the levels of TRIM72. Reproduced from McElhanon KE et al. JCI. 2020 Aug 3; 130(8):4440-4455.FIG. 6 shows that autoantibodies targeting plasma membrane repair proteins may contribute to a feedback loop IIM. Reproduced from McElhanon KE et al. JCI. 2020 Aug 3; 130(8) : 4440-4455.FIG. 7 shows that low levels of TRIM72 autoantibodies are sufficient to induce a membrane repair defect in human cells. Reproduced from McElhanon KE et al. JCI. 2020 Aug 3; 130(8) : 4440-4455.FIGs. 8A-8B shows that TRIM72 and dysferlin autoantibodies are detected by ELISA in the serum from myositis patients.FIG. 8A shows healthy (n=15), dermatomyositis (DM, n=52), and polymyositis (PM, n=51) subject sera tested for levels of TRIM72 and dysferlin autoantibodies by ELISA. 34.6% and 21.6% respectively of DM and PM subject serum screened had O.D. values greater than 1 SD compared to healthy subject serum. For dysferlin, there was a significant increase (** p>0.01 by T-test) in the average level in pooled myositis patients.Docket No. 103362-068WO1FIG. 8B shows that Pearson’s analysis suggests a moderate correlation (coefficient of 0.341) between dysferlin antibody levels and increasing levels of serum creatine kinase (CK) in pooled DM and PM patient samples (n=30).FIG. 9 shows increased autoantibodies for dysferlin specifically in myositis patients. Custom ELIS As were used to detect the level of antibodies that bind dysferlin in serum samples from various human patient populations. Levels of anti-dysferlin autoantibodies in patients with systemic lupus erythematosus (SLE) or SLE with myocarditis complications (Myocarditis) are similar to that seen in healthy volunteers (Healthy). There is a significantly elevated level of anti-dysferlin autoantibodies in the myositis patients compared to all of these other groups, indicating that the elevated level of dysferlin autoantibodies are specific to myositis rather than an effect seen in autoimmune diseases in general. ANOVA testing with *** p<0.005, **** p<0.001.FIGs. 10A-10B shows that anti-dysferlin antibody is sufficient to compromise membrane repair.FIG. 10A shows flexor digitorum brevis (FDB) muscle bundles from C57B1 mice subjected to laser injury in the presence of pre-immune rabbit serum or anti-dysferlin polyclonal antibodies. Isolated FDB muscles were damaged with a 5 second laser pulse and membrane resealing capacity was assessed. Application of rabbit polyclonal antibody against dysferlin results in increased dye influx into wild type muscle fibers, n = 10-13, indicating compromised membrane repair compared is induced by dysferlin antibodies. P <0.0001 by ANOVA.FIG. 10B shows the area under the curve (AUC) analysis from the FM4-64 dye fluorescence over time traces, n = 10-13. p value <0.0001 by T-test.FIGs. 11A-11C shows that injection of antibodies against membrane repair proteins increases myositis pathology.FIG. 11A shows that the quadriceps muscles of adoptive transfer myositis mice were injected weekly with mouse IgG, TRIM72 antibody, or dysferlin (DYSF) antibody. After 8 weeks representative histopathology images show increased inflammatory inclusions in dysferlin antibody injected animals relative to IgG injected or wild type animals. Bar is 100 um.FIG. 1 IB shows quantification of areas of inflammation show increased inflammation with antibody supplementation. *** P<0.005.Docket No. 103362-068WO1FIG. 11C shows elevated serum creatine kinase (CK) in adoptive transfer myositis mice after dysferlin antibody injection. This shows increased muscle damage is produced by elevated dysferlin antibodies. * P<0.05.DETAILED DESCRIPTIONThe following description of the disclosure is provided as an enabling teaching of the disclosure in its best, currently known embodiment(s). To this end, those skilled in the relevant art will recognize and appreciate that many changes can be made to the various embodiments of the invention described herein, while still obtaining the beneficial results of the present disclosure. It will also be apparent that some of the desired benefits of the present disclosure can be obtained by selecting some of the features of the present disclosure without utilizing other features. Accordingly, those who work in the art will recognize that many modifications and adaptations to the present disclosure are possible and can even be desirable in certain circumstances and are a part of the present disclosure. Thus, the following description is provided as illustrative of the principles of the present disclosure and not in limitation thereof.Reference will now be made in detail to the embodiments of the invention, examples of which are illustrated in the drawings and the examples. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.TerminologyUnless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of’ and “consisting of’ can be used in place of “comprising” and “including” to provide for more specific embodiments and are also disclosed. As used in this disclosure and in the appended claims, the singular forms “a”, “an”, “the”, include plural referents unless the context clearly dictates otherwise.The following definitions are provided for the full understanding of terms used in this specification.Docket No. 103362-068WO1The terms "about" and "approximately" are defined as being “close to” as understood by one of ordinary skill in the art. In one non-limiting embodiment the terms are defined to be within 10%. In another non-limiting embodiment, the terms are defined to be within 5%. In still another non-limiting embodiment, the terms are defined to be within 1%.Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “10” is disclosed the “less than or equal to 10”as well as “greater than or equal to 10” is also disclosed. It is also understood that throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.As used herein, the term “antibody” encompasses, but is not limited to, whole immunoglobulin (i.e., an intact antibody) of any class. Native antibodies are usually heterotetrameric glycoproteins, composed of two identical light (L) chains and two identical heavy (H) chains. Typically, each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (V(H)) followed by a number of constant domains. Each light chain has a variable domain at one end (V(L)) and a constant domain at its other end; the constant domain of the light chain is alignedDocket No. 103362-068WO1with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains. The light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (k) and lambda (1), based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of human immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. One skilled in the art would recognize the comparable classes for mouse. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.As used herein, the terms "may," "optionally," and "may optionally" are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur. Thus, for example, the statement that a formulation "may include an excipient" is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient.An "increase" can refer to any change that results in a greater amount of a symptom, disease, composition, condition, or activity. An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount. Thus, the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increase so long as the increase is statistically significant.A "decrease" can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. A substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance. Also, for example, a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed. A decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount. Thus, the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long as the decrease is statistically significant.Docket No. 103362-068WO1"Inhibit," "inhibiting," and "inhibition" mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.By “reduce” or other forms of the word, such as “reducing” or “reduction,” means lowering of an event or characteristic (e.g., tumor growth). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. For example, “reduces tumor growth” means reducing the rate of growth of a tumor relative to a standard or a control.By “prevent” or other forms of the word, such as “preventing” or “prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed.The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. In one aspect, the subject can be human, non-human primate, bovine, equine, porcine, canine, or feline. The subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.The term “therapeutically effective” refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is,Docket No. 103362-068WO1treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder."Comprising" is intended to mean that the compositions, methods, etc. include the recited elements, but do not exclude others. "Consisting essentially of' when used to define compositions and methods, shall mean including the recited elements, but excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. "Consisting of' shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions provided and / or claimed in this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure.The term “amino acid,” includes but is not limited to amino acids contained in the group consisting of alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (He or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gin or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Vai or V), tryptophan (Trp or W), and tyrosine (Tyr or Y) residues. The term “amino acid residue” also may include amino acid residues contained in the group consisting of homocysteine, 2- Aminoadipic acid, N-Ethylasparagine, 3 -Aminoadipic acid, Hydroxylysine, P-alanine, P-Amino-propionic acid, allo-Hydroxylysine acid, 2-Aminobutyric acid, 3-Hydroxyproline, 4-Aminobutyric acid, 4-Hydroxyproline, piperidinic acid, 6-Aminocaproic acid, Isodesmosine, 2-Aminoheptanoic acid, allo-Isoleucine, 2-Aminoisobutyric acid, N-Methylglycine, sarcosine, 3 -Aminoisobutyric acid, N-Methylisoleucine, 2-Aminopimelic acid, 6-N-Methyllysine, 2,4-Diaminobutyric acid, N-Methylvaline, Desmosine, Norvaline, 2,2'-Diaminopimelic acid, Norleucine, 2,3-Diaminopropionic acid, Ornithine, and N-Ethylglycine. Typically, the amide linkages of theDocket No. 103362-068WO1peptides are formed from an amino group of the backbone of one amino acid and a carboxyl group of the backbone of another amino acid.Reference also is made herein to peptides, polypeptides, proteins, and compositions comprising peptides, polypeptides, and proteins. As used herein, a polypeptide and / or protein is defined as a polymer of amino acids, typically of length>100 amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks / Cole, 110). A peptide is defined as a short polymer of amino acids, of a length typically of 20 or less amino acids, and more typically of a length of 12 or less amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks / Cole, 110).The peptides, polypeptides, and proteins disclosed herein may be modified to include non-amino acid moieties. Modifications may include but are not limited to carboxylation (e.g., N-terminal carboxylation via addition of a di-carboxylic acid having 4-7 straight-chain or branched carbon atoms, such as glutaric acid, succinic acid, adipic acid, and 4,4-dimethylglutaric acid), amidation (e.g., C-terminal amidation via addition of an amide or substituted amide such as alkylamide or dialkylamide), PEGylation (e.g., N-terminal or C-terminal PEGylation via additional of polyethylene glycol), acylation (e.g., O-acylation (esters), N-acylation (amides), S-acylation (thioesters)), acetylation (e.g., the addition of an acetyl group, either at the N-terminus of the protein or at lysine residues), formylation lipoylation (e.g., attachment of a lipoate, a C8 functional group), myristoylation (e.g., attachment of myristate, a C14 saturated acid), palmitoylation (e.g., attachment of palmitate, a C16 saturated acid), alkylation (e.g., the addition of an alkyl group, such as an methyl at a lysine or arginine residue), isoprenylation or prenylation (e.g., the addition of an isoprenoid group such as farnesol or geranylgeraniol), amidation at C-terminus, glycosylation (e.g., the addition of a glycosyl group to either asparagine, hydroxylysine, serine, or threonine, resulting in a glycoprotein). Distinct from glycation, which is regarded as a nonenzymatic attachment of sugars, polysialylation (e.g., the addition of polysialic acid), glypiation (e.g., glycosylphosphatidylinositol (GPI) anchor formation, hydroxylation, iodination (e.g., of thyroid hormones), and phosphorylation (e.g., the addition of a phosphate group, usually to serine, tyrosine, threonine, or histidine).The word “vector” refers to any vehicle that carries a polynucleotide into a cell for the expression of the polynucleotide in the cell. The vector may be, for example, a plasmid, a virus, a phage particle, or a nanoparticle. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may in some instances, integrateDocket No. 103362-068WO1into the genome itself. In some embodiments, the vector is a DNA construct containing a DNA sequence which is operably linked to a suitable control sequence capable of effecting the expression of the DNA in a suitable host cell. Such control sequences can include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control the termination of transcription and translation. In other embodiments, the vector is a lipid nanoparticle. Lipid nanoparticles can be used to deliver mRNA to a host cell for expression of the mRNA in the host cell. In some embodiments, the expression vector comprises a plasmid or a virus or viral vector. A plasmid or a viral vector can be capable of extrachromosomal replication or, optionally, can integrate into the host genome. As used herein, the term "integrated" used in reference to an expression vector (e.g., a plasmid or viral vector) means the expression vector, or a portion thereof, is incorporated (physically inserted or ligated) into the chromosomal DNA of a host cell. As used herein, a “viral vector” refers to a virus-like particle containing genetic material which can be introduced into a eukaryotic cell without causing substantial pathogenic effects to the eukaryotic cell. A wide range of viruses or viral vectors can be used for transduction but should be compatible with the cell type the virus or viral vector are transduced into (e.g., low toxicity, capability to enter cells). Suitable viruses and viral vectors include adenovirus, lentivirus, retrovirus, among others. In some embodiments, the expression vector encoding a chimeric polypeptide is a naked DNA or is comprised in a nanoparticle (e.g., liposomal vesicle, porous silicon nanoparticle, gold-DNA conjugate particle, polyethyleneimine polymer particle, cationic peptides, etc.).The term “administer,” “administering”, or derivatives thereof refer to delivering a composition, substance, inhibitor, or medication to a subject or object by one or more the following routes: oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation or via an implanted reservoir. The term “parenteral” includes subcutaneous, intravenous, intramuscular, intraarticular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injections or infusion techniques.A “vaccine” refers to a biological preparation that provides active acquired immunity to a particular infectious diseases caused by a virus, bacteria, parasite, or any other microorganisms. Vaccines typically comprise an agent or several agents, also referred to as antigens, resembling the disease-causing microorganism and is often made from weakened orDocket No. 103362-068WO1killed forms of the microbe, its toxins, or its surface proteins / peptides. Vaccines are also made to comprise additional components, such as adjuvants, preservatives, and / or stabilizers to boost the immune response, improve safety, and improve vaccine storage.The term “kit” describes a wide variety of bags, containers, carrying cases, and other portable enclosures which may be used to carry and store solid substances, liquid substances, and other accessories necessary to administer the vaccine composition to a subject or express the nucleic acid sequence encoding the non-toxic domains disclosed herein.A “pharmaceutically effective amount” of a drug necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation."Pharmaceutically acceptable carrier" (sometimes referred to as a “carrier”) means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic, and includes a carrier that is acceptable for veterinary and / or human pharmaceutical or therapeutic use. The terms "carrier" or "pharmaceutically acceptable carrier" can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil / water or water / oil emulsion) and / or various types of wetting agents.As used herein, the term “carrier” encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations. The choice of a carrier for use in a composition will depend upon the intended route of administration for the composition. The preparation of pharmaceutically acceptable carriers and formulations containing these materials is described in, e.g., Remington's Pharmaceutical Sciences, 21st Edition, ed. University of the Sciences in Philadelphia, Lippincott, Williams & Wilkins, Philadelphia, PA, 2005. Examples of physiologically acceptable carriers include saline, glycerol, DMSO, buffers such as phosphate buffers, citrate buffer, and buffers with other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN™ (ICI, Inc.; Bridgewater, NewDocket No. 103362-068WO1Jersey), polyethylene glycol (PEG), and PLURONICS™ (BASF; Florham Park, NJ). To provide for the administration of such dosages for the desired therapeutic treatment, compositions disclosed herein can advantageously comprise between about 0.1% and 99% by weight of the total of one or more of the subject compounds based on the weight of the total composition including carrier or diluent.An “adjuvant” refers to a drug, molecule, substance, or a combination thereof that is used to increase the efficacy or potency of certain therapeutic agents, such as for example vaccines and / or antibodies. “Adjuvant(s)” are often at least one ingredient used in some vaccines that help create a stronger immune response in the host receiving said vaccine.MethodsDisclosed herein is a method of treating idiopathic inflammatory myopathies (UM) in a subject.Idiopathic inflammatory myopathies (UM) are a group of disorders in which autoimmune responses produce a chronic state of inflammation resulting in degeneration of skeletal muscle structure and function. These IIM include five major conditions: polymyositis (PM), dermatomyositis (DM), anti synthetase syndrome (ASyS), juvenile dermatomyositis (JDM), inclusion body myositis (IBM), and immune-mediated necrotizing myopathy (IMNM). It should be noted that the definition of these diseases can vary between different clinical sources, but all of these, as well as some other diseases, all fall into the general group of IIM. Symptoms of IIM include muscle weakness that develops gradually, joint pain, fatigue, swelling, and muscle damage, and skin lesions that may include a heliotrope rash around the eyes, a "V" sign rash on the chest, and Gottron papules on the hands. IIM can occur at any age, but usually appears in adults between ages 40 and 60 or in children between ages 5 and 15.In some embodiments, the IIM is selected from polymyositis (PM), dermatomyositis (DM), juvenile dermatomyositis, inclusion body myositis (IBM), immune-mediated necrotizing myopathy (IMNM), or other classifications of IIM.Polymyositis (PM)In some embodiments, the IIM comprises PM. PM occurs in adults greater than 20 years old and is more common in women than in men (Dimachkie etal, Semin. Neurol. 32:227-236 (2012)). Symptoms such as myalgias and tenderness are more common. Dysphagia occurs in one-third of patients, with facial weakness occasionally presenting. Rash or otherDocket No. 103362-068WO1inflammation disorders of the skin do not occur in PM. PM is diagnosed by exclusion of all other types of IIM. For example, serum CK levels can be elevated up to 50-fold the upper limit of normal in the subacute active phase (Mandel et al, Int. J. Mol. Sci. 18: 1084-1094 (2017)). Histopathologic hallmarks of PM include hallmarks of endomysial cytotoxic CD8+ T-cells and widespread upregulation of MHC class I in muscle fibers (Hoogendijk et al, Neuromuscular Dis. 14:337-345 (2004)).Dermatomyositis (DM)In some embodiments, the IIM comprises DM. DM presents with proximal symmetric muscle weakness and associated characteristic rashes of the skin such as Gottron papules on the dorsal sides of the hands and fingers, a periorbital oedema, and erythema of the face (heliotrope rash), the anterior upper chest (V-sign) or the posterior neck (shawl sign). Muscle inflammation of DM causes proximal weakness which can develop acutely (within several days) or subacutely (within several weeks up to a few months). Subjects having DM suffer from impairment of walking and stair climbing, as well as lifting their arms and heavy objects (Schmidt, J. Neuromuscular Dis. 5:109-129 (2018)). Histopathologic hallmarks of DM include perimysial inflammation, perifascicular atrophy, and perifascicular elevation of MHC class I, binding of complement to capillaries and the surface of the sarcolemma and reduction of capillaries.Certain variants of classical DM include clinically amyopathic DM (CADM) (Sun Y et al, Rheumatol. Int. 33:1295- 1302 (2013)), adermatopathic DM ("dermatomyositis sine dermatitis") (laccarino et al, J. Autoimmun. 48-49:122-127 (2014)), or juvenile dermatomyositis (JDM). In some embodiments, the DM is selected from CADM, adermatopathic DM, or JDM.Antisynthetase syndrome (ASyS)In some embodiments, the IIM comprises ASyS. Patients having ASyS have autoantibodies recognizing one of the aminoacyl -tRNA synthetases including Joi. The disease is more common in women and is characterized by muscle inflammation, interstitial lung disease, inflammatory arthritis, raynaud’s phenomenon, mechanic’s hands and fever (Musai J et al. Recent Updates on the Pathogenesis of Inflammatory Myopathies. Curr Rheumatol Rep.2024 Dec;26(12):421-430; Epub 2024 Sep 24).Docket No. 103362-068WO1Juvenile Dermatomyositis (JDM)In some embodiments, the IIM comprises JDM. JDM is associated with multisystemic vasculitis, a high frequency of calcinosis, Gottron papules, symmetrical proximal muscle weakness, and heliotrope rash. The mean age of onset of JDM is approximately 7 years of age. The hallmark pathological feature of perifascicular atrophy is more common in JDM than in DM (Dalakas, New Engl. J. Med. 372:1734-1747 (2015)). Abnormal elevation of serum activities of certain muscle enzymes (e.g., creatine kinase (CK)) are also common in JDM.Inclusion Body Myositis (IBM)In some embodiments, the IIM comprises IBM. IBM is an autoimmune myopathy that is the most common acquired muscle disease in people over 50 years of age. IBM is a chronic and debilitating condition that affects an increased number of men relative to women at a 3 : 1 ratio (Price et al, J. Neuromuscular Dis. 3 :67-75 (2016)). IBM is characterized by slowly progressive weakness and muscle wasting (relative to other forms of myositis), typically of the quadriceps and long finger flexor muscles (Rothwell et al, Curr. Opin. Rheumatol. 29-639-644 (2017)). Patterns of muscle involvement includes weakness of the long finger flexors, the quadriceps, the tibialis anterior and other arm and leg muscles. IBM leads to profound muscle atrophy and, as the disease progresses, patients progressively lose muscle strength at a rate of 3.5-16.8% per year. Most patients having IBM require daily assistance, as well as needing ambulatory aids such as a cane, walker, and / or wheelchair (Price etal, J. Neuromuscular Dis.3:67-75 (2016)). Weakening of the pharyngeal and esophageal muscles often lead to dysphagia, experienced by a majority of IBM patients. Dysphagia can result in aspiration, choking, weight loss, and pneumonia. These symptoms explain a relatively high rate of mortality in IBM patients relative to other forms of IIM.Immune-Mediated Necrotizing Myopathy (IMNM)In some embodiments, the IIM comprises IMNM. IMNM leads to an acute or subacute proximal weakness of the arms and legs. The disease course of IMNM is rapid and severe relative to DM and PM. Muscle enzymes are usually very high, for example 20-50-fold elevated CK. IMNM is characterized histologically by scattered necrotic myofibers, moderate and focal upregulation of MHC class I (e.g., areas comprising necrotic fibers), and binding of complement to the sarcolemma (Allenbach et al, Neuropath. Appl. Neurobiol. 43:62-81Docket No. 103362-068WO1(2017)). Necrotic fibers typically display a secondary invasion by macrophages for clearance of the cell debris.The methods of treating IIM of any aspect disclosed herein comprises obtaining a sample from the subject, wherein the sample is blood or any blood component. In some embodiments, the sample is serum isolated from the blood obtained from the subject. The method further comprises measuring the level of one or more dysferlin autoantibodies.DysferlinDysferlin (UniProt ID: 075923; HGNC: 3097; Ensembl: ENSG00000135636) is a protein that helps repair damaged muscle fibers and regulates cellular interactions. Dysferlin is a transmembrane protein found in the sarcolemma, the thin membrane that surrounds muscle fibers. Dysferlin assists in the repair of the sarcolemma when it is damaged by muscle strain, and may also be involved in muscle fiber regeneration and inflammation. As shown herein, TRIM72 / MG53 and dysferlin proteins are critical components of the membrane repair process. It is also shown in Example 1 herein that autoantibodies against TRIM72 / MG53 are found in IIM patients and that these antibodies could compromise sarcolemmal membrane repair and contribute to the progression of IIM. Further shown in Example 1 is that autoantibodies against dysferlin are very common in IIM patients and that the levels of dysferlin antibodies are linked with the severity and the progression of the disease.In some embodiments, an increase in the level of one or more dysferlin autoantibodies compared to a control indicates the presence of IIM or risk thereof in the subject. In some embodiments, the control comprises a standardized threshold level of one or more dysferlin autoantibodies from subjects without IIM. As used herein, the term “threshold level” refers to the measurement of the level of one or more autoantibodies against the tested antigen (dysferlin) in a given control subject, or for a given population of control subjects of known outcome (diagnosis, prognosis or stratification) with respect to IIM. In some embodiments, the control comprises a sample from the subject at an earlier time period or comparison to values produced by natural history studies on the progression of IIM.In some embodiments, the method reduces the level of one or more dysferlin autoantibodies in the subject. In some embodiments, the method reduces the level of one or more dysferlin autoantibodies in the subject by at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%.Docket No. 103362-068WO1In some embodiments, among a plurality of subjects treated according to the method, the level of one or more dysferlin autoantibodies in the subjects is reduced by an average of at least about 80%, at least about 90%, at least about 95%, or at least about 99%. In some embodiments, among a plurality of subjects treated according to the method, the level of one or more dysferlin autoantibodies in the subjects is reduced by an average of at least about 50%. In some embodiments, among a plurality of subjects treated according to the method, the level of one or more dysferlin autoantibodies in the subjects is reduced by an average of at least about 60%. In some embodiments, among a plurality of subjects treated according to the method, the level of one or more dysferlin autoantibodies in the subjects is reduced by an average of at least about 70%. In some embodiments, among a plurality of subjects treated according to the method, the level of one or more dysferlin autoantibodies in the subjects is reduced by an average of at least about 80%. In some embodiments, among a plurality of subjects treated according to the method, the level of one or more dysferlin autoantibodies in the subjects is reduced by an average of at least about 90%. In some embodiments, among a plurality of subjects treated according to the method, the level of one or more dysferlin autoantibodies in the subjects is reduced by an average of at least about 95%. In some embodiments, among a plurality of subjects treated according to the method, the level of one or more dysferlin autoantibodies in the subjects is reduced by an average of at least about 99%.In some embodiments, the level of one or more dysferlin autoantibodies in the subjects is significantly reduced as compared to subjects having the disease or condition and not treated according to the method.It is understood and herein contemplated that levels of one or more dysferlin autoantibodies in samples obtained from a subject can be detected and measured using any detection immunoassays known in the art, which include but are not limited to ELISA, RIA, radioimmunoprecipitation assays, surface plasmon resonance (SPR), immunohistochemistry, chemiluminescence, ELISpot (including, but not limited to plasma cell ELISpot), electrochemiluminescence, immunoblotting, mass spectrometry, hospital modular analyzers, flow cytometry, or multiplex assay including, but not limited to, Luminex, Mesoscale, MultiArray, A2, FAST Quant, Flow Cytomix, multiplex lateral flow immunoassay, multiplex immunohistochemistry, MILLIPLEX®, Bio-Plex, Cytometric Bead Array, and ImmuneD.The steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g., Maggio et al. Enzyme-Immunoassay, (1987) and Nakamura et al. Enzyme Immunoassays: Heterogeneous and Homogeneous Systems, Handbook ofDocket No. 103362-068WO1Experimental Immunology, Vol. 1: Immunochemistry, 27.1-27.20 (1986), each of which is incorporated herein by reference in its entirety and specifically for its teaching regarding immunodetection methods. Immunoassays, in their most simple and direct sense, are binding assays involving binding between antibodies and antigen. Many types and formats of immunoassays are known and all are suitable for detecting the anti-dysferlin autoantibodies as disclosed herein. Examples of immunoassays are enzyme linked immunosorbent assays (ELISAs), enzyme linked immunospot assay (ELISpot), radioimmunoassays (RIA), radioimmune precipitation assays (RIP A), immunobead capture assays, Western blotting, dot blotting, gel-shift assays, Flow cytometry, protein arrays, multiplexed bead arrays, magnetic capture, in vivo imaging, fluorescence resonance energy transfer (FRET), and fluorescence recovery / localization after photobleaching (FRAP / FLAP).In general, immunoassays involve contacting a sample suspected of containing a molecule of interest (such as the disclosed one or more anti-dysferlin autoantibodies) with an antibody to the molecule of interest or contacting an antibody to a molecule of interest (such as the disclosed one or more anti-dysferlin autoantibodies) with a molecule that can be bound by the antibody, as the case may be, under conditions effective to allow the formation of immunocomplexes. Contacting a sample with an antibody to the molecule of interest or with the molecule that can be bound by an antibody to the molecule of interest under conditions effective and for a period of time sufficient to allow the formation of immune complexes (primary immune complexes) is generally a matter of bringing into contact the molecule or antibody and the sample and incubating the mixture for a period of time long enough for the antibodies to form immune complexes with, i.e., to bind to, any molecules (e.g., antigens) present to which the antibodies can bind. In many forms of immunoassay, the sample-antibody composition, such as a tissue section, ELISA plate, dot blot or Western blot, can then be washed to remove any non-specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected.Immunoassays can include methods for detecting or quantifying the amount of a molecule of interest (such as the disclosed dysferlin biomarker or their autoantibodies) in a sample, which methods generally involve the detection or quantitation of any immune complexes formed during the binding process. In general, the detection of immunocomplex formation is well known in the art and can be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as any radioactive, fluorescent, biological or enzymatic tags or any other known label. See, forDocket No. 103362-068WO1example, U. S. Patents 3,817,837; 3,850, 752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241, each of which is incorporated herein by reference in its entirety and specifically for teachings regarding immunodetection methods and labels.As used herein, a label can include a fluorescent dye, a member of a binding pair, such as biotin / streptavidin, a metal (e.g., gold), or an epitope tag that can specifically interact with a molecule that can be detected, such as by producing a colored substrate or fluorescence. Substances suitable for detectably labeling proteins include fluorescent dyes (also known herein as fluorochromes and fluorophores) and enzymes that react with colorimetric substrates (e.g., horseradish peroxidase). The use of fluorescent dyes is generally preferred in the practice of the invention as they can be detected at very low amounts. Furthermore, in the case where multiple antigens are reacted with a single array, each antigen can be labeled with a distinct fluorescent compound for simultaneous detection. Labeled spots on the array are detected using a fluorimeter, the presence of a signal indicating an antigen bound to a specific antibody.Fluorophores are compounds or molecules that luminesce. Typically fluorophores absorb electromagnetic energy at one wavelength and emit electromagnetic energy at a second wavelength. Representative fluorophores include, but are not limited to, 1,5 IAEDANS; 1,8-ANS; 4- Methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM); 5-Carboxynapthofluorescein; 5-Carboxytetramethylrhodamine (5-TAMRA); 5-Hydroxy Tryptamine (5-HAT); 5-ROX (carboxy-X-rhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE; 7-Amino-4-methylcoumarin; 7- Aminoactinomycin D (7-AAD); 7-Hydroxy-4-I methylcoumarin; 9-Amino-6-chloro-2-methoxyacridine (ACMA); ABQ; Acid Fuchsin; Acridine Orange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin Feulgen SITSA; Aequorin (Photoprotein); AFPs - AutoFluorescent Protein - (Quantum Biotechnologies) see sgGFP, sgBFP; Alexa Fluor 350™; Alexa Fluor 430™; Alexa Fluor 488™; Alexa Fluor 532™; Alexa Fluor 546™; Alexa Fluor 568™; Alexa Fluor 594™; Alexa Fluor 633™; Alexa Fluor 647™; Alexa Fluor 660™; Alexa Fluor 680™; Alizarin Complexon; Alizarin Red; Allophycocyanin (APC); AMC, AMCA-S; Aminomethylcoumarin (AMCA); AMCA-X; Aminoactinomycin D; Aminocoumarin; Anilin Blue; Anthrocyl stearate; APC-Cy7; APTRA-BTC; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrine; ATTO- TAG™ CBQCA; ATTO-TAG™ FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9 (Bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH); Berberine Sulphate; Beta Lactamase; BFP blue shifted GFP (Y66H); Blue Fluorescent Protein; BFP / GFP FRET; Bimane; Bisbenzemide; Bisbenzimide (Hoechst); bis-Docket No. 103362-068WO1BTC; Blancophor FFG; Blancophor SV; BOBO™ -1; BOBO™-3; Bodipy492 / 515; Bodipy493 / 503; Bodipy500 / 510; Bodipy; 505 / 515; Bodipy 530 / 550; Bodipy 542 / 563; Bodipy 558 / 568; Bodipy 564 / 570; Bodipy 576 / 589; Bodipy 581 / 591; Bodipy 630 / 650-X; Bodipy 650 / 665-X; Bodipy 665 / 676; Bodipy Fl; Bodipy FL ATP; Bodipy Fl-Ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE; BO-PRO™ -1; BO-PRO™ -3; Brilliant Sulphoflavin FF; BTC; BTC-5N; Calcein; Calcein Blue; Calcium Crimson - ; Calcium Green; Calcium Green- 1 Ca2+Dye; Calcium Green-2 Ca2+; Calcium Green-5N Ca2+; Calcium Green-C18 Ca2+; Calcium Orange; Calcofluor White; Carboxy-X-rhodamine (5-ROX); Cascade Blue™; Cascade Yellow; Catecholamine; CCF2 (GeneBlazer); CFDA; CFP (Cyan Fluorescent Protein); CFP / YFP FRET; Chlorophyll; Chromomycin A; Chromomycin A; CL-NERF; CMFDA; Coelenterazine; Coelenterazine cp; Coelenterazine f; Coelenterazine fcp; Coelenterazine h; Coelenterazine hep; Coelenterazine ip; Coelenterazine n; Coelenterazine O; Coumarin Phalloidin; C-phycocyanine; CPM I Methylcoumarin; CTC; CTC Formazan; Cy2™; Cy3.1 8; Cy3.5™; Cy3™; Cy5.1 8; Cy5.5™; Cy5™; Cy7™; Cyan GFP; cyclic AMP Fluorosensor (FiCRhR); Dabcyl; Dansyl; Dansyl Amine; Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3’DCFDA; DCFH (Diehl orodihydrofluorescein Diacetate); DDAO; DHR (Dihydorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-Di 16-ASP); Diehl orodihydrofluorescein Diacetate (DCFH); DiD- Lipophilic Tracer; DiD (DilC18(5)); DIDS; Dihydorhodamine 123 (DHR); Dil (DilC18(3)); I Dinitrophenol; DiO (DiOC18(3)); DiR; DiR (DilC 18(7)); DM-NERF (high pH); DNP; Dopamine; DsRed; DTAF; DY-630-NHS; DY-635-NHS; EBFP; ECFP; EGFP; ELF 97; Eosin; Erythrosin; Erythrosin ITC; Ethidium Bromide; Ethidium homodimer-1 (EthD-1); Euchrysin; EukoLight; Europium (111) chloride; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline); FIF (Formaldehyd Induced Fluorescence); FITC; Flazo Orange; Fluo-3; Fluo-4; Fluorescein (FITC); Fluorescein Diacetate; Fluoro-Emerald; Fluoro-Gold (Hydroxy stilbamidine); Fluor-Ruby; FluorX; FM 1-43™; FM 4-46; Fura Red™ (high pH); Fura Red™ / Fluo-3; Fura-2; Fura-2 / BCECF; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow 5GF; GeneBlazer; (CCF2); GFP (S65T); GFP red shifted (rsGFP); GFP wild type’ non-UV excitation (wtGFP); GFP wild type, UV excitation (wtGFP); GFPuv; Gloxalic Acid; Granular blue; Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; Hydroxy coumarin; Hydroxy stilbamidine (FluoroGold); Hydroxytryptamine; Indo-1, high calcium; Indo-1 low calcium; Indodicarbocyanine (DiD); Indotricarbocyanine (DiR);Docket No. 103362-068WO1Intrawhite Cf; JC-1; JO JO-1; JO-PRO-1; LaserPro; Laurodan; LDS 751 (DNA); LDS 751 (RNA); Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine Rhodamine; Lissamine Rhodamine B; Calcein / Ethidium homodimer; LOLO-1; LO-PRO-1; ; Lucifer Yellow; Lyso Tracker Blue; Lyso Tracker Blue-White; Lyso Tracker Green; Lyso Tracker Red; Lyso Tracker Yellow; LysoSensor Blue; LysoSensor Green; LysoSensor Yellow / Blue; Mag Green; Magdala Red (Phloxin B); Mag-Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-lndo-1; Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue; I Maxiion Brilliant Flavin 10 GFF; Maxiion Brilliant Flavin 8 GFF; Merocyanin; Methoxy coumarin; Mitotracker Green FM; Mitotracker Orange; Mitotracker Red; Mitramycin; Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD Amine; Nile Red; Nitrobenzoxedidole; Noradrenaline; Nuclear Fast Red; i Nuclear Yellow; Nylosan Brilliant lavin E8G; Oregon Green™; Oregon Green™ 488; Oregon Green™ 500; Oregon Green™ 514; Pacific Blue; Pararosaniline (Feulgen); PBFI; PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-TexasRed (Red 613); Phloxin B (Magdala Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; PhotoResist; Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26 (Sigma); PKH67; PMIA; Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO- 1 PRO-3; Primuline; Procion Yellow; Propidium lodid (Pl); PyMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Resorufin; RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G; Rhodamine B; Rhodamine B 200; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine: Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal; R-phycocyanine; R-phycoerythrin (PE); rsGFP; S65A; S65C; S65L; S65T; Sapphire GFP; SBFI; Serotonin; Sevron Brilliant Red 2B; Sevron Brilliant Red 4G; Sevron I Brilliant Red B; Sevron Orange; Sevron Yellow L; sgBFP™ (super glow BFP); sgGFP™ (super glow GFP); SITS (Primuline; Stilbene Isothiosulphonic Acid); SNAFL calcein; SNAFL-1; SNAFL-2; SNARF calcein; SNARF1; Sodium Green; SpectrumAqua; SpectrumGreen; SpectrumOrange; Spectrum Red; SPQ (6-methoxy- N-(3 sulfopropyl) quinolinium); Stilbene; Sulphorhodamine B and C; Sulphorhodamine Extra; SYTO 11; SYTO 12; SYTO 13; SYTO 14; SYTO 15; SYTO 16; SYTO 17; SYTO 18; SYTO 20; SYTO 21; SYTO 22; SYTO 23; SYTO 24; SYTO 25; SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO 44; SYTO 45; SYTO 59; SYTO 60; SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO 80; SYTO 81; SYTO 82; SYTO 83; SYTO 84; SYTO 85; SYTOX Blue; SYTOX Green; SYTOX Orange; Tetracycline; Tetramethylrhodamine (TRITC); Texas Red™; TexasDocket No. 103362-068WO1Red-X™ conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TON; Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TIER; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITC TetramethylRodaminelsoThioCyanate; True Blue; Tru Red; Ultralite; Uranine B; Uvitex SFC; wt GFP; WW 781; X-Rhodamine; XRITC; Xylene Orange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO- PRO 3; YOYO- 1; YOYO-3; Sybr Green; Thiazole orange (interchelating dyes); semiconductor nanoparticles such as quantum dots; or caged fluorophore (which can be activated with light or other electromagnetic energy source), or a combination thereof.A modifier unit such as a radionuclide can be incorporated into or attached directly to any of the compounds described herein by halogenation. Examples of radionuclides useful in this embodiment include, but are not limited to, tritium, iodine-125, iodine-131, iodine-123, iodine-124, astatine-210, carbon-11, carbon-14, nitrogen-13, fluorine-18. In another aspect, the radionuclide can be attached to a linking group or bound by a chelating group, which is then attached to the compound directly or by means of a linker. Examples of radionuclides useful in the aspect include, but are not limited to, Tc-99m, Re-186, Ga-68, Re-188, Y-90, Sm-153, Bi-212, Cu-67, Cu-64, and Cu-62. Radiolabeling techniques such as these are routinely used in the radiopharmaceutical industry. Radiolabeled compounds described herein can be used in conjunction with imaging techniques such as positron emission tomography (PET) or single photon emission computerized tomography (SPECT).Labeling can be either direct or indirect. In direct labeling, the detecting antibody (the antibody for the molecule of interest) or detecting molecule (the molecule that can be bound by an antibody to the molecule of interest) include a label. Detection of the label indicates the presence of the detecting antibody or detecting molecule, which in turn indicates the presence of the molecule of interest or of an antibody to the molecule of interest, respectively. In indirect labeling, an additional molecule or moiety is brought into contact with, or generated at the site of, the immunocomplex. For example, a signal-generating molecule or moiety such as an enzyme can be attached to or associated with the detecting antibody or detecting molecule. The signal-generating molecule can then generate a detectable signal at the site of the immunocomplex. For example, an enzyme, when supplied with suitable substrate, can produce a visible or detectable product at the site of the immunocomplex. ELISAs use this type of indirect labeling.Docket No. 103362-068WO1As another example of indirect labeling, an additional molecule (which can be referred to as a binding agent) that can bind to either the molecule of interest or to the antibody (primary antibody) to the molecule of interest, such as a second antibody to the primary antibody, can be contacted with the immunocomplex. The additional molecule can have a label or signalgenerating molecule or moiety. The additional molecule can be an antibody, which can thus be termed a secondary antibody. Binding of a secondary antibody to the primary antibody can form a so-called sandwich with the first (or primary) antibody and the molecule of interest. The immune complexes can be contacted with the labeled, secondary antibody under conditions effective and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes can then be generally washed to remove any non-specifically bound labeled secondary antibodies, and the remaining label in the secondary immune complexes can then be detected. The additional molecule can also be or include one of a pair of molecules or moieties that can bind to each other, such as the biotin / avadin pair. In this mode, the detecting antibody or detecting molecule should include the other member of the pair.Other modes of indirect labeling include the detection of primary immune complexes by a two step approach. For example, a molecule (which can be referred to as a first binding agent), such as an antibody, that has binding affinity for the molecule of interest or corresponding antibody can be used to form secondary immune complexes, as described above. After washing, the secondary immune complexes can be contacted with another molecule (which can be referred to as a second binding agent) that has binding affinity for the first binding agent, again under conditions effective and for a period of time sufficient to allow the formation of immune complexes (thus forming tertiary immune complexes). The second binding agent can be linked to a detectable label or signal-generating molecule or moiety, allowing detection of the tertiary immune complexes thus formed. This system can provide for signal amplification.Immunoassays that involve the detection of a substance, such as a protein or an antibody to a specific protein, include label-free assays, protein separation methods (i.e., electrophoresis), solid support capture assays, or in vivo detection. Label-free assays are generally diagnostic means of determining the presence or absence of a specific protein, or an antibody to a specific protein, in a sample. Protein separation methods are additionally useful for evaluating physical properties of the protein, such as size or net charge. Capture assays are generally more useful for quantitatively evaluating the concentration of a specific protein, orDocket No. 103362-068WO1antibody to a specific protein, in a sample. Finally, in vivo detection is useful for evaluating the spatial expression patterns of the substance, i.e., where the substance can be found in a subject, tissue or cell.Provided that the concentrations are sufficient, the molecular complexes ([Ab-Ag] / / ) generated by antibody-antigen interaction are visible to the naked eye, but smaller amounts may also be detected and measured due to their ability to scatter a beam of light. The formation of complexes indicates that both reactants are present, and in immunoprecipitation assays a constant concentration of a reagent antibody is used to measure specific antigen ([Ab-Ag] / / ), and reagent antigens are used to detect specific antibody ([Ab-Ag] / / ). If the reagent species is previously coated onto cells (as in hemagglutination assay) or very small particles (as in latex agglutination assay), “clumping” of the coated particles is visible at much lower concentrations. A variety of assays based on these elementary principles are in common use, including Ouchterlony immunodiffusion assay, rocket immunoelectrophoresis, and immunoturbidometric and nephelometric assays. The main limitations of such assays are restricted sensitivity (lower detection limits) in comparison to assays employing labels and, in some cases, the fact that very high concentrations of analyte can actually inhibit complex formation, necessitating safeguards that make the procedures more complex. Some of these Group 1 assays date right back to the discovery of antibodies and none of them have an actual “label” (e.g., Ag-enz). Other kinds of immunoassays that are label free depend on immunosensors, and a variety of instruments that can directly detect antibody-antigen interactions are now commercially available. Most depend on generating an evanescent wave on a sensor surface with immobilized ligand, which allows continuous monitoring of binding to the ligand. Immunosensors allow the easy investigation of kinetic interactions and, with the advent of lower-cost specialized instruments, may in the future find wide application in immunoanalysis.The use of immunoassays to detect a specific protein can involve the separation of the proteins by electrophoresis. Electrophoresis is the migration of charged molecules in solution in response to an electric field. Their rate of migration depends on the strength of the field; on the net charge, size and shape of the molecules and also on the ionic strength, viscosity and temperature of the medium in which the molecules are moving. As an analytical tool, electrophoresis is simple, rapid and highly sensitive. It is used analytically to study the properties of a single charged species, and as a separation technique.Docket No. 103362-068WO1Generally the sample is run in a support matrix such as paper, cellulose acetate, starch gel, agarose or polyacrylamide gel. The matrix inhibits convective mixing caused by heating and provides a record of the electrophoretic run: at the end of the run, the matrix can be stained and used for scanning, autoradiography or storage. In addition, the most commonly used support matrices - agarose and polyacrylamide - provide a means of separating molecules by size, in that they are porous gels. A porous gel may act as a sieve by retarding, or in some cases completely obstructing, the movement of large macromolecules while allowing smaller molecules to migrate freely. Because dilute agarose gels are generally more rigid and easy to handle than polyacrylamide of the same concentration, agarose is used to separate larger macromolecules such as nucleic acids, large proteins and protein complexes. Polyacrylamide, which is easy to handle and to make at higher concentrations, is used to separate most proteins and small oligonucleotides that require a small gel pore size for retardation.Proteins are amphoteric compounds; their net charge therefore is determined by the pH of the medium in which they are suspended. In a solution with a pH above its isoelectric point, a protein has a net negative charge and migrates towards the anode in an electrical field. Below its isoelectric point, the protein is positively charged and migrates towards the cathode. The net charge carried by a protein is in addition independent of its size - i.e., the charge carried per unit mass (or length, given proteins and nucleic acids are linear macromolecules) of molecule differs from protein to protein. At a given pH therefore, and under non-denaturing conditions, the electrophoretic separation of proteins is determined by both size and charge of the molecules.Sodium dodecyl sulphate (SDS) is an anionic detergent which denatures proteins by “wrapping around” the polypeptide backbone - and SDS binds to proteins fairly specifically in a mass ratio of 1.4:1. In so doing, SDS confers a negative charge to the polypeptide in proportion to its length. Further, it is usually necessary to reduce disulfide bridges in proteins (denature) before they adopt the random-coil configuration necessary for separation by size; this is done with 2-mercaptoethanol or dithiothreitol (DTT). In denaturing SDS-PAGE separations therefore, migration is determined not by intrinsic electrical charge of the polypeptide, but by molecular weight.Determination of molecular weight is done by SDS-PAGE of proteins of known molecular weight along with the protein to be characterized. A linear relationship exists between the logarithm of the molecular weight of an SDS-denatured polypeptide, or native nucleic acid, and its Rf. The Rf is calculated as the ratio of the distance migrated by theDocket No. 103362-068WO1molecule to that migrated by a marker dye-front. A simple way of determining relative molecular weight by electrophoresis (Mr) is to plot a standard curve of distance migrated vs. loglOMW for known samples, and read off the logATr of the sample after measuring distance migrated on the same gel.In two-dimensional electrophoresis, proteins are fractionated first on the basis of one physical property, and, in a second step, on the basis of another. For example, isoelectric focusing can be used for the first dimension, conveniently carried out in a tube gel, and SDS electrophoresis in a slab gel can be used for the second dimension. One example of a procedure is that of O’Farrell, P.H., High Resolution Two-dimensional Electrophoresis of Proteins, J. Biol. Chem. 250:4007-4021 (1975), herein incorporated by reference in its entirety for its teaching regarding two-dimensional electrophoresis methods. Other examples include but are not limited to, those found in Anderson, L & Anderson, NG High resolution two-dimensional electrophoresis of human plasma proteins, Proc. Natl. Acad. Sci. 74:5421-5425 (1977), and Ornstein, L. Disc electrophoresis, L. Ann. N.Y. Acad. Sci. 121:321349 (1964), each of which is herein incorporated by reference in its entirety for teachings regarding electrophoresis methods.Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227:680 (1970), which is herein incorporated by reference in its entirety for teachings regarding electrophoresis methods, discloses a discontinuous system for resolving proteins denatured with SDS. The leading ion in the Laemmli buffer system is chloride, and the trailing ion is glycine. Accordingly, the resolving gel and the stacking gel are made up in Tris-HCl buffers (of different concentration and pH), while the tank buffer is Trisglycine. All buffers contain 0.1% SDS.One example of an immunoassay that uses electrophoresis that is contemplated in the current methods is Western blot analysis. Western blotting or immunoblotting allows the determination of the molecular mass of a protein and the measurement of relative amounts of the protein present in different samples. Detection methods include chemiluminescence and chromagenic detection. Standard methods for Western blot analysis can be found in, for example, D.M. Bollag et al., Protein Methods (2d edition 1996) and E. Harlow & D. Lane, Antibodies, a Laboratory Manual (1988), U.S. Patent 4,452,901, each of which is herein incorporated by reference in their entirety for teachings regarding Western blot methods. Generally, proteins are separated by gel electrophoresis, usually SDS-PAGE. The proteins are transferred to a sheet of special blotting paper, e.g., nitrocellulose, though other types of paper,Docket No. 103362-068WO1or membranes, can be used. The proteins retain the same pattern of separation they had on the gel. The blot is incubated with a generic protein (such as milk proteins) to bind to any remaining sticky places on the nitrocellulose. An antibody is then added to the solution which is able to bind to its specific protein.The attachment of specific antibodies to specific immobilized antigens can be readily visualized by indirect enzyme immunoassay techniques, usually using a chromogenic substrate (e.g. alkaline phosphatase or horseradish peroxidase) or chemiluminescent substrates. Other possibilities for probing include the use of fluorescent or radioisotope labels (e.g., fluorescein,125I). Probes for the detection of antibody binding can be conjugated anti-immunoglobulins, conjugated staphylococcal Protein A (binds IgG), or probes to biotinylated primary antibodies (e.g., conjugated avidin / streptavidin).The power of the technique lies in the simultaneous detection of a specific protein by means of its antigenicity, and its molecular mass. Proteins are first separated by mass in the SDS-PAGE, then specifically detected in the immunoassay step. Thus, protein standards (ladders) can be run simultaneously in order to approximate molecular mass of the protein of interest in a heterogeneous sample.The gel shift assay or electrophoretic mobility shift assay (EMSA) can be used to detect the interactions between DNA binding proteins and their cognate DNA recognition sequences, in both a qualitative and quantitative manner. Exemplary techniques are described in Ornstein L., Disc electrophoresis - 1: Background and theory, Ann. NY Acad. Sci. 121:321-349 (1964), and Matsudiara, PT and DR Burgess, SDS microslab linear gradient polyacrylamide gel electrophoresis, Anal. Biochem. 87:386-396 (1987), each of which is herein incorporated by reference in its entirety for teachings regarding gel-shift assays.In a general gel-shift assay, purified proteins or crude cell extracts can be incubated with a labeled (e.g.,32P-radiolabeled) DNA or RNA probe, followed by separation of the complexes from the free probe through a nondenaturing polyacrylamide gel. The complexes migrate more slowly through the gel than unbound probe. Depending on the activity of the binding protein, a labeled probe can be either double-stranded or single-stranded. For the detection of DNA binding proteins such as transcription factors, either purified or partially purified proteins, or nuclear cell extracts can be used. For detection of RNA binding proteins, either purified or partially purified proteins, or nuclear or cytoplasmic cell extracts can be used. The specificity of the DNA or RNA binding protein for the putative binding site is established by competition experiments using DNA or RNA fragments or oligonucleotides containing aDocket No. 103362-068WO1binding site for the protein of interest, or other unrelated sequence. The differences in the nature and intensity of the complex formed in the presence of specific and nonspecific competitor allows identification of specific interactions.Gel shift methods can include using, for example, colloidal forms of COOMASSIE (Imperial Chemicals Industries, Ltd) blue stain to detect proteins in gels such as polyacrylamide electrophoresis gels. Such methods are described, for example, in Neuhoff et al., Electrophoresis 6:427-448 (1985), and Neuhoff et al. , Electrophoresis 9:255-262 (1988), each of which is herein incorporated by reference in its entirety for teachings regarding gel shift methods. In addition to the conventional protein assay methods referenced above, a combination cleaning and protein staining composition is described in U.S. Patent 5,424,000, herein incorporated by reference in its entirety for its teaching regarding gel shift methods. The solutions can include phosphoric, sulfuric, and nitric acids, and Acid Violet dye.Radioimmune Precipitation Assay (RIP A) is a sensitive assay using radiolabeled antigens to detect specific antibodies in serum / plasma. The antigens are allowed to react with the serum and then precipitated using a special reagent such as, for example, protein A sepharose beads. The bound radiolabeled immunoprecipitate is then commonly analyzed by gel electrophoresis. Radioimmunoprecipitation assay (RIP A) is often used as a confirmatory test for diagnosing the presence of HIV antibodies. RIPA is also referred to in the art as Farr Assay, Precipitin Assay, Radioimmune Precipitation Assay; Radioimmunoprecipitation Analysis; Radioimmunoprecipitation Analysis, and Radioimmunoprecipitation Analysis.While the above immunoassays that utilize electrophoresis to separate and detect the specific proteins of interest allow for evaluation of protein size, they are not very sensitive for evaluating protein concentration. However, also contemplated are immunoassays wherein the protein or antibody specific for the protein is bound to a solid support (e.g., tube, well, bead, or cell) to capture the antibody or protein of interest, respectively, from a sample, combined with a method of detecting the protein or antibody specific for the protein on the support. Examples of such immunoassays include Radioimmunoassay (RIA), Enzyme-Linked Immunosorbent Assay (ELISA), Flow cytometry, protein array, multiplexed bead assay, and magnetic capture.Radioimmunoassay (RIA) is a classic quantitative assay for detection of antigenantibody reactions using a radioactively labeled substance (radioligand), either directly or indirectly, to measure the binding of the unlabeled substance to a specific antibody or other receptor system. Radioimmunoassay is used, for example, to test hormone levels in the bloodDocket No. 103362-068WO1without the need to use a bioassay. Non-immunogenic substances (e.g., haptens) can also be measured if coupled to larger carrier proteins (e.g., bovine gamma-globulin or human serum albumin) capable of inducing antibody formation. RIA involves mixing a radioactive antigen (because of the ease with which iodine atoms can be introduced into tyrosine residues in a protein, the radioactive isotopes125I or131I are often used) with antibody to that antigen. The antibody is generally linked to a solid support, such as a tube or beads. Unlabeled or “cold” antigen is then adding in known quantities and measuring the amount of labeled antigen displaced. Initially, the radioactive antigen is bound to the antibodies. When cold antigen is added, the two compete for antibody binding sites - and at higher concentrations of cold antigen, more binds to the antibody, displacing the radioactive variant. The bound antigens are separated from the unbound ones in solution and the radioactivity of each used to plot a binding curve. The technique is both extremely sensitive, and specific.Enzyme-Linked Immunospot Assay (ELISpot is an immunoassay that can detect an antibody specific for a protein or antigen. In such an assay, a detectable label bound to either an antibody-binding or antigen-binding reagent is an enzyme. When exposed to its substrate, this enzyme reacts in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or visual means. Enzymes which can be used to detectably label reagents useful for detection include, but are not limited to, horseradish peroxidase, alkaline phosphatase, glucose oxidase, P-galactosidase, ribonuclease, urease, catalase, malate dehydrogenase, staphylococcal nuclease, asparaginase, yeast alcohol dehydrogenase, alpha. -glycerophosphate dehydrogenase, triose phosphate isomerase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. In this assay a nitrocellulose microtiter plate is coated with antigen. The test sample is exposed to the antigen and then reacted similarly to an ELISA assay. Detection differs from a traditional ELISA in that detection is determined by the enumeration of spots on the nitrocellulose plate. The presence of a spot indicates that the sample reacted to the antigen. The spots can be counted and the number of cells in the sample specific for the antigen determined.Enzyme-Linked Immunosorbent Assay (ELISA), or more generically termed EIA (Enzyme ImmunoAssay), is an immunoassay that can detect an antibody specific for a protein. In such an assay, a detectable label bound to either an antibody-binding or antigen-binding reagent is an enzyme. When exposed to its substrate, this enzyme reacts in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or visual means. Enzymes which can be used to detectably label reagents usefulDocket No. 103362-068WO1for detection include, but are not limited to, horseradish peroxidase, alkaline phosphatase, glucose oxidase, P-galactosidase, ribonuclease, urease, catalase, malate dehydrogenase, staphylococcal nuclease, asparaginase, yeast alcohol dehydrogenase, alpha. -glycerophosphate dehydrogenase, triose phosphate isomerase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. For descriptions of ELISA procedures, see Voller, A. et al., J. Clin. Pathol. 31:507-520 (1978); Butler, J. E., Meth. Enzymol. 73:482-523 (1981); Maggio, E. (ed.), Enzyme Immunoassay, CRC Press, Boca Raton, 1980; Butler, J. E., In: Structure of Antigens, Vol. 1 (Van Regenmortel, M., CRC Press, Boca Raton, 1992, pp. 209-259; Butler, J. E., In: van Oss, C. J. et al., (eds), Immunochemistry, Marcel Dekker, Inc., New York, 1994, pp. 759-803; Butler, J. E. (ed. ), Immunochemistry of Solid-Phase Immunoassay, CRC Press, Boca Raton, 1991); Crowther, ‘ELISA: Theory and Practice, ” In: Methods in Molecule Biology, Vol. 42, Humana Press; New Jersey, 1995;U.S. Patent 4,376,110, each of which is incorporated herein by reference in its entirety and specifically for teachings regarding ELISA methods.Variations of ELISA techniques are know to those of skill in the art. In one variation, antibodies that can bind to proteins can be immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing a marker antigen can be added to the wells. After binding and washing to remove non-specifically bound immunocomplexes, the bound antigen can be detected. Detection can be achieved by the addition of a second antibody specific for the target protein, which is linked to a detectable label. This type of ELISA is a simple “sandwich ELISA.” Detection also can be achieved by the addition of a second antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.Another variation is a competition ELISA. In competition ELISA’ s, test samples compete for binding with known amounts of labeled antigens or antibodies. The amount of reactive species in the sample can be determined by mixing the sample with the known labeled species before or during incubation with coated wells. The presence of reactive species in the sample acts to reduce the amount of labeled species available for binding to the well and thus reduces the ultimate signal.Regardless of the format employed, ELISAs have certain features in common, such as coating, incubating or binding, washing to remove non-specifically bound species, and detecting the bound immunecomplexes. Antigen or antibodies can be linked to a solid support,Docket No. 103362-068WO1such as in the form of plate, beads, dipstick, membrane or column matrix, and the sample to be analyzed applied to the immobilized antigen or antibody. In coating a plate with either antigen or antibody, one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period of hours. The wells of the plate can then be washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells can then be “coated” with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine serum albumin (BSA), casein and solutions of milk powder. The coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.In ELISAs, a secondary or tertiary detection means rather than a direct procedure can also be used. Thus, after binding of a protein or antibody to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the control clinical or biological sample to be tested under conditions effective to allow immunecomplex (antigen / antibody) formation. Detection of the immunecomplex then requires a labeled secondary binding agent or a secondary binding agent in conjunction with a labeled third binding agent.The term “under conditions effective to allow immunecomplex (antigen / antibody) formation” as used herein means that the conditions include diluting the antigens and antibodies with solutions such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS) / Tween so as to reduce non-specific binding and to promote a reasonable signal to noise ratio. The suitable conditions also mean that the incubation is at a temperature and for a period of time sufficient to allow effective binding. Incubation steps can typically be from about 1 minute to twelve hours, at temperatures of about 20° to 30° C, or can be incubated overnight at about 0° C to about 10° C. Following all incubation steps in an ELISA, the contacted surface can be washed so as to remove non-complexed material. A washing procedure can include washing with a solution such as PBS / Tween or borate buffer. Following the formation of specific immunecomplexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immunecomplexes can be determined.To provide a detecting means, the second or third antibody can have an associated label to allow detection, as described above. This can be an enzyme that can generate color development upon incubating with an appropriate chromogenic substrate. Thus, for example,Docket No. 103362-068WO1one can contact and incubate the first or second immunecomplex with a labeled antibody for a period of time and under conditions that favor the development of further immunecomplex formation (e.g., incubation for 2 hours at room temperature in a PBS-containing solution such as PBS-Tween).After incubation with the labeled antibody, and subsequent to washing to remove unbound material, the amount of label can be quantified, e.g., by incubation with a chromogenic substrate such as urea and bromocresol purple or 2,2’-azido-di-(3-ethyl-benzthiazoline-6-sulfonic acid [ABTS] and H2O2, in the case of peroxidase as the enzyme label. Quantitation can then be achieved by measuring the degree of color generation, e.g., using a visible spectra spectrophotometer.Protein arrays are solid-phase ligand binding assay systems using immobilized proteins on surfaces which include glass, membranes, microtiter wells, mass spectrometer plates, and beads or other particles. The assays are highly parallel (multiplexed) and often miniaturized (microarrays, protein chips). Their advantages include being rapid and automatable, capable of high sensitivity, economical on reagents, and giving an abundance of data for a single experiment. Bioinformatics support is important; the data handling demands sophisticated software and data comparison analysis. However, the software can be adapted from that used for DNA arrays, as can much of the hardware and detection systems.One of the chief formats is the capture array, in which ligand-binding reagents, which are usually antibodies but can also be alternative protein scaffolds, peptides or nucleic acid aptamers, are used to detect target molecules in mixtures such as plasma or tissue extracts. In diagnostics, capture arrays can be used to carry out multiple immunoassays in parallel, both testing for several analytes in individual sera for example and testing many serum samples simultaneously. In proteomics, capture arrays are used to quantitate and compare the levels of proteins in different samples in health and disease, i.e. protein expression profiling. Proteins other than specific ligand binders are used in the array format for in vitro functional interaction screens such as protein-protein, protein-DNA, protein-drug, receptor-ligand, enzyme-substrate, etc. The capture reagents themselves are selected and screened against many proteins, which can also be done in a multiplex array format against multiple protein targets.For construction of arrays, sources of proteins include cell-based expression systems for recombinant proteins, purification from natural sources, production in vitro by cell-free translation systems, and synthetic methods for peptides. Many of these methods can be automated for high throughput production. For capture arrays and protein function analysis, itDocket No. 103362-068WO1is important that proteins should be correctly folded and functional; this is not always the case, e.g. where recombinant proteins are extracted from bacteria under denaturing conditions. Nevertheless, arrays of denatured proteins are useful in screening antibodies for crossreactivity, identifying autoantibodies and selecting ligand binding proteins.Protein arrays have been designed as a miniaturization of familiar immunoassay methods such as ELISA and dot blotting, often utilizing fluorescent readout, and facilitated by robotics and high throughput detection systems to enable multiple assays to be carried out in parallel. Commonly used physical supports include glass slides, silicon, microwells, nitrocellulose or PVDF membranes, and magnetic and other microbeads. While microdrops of protein delivered onto planar surfaces are the most familiar format, alternative architectures include CD centrifugation devices based on developments in microfluidics (Gyros, Monmouth Junction, NJ) and specialized chip designs, such as engineered microchannels in a plate (e.g., The Living Chip™, Biotrove, Woburn, MA) and tiny 3D posts on a silicon surface (Zyomyx, Hayward CA). Particles in suspension can also be used as the basis of arrays, providing they are coded for identification; systems include color coding for microbeads (Luminex, Austin, TX; Bio-Rad Laboratories) and semiconductor nanocrystals (e.g., QDots™, Quantum Dot, Hayward, CA), and barcoding for beads (UltraPlex™, SmartBead Technologies Ltd, Babraham, Cambridge, UK) and multimetal microrods (e.g., Nanobarcodes™ particles, Nanoplex Technologies, Mountain View, CA). Beads can also be assembled into planar arrays on semiconductor chips (LEAPS technology, BioArray Solutions, Warren, NJ).Immobilization of proteins involves both the coupling reagent and the nature of the surface being coupled to. A good protein array support surface is chemically stable before and after the coupling procedures, allows good spot morphology, displays minimal nonspecific binding, does not contribute a background in detection systems, and is compatible with different detection systems. The immobilization method used are reproducible, applicable to proteins of different properties (size, hydrophilic, hydrophobic), amenable to high throughput and automation, and compatible with retention of fully functioning protein activity. Orientation of the surface-bound protein is recognized as an important factor in presenting it to ligand or substrate in an active state; for capture arrays the most efficient binding results are obtained with orientated capture reagents, which generally require site-specific labeling of the protein.Both covalent and noncovalent methods of protein immobilization are used and have various pros and cons. Passive adsorption to surfaces is methodologically simple, but allows little quantitative or orientational control; it may or may not alter the functional properties ofDocket No. 103362-068WO1the protein, and reproducibility and efficiency are variable. Covalent coupling methods provide a stable linkage, can be applied to a range of proteins and have good reproducibility; however, orientation may be variable, chemical derivatization may alter the function of the protein and requires a stable interactive surface. Biological capture methods utilizing a tag on the protein provide a stable linkage and bind the protein specifically and in reproducible orientation, but the biological reagent must first be immobilized adequately and the array may require special handling and have variable stability.Several immobilization chemistries and tags have been described for fabrication of protein arrays. Substrates for covalent attachment include glass slides coated with amino- or aldehyde-containing silane reagents. In the Versalinx™ system (Prolinx, Bothell, WA) reversible covalent coupling is achieved by interaction between the protein derivatized with phenyl dib oronic acid, and salicylhydroxamic acid immobilized on the support surface. This also has low background binding and low intrinsic fluorescence and allows the immobilized proteins to retain function. Noncovalent binding of unmodified protein occurs within porous structures such as HydroGel™ (PerkinElmer, Wellesley, MA), based on a 3-dimensional polyacrylamide gel; this substrate is reported to give a particularly low background on glass microarrays, with a high capacity and retention of protein function. Widely used biological coupling methods are through biotin / streptavidin or hexahistidine / Ni interactions, having modified the protein appropriately. Biotin may be conjugated to a poly-lysine backbone immobilised on a surface such as titanium dioxide (Zyomyx) or tantalum pentoxide (Zeptosens, Witterswil, Switzerland).Array fabrication methods include robotic contact printing, ink-jetting, piezoelectric spotting and photolithography. A number of commercial arrayers are available [e.g. Packard Biosciences] as well as manual equipment [V & P Scientific], Bacterial colonies can be robotically gridded onto PVDF membranes for induction of protein expression in situ.At the limit of spot size and density are nanoarrays, with spots on the nanometer spatial scale, enabling thousands of reactions to be performed on a single chip less than 1mm square. BioForce Laboratories have developed nanoarrays with 1521 protein spots in 85sq microns, equivalent to 25 million spots per sq cm, at the limit for optical detection; their readout methods are fluorescence and atomic force microscopy (AFM).Fluorescence labeling and detection methods are widely used. The same instrumentation as used for reading DNA microarrays is applicable to protein arrays. For differential display, capture (e.g., antibody) arrays can be probed with fluorescently labeledDocket No. 103362-068WO1proteins from two different cell states, in which cell lysates are directly conjugated with different fluorophores (e.g. Cy-3, Cy-5) and mixed, such that the color acts as a readout for changes in target abundance. Fluorescent readout sensitivity can be amplified 10-100 fold by tyramide signal amplification (TSA) (PerkinElmer Lifesciences). Planar waveguide technology (Zeptosens) enables ultrasensitive fluorescence detection, with the additional advantage of no intervening washing procedures. High sensitivity can also be achieved with suspension beads and particles, using phycoerythrin as label (Luminex) or the properties of semiconductor nanocrystals (Quantum Dot). A number of novel alternative readouts have been developed, especially in the commercial biotech arena. These include adaptations of surface plasmon resonance (HTS Biosystems, Intrinsic Bioprobes, Tempe, AZ), rolling circle DNA amplification (Molecular Staging, New Haven CT), mass spectrometry (Intrinsic Bioprobes; Ciphergen, Fremont, CA), resonance light scattering (Genicon Sciences, San Diego, CA) and atomic force microscopy [BioForce Laboratories],Capture arrays form the basis of diagnostic chips and arrays for expression profiling. They employ high affinity capture reagents, such as conventional antibodies, single domains, engineered scaffolds, peptides or nucleic acid aptamers, to bind and detect specific target ligands in high throughput manner.Antibody arrays have the required properties of specificity and acceptable background, and some are available commercially (BD Biosciences, San Jose, CA; Clontech, Mountain View, CA; BioRad; Sigma, St. Louis, MO). Antibodies for capture arrays are made either by conventional immunization (polyclonal sera and hybridomas), or as recombinant fragments, usually expressed in E. coli, after selection from phage or ribosome display libraries (Cambridge Antibody Technology, Cambridge, UK; BioInvent, Lund, Sweden; Affitech, Walnut Creek, CA; Biosite, San Diego, CA). In addition to the conventional antibodies, Fab and scFv fragments, single V-domains from camelids or engineered human equivalents (Domantis, Waltham, MA) may also be useful in arrays.The term “scaffold” refers to ligand-binding domains of proteins, which are engineered into multiple variants capable of binding diverse target molecules with antibody -like properties of specificity and affinity. The variants can be produced in a genetic library format and selected against individual targets by phage, bacterial or ribosome display. Such ligand-binding scaffolds or frameworks include ‘Affibodies’ based on Staph, aureus protein A (Affibody, Bromma, Sweden), ‘Trinectins’ based on fibronectins (Phylos, Lexington, MA) and ‘Anticalins’ based on the lipocalin structure (Pieris Proteolab, Freising-Weihenstephan,Docket No. 103362-068WO1Germany). These can be used on capture arrays in a similar fashion to antibodies and may have advantages of robustness and ease of production.Nonprotein capture molecules, notably the single-stranded nucleic acid aptamers which bind protein ligands with high specificity and affinity, are also used in arrays (SomaLogic, Boulder, CO). Aptamers are selected from libraries of oligonucleotides by the Selex™ procedure and their interaction with protein can be enhanced by covalent attachment, through incorporation of brominated deoxyuridine and UV-activated crosslinking (photoaptamers). Photocrosslinking to ligand reduces the cross reactivity of aptamers due to the specific steric requirements. Aptamers have the advantages of ease of production by automated oligonucleotide synthesis and the stability and robustness of DNA; on photoaptamer arrays, universal fluorescent protein stains can be used to detect binding.Protein analytes binding to antibody arrays may be detected directly or via a secondary antibody in a sandwich assay. Direct labelling is used for comparison of different samples with different colors. Where pairs of antibodies directed at the same protein ligand are available, sandwich immunoassays provide high specificity and sensitivity and are therefore the method of choice for low abundance proteins such as cytokines; they also give the possibility of detection of protein modifications. Label- free detection methods, including mass spectrometry, surface plasmon resonance and atomic force microscopy, avoid alteration of ligand. What is required from any method is optimal sensitivity and specificity, with low background to give high signal to noise. Since analyte concentrations cover a wide range, sensitivity has to be tailored appropriately; serial dilution of the sample or use of antibodies of different affinities are solutions to this problem. Proteins of interest are frequently those in low concentration in body fluids and extracts, requiring detection in the picogram range or lower, such as cytokines or the low expression products in cells.An alternative to an array of capture molecules is one made through ‘molecular imprinting’ technology, in which peptides (e.g., from the C-terminal regions of proteins) are used as templates to generate structurally complementary, sequence-specific cavities in a polymerizable matrix; the cavities can then specifically capture (denatured) proteins that have the appropriate primary amino acid sequence (ProteinPrint™, Aspira Biosystems, Burlingame, CA).Another methodology which can be used diagnostically and in expression profiling is the ProteinChip® array (Ciphergen, Fremont, CA), in which solid phase chromatographic surfaces bind proteins with similar characteristics of charge or hydrophobicity from mixturesDocket No. 103362-068WO1such as plasma or tumor extracts, and SELDI-TOF mass spectrometry is used to detection the retained proteins.Large-scale functional chips have been constructed by immobilizing large numbers of purified proteins and used to assay a wide range of biochemical functions, such as protein interactions with other proteins, drug-target interactions, enzyme-substrates, etc. Generally, they require an expression library, cloned into A. coli, yeast or similar from which the expressed proteins are then purified, e.g. via a His tag, and immobilized. Cell free protein transcription / translation is a viable alternative for synthesis of proteins which do not express well in bacterial or other in vivo systems.For detecting protein-protein interactions, protein arrays can be in vitro alternatives to the cell-based yeast two-hybrid system and may be useful where the latter is deficient, such as interactions involving secreted proteins or proteins with disulfide bridges. High-throughput analysis of biochemical activities on arrays has been described for yeast protein kinases and for various functions (protein-protein and protein-lipid interactions) of the yeast proteome, where a large proportion of all yeast open-reading frames was expressed and immobilized on a microarray. Large-scale ‘proteome chips’ promise to be very useful in identification of functional interactions, drug screening, etc. (Proteometrix, Branford, CT).As a two-dimensional display of individual elements, a protein array can be used to screen phage or ribosome display libraries, in order to select specific binding partners, including antibodies, synthetic scaffolds, peptides and aptamers. In this way, ‘library against library’ screening can be carried out. Screening of drug candidates in combinatorial chemical libraries against an array of protein targets identified from genome projects is another application of the approach.A multiplexed bead assay, such as, for example, the BD™ Cytometric Bead Array, is a series of spectrally discrete particles that can be used to capture and quantitate soluble analytes. The analyte is then measured by detection of a fluorescence-based emission and flow cytometric analysis. Multiplexed bead assay generates data that is comparable to ELISA based assays, but in a “multiplexed” or simultaneous fashion. Concentration of unknowns is calculated for the cytometric bead array as with any sandwich format assay, i.e. through the use of known standards and plotting unknowns against a standard curve. Further, multiplexed bead assay allows quantification of soluble analytes in samples never previously considered due to sample volume limitations. In addition to the quantitative data, powerful visual imagesDocket No. 103362-068WO1can be generated revealing unique profiles or signatures that provide the user with additional information at a glance.The method disclosed herein shows that when an increase of IIM was measured in a subject, a therapeutically effective dose of an agent that inhibits one or more dysferlin autoantibodies is administered to the subject.In some embodiments, the agent that inhibits one or more dysferlin autoantibodies comprises a small molecule, a peptide, a protein, an antibody, an antibody fragment, or a combination thereof. In some embodiments, the agent that inhibits one or more dysferlin autoantibodies comprises a small molecule conjugated to an antibody or an antibody fragment. In some embodiments, the agent that inhibits one or more dysferlin autoantibodies comprises a peptide or protein conjugated to an antibody or an antibody fragment.In some embodiments, the agent that inhibits one or more dysferlin autoantibodies comprises an immunosuppressive agent. In some embodiments, the agent that inhibits one or more dysferlin autoantibodies is administered in combination with an additional immunosuppressive agent. In some embodiments, the immunosuppressive agent is selected from corticosteroids, mycophenolate mofetil, azathioprin, methotrexate, cyclophosphamide, rituximab, tocilizumab, or a combination thereof. In some embodiments, the immunosuppressive agent comprises methotrexate. In some embodiments, the immunosuppressive agent comprises azathioprine. In some embodiments, the immunosuppressive agent comprises rituximab.In some embodiments, the immunosuppressive agent comprises a corticosteroid. In some embodiments, the corticosteroid is selected from alclometasone dipropionate, amcinonide, amcinafel, amcinafide, beclamethasone, betamethasone, betamethasone dipropionate, betamethasone valerate, clobetasone propionate, chloroprednisone, clocortelone, cortisol, cortisone, cortodoxone, difluorosone diacetate, descinolone, desonide, defluprednate, dihydroxy cortisone, desoximetasone, dexamethasone, deflazacort, diflorasone, diflorasone diacetate, dichlorisone, esters of betamethasone, fluazacort, flucetonide, flucloronide, fludrotisone, fluorocortisone, flumethasone, flunisolide, fluocinonide, fluocinolone, fluocinolone acetonide, flucortolone, fluperolone, fluprednisolone, fluroandr enol one acetonide, fluocinolone acetonide, flurandrenolide, fluorametholone, fluticasone propionate, hydrocortisone, hydrocortisone butyrate, hydrocortisone valerate, hydrocortamate, loteprendol, medrysone, meprednisone, methylprednisone, methylprednisolone, 6-methylprednisolone, mometasone furoate, paramethasone, paramethasone acetate, prednisone,Docket No. 103362-068WO1prednisolone, prednidone, predni carb ate, triamcinolone acetonide, triamcinolone hexacatonide, tixocortol prednisolone, triamcinolone, or pharmaceutically acceptable salts thereof, derivatives thereof, mixtures thereof, or any combination thereof. In some embodiments, the corticosteroid is selected from prednisolone, methylprednisolone, cortisone, dexamethasone, betamethasone, or hydrocortisone. In some embodiments, the corticosteroid comprises prednisone. In some embodiments, the corticosteroid comprises methylprednisone.In one aspect, provided herein is a method of treating IIM in a subject in need thereof, comprising: (i) obtaining a sample from the subject; (ii) measuring level of one or more dysferlin autoantibodies in the sample, wherein an increase in the level of one or more dysferlin autoantibodies compared to a control indicates the presence of IIM or risk thereof in the subject; and (iii) if an increase in the level of one or more dysferlin autoantibodies is observed in the sample compared to a control, then administering to the subject a therapeutically effective amount of an immunosuppressive agent.In one aspect, provided herein is a method of treating IIM in a subject in need thereof, comprising: (i) obtaining a sample from the subject; (ii) measuring level of dysferlin in the sample, wherein a decrease in the level of dysferlin compared to a control indicates the presence of IIM or risk thereof in the subject; and (iii) if a decrease in the level of dysferlin is observed in the sample compared to a control, then administering to the subject a therapeutically effective amount of an agent that inhibits one or more dysferlin autoantibodies.In one aspect, provided herein is a method of treating IIM in a subject in need thereof, comprising: (i) obtaining a sample from the subject; (ii) measuring level of dysferlin in the sample, wherein a decrease in the level of dysferlin compared to a control indicates the presence of IIM or risk thereof in the subject; and (iii) if a decrease in the level of dysferlin is observed in the sample compared to a control, then administering to the subject a therapeutically effective amount of an immunosuppressive agent.In some embodiments, the level of one or more dysferlin autoantibodies in the sample is increased by at least about 10%, for example, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, or more, compared to a control.In some embodiments, the level of dysferlin in the sample is decreased by at least about 10%, for example, at least about 15%, at least about 20%, at least about 25%, at least aboutDocket No. 103362-068WO130%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, or more, compared to a control.In some embodiments, the method produces at least about a 50% decrease (for example, at least about a 60% decrease, at least about a 70% decrease, at least about an 80% decrease, at least about a 90% decrease, or more) in disease severity of a subject treated with an agent that inhibits one or more dysferlin autoantibodies compared to an untreated subject.In one aspect, disclosed herein is a method of diagnosing IIM in a subject, comprising, obtaining a sample from the subject and measuring level of one or more dysferlin autoantibodies. In some embodiments, an increase in the level of one or more dysferlin autoantibodies compared to a control indicates the presence of IIM or risk thereof in the subject. In some embodiments, at least one additional biomarker is also measured in the subject. In some embodiments, a threshold level is established, and wherein, when the level of one or more dysferlin autoantibodies increases above that threshold level, it is indicative of the presence of IIM or the progression of IIM in a subject who has already been diagnosed with IIM.In one aspect, provided herein is a method of diagnosing IIM in a subject in need thereof, comprising: (i) obtaining a sample from the subject; and (ii) measuring level of dysferlin in the sample, wherein a decrease in the level of dysferlin compared to a control indicates the presence of IIM or risk thereof in the subject.In some embodiments, the threshold level of one or more dysferlin autoantibodies is at least about 0.5 optical density (O.D.) at 650 nanometers (nm) as determined by ELISA, for example at least about 0.55 O.D. at 650 nm, at least about 0.60 O.D. at 650 nm, at least about 0.65 O.D. at 650 nm, at least about 0.70 O.D. at 650 nm, at least about 0.75 O.D. at 650 nm, at least about 0.80 O.D. at 650 nm, at least about 0.85 O.D. at 650 nm, at least about 0.90 O.D. at 650 nm, at least about 0.95 O.D. at 650 nm, at least about 1.00 O.D. at 650 nm, at least about 1.10 O.D. at 650 nm, at least about 1.20 O.D. at 650 nm, at least about 1.30 O.D. at 650 nm, at least about 1.40 O.D. at 650 nm, at least about 1.50 O.D. at 650 nm, or more than 1.50 O.D. at 650 nm.In some embodiments, the agent that inhibits one or more dysferlin autoantibodies when administered to a subject with IIM, increases membrane repair even in the presence of a dysferlin antibody, compared to a control not administered with the agent. In some embodiments, the agent that inhibits one or more dysferlin autoantibodies when administeredDocket No. 103362-068WO1to a subject with EM, decreases the severity of myositis when compared to a control not administered with the agent.In some embodiments, the agent that inhibits one or more dysferlin autoantibodies is formulated in a delivery vehicle.In some embodiments, the delivery vehicle comprises a liposome, a lipid nanoparticle, an exosome, an extracellular vesicle, a viral particle, or a combination thereof.In some embodiments, the delivery vehicle comprises exosomes. Exosomes are cell-derived vesicles that are present in many and perhaps all eukaryotic fluids. Exosomes contain RNA, proteins, lipids and metabolites that is reflective of the cell type of origin. The reported diameter of exosomes is between 30 and 100 nm. Exosomes are either released from the cell when multivesicular bodies fuse with the plasma membrane or released directly from the plasma membrane.In some embodiments, the delivery vehicle comprises extracellular vesicles. As used herein, the term "extracellular vesicle" includes, but is not limited to, all vesicles released from cells by any mechanism. “Extracellular vesicles” includes exosomes which are released from multivesicular bodies and microvesicles that are shed from the cell surface. “Extracellular vesicles” includes vesicles created by exocytosis or ectocytosis. “Extracellular vesicles” encompasses exosomes released from multivesicular bodies, vesicles released by reverse budding, fission of membrane(s), multivesicular endosomes, ectosomes, microvesicles, microparticles, and vesicles released by apoptotic bodies, and hybrid vesicles containing plasma membrane components. Extracellular vesicles can contain proteins, nucleic acids, lipids, and other molecules common to the originating cell.In some embodiments, the agent that inhibits one or more dysferlin autoantibodies is comprised and delivered to a subject via a delivery vehicle comprising lipids such as lipid nanoparticles or liposomes, such as cationic liposomes (e.g., DOTMA, DOPE, DC-cholesterol) or anionic liposomes. Liposomes can further comprise proteins to facilitate targeting a particular cell, if desired. Administration of a composition comprising the agent that inhibits one or more dysferlin autoantibodies as disclosed herein and a cationic liposome can be administered to the blood afferent to a target organ or inhaled into the respiratory tract to target cells of the respiratory tract. Regarding liposomes, see, e.g., Brigham et al. Am. J. Resp. Cell. Mol. Biol. 1 :95-100 (1989); Feigner et al. Proc. Natl. Acad. Sci USA 84:7413-7417 (1987); U.S. Pat. No. 4,897,355. Furthermore, the agent that inhibits one or more dysferlin autoantibodies can be administered as a component of a microcapsule that can be targeted toDocket No. 103362-068WO1specific cell types, or where the diffusion of the agent that inhibits one or more dysferlin autoantibodies or delivery of the agent that inhibits one or more dysferlin autoantibodies from the microcapsule is designed for a specific rate or dosage.In some embodiments, the delivery of the agent that inhibits one or more dysferlin autoantibodies can be via a liposome, using commercially available liposome preparations such as LIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, MD), SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison, WI), as well as other liposomes developed according to procedures standard in the art.In some embodiments, the agent that inhibits one or more autoantibodies is formulated in an oil-in-water emulsion, a nano-emulsion, a nanoparticle, an excipient, a diluent, a salt, a buffer, a stabilizer, a lipid, or combinations thereof.In some embodiments, the subject is a human. In some embodiments, the human subject is a child. In some embodiments, the human subject is an adult.A number of embodiments of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.By way of non-limiting illustration, examples of certain embodiments of the present disclosure are given below.EXAMPLESThe following examples are set forth below to illustrate the compositions, devices, methods, and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled in the art.Example 1: Antibody biomarker to diagnose idiopathic inflammatory myopathies Across the IIMs the average range of symptom onset is 45-60 years for adults and 5-15 years for juvenile dermatomyositis. The prevalence of IIM varies in different population, with a range of 14 to 21 per 100,000 patients with higher prevalence for older patients and particularly in women who comprise approximately two thirds of all IIM patients.Docket No. 103362-068WO1One challenge stemming from the heterogeneity of these diseases is the variation in the autoantibodies that appear in patient samples. Even the most commonly known myositis associated autoantibodies, such as Jo-1, are seen in approximately 20-25% of myositis patients. Finding new antibodies that appear in a majority of IIM patients would provide a much more effective diagnostic tool to diagnose IIM patients and get them the treatment they require more rapidly. To find new autoantibodies that might be more common in IIM patients, it was tested to see if antibodies against proteins involved in the sarcolemmal membrane repair response appear in IIM patients and if such antibodies could contribute to progression of IIM. The sarcolemmal membrane repair response is a conserved response necessary to restore membrane integrity in myocytes in a variety of muscle diseases. Previous published studies helped identify TRIM72 / MG53 and dysferlin proteins as critical components of the membrane repair process. Additional published studies showed that autoantibodies against TRIM72 / MG53 are found in IIM patients and that these antibodies could compromise sarcolemmal membrane repair and contribute to the progression of IIM.It is surprisingly shown herein that autoantibodies against dysferlin are very common in IIM patients and that the levels of dysferlin antibodies are linked with the severity and the progression of the disease. These findings support:1) Detection dysferlin antibodies could be used as a biomarker for diagnosis of IIM. Since most of the existing biomarkers are found in 25% of patients or less, these highly abundant antibodies that show at least some specificity towards myositis would be a significant step forward in diagnosing IIM and improve patient health by allowing for earlier and more effective treatment.2) Dysferlin autoantibodies could be targeted for the treatment of IIM.Dysferlin autoantibodies can contribute to the progression of IIM. Efforts to block the action of, or reduce the levels of, dysferlin autoantibodies are an effective treatment for the IIM.These uses for dysferlin autoantibodies are supported by data presented herein, where it is established by direct ELISA that autoantibodies against dysferlin are elevated in IIM patient sera and find that exogenous delivery of IIM positive patient serum compromises sarcolemma repair in healthy skeletal muscle. Moreover, the injection of exogenous antibodies against membrane repair proteins can increase muscle pathology in this adoptive transfer mouse model of IIM. These findings represent a novel mechanism contributing to theDocket No. 103362-068WO1pathogenesis of IIM when decreased sarcolemma integrity induces a vicious cycle of antigen presentation that directly contributes to the pathophysiology of idiopathic immune myopathies.Results:Autoantibodies against dysferlin are specifically found in myositis patient serum samples.To expand on the findings that TRIM72 antibodies are present in IIM patients, an ELISA was developed to measure the levels of dysferlin autoantibodies in human myositis patient serum samples by first coating wells with full length recombinant human dysferlin protein and then applying myositis patient and healthy control serum samples in various dilutions. ELISA screening showed a remarkable elevation in the level of dysferlin antibodies in IIM patients (FIG. 8A). This percentage of patients harboring dysferlin antibodies are far in excess of the percentage of patients that are positive for the known myositis associated autoantibodies, such as Jo-1 which are seen in just over 20% of myositis patients. It is also seen that these higher levels of dysferlin autoantibodies in myositis patient serum correlate with higher levels of creatine kinase (CK) (FIG. 8B). This directly links elevated dysferlin antibodies with the most widely used diagnostic enzyme marker for muscle disease pathology, further supporting the hypothesis that antibodies against repair proteins accelerate myositis development. These results show that dysferlin autoantibodies are highly abundant in IIM patients at levels in excess of other known clinical markers of IIM. It is also found that this increase in dysferlin antibodies is specifically linked to IIM. Using the same ELISA approach to measure the levels of dysferlin autoantibodies, the level of dysferlin autoantibodies was tested in the serums of patients with systemic lupus erythematosus (SLE) and another set of SLE patients with myocarditis complications. It was found that high levels of dysferlin autoantibodies are only found in the IIM patients (FIG. 9), indicating that elevated dysferlin auto antibodies is not a general effect of autoimmune disease but is rather a more specific marker of IIM that can be linked to the severity of the disease progression.Dysferlin antibodies are sufficient to compromise membrane repair in normal muscle. These data show that antibodies against dysferlin are present in myositis patient serum (FIG. 8A-8B). Given the importance of dysferlin in skeletal muscle sarcolemmal membrane repair, it was hypothesized that these autoantibodies could be one mechanism that contributes to compromised membrane repair in myositis muscle. To begin to test this hypothesis, first examined were the effects of antibodies against dysferlin on membrane repair in wild typeDocket No. 103362-068WO1(WT) mouse muscle fibers. Purified commercial rabbit polyclonal antibody against dysferlin was applied to the extracellular buffer solution surrounding FDB muscles dissected from WT C57B1 / 6J mice immediately before multi-photon laser wounding to the sarcolemmal membrane. Application of dysferlin antibody decreased membrane repair, while control pre-immune rabbit serum had no effects on membrane repair (FIG. 10A-10B). These data indicate that antibodies in this mouse model can compromise repair and indicating that anti-dysferlin antibodies are sufficient to compromise membrane repair.Dysferlin antibodies are sufficient to compromise membrane repair in normal muscle. These data show that anti-dysferlin antibodies are sufficient to compromise membrane repair (FIGs. 10A-10B). In order to address the possibility that dysferlin antibodies can accelerate inflammatory myositis phenotype in myositis, additional anti-dysferlin antibodies were injected into a mouse model of IIM (Rag-1- / - mice that were the recipient of IP lymph node cells from FoxP3- / y mice). This injection began at 6 weeks before adoptive transfer as these mice tend to begin to die 4-6 weeks after adoptive transfer. Injected mice were sacrificed at 4 weeks after adoptive transfer. These results indicate that injection of dysferlin antibodies into Rag-1- / - recipient of IP FoxP3- / y lymph node cells further compromises muscle membrane repair, increases the incidence of inflammation in muscle tissues (FIGs. 11 A-l IB) and elevates serum CK levels (FIG. 11C). These findings provide direct evidence that elevated antibodies against the membrane repair protein dysferlin are sufficient to enhance the myositis phenotype in a T reg deficient milieu.It will be apparent to those skilled in the art that various modifications and variations can be made in the present disclosure without departing from the scope or spirit of the invention. Other embodiments of the disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the methods disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Claims

Docket No. 103362-068WO1 CLAIMSWhat is claimed is:

1. A method of treating idiopathic inflammatory myopathy (IIM) in a subject in need thereof, comprising:(i) obtaining a sample from the subject;(ii) measuring level of one or more dysferlin autoantibodies in the sample, wherein an increase in the level of one or more dysferlin autoantibodies compared to a control indicates the presence of IIM or risk thereof in the subject; and(iii) if an increase in the level of one or more dysferlin autoantibodies is observed in the sample compared to a control, then administering to the subject a therapeutically effective amount of an agent that inhibits one or more dysferlin autoantibodies.

2. The method of claim 1, wherein the control comprises a standardized level of dysferlin antibody from a subject without IIM.

3. The method of claim 1 or 2, wherein the control comprises a sample from the subject at an earlier time period.

4. The method of any one of claims 1-3, wherein the agent that inhibits one or more dysferlin autoantibodies is selected from a small molecule, a peptide, a protein, an antibody, an antibody fragment, or a combination thereof.

5. The method of any one of claims 1-4, wherein the agent that inhibits one or more dysferlin autoantibodies is formulated in a delivery vehicle.

6. The method of claim 5, wherein the delivery vehicle comprises a liposome, a lipid nanoparticle, an exosome, an extracellular vesicle, a viral particle, or a combination thereof.

7. The method of any one of claims 1-6, wherein the sample comprises blood or any blood component.Docket No. 103362-068WO18. The method of claim 7, wherein the blood component comprises serum or plasma.

9. The method of any one of claims 1-8, wherein the method produces at least a 50% decrease in disease severity of a subject treated with an agent that inhibits one or more dysferlin autoantibodies compared to an untreated subject.

10. The method of any one of claims 1-9, wherein the method further comprises measuring the level of dysferlin in the sample, wherein a decrease in the level of dysferlin compared to a control indicates the presence of IIM or risk thereof in the subject.

11. A method of diagnosing idiopathic inflammatory myopathy (IIM) in a subject in need thereof, comprising:(i) obtaining a sample from the subject; and(ii) measuring level of one or more dysferlin autoantibody in the sample, wherein an increase in the level of one or more dysferlin autoantibodies compared to a control indicates the presence of IIM or risk thereof in the subject.

12. The method of claim 11, wherein at least one additional biomarker is also measured in the subject.

13. The method of claim 11 or 12, wherein the method further comprises measuring the level of dysferlin in the sample, wherein a decrease in the level of dysferlin compared to a control indicates the presence of IIM or risk thereof in the subject.

14. The method of any one of claims 11-13, wherein a threshold level is established, and wherein, when the level of dysferlin antibody is above that level, it is indicative of the presence of IIM or the progression of IIM in a subject who has already been diagnosed with IIM.

15. The method of any one of claims 1-14, wherein the IIM is selected from polymyositis (PM), dermatomyositis (DM), anti synthetase syndrome (ASyS), juvenile dermatomyositis (JDM), inclusion body myositis (IBM), immune-mediated necrotizing myopathy (IMNM), or other classifications of IIM.Docket No. 103362-068WO116. The method of any one of claims 1-15, wherein the subject is a human.

17. The method of claim 16, wherein the human subject is a child or an adult.

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