TITLE

COMPOSITIONS AND METHODS TO DETECT AUTOIMMUNE ANTIBODIES

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of the U.S. provisional application number 63/358,415 filed July 5, 2022, the disclosure of which is herein incorporated by reference in its entirety.

ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under DA042072, DA047325, and NS120496 awarded by the National Institutes of Health. The government has certain rights in the invention.

SEQUENCE LISTING

[0003] This application contains a Sequence Listing that has been submitted in WIPO ST.26 .xml format via EFS-Web and is hereby incorporated by reference in its entirety. The .xml copy is named “106546-761032 UTSD 4049. xml" and is 248.7 KB in size.

BACKGROUND

[0004] 1. Field

[0005] The present disclosure provides methods, compositions, and kits for diagnosing autoimmune diseases of the nervous system.

[0006] 2. Discussion of Related Art

[0007] Autoimmunity in the nervous system can cause diverse pathologies and diseases including encephalitis, muscle weakness, and seizures. These autoimmune diseases largely result from improper interactions of the immune system with endogenous proteins, for instance antibodies made by self, interacting with endogenous proteins. A subset of autoreactive antibodies targets a family of cell-surface proteins called Cys-loop receptors. These ligand- gated ion channels include for example the cation selective nicotinic acetylcholine and 5-HTs serotonin receptors, and anion-selective GABA _A and glycine receptors. Additional autoimmune targets of the nervous system associated with disease include glutamate family receptors including NMDA, AMPA and kainate receptors. Antibodies targeting glutamate, glycine or GABA _A receptors have been linked to encephalitis and epileptic seizures; antibodies against neuronal nicotinic receptors can cause dysautonomia; antibodies against the muscle- type nicotinic receptor cause Myasthenia gravis.

[0008] However, rapid and accurate diagnosis of these autoimmune conditions has been challenging due to overlapping presentations with other disease conditions caused by infections, tumors, brain damage and various genetic disorders. Easily implementable and optimized diagnostics specifically geared towards autoimmune diseases of the nervous system could greatly inform selection of treatment options and improve outcomes for patients.

SUMMARY

[0009] In some aspects, the current disclosure encompasses a method of diagnosing a subject in need thereof with an autoimmune disease of the nervous system, the method comprising: contacting a fluid sample obtained from the subject with a recombinant receptor molecule, wherein the recombinant reporter molecule specifically binds with an antibody in the sample to comprise an antibody-reporter complex; quantifying a level of the antibody- receptor complex; and diagnosing the subject with the autoimmune disease if the level of the antibody-receptor complex exceeds a threshold.

[0010] in some aspects, the current disclosure also encompasses a method for detecting the presence of an autoantibody in a subject, comprising: contacting a fluid sample obtained from the subject with a recombinant receptor molecule, wherein the recombinant receptor molecule specifically binds with the autoantibody in the sample to comprise an antibody-receptor complex; and detecting a level of specific antibody-receptor complex.

[0011] In some aspects, the current disclosure encompasses methods of monitoring an autoimmune disease of the nervous system, the method comprising: contacting a fluid sample obtained from the subject with a recombinant receptor molecule, wherein the recombinant receptor molecule specifically binds with the autoantibody in the sample to comprise an antibody- receptor complex; quantifying a level of the antibody-receptor complex; and monitoring the subject's autoimmune disease based on the level of the antibody-receptor.

[0012] In some aspects, of the methods provided herein, the recombinant receptor molecule comprises a nicotinic acetylcholine receptor polypeptide (neuronal or muscle-type), an NMDA- type glutamate receptor polypeptide, a GABA _A receptor polypeptide or derivatives, a glycine- receptor polypeptide, an ionotropic serotonin receptor polypeptide or derivatives, a muscle- specific kinase polypeptide, leucine-rich glioma-inactivated 1 polypeptide, fragments, and variants thereof.

[0013] In some aspects, of the methods provided herein, the threshold is set based on levels of non-specific receptor complexes formed with fluid samples from subjects not suffering from the autoimmune disease.

[0014] In some aspects, of the methods provided herein, the recombinant receptor molecule further comprises a reporter, in some aspects, of the methods provided herein the reporter is a fluorescent polypeptide. In some aspects, of the methods provided herein, the reporter is a fluorescent polypeptide for example, Green Fluorescent Protein (GFP), eGFP, Red Fluorescent Protein (RFP), Teal Fluorescent Protein (TFP), Blue Fluorescent Protein (BFP), Yellow Fluorescent Protein (YFP), miRFP, cerulean fluorescent protein (CFP), eCyanFP and functional variants thereof. In some aspects, the reporter is a fluorescent small molecule.

[0015] In some aspects, of the methods provided herein, the recombinant receptor molecule comprises a polypeptide with an amino acid sequence at least 80% identical to any one of SEQ ID NOS. 1-54, or a derivative, fragment, or a variant thereof. In some aspects, the recombinant receptor molecule comprises a polypeptide with an amino acid sequence at least 80% identical to any one of SEQ ID NOS. 62-136 , or a derivative, fragment, or a variant thereof

[0016] In some aspects, of the methods provided herein, the detection step comprises the use of a size exclusion column. In some aspects, the detection step comprises the use of a fluorescence detector. In some aspects, the detection step comprises the use of a fluorescence size exclusion chromatography system (FSEC)

[0017] In some aspects, of the methods provided herein, the subject is a mammal. In some aspects, the mammal is a human. In some aspects, of the methods provided herein, the fluid sample is cerebrospinal fluid, blood, serum or plasma from a human.

[0018] In some aspects, the current disclosure also encompasses a recombinant polypeptide comprising a recombinant receptor sequence or a variant thereof that binds specifically to one or more autoantibody(ies) for use in the diagnosis of autoimmune disorders of the nervous system. In some aspects, the recombinant receptor sequence comprises a neuronal nicotinic acetylcholine receptor polypeptide, muscle nicotinic acetylcholine receptor polypeptide, ganglionic nicotinic acetylcholine receptor polypeptide, an NMDA-type glutamate receptor polypeptide, a GABA _A receptor polypeptide or derivatives, a glycine-receptor polypeptide, an ionotropic serotonin receptor polypeptide or derivatives, a muscle-specific kinase polypeptide, leucine-rich glioma-inactivated 1 polypeptide, fragments, and variants thereof. In some aspects, the recombinant receptor sequence or variant thereof further comprises a reporter.

[0019] In some aspects, the reporter is a fluorescent polypeptide, non-limiting examples of which include GFP, eGFP, RFP, TFP, BFP, YFP, cerulean fluorescent protein, and cyan fluorescent protein and functional variants thereof.

[0020] In some aspects, the polypeptide comprises an amino acid sequence at least 80% identical to any one of SEQ ID NOS. 55-61, or a derivative, fragment, or a variant thereof. In some aspects, the recombinant receptor molecule comprises a polypeptide with an amino acid sequence at least 80% identical to any one of SEQ ID NOS. 1-54, or a derivative, or fragment thereof. In some aspects, the recombinant receptor molecule comprises a polypeptide with an amino acid sequence at least 80% identical to any one of SEQ ID NOS. 62-136, or a derivative, fragment, or a variant thereof. In some aspects, the current disclosure also encompasses use of the recombinant polypeptide for detection of autoantibodies that bind specifically to the recombinant polypeptide in a fluid sample from a subject, wherein the autoantibodies form an antibody-receptor complex and wherein the presence of the antibody-receptor complex above a threshold is used for in vitro diagnosis of an autoimmune disease in the subject.

[0021] In some aspects, the current disclosure also encompasses a kit comprising: a recombinant receptor molecule as disclosed herein; reagents; and instructions for use. In some aspects, the devices and reagents to obtain a fluid sample from a subject in need thereof are also included with the kit. In some aspects, the fluid sample is cerebrospinal fluid, blood, plasma or serum. In some aspects, the subject is a human.

[0022] Other aspects and iterations of the disclosure are recited herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to the drawing in combination with the detailed description of specific embodiments presented herein.

[0024] FIG. 1 A shows that patient-derived antibodies bind to the α1 β2y2 GABA _A receptor. Purified GABA _A receptor was reconstituted in nanodiscs in the absence or presence of each monoclonal antibody (mAb) indicated and analyzed by size exclusion chromatography.

[0025] FIG. 1 B shows binding of Fab ₁₀₁ fragment produced from the mAbs to GABA _A receptor in nanodiscs. Increasing amounts of Fab were added to GABA _A receptor to determine at which point the elution volume shift is saturated.

[0026] FIG. 1C shows binding of Fab ₁₁₅ fragment produced from the mAbs to GABA _A receptor in nanodiscs. Increasing amounts of Fab were added to GABA _A receptor to determine at which point the elution volume shift is saturated.

[0027] FIG. 1 D shows binding of Fab ₁₁₅ fragment produced from the mAbs to GABA _A receptor in nanodiscs. Increasing amounts of Fab were added to GABA _A receptor to determine at which point the elution volume shift is saturated.

[0028] FIG. 1 E shows binding of Fab ₂₀₁ fragment produced from the mAbs to GABA _A receptor in nanodiscs. Increasing amounts of Fab were added to GABA _A receptor to determine at which point the elution volume shift is saturated.

[0029] FIG. 1F shows immunofluorescence staining of selected GABA _A Fabs (green) on PFA- fixed murine brain tissue sections shown in the CA3 region of the hippocampus with DRAQ5 stained nuclei (red). Representative scale bar indicates 50 pm.

[0030] FIG. 2A shows FSEC traces of receptor alone (GABA _A trace) and receptor plus Fab ₁₁₅ (Fab- a trace). A shift to the left indicates an increase in size associated with binding.

[0031] FIG. 2B shows FSEC trace of the final material applied to EM grids. Inset shows Coomassie-stained SDS-PAGE gel of the complex.

[0032] FIG. 3 shows side and top views of the cryo-EM structure of Fab ₁₁₅ : GABA _A complex. Subunits and Fab densities are indicated. The constant region of the Fab is not shown as this peripheral domain is flexible and conformationally disordered.

[0033] FIG. 4A is a cryo-EM structure model showing that Fab ₁₁₅ competes directly with neurotransmitter binding. The figure shows side and top views of receptor and Fab atomic models. Boxes indicate expanded regions shown in Fig. 4B and Fig. 4C

[0034] FIG. 4B shows Fab:receptor interactions in the neurotransmitter binding site. Key amino acids are highlighted; dashed lines indicate electrostatic interactions closer than 4 A.

[0035] FIG. 4C shows the anchoring interaction between Fab ₁₁₅ and GABA _A . Residues shown are an alignment of key interacting residues of a subunits; red letters indicate potential specificity- determining residues.

[0036] FIG. 5 shows alignment of GABA _A receptor subunits contacted by autoantibodies. Amino acid sequence alignments of GABA _A subunits contacted by Fab ₁₁₅ and Fab ₁₇₅ are shown. On the right-hand side of the figure are cartoons indicating the location of the antibodies bound (yellow is Fab ₁₁₅ . red is Fab ₁₇₅ ). Subunits shown in the alignment for each box are colored.

[0037] FIG. 6A shows somatic hypermutations of Fab ₁₁₅ compared to receptor contacts. The alignments between sequences of Fab ₁₁₅ and its presumed germline ancestor are shown. Only the variable domain is shown. CDR1 , CDR2, and CDR3 are indicated in bold for each antibody segment. Somatic hypermutations are indicated by noting the parental sequence in the germline. Residues of the V(D)J junctions are underlined. Note, these residues cannot reliably be differentiated from germline ancestors. Hence assignment of somatic hypermutations is limited to

V and J genes only, and thus not for most part of the highly variable CDR3. Residues of the heavy and light chains that contact the GABA _A receptor are indicated and color coded based on subunit. The NG-KR mutations discussed in the text are indicated by asterisks (*).

[0038] MG. 6B shows somatic hypermutations of Fab ₁₇₅ compared to receptor contacts. The alignments between sequences of Fab ₁₇₅ and its presumed germline ancestor are shown. Only the variable domain is shown. CDR1 , CDR2, and CDR3 are indicated in bold for each antibody segment. Somatic hypermutations are indicated by noting the parental sequence in the germline. Residues of the V(D)J junctions are underlined. Note, these residues cannot reliably be differentiated from germline ancestors. Hence assignment of somatic hypermutations is limited to

V and J genes only, and thus not for most part of the highly variable CDR3. Residues of the heavy and light chains that contact the GABA _A receptor are indicated and color coded based on subunit. The NG-KR mutations discussed in the text are indicated by asterisks (*).

[0039] MG. 7 shows side and top views of the Cryo-EM structure of Fab ₁₇₅ : GABA _A complex. Subunits and Fab densities are indicated. The constant region of the Fab is not shown as this peripheral domain is flexible and conformationally disordered.

[0040] FIG. 8 shows that Fab ₁₇₅ binds the a-y interface of the GABA _A receptor. Panel a is an overview of model with area shown in panel b indicated. Panel b shows the amino acid interactions between Fab ₁₇₅ and the α1-γ2 interface of the GABA _A receptor. Panel c shows the interactions between the lower part of the Fab and the α1 benzodiazepine binding pocket Loop C. [0041] FIG. 9A shows a side view of Fab ₁₁₅ -bound, Fab175-bound , and GABA-bound (gray, PDB 6X3Z). Boxes indicate areas represented in indicated Fig. 9 panels.

[0042] FIG. 9B shows zoomed in image of Loop C displacement at a β2-α1 interface.

[0043] FIG. 9C shows the top view of the ECD comparing Fab ₁₁₅ -bound vs Fab ₁₁₅ -bound. Arrows indicate bindings site of Fab ₁₁₅ and Fab ₁₁₅ .

[0044] FIG. 9D is same as in Fig. 9C, but comparing cylinder representations of the TMD.

[0045] FIG. 9E shows the top view of the ECD comparing Fab ₁₁₅ -bound vs GABA-bound. The box provides cylinder representations of the TMD.

[0046] FIG. 9F shows Fab175 docked into GABA-bound receptor structure (6X3Z); spheres show side chains that clash.

[0047] FIG. 9G shows an ion channel pore radius analysis for a panel of GABA _A receptor structures. GABA _A + Fab ₁₁₅ and GABA _A + Fab ₁₁₅ , this study. 6I53 and 6X3Z (PDB structures: Kim et al., 2020; Laverty et al., 2019) are agonist-bound. 6HUJ and 6X3S are antagonist-bound (PDB structures: Kim et al., 2020; Masiulis et al., 2019).

[0048] FIG. 10 shows an FSEC-based assay to measure binding of patient antibodies to α1β2y2 GABA _A receptor. Titration of laboratory antibody on α1β2y2 GABA _A GFP fusion. Protein without (1) antibody is shown as a reference for pentamer elution time. Dilutions of laboratory antibody (2,3,4) decrease the main pentameric peak height in a dose-dependent manner.

[0049] FIG. 11 shows the use of FSEC assay to measure anti-Cys-loop receptor antibodies from patient blood serum. FSEC profile of the α1β2γ2 GABA _A receptor tagged with GFP on y2 is shown. GABA _A receptors are one known antibody target in autoimmune encephalitis (AE) patients. Black trace in the line graph shows the negative control antibody (unbound). Pink trace corresponds to the suspected GABA _A -AE patient antibody complex. Right-hand bar graph shows quantification from multiple patients including the AE patient shown in traces on left. Each circle represents one experimental replicate.

[0050] FIG. 12 shows an FSEC-based assay to measure binding of patient antibodies to the α3β4 nicotinic acetylcholine receptor. Titration of laboratory antibody on α3β4 GFP fusion is shown. Protein without (1) antibody is shown as a reference for pentamer elution time. Dilutions of laboratory antibody (2,3,4) decrease the pentameric receptor peak height in a dose-dependent manner. [0051] FIG. 13 shows the use of FSEC assay to measure anti-Cys-loop receptor antibodies from patient blood serum. FSEC profile of the α3β4 nicotinic acetylcholine receptor tagged with GFP on 34 is shown. This subtype is targeted by antibodies in patients with autoimmune autonomic ganglionopathy (AAG); decrease of main receptor peak in presence of AAG patient (pink) but not negative control patient (NC, black) is shown. Right panel is quantification of decrease of pentamer peak height normalized to control samples. Each circle represents one experimental replicate.

[0052] FIG. 14 shows FSEC-based assay to measure binding of patient antibodies with Torpedo acetylcholine receptor, a proxy for the human muscle-type nicotinic acetylcholine receptor. Decrease of main receptor peak height after incubation with murine antibody sera from mouse immunized with receptor is shown. In this instance receptor is labeled with the fluorescent dye Oregon-Green 488.

[0053] FIG. 15 shows the use of FSEC assay to measure anti-Cys-icop receptor antibodies from patient blood serum. FSEC profile with the α7 subtype of the nicotinic receptor. At least four AAG patients, and the AE patient, have cross-reactive antibodies to this related but distinct subtype, as shown by decrease of main receptor peak height.

[0054] FIG. 16A is an image of a well of cells transfected with GFP-tagged gAChR on the α3 subunit.

[0055] FIG. 16B shows the first step of the fluorescent detection size-exclusion chromatography assay (FSEC), where clarified cell lysate is mixed with patient serum and incubated.

[0056] FIG. 16C provides the clarification step, where a second centrifugation precipitates receptors cross-linked by antibodies.

[0057] FIG. 16D shows a size exclusion chromatographic separation of complexes bound to antibody that did not precipitate out of solution.

[0058] FIG. 16E shows that only bound receptor fluorescence is measured.

[0059] FIG. 17A provides FSEC traces of fluorescence of receptor in the presence of increasing concentrations of laboratory antibody against α3β4 or with laboratory antibody against related GABA _A receptor (Ctrl, black trace). Elution times of aggregates/void (1), pentameric receptor (2), and small molecules including unbound antibodies (3) are indicated. Dashed line indicates elution timepoint of fluorescence measurement. [0060] FIG. 17B provides quantification of pentamer peak representing gAChR normalized to control antibody. Error bars indicate standard deviation of triplicate measurements.

[0061] FIG. 18A shows specificity of FSEC assay with serum from a representative patient with AAG reducing α3β4 AChR peak in the FSEC assay. The #3 peak is an indication of antibodies and other small molecules present in human serum.

[0062] FIG. 18B shows specificity of FSEC assay with serum from a representative patient with AAG reducing α3β4 AChR peak in the FSEC assay (Fig. 18A) but not the related α7 AChR. The #3 peak is an indication of antibodies and other small molecules present in human serum.

[0063] FIG. 18C shows specificity of FSEC assay with serum from a representative patient with AAG reducing α3β4 AChR peak in the FSEC assay (Fig. 18A) but not GABA _A . The #3 peak is an indication of antibodies and other small molecules present in human serum.

[0064] FIG. 19 shows the sensitivity of FSEC assay to detect AAG-associated antibodies. Graph of FSEC-based assay performed with serial dilutions of AAG patient samples. Sample was diluted with negative control serum.

[0065] FIG. 20A shows the specificity of detection of gAChR antibodies in AAG patients but not other autoimmune diseases. Negative control (NC), postural orthostatic tachycardia syndrome patients (POTS), autoimmune encephalitis patients (AE), and AAG patients were screened via FSEC assay for the presence of antibodies against the gAChR, α3β4. Asterisks indicate statistical significance via one-way ANOVA test of p<0.0001.

[0066] FIG. 20B shows data for AAG patients screened via FSEC for antibodies against the closely related α7 AChR.

[0067] FIG. 20C shows data for AAG patients screened via FSEC for antibodies against the closely related synaptic GABA _A receptor.

[0068] FIG. 21 provides a comparison of the established RIPA assay and FSEC. Patients screened by FSEC were also tested for gAChR by established RIPA. 95% confidence interval is 0.88 to 0.51.

[0069] FIG. 22A provides assay of patient 1 sampled over time. Patient 1 is a male, who first presented with symptoms at 60 years of age.

[0070] FIG. 22B provides assay of patient 2 sampled over time. Patient 2 is a female whose first sample was collected at age 78. [0071] FIG. 22C provides assay of patient 3 sampled over time. Patient 3 is a female whose first sample was collected at age 74.

[0072] FIG. 23A provides subunit specificity of encephalitis antibodies against GABA _A receptors. All three subunits of the major synaptic subtype were expressed and incubated with encephalitis patient sera (Patient 6, 2) or a negative control (1). GFP fluorescence indicates the assembly of all three subunits, as it is known the y subunit cannot assemble as a pentamer.

[0073] FIG. 23B provides subunit specificity of encephalitis antibodies against GABA _A receptors. Both a and p subunits are expressed, but not y. The a subunit is tagged with blue-fluorescent protein (BFP), and after incubation with P6 serum (2) the main pentameric peak is not decreased significantly versus the negative control (1), nor is it shifted to an earlier elution time.

[0074] The drawing figures do not limit the present inventive concept to the specific embodiments disclosed and described herein. The drawings are not necessarily to scale, emphasis instead being placed on clearly illustrating principles of certain embodiments of the present inventive concept.

DETAILED DESCRIPTION

[0075] The following detailed description references the accompanying drawings that illustrate various embodiments of the present inventive concept. The drawings and description are intended to describe aspects and embodiments of the present inventive concept in sufficient detail to enable those skilled in the art to practice the present inventive concept. Other components can be utilized, and changes can be made without departing from the scope of the present inventive concept. The following description is, therefore, not to be taken in a limiting sense. The scope of the present inventive concept is defined only by the appended claims, along with the full scope of equivalents to which such claims are entitled.

[0076] The current disclosure is based on the identification and purification of target antigens for autoimmune diseases of the nervous system and development and optimization of novel methods to use the identified targets for clinical and non-clinical applications. Multiple diseases conditions of the nervous system can now be attributed to malfunctioning of the immune system and presence of antibodies that target specific receptors in the brain. Non-limiting examples of these conditions include multiple sclerosis, acute disseminated encephalomyelitis, autoimmune encephalitis, acute myelitis, neuromyelitis optica, Guillain-Barré syndrome, progressive encephalomyelitis with rigidity and myoclonus, and myasthenia gravis. Rapid and accurate diagnosis of these autoimmune conditions has been challenging due to overlapping presentations with other disease conditions caused by infections, tumors, brain damage and various genetic disorders. The few methods available that can quantitatively identify the autoantibodies in patients are tedious to implement in clinical settings and hence are not used as the first line of diagnosis.

[0077] The current disclosure encompasses novel methods that combine gel-filtration and fluorescent detectors to quantify complexes between patient derived auto-antibodies and recombinant receptor molecules. The disclosure further encompasses compositions of recombinant polypeptides and derivative, fragment, or a variant thereof that can be used in these methods. Additionally, the current disclosure also encompasses kits that can be used in clinical and non-clinical settings for easy implementation of these methods.

I. Compositions: recombinant polypeptides and derivatives, fragments, or variants thereof

[0078] In some aspects, the current disclosure encompasses compositions comprising recombinant polypeptides that comprise epitopes that specifically interact with autoantibodies. In some aspects, the current disclosure encompasses purified recombinant polypeptides that can be used in the diagnosis of autoimmune diseases.

[0079] As used herein, the term “autoimmune disease”, relates to a condition that result from association of antibodies against self-polypeptides and proteins in a subject, resulting in destruction or abnormal functioning of these proteins. In some aspects, the autoimmune diseases of the current disclosure pertain to diseases of the nervous system. Non-limiting examples of autoimmune diseases of the nervous system include multiple sclerosis, acute disseminated encephalomyelitis, autoimmune encephalitis, acute myelitis, neuromyelitis optica, Guillain-Barre syndrome and myasthenia gravis. Patients exhibiting such autoimmune disease suffer from a range of mild to highly debilitating symptoms including fatigue, low grade fever, rashes, memory problems, confusion, lethargy, social anxiety, behavioral changes, psychosis, depression, mutism, cognitive dysfunction, nausea, vertigo, scintillating scotomas, headache, seizures, epilepsia partialis continue, status epilepticus, opsoclonus, opsoclonus-myoclonus, stiff-person syndrome, dystonic movements of the tongue, chorea affecting the limbs and trunk, progressive hemiparesis, ataxia, chorea affecting the limbs and trunk. These symptoms primarily affect the nervous system and, more specifically, are at least in part associated with defects in the central nervous system.

[0080] In some aspects, these diseases stem from the abnormal association or interaction of particular receptor proteins in the brain with the immune system, in particular autoantibodies. As used herein the term "autoantibody” is an antibody produced by the immune system of a subject that is directed against one or more of the subject's own proteins. In some particular aspects of the current disclosure, these antibodies target epitopes present on receptor proteins and polypeptides and fragments thereof, expressed in the nervous system. In some aspects, the receptor proteins may also be expressed in other parts of the body. In some aspects, the receptor protein may be predominantly expressed in the nervous system. In some aspects, the receptor protein may be exclusively expressed to the brain. Non-limiting examples of such receptor protein includes neuronal nicotinic acetylcholine receptor polypeptide, muscle nicotinic acetylcholine receptor polypeptide, ganglionic nicotinic acetylcholine receptor polypeptide, an NMDA-type glutamate receptor polypeptide, a GABA _A receptor polypeptide or derivatives, a glycine-receptor polypeptide, an ionotropic serotonin receptor polypeptide or derivatives, a muscle-specific kinase polypeptide, leucine-rich glioma-inactivated 1 polypeptide, fragments, and variants thereof.

[0081] In some aspects, the receptor may comprise an amino acid sequence corresponding to any one of SEQ ID NOS. 1-54 as provided in Table 1 and fragments, derivatives and variants thereof. In some aspects, the receptor may comprise an amino acid sequence at least about 70% identical to any one of SEQ ID NOS. 1-54 or fragments thereof. In some aspects, the receptor may comprise a sequence at least about 70%, or about 75%, or about 80%, or about 85%, or about 90%, or about 95%, or about 100% identical to SEQ ID NOS. 1-54 or fragments thereof.

Table 1 : Sequence of endogenous receptors

[0082] In some aspects, the current disclosure encompasses compositions comprising recombinant polypeptides comprising receptor protein sequences or fragments, derivatives or variants thereof, that can be used to detect the presence of autoantibodies in a sample from a subject. In some aspects, these receptor molecules are recombinantly expressed and purified from a heterologous source. Techniques for cloning and expressing recombinant polypeptides in appropriate heterologous systems are well known in the art. The term “recombinant receptor molecule” as used herein refers to a recombinantly expressed polypeptide comprising receptor protein sequences or fragments, derivatives or variants thereof, that can be used to detect the presence of autoantibodies in a sample from a subject. These recombinant receptor molecules when specifically bound to an antibody (naturally occurring or recombinant or fragments, derivatives thereof) forms an antibody-receptor complex.

[0083] In some aspects, the recombinant polypeptides may further comprise one or more detector molecules. Non-limiting examples of detector molecules may be radioisotopes, haptens, fluorescent labels, fluorescent polypeptides, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles and/or ligands. [0084] In some aspects, the detector molecule is a fluorescent small molecule. In some aspects, the fluorescent molecule may be a small molecule or dye that is chemically attached to the receptor molecules. Examples of fluorescent dyes are described by Briggs et al “Synthesis of Functionalized Fluorescent Dyes and Their Coupling to Amines and Amino Acids,” J. Chem. Soo., Perkin-Trans. 1 (1997) 1051-1058). Fluorescent labels or fluorophores include rare earth chelates (europium chelates), fluorescein type labels including FITC, 5-carboxyfluorescein, 6-carboxy fluorescein; rhodamine type labels including TAMRA; dansyl; Lissarnine; cyanines; phycoerythrins; Texas Red: Oregon Green and analogs thereof. Fluorescent labels also include reactive and conjugated probes, e.g., Aminocoumarin, Fluorescein and Texas red, Alexa Fluor dyes, Cy dyes and DyLight dyes. In some aspects, the small molecule may be chemically attached to the recombinant polypeptide by methods known in the art. The fluorescent labels can be conjugated to an aldehyde group comprised in target molecule. Fluorescent dyes and fluorescent label reagents include those which are commercially available from Invitrogen/Molecular Probes (Eugene, Oreg., USA) and Pierce Biotechnology, Inc. (Rockford, III.). In some aspects, fluorescent labeling may be accomplished using a chemically reactive derivative of a fluorophore. Common reactive groups include amine reactive isothiocyanate derivatives such as FITC and TRITC (derivatives of fluorescein and rhodamine), amine reactive succinimidyl esters such as NHS- fluorescein, and sulfhydryl reactive maleimide activated fluors such as fluorescein-5-maleimide, many of which are commercially available.

[0085] In some aspects, the recombinant polypeptide may be a fusion protein comprising a receptor polypeptide sequence and a fluorescent polypeptide sequence. In some exemplary aspects, the fluorescent protein is Green Fluorescent Protein (GFP), eGFP, Red Fluorescent Protein (RFP), Teal Fluorescent Protein (TFP), Blue Fluorescent Protein (BFP), Yellow Fluorescent Protein (YFP), miRFP, cerulean fluorescent protein (CFP), eCyanFP and functional variants thereof. In some exemplary aspects, the fluorescent polypeptide may be selected from any one of SEQ ID NOS. 55-61 or derivates or variants thereof. In some aspects, the fluorescent polypeptide is at least 70% identical to any one of SEQ ID NOS. 55-61. In some aspects, the fusion protein comprises a polypeptide sequence of any one of SEQ ID NOS. 1-54 or fragments, derivatives or variants thereof and a fluorescent polypeptide sequence of any one of SEQ ID NOS. 55-61 or derivates or variants thereof. In some aspects, the fusion protein comprises an amino acid sequence at least about 70% identical to one or more of SEQ ID NOS. 1-54 or a fragment thereof and an amino acid sequence at least about 70% identical to any one of SEQ ID NOS. 55-61. Table 2: Sequence of fluorescent polypeptides

[0086] In some aspects, the recombinant protein may further comprise one or more of other sequences that may provide additional benefits like improved expression, stability, resistance to proteolytic degradation, solubility, ease of purification etc. These sequences are known in the art. These include one or more of a linker sequences, solubility enhancing sequences, protease cleavage sites, purification tags, stabilization sequences. Non-limiting examples include his-tag, FLAG tag, Strep tag II, HA-tag, soft-tag 1, soft tag 2, c-myc tag, T7-tag, S-tag, elastin-like peptide, chitin binding domain, thioredoxin, xylanase 10A, GST, MBP, GST, BRIL, Trx, NusA, SUMO, SET, DbsC, Skp, T7PK, GB1 or ZZ.

[0087] In some exemplary aspects, the recombinant polypeptide may comprise a sequence at least about 70% identical to any one of SEQ ID NOS. 62-136. In some aspects, the recombinant polypeptide comprises a sequence at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about a 100% identical to any one of SEQ ID NOS. 62-136 or derivates, variants or fragments thereof.

Table 3: Exemplary recombinant receptor sequences

[0088] In some aspects, the current disclosure also encompasses polynucleotide sequences encoding the recombinant receptor sequences provided here. In some exemplary aspects, polynucleotide sequence may be incorporated into a vector sequence including but not limited to a plasmid, viral vector, a shuttle vector. In some aspects, the polynucleotide sequence may be an isolated sequence.

[0089] In some aspects, the recombinant receptor sequences provided herein comprise one or more epitope binding sequences that interact with autoantibodies associated with autoimmune disorders of the nervous system. In other words, the autoantibodies specifically bind epitopes on the recombinant receptor sequences provided herein. An autoantibody “which binds” an antigen of interest, e.g., receptor sequence, is one that binds the antigen with sufficient affinity such that the receptor protein is usefui as an assay reagent, e.g., as a capture or as a detection agent for an autoantibody. Typically, the autoantibody does not significantly cross-react with other polypeptides. With regard to the binding of a polypeptide to a target molecule, the term “specific binding” or “specifically binds to” or is “specific for” a particular polypeptide or an epitope on a particular polypeptide target means binding that is measurably different from a non-specific interaction. An antibody that “specifically binds” to an antigen or an epitope is a term well under- stood in the art. An autoantibody is said to exhibit “specific binding” if for example it reacts more frequently, with more avidity, more rapidly, with greater duration, and/or with greater affinity with a particular target antigen for example an epitope on the receptor molecules provided herein, than it does with alternative targets. Specific binding can be measured, for example, by determining binding of a target molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity.

[0090] As used herein, “binding affinity” refers to the apparent association constant or KA. The KA is the reciprocal of the dissociation constant (KD). In certain aspects, autoantibodies associated with one or more autoimmune disorders described herein may have a suitable binding affinity for a target antigen for example a natural or recombinant receptor or derivatives, variants or fragment thereof. In some aspects, an autoantibody may have a binding affinity to a receptor molecule described herein of at least about 1000 nM, at least about 100 nM, at least about 10 nM, at least about 0.1 nM, or lower for an epitope on the receptor. In some aspects, an antibody described herein may have a binding affinity (KD) between about 100 nM to about 0.1 nM (e.g., about 100 nM, about 75 nM, about 50 nM, about 25 nM, about 10 nM, about 5 nM, about 1 nM, about 0.75 nM, about 0.5 nM, about 0.25 nM, about 0.1 nM) for a receptor. In some aspects, an antibody described herein may have a binding affinity (KD) between about 50 nM to about 40 nM (e.g., about 50 nM, about 49 nM, about 48 nM, about 47 nM, about 46 nM, about 45 nM, about 44 nM, about 43 nM, about 42 nM, about 41 nM, about 40 nM) for a receptor. In some aspects, an antibody described herein may have a binding affinity (KD) between about 50 nM to about 40 nM (e.g., about 50 nM, about 49 nM, about 48 nM, about 47 nM, about 46 nM, about 45 nM, about 44 nM, about 43 nM, about 42 nM, about 41 nM, about 40 nM) for the receptor protein. In some aspects, binding affinity (or binding specificity) can be determined by a variety of methods including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, and/or spectroscopy (e.g., using a fluorescence assay).

[0091] In some aspects, the current disclosure also encompasses compositions comprising the recombinant receptor molecules disclosed herein and suitable additives or ingredients. In some aspects, these may include one or more of buffers, salts, stabilizers, detergents, preservatives such as sodium azide, or glycerol. In some aspects, the compositions of the current disclosure may be in the form of a solid. In some aspects, the compositions disclosed herein may be in a solution (liquid preparation) or a suspension. In some aspects, the compositions may be lyophilized till further use. In some aspects, the compositions may be immobilized on a solid matrix for example assay plates, paper, discs, or matrix.

II Methods

[0092] in some aspects, the current disclosure encompasses method of diagnosing a subject in need thereof with an autoimmune disease, the method comprising: contacting a fluid sample obtained from the subject with a recombinant receptor molecule, wherein the recombinant receptor molecule specifically binds with an antibody in the sample to comprise an antibody- reporter complex; quantifying a level of the antibody-receptor complex; and diagnosing the subject with the autoimmune disease if the level of the antibody-receptor complex exceeds a threshold.

[0093] In some aspects, the sample method comprises contacting a fluid sample obtained from the subject with a recombinant receptor molecule, wherein the recombinant receptor molecule specifically binds with an antibody in the sample to comprise an antibody-reporter complex, subjecting the complex over a protein profiling technique, detecting the complex using a fluorescence detector, quantifying a level of the antibody-receptor complex; and diagnosing the subject with the autoimmune disease if the level of the antibody-receptor complex exceeds a threshold. In some aspects, the profiling technique may be a chromatographic technique for example liquid chromatography, size exclusion chromatography, high performance liquid chromatography (HPLC), or microfluidic chip-based assays or other methods used in the art.

[0094] In some exemplary aspects, the sample method comprises contacting a fluid sample obtained from the subject with a recombinant receptor molecule, wherein the recombinant receptor molecule specifically binds with an antibody in the sample to comprise an antibody- reporter complex, subjecting the complex over to a size exclusion column, detecting the complex using a fluorescence detector, quantifying a level of the antibody-receptor complex; and diagnosing the subject with the autoimmune disease if the level of the antibody-receptor complex exceeds a threshold.

[0095] In certain aspects, the profiling technique is a size exclusion chromatography. The term “size exclusion chromatography” (SEC) is intended to include a chromatographic method in which molecules In solution are separated based on their size and/or hydrodynamic volume. It is applied to large molecules or macromolecular complexes such as proteins and their conjugates. Typically, when an aqueous solution is used to transport the sample through the column, the technique is known as gel filtration chromatography. The underlying principle of SEC is that particles of different sizes will elute (filter) through a stationary phase at different rates. This results in the separation of a solution of particles based on size. Provided that all the particles are loaded simultaneously or near simultaneously, particles of the same size elute together. Each size exclusion column has a range of molecular weights that can be separated The exclusion limit defines the molecular weight at the upper end of this range and is where molecules are too large to be trapped in the stationary phase. The permeation limit defines the molecular weight at the lower end of the range of separation and is where molecules of a small enough size can penetrate into the pores of the stationary phase completely and all molecules below this molecular mass are so small that they elute as a single band.

[0096] In certain aspects, the eluent is collected in constant volumes, or fractions. The more similar the particles are in size, the more likely they will be in the same fraction and not detected separately. Preferably, the collected fractions are examined by spectroscopic techniques to determine the concentration of the particles eluted. Typically, the spectroscopy detection techniques useful in the present disclosure include, but are not limited to, fluorometry, refractive index (Rl), and ultraviolet (UV). In some exemplary aspects, the detector is a fluorescence detector. In certain instances, the elution volume decreases roughly linearly with the logarithm of the molecular hydrodynamic volume (i.e. , heavier moieties come off first).

[0097] In some aspects of the current disclosure, the recombinant receptor molecule in complex with the autoantibody is detectable by a fluorescence detector. The phrase “fluorescence detection” includes a means for detecting a fluorescent label and/or a fluorescent polypeptide. Means for detection include, but are not limited to, a spectrometer, a fluorimeter, a photometer, a detection device commonly incorporated with a chromatography instrument such as, but not limited to, a size exclusion — high performance liquid chromatography. In some aspects, the compositions provided herein comprise a fluorescence label or is a fusion protein comprising a fluorescent polypeptide. The fluorescently labeled receptor molecule alone or in complex with the autoantibody is detectable by a suitable fluorescent detector. Several such detectors are commercially available. The choice of the detector can vary at least depending on the fluorescent molecule used, the scale of the method and the degree of precision required. [0098] In some aspects, the method provided here comprises the use of a combination of size exclusion chromatography with a fluorescent detector. As used herein the term “fluorescence size exclusion chromatography” or “FSEC” refers to a system comprising a size exclusion column and a fluorescence detector.

[0099] In some aspects, the FSEC method of the current disclosure comprises contacting a fluid sample obtained from the subject with a recombinant receptor molecule, wherein the recombinant receptor molecule specifically binds with an antibody in the sample to comprise an antibody- reporter complex, subjecting the complex to size exclusion chromatography and detecting the presence of the labeled bound and/or the unbound receptor in the eluant using a fluorescence detector. In some aspects, the method comprises determining the elution volume for bound and unbound receptor molecule. In some aspects, this can be inferred from the fluorescence profile of the eluate from the size exclusion column, shifts in elution peak and/or changes in peak height deducible from the profile. In some aspects, the unbound receptor elutes at a later elution time (greater volume) in comparison to the bound sample, in other words the bound receptor (as detected by the fluorescence profile) elutes before the unbound receptor. In some aspects of the method the presence of an antibody-reporter complex may be discerned by a shift in the elution peak in comparison to a sample comprising the receptor alone. Appropriate molecular weight standards are often used in the art to determine the appropriate elution volume that would correspond to particular molecular weights and hence can be used as indicators for bound and unbound peaks.

[00100] In some aspects, the method may further comprise quantifying the level of antibody- receptor complex using a fluorescence detector. Methods for quantifying such complexes are well known in the art and may comprise for example use of appropriate standard curves. In some aspects, the methods of the current disclosure may further comprise determining a threshold or background fluorescence (peak) at the eluate volume that corresponds to the bound complex, when the sample is from an individual who does not have autoimmune antibodies to the receptor. In some aspects, the threshold corresponds to the levels of non-specific receptor complex formed in samples from subjects not suffering from an autoimmune disease disclosed herein. This corresponds to receptor that is non-specifically bound to other proteins in the sample. In some aspects, a peak height greater than the threshold or background may be used to diagnose an autoimmune disorder. In some aspects, a peak height at least greater than 20%, or at least greater than 30%, or at least greater than 40%, or at least greater than 50%, or at least greater than 60%, or at least greater than 70%, or at least greater than 80%, or at least greater than 90%, or at least greater than 100% of the threshold is indicative of a disease state. In some aspects, the comparison is made between two samples from the patient drawn at different times during the course of treatment. In this exemplary aspect a peak height at least greater than 20%, or at least greater than 30%, or at least greater than 40%, or at least greater than 50%, or at least greater than 60%, or at least greater than 70%, or at least greater than 80%, or at least greater than 90%, or at least greater than 100% of the previous sample is indicative of a worsening disease condition while a peak height at least less than 20%, or at least less than 30%, or at least less than 40%, or at least less than 50%, or at least less than 60%, or at least less than 70%, or at least less than 80%, or at least less than 90%, or at least less than 100% is indicative of improvement in the patient’s state.

[00101] In some exemplary aspects, the sample method comprises contacting a sample with a recombinant receptor molecule, wherein the recombinant receptor molecule specifically binds with an antibody in the sample to comprise an antibody-reporter complex.

[00102] Samples as used herein may vary depending on the application. For example, it may be a biological sample or a non-biological sample. The term non-biological sample may include synthetic peptides, buffers, non-clinical fluids, surfaces, etc. that may comprise antibodies that specifically bind to the recombinant receptor. The term "biological sample” includes any biological specimen obtained from an individual. Suitable samples for use in the present disclosure include, without limitation, whole blood, plasma, serum, cerebrospinal fluids (CSF), saliva, urine, stool (i.e. , feces), tears, amniotic fluid and any other bodily fluid, or a tissue sample (i.e., biopsy) such as brain sample, and cellular extracts thereof (e.g., neuronal extract). In a preferred embodiment, the sample is a fluid sample like blood, plasma, serum, urine or CSF. In a more preferred embodiment, the sample is a serum sample. The use of samples such as serum, plasma, blood samples are well known in the art (see, e.g., Hashida et al., J. Clin. Lab. Anal., 11 :267-86 (1997)). One skilled in the art will appreciate that samples such as serum samples can be diluted prior to the analysis of antibody levels. The sample may be a fluid sample, a solid sample or a sample bound to a solid surface like matrices, beads, strips, solid substrate material or membrane (e.g., plastic, nylon, paper), plates etc. The samples may be concentrated or diluted such that the autoantibody levels are within a preferred range based on the application and detection method.

[00103] In some aspects, the fluid sample may be derived from a subject diagnosed with or suspected of having an autoimmune disorder. Exemplary autoimmune diseases include Alopecia Areata, Ankylosing Spondylitis, Antiphosphoiipid Syndrome, Autoimmune Addison's Disease, Autoimmune Hemolytic Anemia, Autoimmune Hepatitis, Behcet's Disease, Bullous Pemphigoid, Cardiomyopathy, Celiac Sprue-Dermatitis, Chronic Fatigue Immune Dysfunction Syndrome (CFIDS), Chronic Inflammatory Demyelinating Polyneuropathy, Churg-Strauss Syndrome, Cicatricial Pemphigoid, CREST Syndrome, Cold Agglutinin Disease, Crohn's Disease, Discoid Lupus, Essential Mixed Cryoglobulinemia, Fibromyalgia-Fibromyositis, Graves' Disease, Guillain- Barre, Hashimoto's Thyroiditis, Hypothyroidism, Idiopathic Pulmonary Fibrosis, Idiopathic Thrombocytopenia Purpura (ITP), IgA Nephropathy, Insulin dependent Diabetes, Juvenile Arthritis, Lichen Planus, Lupus, Meniere's Disease, Mixed Connective Tissue Disease, Multiple Sclerosis, Myasthenia Gravis, Pemphigus Vulgaris, Pernicious Anemia, Polyarteritis Nodosa, Polychondritis, Polyglandular Syndromes, Polymyalgia Rheumatica, Polymyositis and Dermatomyositis, Primary Agammaglobulinemia, Primary Biliary Cirrhosis, Psoriasis, Raynaud's Phenomenon, Reiter's Syndrome, Rheumatic Fever, Rheumatoid Arthritis, Sarcoidosis, Scleroderma, Sjogren's Syndrome, Stiff-Man Syndrome, Takayasu Arteritis, Temporal Arteritis/Giant Cell Arteritis, Ulcerative Colitis, Uveitis, Vasculitis, Vitiligo, Wegener's Granulomatosis, and myasthenia gravis. In some aspects, the autoimmune disorder is of the nervous system. In some aspect the subject is a mammal. In some aspects, the subject is a human.

[00104] Recombinant receptor molecules as used herein are as provided herein (e.g., Section I). In some exemplary aspects, the recombinant receptor may comprise a sequence at least about 70% identical to any one of SEQ ID NOS. 62-136. In some aspects, the recombinant polypeptide comprises a sequence at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about a 100% identical to any one of SEQ ID NOS. 62-136 or derivates, variants or fragments thereof. In some aspects, the recombinant receptor molecule specifically binds with an antibody in the sample to comprise an antibody-reporter complex that can be detected using an FSEC assay.

[00105] In some aspects, the current disclosure also encompasses methods of monitoring an autoimmune disease of the nervous system, the method comprising: contacting a fluid sample obtained from the subject with a recombinant receptor molecule, wherein the recombinant receptor molecule specifically binds with the autoantibody in the sample to comprise an antibody- receptor complex; quantifying a level of the antibody-receptor complex; and monitoring the subject’s autoimmune disease based on the level of the antibody-receptor. In some aspects, the monitoring may require determining the level of the antibody-receptor complex at multiple time- point separated over an interval of time (for example, weekly, or bi-weekly, or monthly, or 2- monthly or 3-monthly, or 4-monthly, or 5-monthly, or 6-monthly or more), and comparing the change in the level of the antibody- receptor complex over time. The data can then be used to determine treatment regimens and predict outcomes.

[00106] In some aspects, the current disclosure also encompasses using the assays and methods provided herein to diagnose or confirm diagnosis of or monitor an autoimmune disorder in a subject in need thereof. In some aspects, the assays are conducted in vitro using FSEC using appropriate labeled receptors as disclosed herein and contacting the receptor with the patient sample. The complex can then be run on a size exclusion column as disclosed herein and the level of the complex quantified and compared with a threshold to diagnose a disease state.

[00107] III. Kits

[00108] In some aspects, the assay methods and compositions of this disclosure can be provided in the form of a kit. In an exemplary aspect the kit comprises a recombinant receptor molecule as provided herein, one or more reagents to facilitate formation of the antibody-receptor complex and subjecting the complex to a chromatographic method. In some aspects, the reagents may comprise salts, buffers, detergents, or glycerol. In some aspects, the kit may further comprise appropriate molecular weight standards. In some aspects, the kit may further comprise means for drawing a patient sample, for example a syringe, appropriate tubing etc.

[00109] IV. Definitions

[00110] In order that the present disclosure can be more readily understood, certain terms are first defined. As used in this application, except as otherwise expressly pro-vided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the application.

[00111] The term “and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and/or as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following aspects: A, B, and C: A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[00112] It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of” and/or “consisting essentially of are also provided. [00113] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pel- Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[00114] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. The headings provided herein are not limitations of the various aspects of the disclosure, which is to be understood by reference to the specification as a whole. The terms defined immediately below are to be understood by reference to the specification in its entirety.

[00115] As used herein, the term “corresponding” as used generally with reference to antibody- antigen binding complexes, for example, an antibody corresponding to an anti-gen will bind to the antigen under physiologic conditions. The bound antibody-antigen is referred to as an antibody- antigen binding complex.

[00116] The terms “derivative,” “variant,” and “fragment,” when used herein with reference to a polypeptide, refers to a polypeptide related to a wild type polypeptide or recombinant polypeptide, for example either by amino acid sequence, structure (e g., secondary and/or tertiary), activity (e.g., enzymatic activity) and/or function. Derivatives, variants and fragments of a polypeptide can comprise one or more amino acid variations (e.g., mutations, insertions, and deletions), truncations, modifications, or combinations thereof compared to a wild type polypeptide. A part or fragment of a polypeptide may correspond to at least 1%, at least 2%, at least 3 %, at least 4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40% of the length of a polypeptide, such as a polypeptide having an amino acid sequence having at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 99% of the length (in amino acids) of the polypeptide.

[00117] The term “size exclusion chromatography” (SEC) is intended to include a chromatographic method in which molecules in solution are separated based on their size and/or hydrodynamic volume. It is applied to large molecules or macromolecular complexes such as proteins and their conjugates. Typically, when an aqueous solution is used to transport the sample through the column, the technique is known as gel filtration chromatography. EXAMPLES

[00118] The following examples are included to demonstrate preferred embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the present disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.

Example 1 : GABA _A receptor expression, purification, and reconstitution into nanodiscs

[00119] The current disclosure resulted from the finding that purified receptors can be used to quantitatively determine the level of clinically relevant autoantibodies in patient sample using fluorescence size exclusion chromatography (FSEC).

[00120] In order to determine the feasibility of this approach GABA _A receptors were purified and reconstituted into nanodiscs. The tricistronic construct encoding the α1, [32 and y2 subunits of the GABA _A receptor, and its purification and reconstitution into nanodiscs was achieved essentially as provided in Kim et al., 2020. Purification of the GABA _A receptor was performed as described but with the exception that all purifications were done in the absence of exogenous ligand. For FSEC purified receptor reconstituted in lipidic nanodiscs were aliquoted and flash frozen in liquid nitrogen until required. For antibody tests, a GABA _A receptor aliquot was thawed on ice, and ~2 pg of receptor was incubated with ~0.5-2 pg of each Fab or mAb at room temperature for 10 min. Protein was diluted in TBS (20 mM Tris, pH 7.4, 150 mM NaCI) and centrifuged to pellet aggregated receptor before analysis by FSEC.

Example 2: Purification of patient derived antibodies against GABA _A receptor

[00121] Fab fragments were produced using cDNA sequences isolated for patients. Briefly, cDNA encoding the heavy and light chains of immunoglobulin were cloned from single cells isolated from an encephalitis patient's cerebrospinal fluid. These sequences were cloned into mammalian expression vectors, from which monoclonal antibodies were produced for initial characterization. Fab fragment was generated by replacing the Fc domains CH2 and CH3 of the heavy chain expression vector with affinity tags and synthesized, expressed and purified by an external provider (inVivo BioTech Germany). A panel of 5 patient derived antibodies for binding to the purified α1β2y2 GABA _A receptor were tested. Example 3: Fluorescence size exclusion chromatography

[00122] Purified monoclonal antibodies were first screened for receptor binding by fluorescence- detection size exclusion chromatography (FSEC). Purified GABA _A receptor as described in Example 1 was mixed at a molar ratio of 1 :1 with purified monoclonal antibodies. Approximately 1 pg of purified receptor was mixed with 0.7 pg of mAb in a 40 μl reaction of 1mM DDM/150 mM NaCI/20 mM Tris pH 7.4 (FSEC buffer). After incubation for 15’ on ice, Tryptophan fluorescence was used to detect samples by examining shift of the main pentameric peak to an earlier elution volume, indicating cross-linking and/or precipitation. This assay detected binding by all antibodies tested except for mAb ₁₉₈ . Fab fragments were then generated from antibodies mAb ₁₀₁ , mAb ₁₁₅ , mAb ₁₇₅ and mAb ₂₀₁ for structural studies by in vitro expression and purification (performed by a commercial company, inVivo Biotech). Binding stoichiometries were estimated by FSEC with increasing molar ratios of Fab:receptor of 1 :1 , 1 :2, and 1:3 (FIG. 1A-E) and reactivity to native receptors was analyzed in murine brain tissues (FIG. 1F) before large-scale complex formation and purification. Fab ₂₀₁ failed to bind detectably to the GABA _A receptor and was excluded from structural studies. Fab ₁₀₁ no longer bound the GABA _A receptor in an affinity suitable for structural work; it may require bivalency to bind appreciably. Fab ₁₁₅ and Fab ₁₁₅ were pursued for structural studies.

Example 4: Structure of the GABA _A receptor bound to an inhibitory autoantibody

[00123] In contrast to mechanisms of autoantibody-induced neurological disease described thus far in literature, mAb ₁₁₅ and Fab ₁₁₅ directly inhibit GABA _A function without causing receptor internalization from the cell surface. To understand its mechanism of inhibition the structure of Fab ₁₁₅ bound to the GABA _A receptor was determined. The oiβ2y2 synaptic GABA _A receptor was purified and reconstituted into brain lipid nanodiscs. Reconstituted receptors were incubated with Fab ₁₁₅ before further polishing by size-exclusion chromatography (FIG. 2A and 2B). The purified complex was applied to cryo-electron microscopy (cryo-EM) grids and a dataset was collected that allowed the reconstruction of a 3.0 A resolution map (FIG. 3). The map quality allowed modelling of the receptor and the variable domains of the Fab (V _H and V _L for heavy and light chain variable domains, respectively). Local resolution in the areas of greatest interest, including side- chain interactions between Fab ₁₁₅ and receptor, was ~2.9 Å.

[00124] The complex structure reveals one Fab ₁₁₅ bound at each of the two 13-a subunit interfaces in the ECD (FIG. 4A). The complementarity determining region (CDR) 3 of the Fab heavy chain penetrates the neurotransmitter binding pocket and contacts conserved residues known to impact agonist binding (FIG. 4B) In particular, R109 of the V _H CDR3 is positioned to make electrostatic and cation-Tr interactions with several residues on the 132 and α1 subunits (FIG. 4). Of special interest is the contact between V _H R105 and E155. E155 forms a salt bridge with the primary amine of the neurotransmitter GABA and is critical for binding and gating. Thus, the Fab ₁₁₅ V _H CDR3 mimics an aspect of neurotransmitter chemistry and directly competes with GABA binding in a manner analogous to a competitive antagonist.

[00125] in the initial antibody characterization, mAb ₁₁₅ was found to be selective for binding among different GABA _A receptor subunits. The structure reveals the basis of this exquisite selectivity. First, although GABA _A 13 subunits can form a homopentamer, 13 alone did not bind antibody despite its numerous contacts with Fab ₁₁₅ in the neurotransmitter binding pocket. Examination of the Fab ₁₁₅ : receptor structure reveals that most of the interactions between Fab ₁₁₅ and receptor are with the receptor α1 subunit contacts with 13 alone cannot support binding. Furthermore, mAb ₁₁₅ is only able to bind GABA _A receptors containing the α1, but not α2-5 subunits. This specificity may arise from interactions between Fab ₁₁₅ and four key residues on the α1 subunit: E170, R173, E174, and R177 (FIG. 4 panel c). Evidence for the contribution of these residues to specificity is found in an examination of contacts between Fab ₁₁₅ and GABA _A (FIG. 5, yellow highlights). Most contacted residues are conserved between α1-a6. The exceptions are T122-D124 and E170-V180. In the case of T122-D124, Fab ₁₁₅ only makes electrostatic interactions with backbone carbonyl oxygens, and therefore the identity of the residues present in the receptor are less important. The string of 11 residues beginning at E170 exhibit variable conservation (FIG. 4C ); however, the tetrad of E170, R173, E174 and R177 are only found in α1 and are all involved in salt bridges. These electrostatic contacts stabilize the Fab: receptor interaction and likely determine Fab ₁₁₅ ’ s specificity for α1.

[00126] Structural and sequence analyses suggest the ways in which mAb ₁₁₅ evolved to increase not just its selectivity but also its affinity for the GABA _A receptor. Somatic hypermutation is a process unique to B cells and antibody secreting cells that enables diversification of immunoglobulins in their variable gene regions as an adaptive immune response to enhance affinity for their antigenic target. The Fab ₁₁₅ antibody sequence was compared to an immunoglobulin gene database to derive a germline ancestor with highest sequence identity. This approach identifies somatically hypermutated residues within the variable V(D)J gene segments that have evolved for affinity maturation (FIG. 6A). Both Fab ₁₁₅ . V _H and V _L contain somatically hypermutated residues that are involved in binding the GABA _A receptor (FIG. 6A). These include R97 and K98 of the Fab ₁₁₅ V _L , which are encoded in the germline sequence as an asparagine and a glycine. These mutations contribute two new positive charges that are stabilized at the interface through salt bridges to help anchor Fab ₁₁₅ to the α1 subunit (FIG. 4C). Thus, somatic hypermutations are predicted to enhance the affinity of Fab ₁₁₅ for its target.

Example 5: Structure of the GABA _A receptor in complex with a non-competitive autoantibody

[00127] The mAb ₁₁₅ autoantibody has been shown to trigger seizure activity in rodents and was shown to require both the α1 subunit and the γ2 subunit for binding to the GABA _A receptor in cell- based assays. The a-y interface is of special interest because it forms the binding site for benzodiazepines, a modulatory class of GABA _A drugs that includes diazepam, zolpidem, and other prescribed anxiolytics and sedatives. In accordance with requirement of the α-γ interface for mAb ₁₇₅ binding, FSEC experiments indicated a stoichiometry of 1 :1 for receptor: Fab ₁₁₅ (FIG. 1D). We used cryo-EM to obtain a structure of the Fab175:GABA _A complex at an overall resolution of 2.7 A (FIG. 7). The map quality allowed confident modelling of the receptor and the variable domains of the Fab. Fab175 interacts at the α1-y2 subunit interface, consistent with cell-based and FSEC experiments (FIG. SB). In a manner similar to Fab ₁₁₅ , the majority of surface area contacted by Fab ₁₁₅ is with the α1 subunit. Fab ₁₁₅ V ^H contains a methionine (IM 103) that buries itself into a hydrophobic pocket composed of residues from both sides of the α1:y2 interface, above the benzodiazepine site (FIG. 8 panel b). The Fab ₁₁₅ V _L docks onto Loop C of the α1 subunit: this loop caps the benzodiazepine-binding pocket of the receptor This V _L attachment site centers upon α1 R164 of the GABA _A receptor, which forms a network of five electrostatic interactions connecting the two proteins (FIG. 8 panel C). This arginine is unique to α1; while some of the other a subunits contain a lysine at this position, a primary amine would not be able to recapitulate the elaborate network of electrostatic interactions. This arginine likely contributes to subunit specificity, as most other receptor residues contacted by Fab ₁₁₅ are conserved (FIG. 5). Fab ₁₁₅ underwent somatic hypermutation to a lesser degree than Fab ₁₁₅ , changing six amino acids in the heavy chain and one in the light chain (FIG. 6). The physiological consequences of the Fab ₁₇₅ :GABA _A receptor interaction are less straightforward than for Fab ₁₁₅ . Fab ₁₁₅ behaves as a competitive antagonist, stabilizing the ion pore in a resting-like, non-conducting state (FIG. 9), which correlates with its direct functional inhibition in electrophysiology experiments. When the antibody was injected into mouse CNS, it triggered spontaneous epileptic events, insight into the disease mechanism comes from the conformation of the receptor bound to Fab ₁₁₅ . Unlike the Fab ₁₁₅ -bound receptor, the Fab ₁₁₅ -bound receptor contains density consistent with GABA in its binding site, even though the agonist was not added to the preparation. The presence of GABA induces a partial closure of Loop C in the neurotransmitter binding pockets (FIG. 9 panel b). Normally, closure of Loop C by agonist triggers a rotation and contraction of the ECD, which is then translated into opening of the transmembrane pore. After channel opening, the agonist remains bound but a lower gate in the pore closes, a process termed desensitization. However, the Fab175-bound GABA _A structure reveals a conflict in this paradigm. Although Loop C is mostly closed, the receptor pore is in a resting-like, closed conformation (FIG. 9). Closer examination of the structure in comparison with a GABA-bound receptor (PDB 6X3Z, reveals a failure of the Fab ₁₁₅ -bound receptor to contract its ECD (FIG. 9C and 9E). Fab ₁₇₅ blocks this contraction by wedging between the α1 and y2 subunits in the upper part of the ECD (FIG. 8 panel C).

[00128] Superposition of the Fab ₁₇₅ bound structure on the GABA-bound structure suggests many atomic clashes would prevent the ECD compaction that is an initial step in channel gating in the pentameric receptor superfamily (FIG. 9E). The consequence is that GABA is positioned farther away from the residues of the neurotransmitter binding pocket. Specifically, the GABA amino nitrogen is 4.2 A from E155, as opposed to 2.5 A in the GABA-bound structure in the absence of Fab. The GABA: E155 interaction is required for converting agonist binding into channel gating and is weakened in the presence of Fab ₁₁₅ . Together these findings reveal distinct mechanisms leveraged by the two antibodies to cause encephalitis, psychiatric abnormalities and seizures.

Example 6: FSEC based assay system to test binding of lab generated and patient antibodies to GABA _A receptor-GFP fusion protein

[00129] To measure control laboratory-generated antibodies or patient serum samples containing antibodies reactive to neuronal receptors, GFP was fused to the y2 subunits of the GABA _A receptor. These fusion proteins were co-expressed with their partner subunits to assemble recombinant receptor targets in human embryonic kidney 293 cells. Co-expression was achieved in one of three ways: transient transfection using established methods common to the art, stable transfection using a transposon-based system well known to the community, or bacmam transduction, a baculovirus that has been modified to infect mammalian cells in a nonreplicative manner. Celis were solubilized with 40 mM dodecyl-maltoside for 1 hour in the presence of 1mM phenylmethylsulfonylfluoride to prevent proteolytic digestion. Insoluble material was cleared by ultracentrifugation at 40,000 g for 40 minutes.

[00130] Monoclonal antibodies raised against α3β4 nicotinic receptor in the laboratory were used for proof-of-concept experiments to confirm that a clear change in peak height was discernable for bound complex in comparison to unbound protein using fluorescence detection (FIG. 10). [00131] Initially, proof of concept experiments with laboratory-generated antibodies against receptors were utilized to determine appropriate ratios of antibody: receptor. Molar ratios of 1 :1 , 1 :2, and 1 :3 were tested.

[00132] Forty μl of cell lysate containing the GFP-tagged receptors was mixed with 5 μl of freshly thawed patient serum or negative control human serum. Receptor: serum was incubated for 1 hour on ice before ultracentrifugation to clear large aggregates. Twenty μl of receptor: anti body mixture was injected on a Shimadzu HPLC system over a Sepax SRT-500 column connected to an in-line fluorescence detector. Receptor fluorescence was measured at an excitation wavelength of 483 nm and emission wavelength of 505 nm. It was found that for the α3β4 nicotinic receptor, α7 nicotinic receptor, and GABA _A receptor, receptor fluorescence at the peak pentameric elution time should be around 4-8000uV to allow appropriate detection of patient antibodies. Each receptorserum sample was performed in triplicate to reduce variability resulting from working with natural products. Fluorescence was measured at a constant elution time determined by incubation of the fluorescent receptor fusion with negative control antibodies; fluorescence in the presence of patient antibodies was then measured and a ratio of patient/control fluorescence was reported. FSEC assays could successfully detect the presence of patient derived autoantibodies in blood serum samples (FIG. 11).

Example 7: FSEC based assay system to test binding of lab generated and patient antibodies to α3β4 nicotinic receptor -GFP fusion protein

[00133] To measure patient serum samples containing antibodies reactive to neuronal receptors, GFP was fused to one of the subunits of the α3β4 nicotinic acetylcholine receptor. These fusion proteins were co-expressed with their partner subunits to assemble recombinant receptor targets in human embryonic kidney 293 cells. Co-expression was achieved in one of three ways: transient transfection using established methods common to the art, stable transfection using a transposon- based system well known to the community, or bacmam transduction, a baculovirus that has been modified to infect mammalian cells in a non-replicative manner. Cells were solubilized with 40 mM dodecyl-maltoside for 1 hour in the presence of 1mM phenylmethylsulfonylfluoride to prevent proteolytic digestion. Insoluble material was cleared by ultracentrifugation at 40,000 g for 40 minutes.

[00134] Monoclonal antibodies raised against α3β4 nicotinic receptor in the laboratory were used for proof-of-concept experiments to confirm that a clear change in peak height was discernable for bound complex in comparison to unbound protein using fluorescence detection (FIG. 12). [00135] For patient samples, 40 μl of cell lysate containing the GFP-tagged receptors was mixed with 5 μl of freshly thawed patient serum or negative control human serum. Receptor: serum was incubated for 1 hour on ice before ultracentrifugation to clear large aggregates. Twenty μl of receptorantibody mixture was injected on a Shimadzu HPLC system over a Sepax SRT-500 column connected to an in-line fluorescence detector. Receptor fluorescence was measured at an excitation wavelength of 483 nm and emission wavelength of 505 nm. Each receptor: serum sample was performed in triplicate to reduce variability resulting from working with natural products (FIG. 13).

Example 8: FSEC based assay system to test binding of lab generated and patient antibodies to muscle-type nicotinic receptor labeled with Oregon green

[00136] The muscle-type nicotinic AChR was prepared from native tissue of the Torpedo electric ray. For the preparation of AChR-rich membranes, ~ 100 g of frozen organ from Torpedo californica (EastCoast Bio, Catalog # DZ800) was thawed in 300 ml of buffer A (400 mM NaCI, 20 mM aH2PO4, pH 7.4) with 150 mg of NEM (N-ethylmaleimide) at room temperature. Subsequent operations were carried out at 4° C or on ice. The organ was homogenized for 2 min at top speed with Kinematica Polytron Homogenizer (GmbH). The mix was centrifuged for 15 min at 4000 rpm in the centrifuge 5810 R. Supernatants was decanted through cheesecloth in 300 mL beaker on ice; 1 complete protease inhibitor mini tablet was added and gently stirred for 5 min at cold room, the mix was put into 65 ml. ultracentrifuge tubes and spun at 30,000 rpm in the Ti45 rotor for 30 min at 4 °C. Pellets were combined, resuspended in buffer E (80 mM NaCI, 20 mM Tris pH 11.0, 1 mM EDTA, 20% sucrose), gently homogenized and placed on ice for 30 minutes. The supernatant was centrifuged at 30.000/Ti45/30 min, the supernatants were discarded, and the pellets washed 3 times with buffer B (20 mM NaH ₂ PO ₄ , 80 mM NaCI pH 7.4). The pellets were aliquoted, weighed, and stored at -80 °C. To prepare the affinity column, TDAC-2 resin: NHS Sepharose 25mL (GE life sciences, 17090601 , 16-23 pmol NHS/ml drained medium, total -575 pmol) was gently vacuum filtered using a 50mL disposable vacuum filter (0.22 pm Millipore Express) and washed with 2x100 mL of cold MilliQ water using a 100 mL 0.2 micron disposable filter, ensuring that the slurry does not completely dry out. The slurry was transferred to a 250mL centrifuge bottle and 0.7 grams (2.2 mMoles) of TDAC2 (2-(4-Aminobutanamido)-N, N, N- trimethyl-ethanaminium iodide, Molecular Weight 351.66) dissolved in 75 mL of 100 mM HEPES, pH 8.0 was added. The slurry was gently shaken and placed on a rotary shaker for 2 hours. The slurry was gently vacuum filtered, and gently vacuum filter washed with 4 x 100 mL of cold MilliQ water. The slurry was then resuspended in 20% ethanol for 4 °C storage or used directly to pack an HR 16/10 column and stored at 4 °C in 20% ethanol. TDAC2 was provided by Dr. Michael Stowell, University of Colorado.

[00137] To purify the receptors via affinity, pellets were resuspended (~ 2 gm in 25x volume/wt (previously I used 10x volume/wt)) in buffer B and homogenized. PMSF was added to a final cone, of 1 mM, 10% Triton X-100 was added to a final concentration of 1.5% and stirred for 1 hour. The sample was centrifuged at 30K rpm in a Ti45 rotor for 30 min at 4 °C and supernatant was recovered, and diluted 2X with cold MilliQ water. The diluted supernatant was batch bound on a TDAC2 resin (~5 mL bed volume) for 1 hour, rotating at 4 °C. The resin was washed with 100 mL of buffer C (80 mM NaCI, 20 mM Tris pH 7.4, 1 mM EDTA, 1 mM DDM), then eluted with 25 ml (previously I used 15 ml) buffer F (80 mM NaCI, 20mM Tris 7.4, 1mM EDTA, 1 mM DDM, 50 mM Carbachol, 50 mM pME). The absorbance of the eluted sample was measured and concentrated with 100kDa cutoffs to A280 ~8 mg/mL. For reconstitution in nanodiscs, the affinity elution fractions were concentrated to A280 = -8. For reconstitution in saposin, the receptor was mixed with saposin and soy polar lipid in a molar ratio of 1 :18:90 and for reconstitution in MSP1 E2 and MSP1 E3, the receptor protein was mixed with respective MSP and soy polar lipid in a molar ratio of 1:2.5:20. The detergents (DDM) were removed using Bio-Beads (Bio-Rad) rotating overnight at 4 °C and exchanging with the fresh Bio-beads at morning and rotating at 4 °C for another 1 hour. The reconstituted receptors were ultracentrifuged (40 min at 40k rpm in the table-top ultracentrifuge), and run through a size-exclusion chromatography equilibrated in TBS to remove empty nanodiscs and BME from the sample.

[00138] To label the purified protein with reactive Oregon-Green488, approximately 10 mg of protein was mixed with 1mg of Oregon Green as per manufacturer’s instructions. The labeled receptor was purified via size-exclusion chromatography on a Superose 6 10/300 GL column with TBS as the mobile phase. Fractions correlating to pentamer were analyzed via FSEC to confirm appropriate morphology and behavior were retained during the labeling process.

[00139] Monoclonal antibodies raised against the torpedo receptor in mice were used for proof- of-concept experiments to confirm that a clear change in peak height was discernable for bound complex in comparison to unbound protein using fluorescence detection (FIG. 14).

[00140] To examine autoantibody reactivity against the related α7 nicotinic acetylcholine receptor, which has been implicated in autoimmune encephalitis, possibly schizophrenia, and a related Alzheimer’s like dementia, HEK 293 cells were transduced with a bacmam virus expressing the human o:7 gene fused to YFP in the M3-M4 loop. This virus contained a porcine retrovirus T2A signal that allowed for single transcription of the receptor followed by its protein chaperone NACHO. Transduction of suspension cell culture at an MOI of 1 was followed by incubation at 37 degrees C for 3 days. Cells were pelleted by gentle centrifugation at 4,000 rpm for 10 minutes, followed by solubilizing with 40 mM DDM/TBS/1 mM PMSF as before. After clearing of insoluble material by ultracentrifugation at 40,000 rpm for 40 minutes, cell lysate was aliquotted into 40 μl portions and mixed with 5 μl of human serum from negative control and potential patient samples. As before, serum/receptor mixtures were incubated on ice for 1 h, then massive aggregates were cleared by an ultracentrifugation spin for 20 minutes before transfer of lysate and injection via FSEC.

[00141] As the α3β4, muscle-type nicotinic acetylcholine, and GABA _A receptor all share a similar overall architecture, there has been suggestion that antibodies against related receptors could also have pathological effects. Thus, we screened several AAG and AE patients against this receptor and found cross- reactivity in a subset. FSEC assays could successfully detect the presence of patient derived autoantibodies in blood serum samples (FIG. 15). The pathological consequences of antibodies against the o7 subtype remain to be fully characterized, but the role of this protein in the anti-inflammatory immune response, memory, cognition, and auditory gating suggest that they could be multi-factorial and thus this receptor is an important target to consider in surveying for autoantibody mechanisms of disease.

Example 9: Expression of Glycine receptor using a pEZT plasmid

[00142] The polynucleotide sequence encoding glycine receptors B, glycine receptor ct1, glycine receptor a2 or glycine receptor α3 was cloned into a pEZT plasmid vector (SEQ ID NO. 137) comprising a polynucleotide sequence encoding GFP downstream of the multiple cloning site (SEQ ID NOS. 137-141). This construct was used to express glycine receptors as a fusion protein in HEK 293T GnTI' cells. The protein was purified and used in FSEC experiments.

[00143] The same vector backbone can be used to clone polynucleotides to express other recombinant receptor proteins - GFP fusion proteins. These can then be contacted with patient samples and used in FSEC assay to determine the presence of autoantibodies in a patient in need thereof. Example 10: Development of specific method to detect autoantibodies against the ganglionic acetylcholine receptor.

[00144] In order to test the utility of FSEC to clinical patient samples by detecting autoantibodies specific for transmembrane ion channels, as a test case, patient samples for antibodies against the ganglionic nicotinic acetylcholine receptor (gAChR), which is targeted in autoimmune autonomic ganglionopathy (AAG), were examined (see FIG. 16A-16E for workflow).

[00145] Cys-loop receptors are pentameric ligand-gated ion channels that include the cation- selective nicotinic acetylcholine receptors. These receptors are found in and near neuronal synapses and between motor neurons and muscle fibers, where they mediate fast chemical neurotransmission. Subunit composition and stoichiometry of the receptors influences their physiological function and location. At the synapses in the autonomic ganglia, the α3β4 nicotinic acetylcholine receptor (gAChR) is the principal postsynaptic receptor and is required for autonomic function. Autoantibodies against the gAChR are found in 50-60% of AAG cases ² . Patients with AAG present with severe autonomic failure; characteristic features include orthostatic hypotension, constipation, urinary retention, loss of sweating, and impaired pupillary reflexes. These problems can be life-threatening.

[00146] Detection of autoantibodies against membrane receptors like the gAChR currently relies on one of three methods: cell-based immunofluorescence assays, enzyme-linked immunosorbent assays, or radioligand-immunoprecipitation assays. Each has its limitations; in cell-based assays (CBA), conformational epitopes may be distorted as a result of treatment with formaldehyde- based fixatives. A recent study comparing the most commonly used commercial CBA (fixed cells from Eurolmmun) noted that this assay missed up to 18% of neuroreactive autoantibodies. These autoantibodies were missed due to poor sensitivity or poor antigen expression in the cells. They were subsequently detected in “in-house" assays and on immunohistochemistry using rat brain slices, neither of which are widely available. Live CBA, which is considered the “gold standard”, requires a BSL-2 tissue culture laboratory. Enzyme-linked immunosorbent assays (ELISA) depend upon the ability to purify appropriately folded protein and immobilize it upon a substrate. In the context of membrane proteins, purification yields are typically very low and essential detergents can lead to non-native protein conformations. These hurdles limit the utility of ELISA for neuronal receptors, as the purification process can cause aggregation and inaccessibility of epitopes. Furthermore, ELISA has been consistently shown to be the least sensitive and most poorly reproducible assay for several other targets, including aquaporin-4 antibodies and myelin oligodendrocyte glycoprotein antibodies.

[00147] The other major technique, radioligand immunoprecipitation assays (RIPA), involves the use of specialized laboratory equipment and radioactive reagents. These also require appropriate (high affinity, highly selective) radioligands that are not readily available for many neuronal receptors. RIPA may also have high background signal or be overly sensitive, detecting antibodies of questionable relevance to disease. Furthermore, RIPA for AAG and related diseases such as myasthenia gravis uses radioactive ligands that bind to the neurotransmitter site, which can preclude binding by subsets of antibodies via direct competition or by conformational changes. Detection by RIPA depends upon precipitation by patient antibodies via extensive cross- linking. Antibodies that do not induce cross-linking do not precipitate receptors and fail to be detected in RIPA. Ail current assays may suffer from effects of treatments for autoimmune disease. For example, intravenous immunoglobulin can interfere with serological testing and trigger false positive results.

[00148] In contrast, FSEC as provided herein does not require expensive and hazardous radioactive reagents. Nor does it require a tissue culture laboratory. Furthermore, it ensures the receptor target is appropriately assembled, and does not appear to be affected by immunosuppressive treatments, such as high levels of exogenous non-specific immunoglobulin. Provided herein is data showing FSEC is a viable option for specific detection of autoantibodies in serum from patients.

Methods

Standard Protocol Approvals, Registrations, and Patient Consents

[00149] Patient sample collection was approved by the institutional Review Board at UT Southwestern Medical Center (IRB protocols 092004-041 and 012011-182). With informed consent, serum was collected from patients with autoimmune autonomic ganglionopathy, suspected GABA _A encephalitis, suspected NMDA receptor encephalitis, postural orthostatic tachycardia syndrome, or healthy controls. Serum samples were collected and processed through the Neuroscience Biorepository at University of Texas Southwestern Medical Center using previously established protocols. Patient data was stored via REDCap (Research Electronic Data Capture). For the purposes of this study, AAG was defined by typical clinical symptoms and the presence of antibodies against the gAChR was determined by RIPA prior to selection for screening by FSEC. Suspected autoimmune encephalitis (AE) patients were identified clinically. Recombinant expression of target proteins

[00150] Genes encoding the α3GFP β4 nicotinic acetylcholine receptor and the o1β2y2GFP GABA _A receptor were cloned into the pEZT plasmid. Transient transfections of HEK 293 GnTI- adherent cells were performed using Lipofectamine 2000 as per manufacturers protocols. Cells were grown in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum. Cells were incubated for 72 hours at 30 ^c C before harvesting. To produce the α3GFPβ4 nicotinic acetylcholine receptor lysate, GnT1- cells (ATCC) were grown in suspension and transduced with bacmam virus generated from the pEZT plasmids. Suspension cells were transduced with bacmam virus at an MOI of 1 for each subunit and supplemented with 3mM sodium butyrate to enhance expression. For the α7YFP nicotinic acetylcholine receptor lysate, the pEZT plasmid containing α7YFP was used to generate bacmam virus as for α3GFPβ4 . However, transductions were performed in the absence of sodium butyrate and cells were kept at 37°C instead of moving to 30°C as for α3GFPβ4 .

Solubilization to obtain lysate preparation

[00151] Transiently transfected or transduced cells were harvested by centrifugation. Resuspended cells were solubilized with 40 mM dodecyl-maltoside (DDM, Anatrace) in TBS (20 mM Tris pH 7.4, 150 mM NaCI) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF). Lysate was rocked for 40 minutes at 4 °C. Insoluble material was pelleted by ultracentrifugation for 40 minutes at 71,000g at 4 °C. Supernatant was quickly transferred to a fresh tube and kept on ice for immediate analysis via FSEC before aliquoting and storing at -20 °C. In the case of the GABA _A receptor, lysate did not perform well after freezing and thawing and therefore was used immediately after preparation for each experiment.

Fluorescence-detection size-exclusion chromatography

[00152] To perform the FSEC assay, 5 μl of patient serum were mixed with 40 μ ol f lysate and incubated on ice for 1 h. Three separate tubes of serum plus lysate were generated for each patient. After precipitation of large aggregates by ultracentrifugation at 21,130 g for 15 minutes, supernatant was transferred into fresh tubes. Twenty μl of lysate and serum were injected over an SRT SEC-500 column (Sepax Technologies) with 1 mM DDM/TBS as the mobile phase. FSEC trace fluorescence was measured at excitation/emission wavelengths of 483 nm and 510 nm for GFP-tagged proteins. Receptor fluorescence was measured at a time determined by injecting receptor alone, without any control or patient serum. Initial optimizations determined amount of fluorescence signal with maximal signal to noise for receptor lysate. Radioimmunoprecipitation assay

[00153] The RIPA for gAChR antibodies was performed. Briefly, solubilized membranes derived from IMR-32 neuroblastoma cells were incubated with 1251-radiolabeled epibatidine, a high affinity agonist of nicotinic acetylcholine receptors. After mixing with patient serum, samples were incubated overnight at 4°C. Precipitation with anti-human serum from goat was measured for radioactivity in comparison to negative control samples.

Statistical analysis

[00154] gAChR FSEC assay results shown in FIG. 19 were compared using a one-way ANOVA; results from the o7 and GABAA data were compared using Student’s t-test. Correlation between FSEC and RIPA was measured using the correlation coefficient r2. All statistical analyses were performed in GraphPad Prism (Prism v9.5.1).

Results

Testing and optimization of the detection assay

[00155] FSEC was adapted to measure interactions between patient antibodies and target receptors as outlined in FIG 16. Each receptor protein was expressed recombinantly in mammalian cells via transient transduction with a bacMam virus. As these are pentameric receptors, each target protein contained at least one subunit fused to a fluorescent reporter protein. The fluorescent protein facilitates detection via FSEC without purification, and is located in an intracellular domain not accessible to autoantibodies. After transient expression in mammalian ceils, receptors were solubilized in a mild, non-ionic detergent that preserves the pentameric integrity of the receptor. Ultracentrifugation to clear insoluble material was performed prior to addition of antibodies.

[00156] Optimization of the assay included reducing variability by ensuring that the receptor lysates were diluted to an equal fluorescence intensity across experimental days. Ratio of lysate to serum were also optimized to most efficiently detect effects of patient antibody cross-linking and precipitation. We determined the amount of receptor fluorescence able to detect patient antibodies to be a range of 2,000-10,000 μV of fluorescence. However, detectors have different sensitivities and users should perform pilot experiments to determine the best range for their systems. Furthermore, the baseline of cell lysate should be established before determining optimal ratios of lysate and serum. Incubation time and injection volume were also optimized to reduce the time the assay requires, and increase the signal-to-noise ratio. The final parameters are described in detail in the methods.

[00157] In size-exclusion chromatography, proteins elute based on their molecular size, and antibody binding shifts this elution time earlier. Cross-linking of receptors by bivalent immunoglobulin antibodies can also cause target proteins to precipitate out of solution; these are removed via centrifugation prior to injection over the column. In either case, antibody binding to its target results in a decrease in peak amplitude at the normal target elution time (FIG 16E). To establish a quantifiable result, dose-response experiments were first performed using increasing amounts of laboratory generated monoclonal antibodies (see FIG. 17A and 17B). With increasing antibody concentrations, the main pentameric peak amplitude further decreases. The measurement is taken from the fluorescence of the main pentameric peak, and exhibits a dose- dependent decline in correlation to increased antibody concentrations (FIG. 17B).

Application of FSEC assay to patients

[00158] To examine if FSEC was suitable for detection of patient autoantibodies, negative control samples were first procured to rule out nonspecific effects of human blood serum. As seen in FIG 18A-18C (black traces), negative control patient samples with no known autoimmune disease had minimal to no effect on receptor behavior. FSEC assay was utilized to test patients known to have antibodies against the gAChR (α3β4 type). These patients exhibit symptoms of AAG and had previously been tested for antibodies against this receptor by RIPA. As seen in FIG. 18A, a representative patient sample decreased the main pentameric peak amplitude compared to healthy control samples by approximately 50%. As a control for specificity, patient samples were also tested for autoantibodies against the related α7 nicotinic acetylcholine receptor and GABA _A receptor. No cross-reactivity was detected for the α7 nicotinic acetylcholine receptor, nor for the GABA _A receptor (FIG. 18B-18C). This confirms that the assay is specific for receptor subtype. To examine the sensitivity of the assay, serial dilutions of serum were performed with known AAG+ patients. The assay appears sensitive up to a 1:10 dilution of serum (FIG. 19). While less sensitive than RIPA, the FSEC assay may be more specific and decrease false-positive results, as very low levels of α3β4 antibodies may be not be pathogenic.

[00159] A large pool of AAG patient samples, and samples from patients with other autoimmune diseases were screened next using the FSEC assay for the α3β4 gAChR (FIG. 20A). These patients included those experiencing postural orthostatic tachycardia syndrome (POTS), a common form of dysautonomia. POTS patients can occasionally test positive for antibodies against the gAChR, but those samples screened here had tested negative by RIPA and this was confirmed using the FSEC assay. Three patient samples were screened from cases of NMDA- receptor and GABA _A receptor autoimmune encephalitis (FIG 20A, “AE”). A previous study suggested that a small percentage of NMDA autoimmune encephalitis cases also have antibodies against the GABA _A receptor. Patients in the cohort were tested for expression of these cross- reactive antibodies against the related α3β4 gAChR, however none appeared to exhibit this phenotype. Thus, detection of gAChR antibodies was restricted to AAG patients. These AAG patient samples were next examined for cross-reactivity to the related o7 nicotinic receptors (FIG. 20C), but again no reactivity for these related subtypes was seen. The AAG samples described in FIG. 20A-C were tested concurrently via RIPA to determine correlation between the novel and established techniques. They correlated reasonably well, with an r2 of 0.55 and a 95% confidence interval of 0.88-0.51 (FiG. 21). Given the lower detection limit of FSEC, it is possible the correlation between the two assays cannot be improved further as the FSEC assay may hit a maximum detection level at the highest levels of RIPA detection.

Utilizing FSEC to measure responses during treatment

[00160] During assay development several patients were identified for whom there were multiple samples collected over the course of a decade (FIG. 22A-22C). These patients received various medical treatments during sample collection, including intravenous immunoglobulin (IVIG), prednisone, mycophenolate, and rituximab. The lack of change in the FSEC detection result is not wholly unexpected. In a comparison of treatment efficacy across six AAG patients, all but one retained detectable gAChR antibodies assayed by RIPA despite improvement in their symptoms. A strength of the FSEC assay observed over the course of the medical treatments is that none of the results appeared to be confounded by IVIG, which can interfere with RIPA and other laboratory tests.

[00161] In conclusion, the feasibility of using FSEC for the α3β4 nicotinic acetylcholine receptor, a target in AAG was confirmed. Comparison to existing methods illustrated several advantages of FSEC. 1) It does not require the use of expensive, hazardous, and difficult to acquire radioligands. 2) It uses less patient sample than cell-based diagnostic assays. 3) It does not suffer from high background due to nonspecific/off-target binding or nonspecific precipitation. 4) it is readily expandable to other targets, including the muscle nicotinic acetylcholine receptor, a target in myasthenia gravis, and the NMDA-type glutamate receptor, the most common target in autoimmune encephalitis. 5) As the cell lysate is prepared in large quantities in advance and stored frozen, it does not require live cell-based assays or live mouse brain slice culture. Example 11 : Testing of specificity of patient encephalitis antibodies against GABA _A receptors

[00162] Next, encephalitis patient samples were tested for specificity by testing the ability of serum antibodies to bind to GABA _A receptors. Two sets of experiments were conducted. First, all three subunits of the major synaptic subtype were expressed and incubated with encephalitis patient sera (P6) or a negative control. GFP fluorescence indicates the assembly of all three subunits, as it is known the y subunit cannot assemble as a pentamer (see FIG. 23A).

[00163] Next, both α and β subunits are expressed, but not y. The α subunit was tagged with biue-fiuorescent protein (BFP) After incubation with P6 serum the main pentameric peak was not decreased significantly versus the negative control (1), nor is it shifted to an earlier elution time (see FIG. 23B).

[00164] This suggests that the patient antibodies could interact with GABA _A receptor with all three subunits but did not with a and β subunits alone. For example, the epitope of the patient antibody may consist of subunit interfaces, included a combination of α, β, or γ subunits.

[00165] The examples are included to demonstrate preferred embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the present disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.