WO2020037378A1 - Detection of functional autoantibodies - Google Patents

Detection of functional autoantibodies Download PDF

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Publication number
WO2020037378A1
WO2020037378A1 PCT/AU2019/050895 AU2019050895W WO2020037378A1 WO 2020037378 A1 WO2020037378 A1 WO 2020037378A1 AU 2019050895 W AU2019050895 W AU 2019050895W WO 2020037378 A1 WO2020037378 A1 WO 2020037378A1
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Prior art keywords
functional
autoantibody
cell
certain embodiments
antigenic target
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PCT/AU2019/050895
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French (fr)
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Michael Warren Jackson
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The Flinders University Of South Australia
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Priority claimed from AU2018903120A external-priority patent/AU2018903120A0/en
Application filed by The Flinders University Of South Australia filed Critical The Flinders University Of South Australia
Publication of WO2020037378A1 publication Critical patent/WO2020037378A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0058—Antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2123/00—Preparations for testing in vivo
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/70—Mechanisms involved in disease identification
    • G01N2800/7004—Stress
    • G01N2800/7009—Oxidative stress

Definitions

  • the present disclosure relates to methods for detecting functional autoantibodies and to diagnostic and prognostic applications of the methods.
  • Autoantibodies are antibodies produced by the immune system that are directed against one or more of the individual's own proteins. A number of autoimmune diseases are caused by such autoantibodies, or the presence of autoantibodies contributes to disease pathology.
  • type 1 diabetes mellitus is a chronic autoimmune disease characterised by insulin deficiency arising at least in part from immune-mediated destruction of insulin-producing beta cells of the pancreas. While the aetiology of autoimmune-mediated destruction of beta cells is currently under investigation, it is believed that autoreactive T cells play an important role. Evidence suggests that the production of autoantibodies against beta cell antigens plays a significant role in the development of type 1 diabetes.
  • Dysautonomia is a disorder of autonomic nervous system (ANS) function that generally involves dysfunction of the sympathetic or parasympathetic components of the AN, and which may have an autoimmine component.
  • Dysautonomia can be local, as in reflex sympathetic dystrophy, or generalized, as in pure autonomic failure. It can also be acute and reversible, as in Guillain-Barre syndrome, or chronic and progressive.
  • Dysautonomia can occur as a primary condition or in association with degenerative neurological diseases such as Parkinson's disease.
  • Other diseases with generalized, primary dysautonomia include multiple system atrophy and familial dysautonomia.
  • the diagnosis of dysautonomia is generally achieved through functional testing of the ANS, focusing on the affected organ system.
  • the present disclosure relates to methods for detecting functional autoantibodies, to methods for identifying subjects suffering from, or susceptible to, diseases, conditions or states associated with functional autoantibodies, to methods for identifying functional autoantibodies and their antigenic targets, and to methods for identifying agents that modulate binding of a functional autoantibody to an antigenic target.
  • Certain embodiments of the present disclosure provide a method of detecting a functional autoantibody against an antigenic target, the method comprising exposing a cell expressing the antigenic target to the autoantibody, wherein the antigenic target has an activity to alter the level of reactive oxygen or nitrogen species in the cell and binding of the autoantibody to the antigenic target alters the activity of the antigenic target, and detecting the functional antibody on the basis of a change in the level of the reactive oxygen or nitrogen species in the cell.
  • Certain embodiments of the present disclosure provide a method of detecting a functional autoantibody against a L-voltage gated calcium channel in a subject suspected of suffering from, or being susceptible to, type 1 diabetes, the method comprising exposing a cell expressing a L-voltage gated calcium channel to a sample containing antibodies from the subject and detecting the functional antibody in the subject on the basis of a change in the level of the reactive oxygen or nitrogen species in the cell.
  • Certain embodiments of the present disclosure provide a method of detecting a functional autoantibody against a L-voltage gated calcium channel in a subject suspected of suffering from, or being susceptible to, an autonomic disorder, the method comprising exposing a cell expressing a L-voltage gated calcium channel to a sample containing antibodies from the subject and detecting the functional antibody in the subject on the basis of a change in the level of the reactive oxygen or nitrogen species in the cell.
  • Certain embodiments of the present disclosure provide a method of identifying a subject suffering from, or susceptible to type 1 diabetes, the method comprising exposing a cell expressing a L-voltage gated calcium channel to a sample containing antibodies from the subject, and identifying the subject as suffering from, or being susceptible to, type 1 diabetes on the basis of an increase in the level of reactive oxygen species detected in the cell.
  • Certain embodiments of the present disclosure provide a method of identifying a subject suffering from, or susceptible to an autonomic disorder associated with functional autoantibodies to a L-voltage gated calcium channel, the method comprising exposing a cell expressing a L-voltage gated calcium channel to a sample containing antibodies from the subject and identifying the subject as suffering from, or being susceptible to the autonomic disorder on the basis of an increase in the level of reactive oxygen species detected in the cell.
  • Certain embodiments of the present disclosure provide a method of identifying an antigenic target for a selected functional autoantibody, the method comprising:
  • Certain embodiments of the present disclosure provide a method of identifying a functional autoantibody for a selected antigenic target, the method comprising:
  • Certain embodiments of the present disclosure provide a method of identifying an antigenic target for a functional autoantibody associated with type 1 diabetes, the method comprising:
  • Certain embodiments of the present disclosure provide a method of identifying an antigenic target for a functional autoantibody associated with an autonomic disorder, the method comprising:
  • Certain embodiments of the present disclosure provide a method of identifying a functional autoantibody associated with type 1 diabetes, the method comprising:
  • Certain embodiments of the present disclosure provide a method of identifying a functional autoantibody associated with an autonomic disorder, the method comprising:
  • Certain embodiments of the present disclosure provide a method of identifying an agent that modulates the binding of a functional autoantibody to an antigenic target, the antigenic target having an activity to alter the level of reactive oxygen or nitrogen species in a cell, the method comprising:
  • identifying the candidate agent as an agent that modulates the binding of the functional autoantibody to antigenic target.
  • Certain embodiments of the present disclosure provide a method of identifying a therapeutic agent for a disease, condition or state associated with binding of a functional autoantibody to an antigenic target, the antigenic target having an activity to alter the level of reactive oxygen or nitrogen species in a cell, the method comprising: exposing a cell expressing the antigenic target to the functional autoantibody and a candidate agent;
  • identifying the candidate agent as a therapeutic agent for the disease, condition or state associated with binding of a functional autoantibody to an antigenic target is a condition or state associated with binding of a functional autoantibody to an antigenic target.
  • Certain embodiments of the present disclosure provide an agent identified by a method as described herein.
  • kits for performing a method as described herein provide a kit for performing a method as described herein.
  • Certain embodiments of the present disclosure provide use of a cell expressing an antigenic target for a functional autoantibody for identifying an agent that modulates the binding of the functional antibody to the antigenic target, wherein the antigenic target has an activity to alter the level of reactive oxygen or nitrogen species in the cell and binding of the autoantibody to the antigenic target alters the activity of the antigenic target.
  • Certain embodiments of the present disclosure provide use of a cell expressing an antigenic target for a functional autoantibody for identifying a therapeutic agent for a disease, condition or state associated with the binding of the functional autoantibody to the target, wherein the antigenic target has an activity to alter the level of reactive oxygen or nitrogen species in the cell and binding of the autoantibody to the antigenic target alters the activity of the antigenic target.
  • kits for detecting a functional autoantibody comprising:
  • control reagents for the level of reactive oxygen or nitrogen species in the cell.
  • Certain embodiments of the present disclosure provide a complex comprising a cell expressing an antigenic target that has an activity to alter the level of reactive oxygen or nitrogen species in the cell, a functional autoantibody bound to the antigenic target on the cell, and a marker in the cell for detecting the level of reactive oxygen or nitrogen species.
  • Figure 1 shows optimisation of a 2’, 7’ -Dichlorofluorescein diacetate (DCF- DA) assay of reactive species generation in the rat insulinoma cell line, RinAl2.
  • DCF- DA dichlorofluorescein diacetate
  • Figure 2 shows IgG with anti-VGCC activity from patients with type 1 diabetes mediates increased stress as determined by DCF-DA positivity in RinAl2 cells.
  • incubation of cells with equivalent concentrations of IgG from a healthy donor did not significantly alter DCF-DA positivity values.
  • Patient codes refer to Table 1. *Significance refers to P ⁇ 0.05 by two-way ANOVA, experimental condition verses baseline.
  • FIG. 3 shows the DCF-DA-based Rin A12 cell stress assay is suitable for screening patient IgG samples for the presence of anti-VGCC Abs.
  • A Pooled IgG- mediated (at 1 mg/ml) DCF-DA positivity results (4 hrs incubation) from patients previously characterised for the presence (red triangles) or absence (black diamonds) of anti-VGCC Abs (see Table 1).
  • Mean DCF-DA positivity values obtained using individual IgG preparations at 1 mg/ml from all 6 patients positive for anti-VGCC Abs were above the mean plus 2 standard deviations (see Table 2) (dotted line) of results obtained using IgG from 6 healthy individuals (black circles).
  • FIG. 5 shows Rotenone-mediated ROS generation in RinAl2, is dependent on exogenous calcium.
  • Incubation of DCF-DA primed Rin A12 cells over 4 h with EGTA results in no significance change in ROS generation compared to the baseline (cells without EGTA incubation) (red dots).
  • Addition of ImM Rotenone results in a significance increase compared to baseline, which is reversed in the presence of EGTA (hatched bars).
  • *Significance refers to P ⁇ 0.05 by two-way ANOVA, experimental condition verses baseline.
  • FIG. 6 shows the interaction of dihydropyridine (DHP) agonist and antagonist drugs at DHP-sensitive voltage-gated calcium channels (VGCCs) modify ROS stress generation in RinAl2 cells.
  • DHP dihydropyridine
  • VGCCs DHP-sensitive voltage-gated calcium channels
  • Figure 7 shows inhibition of DHP-sensitive VGCCs does not reverse Rotenone-mediated ROS generation in RinAl2 cells.
  • Incubation of DCF-DA primed Rin A12 cells over 4 h with 30 nM Nicardipine (black bar) results in no significance change in ROS generation compared to the baseline (untreated cells).
  • Addition of 1 mM Rotenone (silver bar) results in a significance increase in ROS compared to baseline, with co-incubation of cells with both Rotenone and Nicardipine similarly resulting in a significant increase in ROS (grey bars).
  • *Significance refers to P ⁇ 0.05 by two-way ANOVA, experimental condition verses baseline. .
  • FIG. 8 shows anti-VGCC Ab-mediated ROS generation in RinAl2 cells is inhibited by the DHP antagonist, Nicardipine.
  • a significant increase compared to baseline is achieved in ROS generation incubated with T1D IgG in the absence of Nicardipine. All other incubations do not significantly alter ROS generation.
  • *Significance refers to P ⁇ .05 by two-way ANOVA
  • FIG. 9 shows neutralization of anti-VGCC Abs by anti -idiotypic antibodies in IgG from healthy individuals or functional antibody negative patients.
  • ROS generation in DCF-DA primed Rin A12 cells incubated with 1 mg/ml IgG derived from healthy donor (CTR) or preincubated with an equimolar equivalent of IgG from a separate healthy individual (CTR + CTR) is not significantly different to baseline (cells without IgG incubation).
  • IgG from a patient with anti-VGCC Abs induces a significant increase in ROS expression compared to baseline, which is reduced by pre-incubation with IgG either from a healthy donor (T1D + CTR) or a patient negative for anti-VGCC Abs (T!D + T1D neg), resulting in ROS levels not significantly different compared to baseline.
  • Ig pre-incubation of anti-VGCC Abs can neutralize the functional effect of the channel antibodies on ROS generation in RinAl2 cells. Values are the mean and SEM from at least 2 independent assays incorporating duplicate samples. * Significance refers to P ⁇ .05 by two-way ANOVA.
  • Figure 10 shows ROS stress generation as a percenatge of untreated baseline. Time indicates incubation period. Assay conducted using caprylic acid precipitated IgG. Cell line: Rin A12. [0040] Figure 11 shows PI positivity as a percentage of untreated baseline. Time indicates incubation period. Assay conducted using caprylic acid precipitated IgG. Cell line: Rin A12.
  • the present disclosure relates to methods for detecting functional autoantibodies, methods for identifying subjects suffering from, or susceptible to, diseases, conditions or states associated with functional autoantibodies, methods for identifying functional autoantibodies and their antigenic targets, and methods for identifying agents that modulate binding of a functional autoantibody to an antigenic target.
  • the present disclosure is based, at least in part, on the use of a neuroendocrine cell line, RinAl2, which is a derivative of the rat insulinoma 5F line.
  • This cell line has glucose sensitivity, and is capable of producing low levels of insulin release.
  • beta cell lines such as Rin cells include low free radical scavenger production, and ROS generation under stimulation, factors which benefit their use in an assay based on measuring ROS generation.
  • the time of the assay duration described herein is based largely on a requirement for the build up of detectible ROS in response to antibody- mediated intracellular ion disruption, which may not occur in other cell types less susceptible to accumulation of ROS.
  • the assay has successfully been conducted with a second insulin cell line, that being the murine BTC3 cell.
  • the present disclosure provides the ability to detect different anti-channel autoantibodies. For example, studies conducted as part of this study demonstrate that the anti-VGCC antibody associated with type 1 diabetes of the present disclosure is phenotypically distinguished from an anti-neuronal ion channel antibody in human narcolepsy with cataplexy.
  • the assay of the present disclosure is a pan-anti-ion channel (or G-Coupled Protein Receptor) assay, in that it will detect functional (target modifying) antibodies where the functional modification results in intracellular ion derangement resulting in stress generation.
  • the successful detection of an anti-ion channel antibody in a patient with idiotypic dysautonomia resulted in a phenotypic assay presentation distinct from that produced by the anti-VGCC antibody associated with T1D.
  • Certain embodiments of the present disclosure provide a method of detecting a functional autoantibody against an antigenic target.
  • the method permits the detection of functionally active autoantibodies, for example as present in the IgG fraction of patient serum.
  • the method can be used in some embodiments as a real time assay for detecting autoantibody-mediated stress generation, and for assessing the degree of potential autoantibody pathogenicity, and in some embodiments has clinical utility in diagnosis or prognosis of type 1 diabetes and/or in autonomic dysfunction.
  • the present disclosure provides a method of detecting a functional autoantibody against an antigenic target, the method comprising exposing a cell expressing the antigenic target to the autoantibody, wherein the antigenic target has an activity to alter the level of reactive oxygen or nitrogen species in the cell and binding of the autoantibody to the antigenic target alters the activity of the antigenic target, and detecting the functional antibody on the basis of a change in the level of the reactive oxygen or nitrogen species in the cell.
  • the term“functional autoantibody” as used herein refers to an auto-reactive antibody against an antigenic target in a host, the presence of which has some form of pathophysiologic effect in the host.
  • the present disclosure permits the detection of a variety of different autoantibodies, as demonstrated by the studies described herein.
  • the functional autoantibody is an IgG antibody. In certain embodiments, the functional autoantibody is an IgM antibody.
  • the functional autoantibody is present in a biological sample from a subject.
  • the functional antibody is present in a mix of other biological factors.
  • the functional antibody is a semi-purified or a purified antibody.
  • the functional autoantibody is an isolated antibody.
  • the functional antibody is present in a sample enriched for immunoglobulins.
  • the present method contemplates detecting autoantibodies in crude samples, in processed samples, in enriched samples and/or as semi-purified or purified antibodies.
  • antigenic target refers to a target for an autoantibody.
  • the antigenic target is an extracellular target. In certain embodiments, the antigenic target is a membrane associated target.
  • the antigenic target comprises an endogenous target.
  • the antigenic target comprises an exogenous target introduced into a cell, for example for the purposes of establishing a cell line for detecting functional autoantibodies.
  • the antigenic target is a cloned antigenic target.
  • the antigenic target comprises a membrane bound ion channel. In certain embodiments, the antigenic target comprises a membrane bound receptor.
  • the antigenic target comprises a calcium ion channel, a potassium ion channel, a sodium ion channel or a chloride channel.
  • the antigenic target comprises a L-type voltage gated calcium channel (VGCC).
  • VGCC L-type voltage gated calcium channel
  • the L-type voltage gated calcium channel is the ion channel that is defined by the genes for the subunits CACNA1C, CACNA1D , CACNA1S and CACNA1F.
  • the equivalent ion channel in other species may be readily identified by a method known in the art.
  • the antigenic target comprises a G protein-coupled receptor, such as GPR40, GLP-l and a2 adrenoreceptor.
  • the antigenic target comprises a tyrosine kinase receptor, such as an EGF receptor.
  • the identity of the antigenic target is known. In certain embodiments, the identity of the antigenic target is not known. Indeed, the present disclosure does not rely on needing to know the identity of the antigenic target.
  • reactive oxygen species refers to reactive species and free radical containing species derived from oxygen or containing oxygen.
  • reactive nitrogen species refers to reactive species and free radical nitrogenous containing species.
  • the level of reactive oxygen or nitrogen species in the cell increases upon binding of the autoantibody to the antigenic target.
  • the level of reactive oxygen or nitrogen species decreases upon binding of the autoantibody to the antigenic target.
  • the level of the reactive oxygen or reactive nitrogen species is determined using a fluorescent marker or probe to determine the level.
  • Fluorescent markers or probes for detecting reactive oxygen species are described, for example, in Gomes et al. (2005). Journal of Biochemical and Biophysical Methods 65(2-3): 45-80, hereby incorporated by reference. Fluorescent probes for detecting reactive nitrogen species are also described in Gomes et al. (2006). Journal of Fluorescence 16(1): 119-139, hereby incorporated by reference.
  • the level of the reactive oxygen species is determined using the marker 2',7'-dichlorofluorescein diacetate.
  • the method of detecting a functional autoantibody against an antigenic target comprises use of flow cytometry.
  • Other methods are also contemplated, such as fluorescent microscopy or microplate analysis, which are known in the art.
  • the method comprises determining or assessing the level of reactive oxygen species or reactive nitrogen species in the cell.
  • Qualitative and quantitative methods for determining or assessing the level of reactive oxygen species or reactive nitrogen species in a cell are known in the art.
  • the method of detecting a functional autoantibody against an antigenic target comprises comparison of the level of reactive oxygen species or nitrogen species with a control and/or reference level.
  • the level of reactive oxygen species or nitrogen species known to be associated with a basal level of the species, or when induced by the particular autoantibody or other agent may be used for comparison.
  • the method is used to identify the presence of functional autoantibodies in a subject. In certain embodiments, the method is used to detect the presence or level of functional autoantibodies in a subject. In certain embodiments, the method is used to assess the presence or level of functional autoantibodies in a subject. In certain embodiments, the method is used to confirm the presence of functional autoantibodies in the subject. In certain embodiments, the method is used to confirm the absence of functional autoantibodies in the subject.
  • the method comprises detecting the functional autoantibody in a biological sample from a subject.
  • the biological sample is a biological fluid.
  • the biological fluid comprises urine, saliva or blood, plasma or serum.
  • the biological sample comprises saliva or blood.
  • the method comprises processing the biological sample to allow detection of autoantibodies in the biological sample.
  • the method comprises obtaining a biological sample from a subject and processing the sample to detect functional autoantibodies.
  • the biological sample comprises a processed biological sample.
  • the processed biological sample is enriched for IgG and/or IgM.
  • the method comprises enriching for immunoglobulins. Methods for enriching for immunoglobulins are known in the art.
  • the method comprises exposing the cells to at least 0.3 mg/ml IgG, at least 0.6 mg/ml IgG, or at least 1 mg/ml IgG.
  • the subject is a human subject.
  • the subject is suffering from, or susceptible to, a disease, condition or state, as described herein.
  • the functional autoantibody is associated with a disease, condition or state.
  • the subject is a human subject suffering from, or susceptible to, type 1 diabetes.
  • the functional autoantibody is associated with type 1 diabetes
  • the subject is a human subject suffering from, or susceptible to, an autonomic dysfunction.
  • the autonomic dysfunction comprises gastrointestinal and/or bladder autonomic neuropathy, or a cardiovascular autonomic disorder.
  • the functional autoantibody is associated with an autonomic disorder.
  • the subject is a mammalian subject.
  • the subject is an animal, such as a livestock animal (such as a horse, a cow, a sheep, a goat, a pig), a domestic animal (such as a dog or a cat) and other types of animals, such as monkeys, rabbits, mice, rats and laboratory animals.
  • livestock animal such as a horse, a cow, a sheep, a goat, a pig
  • a domestic animal such as a dog or a cat
  • other types of animals such as monkeys, rabbits, mice, rats and laboratory animals.
  • the cell expressing the antigenic target is a cell naturally expressing the antigenic target.
  • the cell expressing the antigenic target is a cell engineered to express the antigenic target.
  • Methods of introducing and expressing exogenous proteins in cells are known in the art, for example as described in Sambrook et al. Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)) and Ausubel et al. Current Protocols in Molecular Biology (2003) John Wiley & Sons, both of which are herein incorporated by reference.
  • the cell may express one or more subunits of an exogenous VGCC introduced by transformation or transfection into the cell, such as by viral transfection.
  • the cell is one or more cells. In certain embodiments, the cell is a plurality of cells. In certain embodiments, the cell is an isolated cell, or a cell present in a population of other cells, which may be substantially similar or different to the cell. Methods for isolating and culturing cells are known in the art.
  • Suitable cells expressing the antigenic target may be selected.
  • the cell expressing the antigenic target to the autoantibody is a cell of neuroendocrine origin.
  • a cell of neuroendocrine origin expresses a large range of neuro-receptors and neuroendocrine associated ion channels also found in nerve cells and other excitable cells.
  • the cell is an insulinoma cell.
  • the cell is a Rin A12 cell, or a variant or derivative thereof. In certain embodiments, the cell is a murine BTC cell.
  • the cell may be exposed to the autoantibody for a suitable amount of time.
  • the method comprises exposing the cells to the functional autoantibody for a period of at least 2 hours. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of 2 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of at least 3 hours. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of 3 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of at least 4 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 4 hours or greater.
  • the method comprises exposing the cells to the functional antibody for a period of at least 5 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 5 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of at least 6 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 6 hours or greater. Other time periods are contemplated.
  • the method comprises exposing the cells to the functional autoantibody for a period of time in one of the following ranges: 2 to 6 hours, 2 to 5 hours, 2 to 4 hours, 2 to 3 hours, 3 to 6 hours, 3 to 5 hours, 3 to 4 hours, 4 to 6 hours, 4 to 5 hours and 5 to 6 hours. Other time periods are contemplated.
  • a level of cells positive for changed reactive oxygen species and/or changed reactive nitrogen species one standard deviation above the mean value for a control is indicative of the presence of a disease, condition or state associated with the functional autoantibodies.
  • a level of cells positive for changed reactive oxygen species or changed reactive nitrogen species two standard deviations above the mean value for a control is indicative of the presence of a disease, condition or state associated with the functional autoantibodies.
  • the method is used to identify a subject suffering from an autoimmune disease, condition or state, to identify a subject suffering from, or being susceptible to, type 1 diabetes, for diagnosis or prognosis of type 1 diabetes, for assessing the risk or likelihood of a subject suffering from, or being susceptible to, type 1 diabetes, to identify an antigenic target on a cell associated with the binding of a functional autoantibody, or to identify a therapeutic agent.
  • the method is used to screen for functional autoantibodies, to identify a subject suffering from an autoimmune disease, condition or state, to identify a subject suffering from, or being susceptible to, an autonomic disorder, for diagnosis or prognosis of an autoimmune disease, condition or state, for diagnosis or prognosis of an autonomic disorder, for assessing the risk or likelihood of a subject suffering from, or being susceptible to, an autoimmune disease, condition or state, for assessing the risk or likelihood of a subject suffering from, or being susceptible to, an autonomic disorder.
  • the present disclosure provides a method of detecting a functional autoantibody against a L-voltage gated calcium channel in a subject. In certain embodiments, the present disclosure provides a method of detecting a functional autoantibody against a L-voltage gated calcium channel in a subject suffering from, or susceptible to, a disease condition or state.
  • the present disclosure provides a method of detecting a functional autoantibody against a L-voltage gated calcium channel in a subject suspected of suffering from, or being susceptible to, type 1 diabetes.
  • the present disclosure provides a method of detecting a functional autoantibody against a L-voltage gated calcium channel in a subject suspected of suffering from, or being susceptible to, type 1 diabetes, the method comprising exposing a cell expressing a L-voltage gated calcium channel to a sample containing antibodies from the subject and detecting the functional antibody in the subject on the basis of a change in the level of the reactive oxygen species in the cell.
  • the level of reactive oxygen species in the cell increases upon binding of the autoantibody to the L-voltage gated calcium channel.
  • the cell expressing the L-voltage gated calcium channel is a cell of neuroendocrine origin.
  • the cell is an insulinoma cell.
  • the cell is a Rin A12 cell, or a variant or derivative thereof.
  • Other cells are as described herein.
  • the cell may be exposed to the autoantibody for a suitable amount of time.
  • the method comprises exposing the cells to the functional autoantibody for a period of at least 2 hours. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of 2 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of at least 3 hours. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of 3 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of at least 4 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 4 hours or greater.
  • the method comprises exposing the cells to the functional antibody for a period of at least 5 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 5 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of at least 6 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 6 hours or greater. Other time periods are contemplated.
  • the method comprises exposing the cells to the functional autoantibody for a period of time in one of the following ranges: 2 to 6 hours, 2 to 5 hours, 2 to 4 hours, 2 to 3 hours, 3 to 6 hours, 3 to 5 hours, 3 to 4 hours, 4 to 6 hours, 4 to 5 hours and 5 to 6 hours. Other time periods are contemplated.
  • a level of cells positive for changed reactive oxygen species one standard deviation above the mean value for a control is indicative of the subject suffering from, or susceptible to type 1 diabetes.
  • a level of cells positive for changed reactive oxygen species two standard deviations above the mean value for a control is indicative of the subject suffering from, or susceptible to type 1 diabetes.
  • the method is used for the diagnosis or prognosis of type 1 diabetes.
  • the present disclosure provides a method of detecting a functional autoantibody against a L-voltage gated calcium channel in a subject suspected of suffering from, or being susceptible to, an autonomic disorder.
  • the present disclosure provides a method of detecting a functional autoantibody against a L-voltage gated calcium channel in a subject suspected of suffering from, or being susceptible to, an autonomic disorder, the method comprising exposing a cell expressing a L-voltage gated calcium channel to a sample containing antibodies from the subject and detecting the functional antibody in the subject on the basis of a change in the level of the reactive oxygen species in the cell.
  • the method is used for diagnosis or prognosis of an autonomic disorder.
  • the autonomic disorder comprises a gastrointestinal or bladder autonomic neuropathy, or cardiovascular autonomic complications.
  • the level of reactive oxygen species in the cell increases upon binding of the autoantibody to the L-voltage gated calcium channel.
  • the cell expressing the L-voltage gated calcium channel is a cell of neuroendocrine origin.
  • the cell is an insulinoma cell.
  • the cell is a Rin A12 cell, or a variant thereof.
  • the cell may be exposed to the autoantibody for a suitable amount of time.
  • the method comprises exposing the cells to the functional autoantibody for a period of at least 2 hours. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of 2 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of at least 3 hours. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of 3 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of at least 4 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 4 hours or greater.
  • the method comprises exposing the cells to the functional antibody for a period of at least 5 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 5 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of at least 6 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 6 hours or greater. Other time periods are contemplated.
  • the method comprises exposing the cells to the functional autoantibody for a period of time in one of the following ranges: 2 to 6 hours, 2 to 5 hours, 2 to 4 hours, 2 to 3 hours, 3 to 6 hours, 3 to 5 hours, 3 to 4 hours, 4 to 6 hours, 4 to 5 hours and 5 to 6 hours. Other time periods are contemplated.
  • a level of cells positive for changed reactive oxygen species one standard deviation above the mean value for a control is indicative of the subject suffering from, or susceptible to an autonomic disorder.
  • a level of cells positive for changed reactive oxygen species two standard deviations above the mean value for a control is indicative of the subject suffering from, or susceptible to an autonomic disorder.
  • Certain embodiments of the present disclosure provide a method of identifying a subject suffering from, or susceptible to type 1 diabetes.
  • the present disclosure provides a method of identifying a subject suffering from, or susceptible to type 1 diabetes, the method comprising exposing a cell expressing a L-voltage gated calcium channel to a sample containing antibodies from the subject, and identifying the subject as suffering from, or being susceptible to, type 1 diabetes on the basis of an increase in the level of reactive oxygen species detected in the cell.
  • the cell expressing the L-voltage gated calcium channel is a cell of neuroendocrine origin.
  • the cell is an insulinoma cell.
  • the cell is a Rin A12 cell, or a variant thereof.
  • the cell may be exposed to the autoantibody for a suitable amount of time.
  • the method comprises exposing the cells to the functional autoantibody for a period of at least 2 hours. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of 2 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of at least 3 hours. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of 3 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of at least 4 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 4 hours or greater.
  • the method comprises exposing the cells to the functional antibody for a period of at least 5 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 5 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of at least 6 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 6 hours or greater. Other time periods are contemplated.
  • the method comprises exposing the cells to the functional autoantibody for a period of time in one of the following ranges: 2 to 6 hours, 2 to 5 hours, 2 to 4 hours, 2 to 3 hours, 3 to 6 hours, 3 to 5 hours, 3 to 4 hours, 4 to 6 hours, 4 to 5 hours and 5 to 6 hours. Other time periods are contemplated.
  • a level of cells positive for changed reactive oxygen species one standard deviation above the mean value for a control is indicative of the subject suffering from, or susceptible to type 1 diabetes.
  • a level of cells positive for changed reactive oxygen species two standard deviations above the mean value for a control is indicative of the subject suffering from, or susceptible to type 1 diabetes.
  • Certain embodiments of the present disclosure provide a method of identifying a subject suffering from, or susceptible to an autonomic disorder associated with functional autoantibodies to a L-voltage gated calcium channel.
  • the present disclosure provides a method of identifying a subject suffering from, or susceptible to an autonomic disorder associated with functional autoantibodies to a L-voltage gated calcium channel, the method comprising exposing a cell expressing a L-voltage gated calcium channel to a sample containing antibodies from the subject and identifying the subject as suffering from, or being susceptible to the autonomic disorder on the basis of an increase in the level of reactive oxygen species detected in the cell.
  • the cell expressing the L-voltage gated calcium channel is a cell of neuroendocrine origin.
  • the cell is an insulinoma cell.
  • the cell is a Rin A12 cell, or a variant thereof.
  • the cell may be exposed to the autoantibody for a suitable amount of time.
  • the method comprises exposing the cells to the functional autoantibody for a period of at least 2 hours. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of 2 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of at least 3 hours. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of 3 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of at least 4 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 4 hours or greater.
  • the method comprises exposing the cells to the functional antibody for a period of at least 5 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 5 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of at least 6 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 6 hours or greater. Other time periods are contemplated.
  • the method comprises exposing the cells to the functional autoantibody for a period of time in one of the following ranges: 2 to 6 hours, 2 to 5 hours, 2 to 4 hours, 2 to 3 hours, 3 to 6 hours, 3 to 5 hours, 3 to 4 hours, 4 to 6 hours, 4 to 5 hours and 5 to 6 hours. Other time periods are contemplated.
  • a level of cells positive for changed reactive oxygen species one standard deviation above the mean value for a control is indicative of the subject suffering from, or susceptible to an autonomic disorder.
  • a level of cells positive for changed reactive oxygen species two standard deviations above the mean value for a control is indicative of the subject suffering from, or susceptible to an autonomic disorder.
  • Other levels are contemplated.
  • Certain embodiments of the present disclosure provide a method of identifying an antigenic target for a selected functional autoantibody.
  • the present disclosure provides a method of identifying an antigenic target for a selected functional autoantibody, the method comprising:
  • the antigenic target comprises a target for a functional autoantibody associated with a disease, condition or state, as described herein.
  • the antigenic target comprises a target for a functional autoantibody associated with type 1 diabetes.
  • the antigenic target comprises a target for a functional autoantibody associated with an autonomic disorder.
  • Functional autoantibodies are as described herein.
  • the functional autoantibody is an IgG antibody.
  • the functional autoantibody is an IgM antibody.
  • the functional autoantibody is present in a biological sample from a subject. In certain embodiments, the functional antibody is present in a biological sample from a subject suffering a disease, condition or state as described herein.
  • the functional antibody is present in a mix of other biological factors.
  • the functional antibody is a semi-purified or a purified antibody.
  • the functional autoantibody is an isolated antibody.
  • the functional antibody is present in a sample enriched for immunoglobulins.
  • the identity of the functional antibody is unknown. In certain embodiments, the identity of the functional antibody is known. In this case, the method does not relay on needing to know the identity of the functional antibody.
  • Antigenic targets are as described herein.
  • the antigenic target is an extracellular target. In certain embodiments, the antigenic target is a membrane associated target.
  • the antigenic target comprises an endogenous target.
  • the antigenic target comprises an exogenous target introduced into a cell. In certain embodiments, the antigenic target is a cloned antigenic target. [00159] In certain embodiments, the antigenic target comprises a membrane bound ion channel. In certain embodiments, the antigenic target comprises a membrane bound receptor.
  • the antigenic target comprises a calcium ion channel, a potassium ion channel or a sodium ion channel. In certain embodiments, the antigenic target comprises a L-voltage gated calcium channel.
  • the antigenic target comprises a G protein-coupled receptor.
  • the level of reactive oxygen or nitrogen species in the cell increases upon binding of the autoantibody to the antigenic target.
  • the level of reactive oxygen or nitrogen species decreases upon binding of the autoantibody to the antigenic target.
  • the method of identifying an antigenic target comprises use of flow cytometry.
  • Other methods are contemplated, such as fluorescent microscopy or microplate analysis.
  • the method of identifying an antigenic target comprises determining or assessing the level of reactive oxygen species or reactive nitrogen species in the cell.
  • Qualitative and quantitative methods for determining or assessing the level of reactive oxygen species or reactive nitrogen species in a cell are known in the art.
  • the method of identifying an antigenic target comprises comparison of the level of reactive oxygen species or nitrogen species with a control and/or reference level.
  • the method comprises using the functional autoantibody in a biological sample from a subject.
  • Biological samples are as described herein.
  • the method comprises processing the biological sample.
  • the method comprises use of a processed biological sample enriched for IgG and/or IgM. In certain embodiments, the method comprises enriching for immunoglobulins.
  • the method comprises exposing the cells to at least 0.3 mg/ml IgG, at least 0.6 mg/ml IgG, or at least 1 mg/ml IgG.
  • the cell expressing the candidate antigenic target is a cell naturally expressing the candidate antigenic target. In certain embodiments, the cell expressing the candidate antigenic target is a cell engineered to express the candidate antigenic target.
  • the cell is one or more cells. In certain embodiments, the cell is a plurality of cells. In certain embodiments, the cell is an isolated cell, or a cell present in a population of other cells, which may be substantially similar or different to the cell.
  • the cell is a population of cells expressing one or more candidate antigenic targets.
  • a library of possible antigenic targets may be introduced into cells, and the antigenic target for the selected functional autoantibody identified by identifying cells showing a change in the level of reactive oxygen or nitrogen species.
  • the particular cells may then be enriched or purified, and the identity of the antigenic target determined.
  • Suitable cells expressing candidate antigenic targets may be selected.
  • the cell expressing the candidate antigenic target is a cell of neuroendocrine origin.
  • the cell is an insulinoma cell.
  • the cell is a Rin A12 cell, or a variant thereof.
  • Other cells are as described herein.
  • the cell may be exposed to the selected functional autoantibody for a suitable amount of time.
  • the method comprises exposing the cells to the functional autoantibody for a period of at least 2 hours. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of 2 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of at least 3 hours. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of 3 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of at least 4 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 4 hours or greater.
  • the method comprises exposing the cells to the functional antibody for a period of at least 5 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 5 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of at least 6 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 6 hours or greater. Other time periods are contemplated.
  • the method comprises exposing the cells to the functional autoantibody for a period of time in one of the following ranges: 2 to 6 hours, 2 to 5 hours, 2 to 4 hours, 2 to 3 hours, 3 to 6 hours, 3 to 5 hours, 3 to 4 hours, 4 to 6 hours, 4 to 5 hours and 5 to 6 hours. Other time periods are contemplated.
  • the method of identifying the antigenic target may further comprises a variety of other in vitro or in vivo techniques known in the art to assist with the identification of the target, such as immunoprecipitation, Western blotting, functional cloning, and blocking agents.
  • Certain embodiments of the present disclosure provide a method of identifying an antigenic target for a functional autoantibody associated with an autonomic disorder.
  • the present disclosure provides a method of identifying an antigenic target for a functional autoantibody associated with an autonomic disorder, the method comprising:
  • the identity of the functional antibody is known. In certain embodiments, the identity of the functional antibody is unknown.
  • the cell expressing the candidate antigenic target is a cell in a population of cells having different possible antigenic targets.
  • exposing the cell to the functional autoantibody comprises exposing the cell to a sample from a subject known to be suffering from the autonomic disorder.
  • Certain embodiments of the present disclosure provide a method of identifying an antigenic target for a functional autoantibody associated with type 1 diabetes.
  • the present disclosure provides a method of identifying an antigenic target for a functional autoantibody associated with type 1 diabetes, the method comprising:
  • the identity of the functional antibody is known. In certain embodiments, the identity of the functional antibody is unknown.
  • the cell expressing the candidate antigenic target is a cell in a population of cells having different possible antigenic targets.
  • exposing the cell to the functional autoantibody comprises exposing the cell to a sample from a subject known to be suffering from type 1 diabetes.
  • Certain embodiments of the present disclosure provide a method of identifying a functional autoantibody for a selected antigenic target.
  • the present disclosure provides a method of identifying a functional autoantibody for a selected antigenic target, the method comprising:
  • the cell expresses an antigenic target able to bind to bind to a functional autoantibody associated with a disease, condition or state as described herein.
  • the cell expresses an antigenic target able to bind a functional autoantibody associated with type 1 diabetes.
  • the functional antibody is a functional antibody associated with type 1 diabetes.
  • the cell expresses an antigen target able to bind a functional autoantibody associated with type 1 diabetes.
  • the cell expresses an antigenic target able to bind a functional autoantibody associated with an autonomic disorder.
  • the functional antibody is a functional antibody associated with an autonomic disorder.
  • the cell expresses an antigenic target able to bind a functional autoantibody associated with an autonomic disorder.
  • Functional antibodies are as described herein.
  • the functional autoantibody is an IgG antibody.
  • the functional autoantibody is an IgM antibody.
  • the functional autoantibody is present in a biological sample from a subject.
  • the functional autoantibody is present in a mix of other biological factors.
  • the functional autoantibody is a semi- purified or a purified antibody.
  • the functional autoantibody is an isolated antibody.
  • the functional autoantibody is present in a sample enriched for immunoglobulins.
  • the functional autoantibody is present in a subject with a disease, condition or state associated with the presence of the autoantibody. In certain embodiments, the functional autoantibody is present in a subject with type 1 diabetes. In certain embodiments, the functional autoantibody is present in a subject with an autonomic disorder.
  • the identity of the selected antigenic target is known. In certain embodiments, the identity of the selected antigenic target is unknown. In this case, the method does not rely on needing to know the identity of the selected antigenic target. [00203] In certain embodiments, the selected antigenic target is an extracellular target. In certain embodiments, the selected antigenic target is a membrane associated target.
  • the selected antigenic target comprises an endogenous target.
  • the selected antigenic target comprises an exogenous target introduced into a cell.
  • the selected antigenic target is a cloned antigenic target.
  • the selected antigenic target comprises a membrane bound ion channel. In certain embodiments, the selected antigenic target comprises a membrane bound receptor.
  • the selected antigenic target comprises a calcium ion channel, a potassium ion channel or a sodium ion channel. In certain embodiments, the selected antigenic target comprises a L-voltage gated calcium channel.
  • the selected antigenic target comprises a G protein- coupled receptor.
  • the selected antigenic target is associated with a disease, condition or state. In certain embodiments, the selected antigenic target is associated with type 1 diabetes. In certain embodiments, the selected antigenic target is associated with an autonomic disorder.
  • the level of reactive oxygen or nitrogen species in the cell increases upon binding of the functional autoantibody to the selected antigenic target.
  • the level of reactive oxygen or nitrogen species decreases upon binding of the functional autoantibody to the selected antigenic target.
  • the method of identifying the functional autoantibody comprises use of flow cytometry. Other methods are contemplated, such as fluorescent microscopy or microplate analysis.
  • the method of identifying the functional autoantibody comprises determining or assessing the level of reactive oxygen species or reactive nitrogen species in the cell.
  • Qualitative and quantitative methods for determining or assessing the level of reactive oxygen species or reactive nitrogen species in a cell are known in the art.
  • the method of identifying the functional autoantibody comprises comparison of the level of reactive oxygen species or nitrogen species with a control and/or reference level.
  • the method comprises using the functional autoantibody in a biological sample from a subject.
  • Biological samples are as described herein.
  • method comprises processing the biological sample.
  • the method comprises use of a processed biological sample enriched for IgG and/or IgM. In certain embodiments, the method comprises enriching for immunoglobulins.
  • the method comprises exposing the cells to at least 0.3 mg/ml IgG, at least 0.6 mg/ml IgG, or at least 1 mg/ml IgG.
  • the cell expressing the selected antigenic target is a cell naturally expressing the selected antigenic target. In certain embodiments, the cell expressing the selected antigenic target is a cell engineered to express the selected antigenic target.
  • the cell expressing the selected antigenic target is a cell of neuroendocrine origin.
  • the cell is an insulinoma cell.
  • the cell is a Rin A12 cell, or a variant thereof.
  • Other cells are contemplated. Other cells are as described herein.
  • the cell may be exposed to the candidate antibody for a suitable amount of time.
  • the method comprises exposing the cells to the functional autoantibody for a period of at least 2 hours. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of 2 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of at least 3 hours. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of 3 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of at least 4 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 4 hours or greater.
  • the method comprises exposing the cells to the functional antibody for a period of at least 5 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 5 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of at least 6 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 6 hours or greater. Other time periods are contemplated.
  • the method comprises exposing the cells to the functional autoantibody for a period of time in one of the following ranges: 2 to 6 hours, 2 to 5 hours, 2 to 4 hours, 2 to 3 hours, 3 to 6 hours, 3 to 5 hours, 3 to 4 hours, 4 to 6 hours, 4 to 5 hours and 5 to 6 hours. Other time periods are contemplated.
  • a variety of methods for identify a candidate antibody binding to the selected antigenic target are known in the art.
  • the selected antigenic target can be used as a target for affinity purification of the functional autoantibody.
  • blocking peptides may be used to identify the antibody.
  • the present disclosure provides a method of identifying a functional autoantibody associated with type 1 diabetes, the method comprising:
  • the present disclosure provides a method of identifying a functional autoantibody associated with an autonomic disorder, the method comprising:
  • Certain embodiments of the present disclosure provide a method of identifying an agent that modulates the binding of a functional autoantibody to an antigenic target.
  • agents may be potential therapeutic agents and/or agents suitable for research purposes.
  • the present disclosure provides a method of identifying an agent that modulates the binding of a functional autoantibody to an antigenic target, the antigenic target having an activity to alter the level of reactive oxygen or nitrogen species in a cell, the method comprising:
  • identifying the candidate agent as an agent that modulates the binding of the functional autoantibody to antigenic target.
  • the method is used to identify a therapeutic candidate for a disease, condition or state, or to identify an agent for use in a method for detecting the autoantibody.
  • the method may be used to identify an agent that modulates the binding of a functional autoantibody associated with type 1 diabetes to an antigenic target.
  • the functional antibody is a functional autoantibody associated with type 1 diabetes.
  • the method may be used to identify an agent that modulates the binding of a functional autoantibody associated an autonomic disorder to an antigenic target.
  • the functional antibody is a functional autoantibody associated with an autonomic disorder.
  • the functional autoantibody is an IgG antibody. In certain embodiments, the functional autoantibody is an IgM antibody.
  • the functional autoantibody is present in a biological sample from a subject.
  • the functional autoantibody is present in a mix of other biological factors. In certain embodiments, the functional autoantibody is a semi- purified or a purified autoantibody. In certain embodiments, the functional autoantibody is an isolated antibody. In certain embodiments, the functional autoantibody is present in a sample enriched for immunoglobulins. [00237] Methods for enriching and purifying antibodies are known in the art.
  • the identity of the functional antibody is known. In certain embodiments, the identity of the functional antibody is not known. In this case, the method does not rely on needing to know the identity of the selected functional antibody.
  • Antigenic targets are as described herein.
  • the antigenic target is an extracellular target. In certain embodiments, the antigenic target is a membrane associated target.
  • the antigenic target comprises an endogenous target.
  • the antigenic target comprises an exogenous target introduced into a cell.
  • the antigenic target is a cloned antigenic target.
  • the antigenic target comprises a membrane bound ion channel. In certain embodiments, the antigenic target comprises a membrane bound receptor.
  • the antigenic target comprises a calcium ion channel, a potassium ion channel or a sodium ion channel.
  • the antigenic target comprises a L-type voltage gated calcium channel (VGCC).
  • VGCC L-type voltage gated calcium channel
  • the antigenic target comprises a G protein-coupled receptor.
  • the identity of the antigenic target is known. In certain embodiments, the identity of the antigenic target is not known. In this case, the method does not rely on need to know the identity of the antigenic target. [00248] Methods for assessing the level of reactive oxygen or nitrogen species in a cell are as described herein.
  • the agent comprises a drug, a small molecule, a protein, a polypeptide, a nucleic acid, a lipid, a ligand, a lipid, a carbohydrate, a nucleic acid, an oligonucleotide, a ribozyme, a biologic, an aptamer, a peptide, a cofactor, a ligand, a ligand mimetic, a receptor, an enzyme, a metal ion, a chelate, a nucleic acid, an antibody, and an amino acid.
  • Other types of molecules are contemplated.
  • the method of identifying the agent comprises use of flow cytometry.
  • Other methods are contemplated, such as fluorescent microscopy or microplate analysis.
  • the method comprises determining or assessing the level of reactive oxygen species or reactive nitrogen species in the cell.
  • Qualitative and quantitative methods for determining or assessing the level of reactive oxygen species or reactive nitrogen species in a cell are as described herein and known in the art.
  • the method of identify an agent that modulates the binding of a functional autoantibody to an antigenic target comprises comparison of the level of reactive oxygen species or nitrogen species with a control and/or reference level.
  • the method comprises using functional autoantibody in a biological sample from a subject.
  • Biological samples are as described herein.
  • the processed biological sample is enriched for IgG and/or IgM.
  • the method comprises enriching for immunoglobulins. Methods for enriching for immunoglobulins are known in the art.
  • the method comprises exposing the cells to at least 0.3 mg/ml IgG, at least 0.6 mg/ml IgG, or at least 1 mg/ml IgG.
  • the functional autoantibody is from a subject suffering from, or susceptible to, a disease, condition or state associated with functional autoantibodies.
  • the functional autoantibody is from a subject suffering from, or susceptible to, type 1 diabetes.
  • the functional autoantibody is from a subject suffering from, or susceptible to, an autonomic dysfunction.
  • the autonomic dysfunction comprises gastrointestinal and/or bladder autonomic neuropathy, or a cardiovascular autonomic disorder.
  • Cells expressing antigenic targets are as described herein. Suitable cells expressing the antigenic target may be selected.
  • the cell expressing the antigenic target is a cell naturally expressing the antigenic target. In certain embodiments, the cell expressing the antigenic target is a cell engineered to express the antigenic target.
  • the cell expressing the antigenic target to the autoantibody is a cell of neuroendocrine origin.
  • the cell is an insulinoma cell.
  • the cell is a Rin A12 cell, or a variant or derivative thereof.
  • the cell may be exposed to the functional autoantibody for a suitable amount of time.
  • the method comprises exposing the cells to the functional autoantibody for a period of at least 2 hours. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of 2 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of at least 3 hours. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of 3 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of at least 4 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 4 hours or greater.
  • the method comprises exposing the cells to the functional antibody for a period of at least 5 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 5 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of at least 6 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 6 hours or greater. Other time periods are contemplated.
  • the method comprises exposing the cells to the functional autoantibody for a period of time in one of the following ranges: 2 to 6 hours, 2 to 5 hours, 2 to 4 hours, 2 to 3 hours, 3 to 6 hours, 3 to 5 hours, 3 to 4 hours, 4 to 6 hours, 4 to 5 hours and 5 to 6 hours. Other time periods are contemplated.
  • the method of identifying an agent that modulates the binding of a functional autoantibody to an antigenic target may further utilise a variety of other in vitro or in vivo techniques known in the art to assist with the identification of the agent, such as immunoassay (eg ELISA), immunoprecipitation, Western blotting, affinity purification, binding assays, and competition assays.
  • immunoassay eg ELISA
  • immunoprecipitation eg.g ELISA
  • Western blotting e.g., affinity purification of binding assays, and competition assays.
  • Certain embodiments of the present disclosure provide an agent identified according to a method as described herein.
  • agents include a drug, a small molecule, a protein, a polypeptide, a nucleic acid, a lipid, a ligand, a lipid, a carbohydrate, a nucleic acid, an oligonucleotide, a ribozyme, a biologic, an aptamer, a peptide, a cofactor, a ligand, a ligand mimetic, a receptor, an enzyme, a metal ion, a chelate, a nucleic acid, an antibody, and an amino acid.
  • Other types of molecules are contemplated.
  • the agent is a candidate therapeutic agent.
  • the agent may be used to detect the functional autoantibody in other assays using binding of the agent to the antibody.
  • assays are known in the art, and include for example, enzyme-linked immunosorbent assay ELISA (eg direct ELISA, indirect ELISA, and sandwich ELISA).
  • ELISA enzyme-linked immunosorbent assay ELISA
  • Certain embodiments of the present disclosure provide a method of detecting a functional autoantibody, the method comprising using an identified agent to detect the functional autoantibody. Methods of detecting a functional autoantibody using an agent are known in the art.
  • Certain embodiments of the present invention provide a method of identifying a therapeutic agent for a disease, condition or state associated with binding of a functional autoantibody to an antigenic target.
  • the present disclosure provides a method of identifying a therapeutic agent for a disease, condition or state associated with binding of a functional autoantibody to an antigenic target, the antigenic target having an activity to alter the level of reactive oxygen or nitrogen species in a cell, the method comprising:
  • identifying the candidate agent as a therapeutic agent for the disease, condition or state associated with binding of a functional autoantibody to an antigenic target is a condition or state associated with binding of a functional autoantibody to an antigenic target.
  • Agents, and methods for identifying agents, are as described herein.
  • the disease, condition or state is type 1 diabetes.
  • the disease, condition or state is an autonomic disorder.
  • the autonomic disorder comprises a gastrointestinal or bladder autonomic neuropathy, or cardiovascular autonomic complications.
  • Methods for determining the ability of an agent to have a therapeutic ability include for example use of in vitro assays for determining the effect of an agent on cells and/or one or more animal models for a disease, condition or state.
  • kits for performing a method as described herein provide a kit for performing a method as described herein.
  • the kit comprises one or more reagents, cells and instructions as described herein.
  • Certain embodiments of the present disclosure provide use of a cell expressing an antigenic target for a functional autoantibody for identifying an agent that modulates the binding of the functional autoantibody to the antigenic target.
  • Cells, functional autoantibodies and antigenic targets, are as described herein. Methods for using cells expressing an antigenic target are as described herein.
  • the present disclosure provides use of a cell expressing an antigenic target for a functional autoantibody for identifying an agent that modulates the binding of the functional autoantibody to the antigenic target, wherein the antigenic target has an activity to alter the level of reactive oxygen or nitrogen species in the cell and binding of the autoantibody to the antigenic target alters the activity of the antigenic target.
  • Certain embodiments of the present disclosure provide use of a cell expressing an antigenic target for a functional autoantibody for identifying a therapeutic agent for a disease, condition or state associated with the binding of the functional autoantibody to the target.
  • the present disclosure provides use of a cell expressing an antigenic target for a functional autoantibody for identifying a therapeutic agent for a disease, condition or state associated with the binding of the functional autoantibody to the target, wherein the antigenic target has an activity to alter the level of reactive oxygen or nitrogen species in the cell and binding of the autoantibody to the antigenic target alters the activity of the antigenic target.
  • Cells, and antigenic targets, are as described herein. Methods for using cells expressing an antigenic target are as described herein.
  • kits for detecting a functional autoantibody provide a kit for detecting a functional autoantibody.
  • the present disclosure provides a kit for detecting a functional autoantibody, the kit comprising:
  • control reagents for the level of reactive oxygen or nitrogen species in the cell.
  • the present disclosure provide an isolated complex comprising a cell expressing an antigenic target that has an activity to alter the level of reactive oxygen or nitrogen species in the cell, a functional autoantibody bound to the antigenic target on the cell, and a marker in the cell for detecting the level of reactive oxygen or nitrogen species.
  • Antigenic targets are as described herein.
  • Markers for detecting the level of reactive oxygen or nitrogen species in cells are as described herein.
  • Certain embodiments of the present disclosure provide a method of detecting a functional autoantibody to an antigenic target on a cell, the method comprising using a complex as described herein to detect the functional autoantibody.
  • Standard techniques and equipment may be used for recombinant DNA technology, DNA sequencing, DNA arrays, oligonucleotide synthesis, molecular biology, cell biology and enzymatic reactions.
  • the foregoing techniques and procedures may be generally performed according to methods known in the art and/or as commercially available, and are as described for example in Sambrook et al. Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)) and Ausubel et al Current Protocols in Molecular Biology (2003) John Wiley & Sons, both of which are herein incorporated by reference.
  • EXAMPLE 1 A cell-based assay for the detection of pathogenic anti -Voltage-Gated Calcium Channel autoantibodies in immunoglobulin G from patients with Type 1
  • T1D patient-derived IgG lacking anti-VGCC activity or IgG from healthy donors altered ROS or annexin V expression, indicating the new assay is specific for the detection of functional anti-VGCC Abs.
  • the new cell-based assay provides, for the first time, experimental evidence supporting a plausible pathophysiological mechanism underlying anti-VGCC Ab-mediated apoptosis induction in b cells. Additionally, the assay is a considerable advance on previously published methods for detecting and characterising the functional activity of anti-VGCC Abs in patient-derived samples.
  • Type 1 diabetes mellitus is a chronic autoimmune disease characterised by insulin deficiency arising from immune-mediated destruction of insulin-producing beta (b) cells of the pancreas.
  • b insulin-producing beta
  • T1D is a chronic autoimmune disease characterised by insulin deficiency arising from immune-mediated destruction of insulin-producing beta (b) cells of the pancreas.
  • b insulin-producing beta
  • T1D is a chronic autoimmune disease characterised by insulin deficiency arising from immune-mediated destruction of insulin-producing beta (b) cells of the pancreas.
  • b insulin-producing beta
  • the cell-based stress induction assay is suitable for screening patients for the presence of these functional autoantibodies, thereby offering for the first time, a diagnostic assay capable of demonstrating both the presence and activity of these functional antibodies in larger patient cohorts, potentially improving the understanding of the role these antibodies play in the pathogenesis of T1D.
  • Table 2 Codes and demographic information for patients and controls.
  • Anti-VGCC Ab positive or negative patients indicate patients where anti-VGCC Abs have been detected in the IgG fraction of serum using a colon assay of colonic migrating motor activity and pharmacological confirmation, as reported in Jackson, M.W. & Gordon, T.P. (2010) A novel impedance-based cellular assay for the detection of anti-calcium channel autoantibodies in type 1 diabetes. J Immunol Methods, 361, 31-36. Uncharacterised patients refers to patients recruited to the study where anti-VGCC Ab status is unknown. [00304] 2.2 Preparation of IgG
  • IgG was prepared from individual serum samples using the caprylic acid precipitation technique (Steinbuch and Audran, Arch Biochem Biophys. 1969 134(2): 279-84.) as described previously (Jackson et al. J Neurosci. 2008 28(49): 13303-9). Briefly, serum was lowered to a pH of 4.8 using a solution of acetic acid (1M) and sodium hydroxide (0.5M), and stirred vigorously for 30 mins at room temperature. Caprylic acid was added while stirring the solution, to achieve a final caprylic acid to serum ratio of 0.74: 10.
  • Rat insulinoma cells were cultured at 37 °C in an atmosphere of 5% C02 in air, in RPMI 1640 (Sigma- Aldrich, Australia) containing penicillin/streptomycin, 5% L-glutamine and supplemented with 10% fetal calf serum (Sigma-Aldrich, Australia). The cells were harvested at confluences of between 60-70 % by trypsin digestion, washed twice and re-suspended in HBSS.
  • Rin A12 cells were primed by incubation with 40 mM DCF-DA for 5 mins at 37°C, using a modification of previously reported methodology (Tampo et al. (2003) Circulation Research , 92: 56-63).
  • DCF-DA primed cells were then washed and re-suspended in individual vials in HBSS and incubated as follows: i) for establishment of positive controls for ROS induction, cells were incubated in HBSS for 4 hrs, followed by 5 mins of incubation in the presence or absence of Rotenone (Sigma-Aldrich, Australia) at between 0.1 and 2 mM, or for 4 hours in HBSS either with or without 30 pM Bay K8644 (Alomone Labs, Hadassah Ein Kerem, Israel), with addition or not of between 1 to 30 pM of nicardipine (Sigma-Aldrich, Australia); ii) in HBSS in the presence or absence of patient or control IgG at final concentrations of between 0.3 and 5 mg/mL.
  • Rotenone Sigma-Aldrich, Australia
  • IgG Experiments conducted with IgG also included duplicate vials of cells incubated in HBSS alone for 4 hours, followed by incubated for 5 mins in the presence of 1 pM Rotenone for confirmation of stress induction in each individual experimental run. Following incubations, cells were washed three times, and resuspended in HBSS (200 pl) at room temperature. Propidium iodide (PI) (Sigma-Aldrich) (50 pg/ml) was added just prior to flow cytometric analysis. All samples were assessed in triplicate and on at least 2 separate occasions. DCF-DA incubation, and all subsequent incubations were performed in the dark.
  • PI Propidium iodide
  • cells (2.5 X l0 5 /ml) were incubated in HBSS in the presence or absence of 1 mg/ml of patient or control IgG for 4 or 16 hrs. Following incubation, cells were washed in binding buffer (eBioscience, Invitrogen, Australia) and re-suspended in 100 pl of the same buffer, and a FITC-conjugated antibody against annexin V (BD Biosciences, Maryland) added at a ratio of 5 parts per 100 of binding buffer, and the cells incubated at room temperature in the dark for 10 mins.
  • binding buffer eBioscience, Invitrogen, Australia
  • a FITC-conjugated antibody against annexin V BD Biosciences, Maryland
  • Controls included cells incubated for 4 or 16 hrs in binding buffer alone, either with or without annexin V labelling, and cells incubated in the presence of 0.1 pM staurosporine (Sigma-Aldrich, Australia) as a positive control for induction of apoptosis. Following incubation with the annexin V antibody, cells were washed 3 times in binding buffer, and PI (50 pg/ml) was added just prior to flow cytometric analysis. [00312] 2.6 Flow cytometric analysis
  • Annexin V For determination of positivity for Annexin V, untreated but Annexin V antibody-labelled cells were acquired and used as the baseline for subsequent comparisons with treated cells from the same experimental run. As with DCF-DA primed cells, PI positive cells were excluded from analysis for Annexin V positivity.
  • DCF-DA is a suitable marker of reactive species generation, and the L-type VGCC agonist, Bay K8466, mediates reversible stress generation in Bin A 12 cells.
  • incubation of cells for 4 hrs with concentrations of IgG ranging from 0.3-2 mg/mL produced no significant change in DCF-DA positivity, however, both 3 and 5 mg/ml of IgG resulted in significantly increased values when compared to no IgG addition (by 2- way ANOVA) ( Figure 1 C), likely resulting from non-specific effects of high IgG concentrations.
  • IgG incubation of cells using patient-derived samples was performed using IgG concentration of between 0.3-2 mg/ml.
  • IgG at between 0.3-2 mg/ml from control subjects was incubated with primed Rin A12 cells for between 1 and 7 hrs, and DCF-DA positivity assessed.
  • DCF-DA positivity values were not significantly altered by the presence of IgG, but were significantly higher following 7 hrs incubation with IgG at all concentrations when compared to cells incubated without IgG ( Figure 1 D). Therefore, incubation with patient IgG was performed for 4 hrs.
  • the DCF-DA cell-based stress assay is a suitable screening assay for determining the presence in patient IgG of functional anti-VGCC Abs.
  • 1 mg/ml appears to be a suitable IgG incubation concentration for detecting the presence of anti- VGCC Abs in IgG derived from T1D patients.
  • analysis of results obtained using 2 mg/ml of IgG from T1D+ patients resulted in mean values above corresponding control means plus 2 SDs in only 2 of 6 cases.
  • the optimum incubation time for the cell-based stress assay is 4 hrs.
  • Footnotes Values are derived from Rin A12 cells incubated with 1 mg/ml IgG, and are expressed as percentage of baseline (cells incubated without IgG). Values above the mean + 2 SD of pooled values derived from control IgG indicate positivity for stress induction
  • Annexin positivity as determined by flow cytometric analysis of annexin V-antibody labelled cells, was significantly higher in cells incubated with T1D+ IgG compared to controls following 16 hrs of incubation (by 2-way ANOVA, pooled means of annexin V positivity values; T1D+ verses control), but not at 4 hrs, while T1D- IgG did not significantly alter annexin positivity at either time point (Fig. 4).
  • the current assay is a suitable substitute for the CMMC assay.
  • the RS cell assay was used to screen IgG from 12 previously untested patients with T1D. Within this cohort, IgG samples from 9 individual patients were identified as having the ability to induce ROS stress induction. Hence, in the total cohort used for the current study, 15 of 20 individual patient IgG samples screened contained functional anti-VGCC Abs capable of mediating RS stress induction in pancreatic b cells.
  • the new assay incorporating a functional assessment of stress induction has the ability to directly investigate the pathogenic effect of these Abs on b cells, the main target of autoimmune responses in T1D.
  • Induction of RS-associated stress is particularly harmful for pancreatic b cells due to a low anti -oxidative capacity including low expression of superoxide dismutase (SOD), catalase, and glutathione peroxidas, leaving these cells vulnerable to dysfunction and apoptosis.
  • SOD superoxide dismutase
  • catalase catalase
  • glutathione peroxidas leaving these cells vulnerable to dysfunction and apoptosis.
  • the current study which assesses RS generation, provides for the first time a potential pathogenic mechanism underlying anti-VGCC Ab-mediated b cell apoptosis.
  • EGTA ethylene glycol tetra-acetic acid
  • Exogenous Ca 2+ is required for rotenone-mediated ROS generation
  • rotenone was used to stimulate ROS generation in cells following incubation with media containing either free Ca 2+ or the chelating agent, EGTA.
  • assessment of DCF-DA positivity for cells incubated in chelator-containing media resulted in unchanged values compared to cells incubated without EGTA (baseline DCF-DA positivity).
  • assessment of DCF-DA positivity for cells incubated in Ca 2+ -containing media with acute addition of rotenone resulted in a significant increase of 538% compared to baseline.
  • the rotenone-mediated increase in DCF-DA positivity was reversed in cells incubated with EGTA, with positivity values not significantly different to baseline.
  • Bay K8644 stimulates ROS generation in Rin A12 cells
  • DCF-DA positivity was assessed following treatment by Bay K8644 and nicardipine, at concentrations ranging from 1-100 nM.
  • Assessment of DCF-DA positivity for cells incubated with Bay K8644 at concentrations of between 1-10 nM or 100 nM resulted in no significant increase compared to untreated cells (baseline DCF-DA positivity), however incubation with 30 nM resulted in a significant increase of DCF-DA positivity of 477% of baseline ( Figure 6A).
  • assessment of DCF-DA positivity for cells incubated with nicardipine (1-100 nM) displayed no statistical change across compared to baseline the concentration range (Figure 6B).
  • Nicardipine does not reverse rotenone -mediated ROS generation
  • nicardipine was used at a concentration of 10 nM to assess the b cell stress response to rotenone.
  • assessment of DCF-DA positivity in unstimulated cells incubated with nicardipine resulted in unchanged values compared to untreated cells (baseline DCF-DA positivity).
  • assessment of DCF-DA positivity for cells incubated without nicardipine and stimulated with rotenone resulted in a significant increase of 891% compared to the baseline.
  • Anti-VGCC Abs-mediated ROS generation is reversed by nicardipine
  • DCF-DA positivity was assessed in cells incubated with IgG from T1D+ROS patients, or healthy donor controls, either alone or with 10 nM of nicardipine.
  • the autoantibodies are able to bind and exert a functional effect depend on the cell types and membrane potential.
  • LEMS Lambert-Eaton myasthenic syndrome
  • nicotinic acetylcholinergic receptors which inhibit skeletal muscle contraction
  • VGCC voltage gated Ca 2 + channels
  • Figure 10 shows ROS the stress generation as a % of untreated baseline.
  • Figure 11 shows the PI positivity as a percentage of untreated baseline.
  • the assays were conducted using caprylic acid precipitated IgG with the Rin A12 cell line.
  • the female patient tested positive on the cell based research assay designed for detection of pathogenic functional auto-antibodies directed against voltage gated calcium channels.
  • the data was generated from at least 2 separate assays, on different days.
  • results produced by the functional antibody in this instance resulted in increased apoptosis at 2, 4 and 6 hours incubation, and an increased stress response across both 4 and 6 hours.
  • the typical profile for anti-VGCC antibodies is an increased stress response at 4 hours with no increase in apoptosis at this time point.
  • the autonomic profile of the dysautonomia patient is substantially more pronounced than that associated with the anti-VGCC antibody.
  • the patient has severe gut dysmotility, atonic/neurogenic bladder, and postural othostatic tachycardia syndrome.
  • the autonomic dysfunction associated with the anti-VGCC antibody in type 1 diabetes appears limited to mild to medium gastrointestinal dysfunction, particularly gastroparesis.
  • Table 5 shows the correlations between the presence in T1D patients of anti- VGCC antibodies and symptoms of gastrointestinal dysfunction (gastroparesis).
  • the table shows the correlation between presence of anti-VGCC Ab and autonomic gastrointestinal tract dysfunction symptoms in 15 patients with T1D screened for the presence of anti-VGCC Abs and for the presence of symptoms of gastrointestinal tract dysfunction (gastroparesis) as determined by treating physician.
  • Footnote The presence of symptoms is positively correlated with positivity for anti-VGCC Abs (P ⁇ 0.05 by 2-tailed Chi Square).
  • results also support the use of the live cell-based bioassay for anti channel antibodies.
  • this assay is based on an excitable neuroendocrine pancreatic beta cell line (Rin A12), and is capable of detecting functional antibodies disrupting ion channels (or GPCRs) present on the cell surface and modifying cellular ion transport.
  • ROS reactive oxygen stress
  • the results indicate the presence in the patient IgG fraction of an anti-ion channel antibody. It is apparent that over multiple time points, IgG from the patient induces increased ROS stress in the cell bioassay, with a corresponding decrease in cell viability (as demonstrated by the increase in PI) up to 6 hours. The data indicates an antibody-mediated stress response leading to the induction of apoptosis in the incubated cells.
  • a kit or combination product for detecting functional autoantibodies may include one or more of the following components:
  • Reagents for processing serum samples from a patient For example solutions for adjusting pH, reagents for enriching immunoglobulins (eg caprylic acid), and Hanks’ Balanced Salt Solution.
  • Reagents for cell labelling to detect reactive oxygen species For example, 2’, 7’ -Dichlorofluorescein diacetate (DCF-DA).
  • Control/Reference Reagents For example, rotenone, nicardipine, control antibodies.
  • a kit or product may also include instructions for processing samples, cell culture, use of controls and reference standards, antibody-antigen binding conditions, and analysis.

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Abstract

The present disclosure relates to methods for detecting functional autoantibodies and to diagnostic and prognostic applications of the methods. In certain embodiments, the present disclosure provides a method of detecting a functional autoantibody against an antigenic target, the method comprising exposing a cell expressing the antigenic target to the autoantibody, wherein the antigenic target has an activity to alter the level of reactive oxygen or nitrogen species in the cell and binding of the autoantibody to the antigenic target alters the activity of the antigenic target, and detecting the functional antibody on the basis of a change in the level of the reactive oxygen or nitrogen species in the cell.

Description

DETECTION OF FUNCTIONAL AUTO ANTIBODIES
PRIORITY CLAIM
[001] This application claims priority to Australian Provisional Patent Application 2018903120 filed on 24 August 2018, the content of which is hereby incorporated by reference in its entirety.
FIELD
[002] The present disclosure relates to methods for detecting functional autoantibodies and to diagnostic and prognostic applications of the methods.
BACKGROUND
[003] Autoantibodies are antibodies produced by the immune system that are directed against one or more of the individual's own proteins. A number of autoimmune diseases are caused by such autoantibodies, or the presence of autoantibodies contributes to disease pathology.
[004] For example, type 1 diabetes mellitus (T1D) is a chronic autoimmune disease characterised by insulin deficiency arising at least in part from immune-mediated destruction of insulin-producing beta cells of the pancreas. While the aetiology of autoimmune-mediated destruction of beta cells is currently under investigation, it is believed that autoreactive T cells play an important role. Evidence suggests that the production of autoantibodies against beta cell antigens plays a significant role in the development of type 1 diabetes.
[005] Dysautonomia (or autonomic dysfunction) is a disorder of autonomic nervous system (ANS) function that generally involves dysfunction of the sympathetic or parasympathetic components of the AN, and which may have an autoimmine component. Dysautonomia can be local, as in reflex sympathetic dystrophy, or generalized, as in pure autonomic failure. It can also be acute and reversible, as in Guillain-Barre syndrome, or chronic and progressive. Dysautonomia can occur as a primary condition or in association with degenerative neurological diseases such as Parkinson's disease. Other diseases with generalized, primary dysautonomia include multiple system atrophy and familial dysautonomia. The diagnosis of dysautonomia is generally achieved through functional testing of the ANS, focusing on the affected organ system.
[006] Unlike standard antibodies, functional autoantibodies bind to their antigens and contribute to a pathophysiologic effect in the host. The detection of autoantibodies typically uses similar methodologies to those that are used for antibodies generally, namely that only detect antibody-antigen interactions. However, the detection of functional autoantibodies is more problematic, as most antibody-antigen based detection methodologies do not provide a functional readout of the binding of antibody to its antigen.
[007] Accordingly, there is a need for improved methods of detecting some types of functional antibodies, particularly for the diagnosis and/or prognosis of some disorders.
SUMMARY
[008] The present disclosure relates to methods for detecting functional autoantibodies, to methods for identifying subjects suffering from, or susceptible to, diseases, conditions or states associated with functional autoantibodies, to methods for identifying functional autoantibodies and their antigenic targets, and to methods for identifying agents that modulate binding of a functional autoantibody to an antigenic target.
[009] Certain embodiments of the present disclosure provide a method of detecting a functional autoantibody against an antigenic target, the method comprising exposing a cell expressing the antigenic target to the autoantibody, wherein the antigenic target has an activity to alter the level of reactive oxygen or nitrogen species in the cell and binding of the autoantibody to the antigenic target alters the activity of the antigenic target, and detecting the functional antibody on the basis of a change in the level of the reactive oxygen or nitrogen species in the cell. [0010] Certain embodiments of the present disclosure provide a method of detecting a functional autoantibody against a L-voltage gated calcium channel in a subject suspected of suffering from, or being susceptible to, type 1 diabetes, the method comprising exposing a cell expressing a L-voltage gated calcium channel to a sample containing antibodies from the subject and detecting the functional antibody in the subject on the basis of a change in the level of the reactive oxygen or nitrogen species in the cell.
[0011] Certain embodiments of the present disclosure provide a method of detecting a functional autoantibody against a L-voltage gated calcium channel in a subject suspected of suffering from, or being susceptible to, an autonomic disorder, the method comprising exposing a cell expressing a L-voltage gated calcium channel to a sample containing antibodies from the subject and detecting the functional antibody in the subject on the basis of a change in the level of the reactive oxygen or nitrogen species in the cell.
[0012] Certain embodiments of the present disclosure provide a method of identifying a subject suffering from, or susceptible to type 1 diabetes, the method comprising exposing a cell expressing a L-voltage gated calcium channel to a sample containing antibodies from the subject, and identifying the subject as suffering from, or being susceptible to, type 1 diabetes on the basis of an increase in the level of reactive oxygen species detected in the cell.
[0013] Certain embodiments of the present disclosure provide a method of identifying a subject suffering from, or susceptible to an autonomic disorder associated with functional autoantibodies to a L-voltage gated calcium channel, the method comprising exposing a cell expressing a L-voltage gated calcium channel to a sample containing antibodies from the subject and identifying the subject as suffering from, or being susceptible to the autonomic disorder on the basis of an increase in the level of reactive oxygen species detected in the cell.
[0014] Certain embodiments of the present disclosure provide a method of identifying an antigenic target for a selected functional autoantibody, the method comprising:
exposing a cell expressing a candidate antigenic target to the selected functional autoantibody, wherein the antigenic target has an activity to alter the level of reactive oxygen or nitrogen species in the cell;
determining the ability of the selected functional antibody to alter the level of reactive oxygen or nitrogen species in the cell; and
identifying the candidate antigenic target as an antigenic target for the selected functional autoantibody.
[0015] Certain embodiments of the present disclosure provide a method of identifying a functional autoantibody for a selected antigenic target, the method comprising:
exposing a cell expressing the selected antigenic target to a candidate antibody, wherein the selected antigenic target has an activity to alter the level of reactive oxygen or nitrogen species in the cell;
determining the ability of the candidate antibody to alter the level of reactive oxygen or nitrogen species in the cell; and
identifying the candidate antibody as a functional autoantibody to the selected antigenic target.
[0016] Certain embodiments of the present disclosure provide a method of identifying an antigenic target for a functional autoantibody associated with type 1 diabetes, the method comprising:
exposing a cell expressing a candidate antigenic target to the functional autoantibody associated with type 1 diabetes, wherein the antigenic target has an activity to alter the level of reactive oxygen or nitrogen species in the cell; determining the ability of the functional antibody to alter the level of reactive oxygen or nitrogen species in the cell; and
identifying the candidate antigenic target as an antigenic target for the functional autoantibody associated with type 1 diabetes.
[0017] Certain embodiments of the present disclosure provide a method of identifying an antigenic target for a functional autoantibody associated with an autonomic disorder, the method comprising:
exposing a cell expressing a candidate antigenic target to the functional autoantibody associated with an autonomic disorder, wherein the antigenic target has an activity to alter the level of reactive oxygen or nitrogen species in the cell;
determining the ability of the functional antibody to alter the level of reactive oxygen or nitrogen species in the cell; and
identifying the candidate antigenic target as an antigenic target for the functional autoantibody associated with the autonomic disorder.
[0018] Certain embodiments of the present disclosure provide a method of identifying a functional autoantibody associated with type 1 diabetes, the method comprising:
exposing a cell expressing a selected antigenic target to a candidate antibody, wherein the antigenic target has an activity to alter the level of reactive oxygen or nitrogen species in the cell;
determining the ability of the candidate antibody to alter the level of reactive oxygen or nitrogen species in the cell; and
identifying the candidate antibody as a functional autoantibody associated with type 1 diabetes..
[0019] Certain embodiments of the present disclosure provide a method of identifying a functional autoantibody associated with an autonomic disorder, the method comprising:
exposing a cell expressing a selected antigenic target to a candidate antibody, wherein the antigenic target has an activity to alter the level of reactive oxygen or nitrogen species in the cell;
determining the ability of the candidate antibody to alter the level of reactive oxygen or nitrogen species in the cell; and
identifying the candidate antibody as a functional autoantibody associated with the autonomic disorder.
[0020] Certain embodiments of the present disclosure provide a method of identifying an agent that modulates the binding of a functional autoantibody to an antigenic target, the antigenic target having an activity to alter the level of reactive oxygen or nitrogen species in a cell, the method comprising:
exposing a cell expressing the antigenic target to the functional autoantibody and a candidate agent; determining the ability of the candidate agent to modulate the change in reactive oxygen or nitrogen species in the cell caused by the functional antibody; and
identifying the candidate agent as an agent that modulates the binding of the functional autoantibody to antigenic target.
[0021] Certain embodiments of the present disclosure provide a method of identifying a therapeutic agent for a disease, condition or state associated with binding of a functional autoantibody to an antigenic target, the antigenic target having an activity to alter the level of reactive oxygen or nitrogen species in a cell, the method comprising: exposing a cell expressing the antigenic target to the functional autoantibody and a candidate agent;
determining the ability of the candidate agent to modulate the change in reactive oxygen or nitrogen species in the cell caused by the functional antibody; and
identifying the candidate agent as a therapeutic agent for the disease, condition or state associated with binding of a functional autoantibody to an antigenic target.
[0022] Certain embodiments of the present disclosure provide an agent identified by a method as described herein.
[0023] Certain embodiments of the present disclosure provide a kit for performing a method as described herein.
[0024] Certain embodiments of the present disclosure provide use of a cell expressing an antigenic target for a functional autoantibody for identifying an agent that modulates the binding of the functional antibody to the antigenic target, wherein the antigenic target has an activity to alter the level of reactive oxygen or nitrogen species in the cell and binding of the autoantibody to the antigenic target alters the activity of the antigenic target.
[0025] Certain embodiments of the present disclosure provide use of a cell expressing an antigenic target for a functional autoantibody for identifying a therapeutic agent for a disease, condition or state associated with the binding of the functional autoantibody to the target, wherein the antigenic target has an activity to alter the level of reactive oxygen or nitrogen species in the cell and binding of the autoantibody to the antigenic target alters the activity of the antigenic target.
[0026] Certain embodiments of the present disclosure a kit for detecting a functional autoantibody, the kit comprising:
a cell expressing an antigenic target to the functional autoantibody, wherein the antigenic target has an activity to alter the level of reactive oxygen or nitrogen species in the cell; and
a marker to determine the level of reactive oxygen or nitrogen species in the cell; and
optionally positive and/or negative control reagents for the level of reactive oxygen or nitrogen species in the cell.
[0027] Certain embodiments of the present disclosure provide a complex comprising a cell expressing an antigenic target that has an activity to alter the level of reactive oxygen or nitrogen species in the cell, a functional autoantibody bound to the antigenic target on the cell, and a marker in the cell for detecting the level of reactive oxygen or nitrogen species.
[0028] Other embodiments are described herein.
BRIEF DESCRIPTION OF THE FIGURES
[0029] Certain embodiments are illustrated by the following figures. It is to be understood that the following description is for the purpose of describing particular embodiments only and is not intended to be limiting with respect to the description.
[0030] Figure 1 shows optimisation of a 2’, 7’ -Dichlorofluorescein diacetate (DCF- DA) assay of reactive species generation in the rat insulinoma cell line, RinAl2. (A) Incubation of Rin A12 cells with between 0.1 and 2 mM of the mitochondrial electron transporter inhibitor, rotenone, for 5 mins results in concentration-dependant induction of DCF-DA positivity, with values achieving statistical significance compared to baseline following addition of 1 or 2 mM rotenone. (B) The dihydroprydine (DHP) acting VGCC agonist, BayK8644 (at 30 nM), mediates a significant increase in DCF- DA positivity over 4 hrs of incubation. Addition of the DHP antagonist, nicardipine, at between 1 and 30 nM results in inhibition of the Bay K8644-mediated effect, with 10 nM of nicardipine resulting in significant inhibition. (C) DCF-DA primed RinAl2 cells incubated for 4 hrs in the presence of increasing concentrations of IgG from healthy donors (n = 4). Concentrations of IgG at between 0.3 and 2 mg/ml do not significantly alter DCF-DA fluorescence compared to baseline values. However, both 3 and 5 mg/ml of IgG resulted in significantly increased DCF-DA values compared to baseline. (D) Incubation of RinAl2 cells with IgG (0.3 mg/ml, dark dotted bars; 0.6 mg/ml, solid bars; 1 mg/ml, light spotted bars; 2 mg/ml, striped bars) from healthy donors (n = 4) over 7 hrs. No significant difference in DCF-DA values were observed at any IgG concentration when compared to equivalent baseline values at incubation times of 1, 2 or 4 hrs. By contrast, 7 hrs of incubation resulted in significant increases in DCF-DA values compared to baseline at 0.3, 0.6, and 1 mg/ml IgG. Note: Baseline values are equivalent cells incubated without IgG. Values are the mean and SEM from at least 2 independent assays incorporating triplicate samples. * Significance refers to P<0.05 by 2-way ANOVA, experimental treatment verses baseline. ** Significance refers to P<0.05 by 2-way ANOVA, Bay K verses Bay K + Nicardipine.
[0031] Figure 2 shows IgG with anti-VGCC activity from patients with type 1 diabetes mediates increased stress as determined by DCF-DA positivity in RinAl2 cells. (A) Individual IgG samples (n = 6; dotted bars) at a concentration of 1 mg/ml, but not other concentrations, from patient Pl previously characterised to produce anti-VGCC Abs mediated a significant increase in stress in Rin A12 cells compared to baseline values following 4 hrs of incubation. By contrast, incubation of cells with equivalent concentrations of IgG from a healthy donor (solid bars; n = 6) did not significantly alter DCF-DA positivity values. (B) Individual IgG samples (n = 6; dotted bars) at a concentration of 0.6 and 1 mg/ml, but not other concentrations, from patient P2, also previously characterised to produce anti-VGCC Abs, mediated significant increased stress in RinAl2 cells, while equivalent concentrations of IgG from a healthy donor (solid bars; n = 6) did not significantly alter DCF-DA positivity values. (C&D) In contrast to the results produced using IgG containing anti-VGCC Abs, incubation of cells with up to 2 mg/ml of individual IgG samples from 2 patients with type 1 diabetes previously characterised to lack anti-VGCC Abs (C = patient P7; D = patient P8) (dotted bars; n = 6) did not significantly alter DCF-DA positivity values compared to baseline values, with DCF-DA values similarly not altered by IgG from healthy donors (solid bars; n = 6 for each individual experiment). (E) An evaluation of anti-VGCC Ab- mediated stress over time. Incubation of cells with 1 mg/ml of individual IgG samples (n = 6) containing anti-VGCC Abs from patient P2 did not alter stress induction at 1 or 2 hrs, but resulted in significant induction compared to baseline after 4 hrs. IgG samples (n = 6) from a healthy individual did not significantly alter DCF-DA values of the same time course. Values are the mean and SEM from at least 2 independent assays incorporating triplicate samples. Baseline values refer to DCF-DA positivity values of cells incubated under equivalent conditions but without IgG addition. Patient codes refer to Table 1. *Significance refers to P < 0.05 by two-way ANOVA, experimental condition verses baseline.
[0032] Figure 3 shows the DCF-DA-based Rin A12 cell stress assay is suitable for screening patient IgG samples for the presence of anti-VGCC Abs. (A) Pooled IgG- mediated (at 1 mg/ml) DCF-DA positivity results (4 hrs incubation) from patients previously characterised for the presence (red triangles) or absence (black diamonds) of anti-VGCC Abs (see Table 1). Mean DCF-DA positivity values obtained using individual IgG preparations at 1 mg/ml from all 6 patients positive for anti-VGCC Abs were above the mean plus 2 standard deviations (see Table 2) (dotted line) of results obtained using IgG from 6 healthy individuals (black circles). In contrast, mean results using IgG from 2 patients lacking anti-VGCC Abs did not exceed the mean + 2 SD of pooled control IgG results. (B) Pooled DCF-DA positivity results mediated by 1 mg/ml of IgG from 10 control (black circles) and 20 type 1 diabetes patients (black triangles). Mean DCF-DA positivity results from 15 of 20 individual patient IgG samples were above the mean plus 2 standard deviations of control results (dotted line). Values represent the mean DCF-DA positivity values obtained from at least n = 6 individual assessments of individual patient or control IgG specimens.
[0033] Figure 4 shows IgG containing anti-VGCC Abs mediates induction of apoptosis in Rin A12 cells, as determined by annexin V. Assessment by flow cytometry of annexin V expression by Rin A12 cells incubated for 4 or 16 hrs with 1 mg/ml of IgG from healthy individuals (solid black bars; n = 4), or with patient IgG without (dotted bars; n = 2) or with (checked bars; n = 2) anti-VGCC Abs. A significant increase in annexin V expression compared to baseline (cells incubated without IgG) was observed at 16 hrs in cells incubated with anti-VGCC positive IgG. Values are the mean and SEM from at least 2 independent assays incorporating triplicate samples. * Significance refers to P < 0.05 by two-way ANOVA, experimental condition verses baseline.
[0034] Figure 5 shows Rotenone-mediated ROS generation in RinAl2, is dependent on exogenous calcium. Incubation of DCF-DA primed Rin A12 cells over 4 h with EGTA (sold bars) results in no significance change in ROS generation compared to the baseline (cells without EGTA incubation) (red dots). Addition of ImM Rotenone (stripped bars) results in a significance increase compared to baseline, which is reversed in the presence of EGTA (hatched bars). *Significance refers to P < 0.05 by two-way ANOVA, experimental condition verses baseline.
[0035] Figure 6 shows the interaction of dihydropyridine (DHP) agonist and antagonist drugs at DHP-sensitive voltage-gated calcium channels (VGCCs) modify ROS stress generation in RinAl2 cells. A. Incubation of DCF-DA primed Rin A12 cells over 4 h with between 1 and 100 nM of the DHP acting VGCC agonist, BayK8644. The addition of 30 nM of BayK8644 results in a significant increase in DCF-DA positivity compared to untreated cells (baseline). B. Incubation of DCF-DA primed Rin A12 cells over 4 h with between 1 and 100 nM of the DHP acting VGCC antagonist, Nicardipine, results in no significant change in DCF-DA positivity compared to untreated cells (baseline). C. The DHP acting VGCC agonist, BayK8644 (at 30 nM) mediates a significant increase in DCF-DA positivity over 4 h of incubation. Co-incubation of BayK8644 and the DHP antagonist, nicardipine, at between 1 and 30 nM results in a decrease in the Bay K8644-mediated effect, with ROS levels not significantly different compared to baseline at 10 nM of nicardipine. *Significance refers to P < 0.05 by two- way ANOVA, experimental condition verses baseline (A and B) and 30 nM Bayk verses BayK + nicardipine (C). .
[0036] Figure 7 shows inhibition of DHP-sensitive VGCCs does not reverse Rotenone-mediated ROS generation in RinAl2 cells. Incubation of DCF-DA primed Rin A12 cells over 4 h with 30 nM Nicardipine (black bar) results in no significance change in ROS generation compared to the baseline (untreated cells). Addition of 1 mM Rotenone (silver bar) results in a significance increase in ROS compared to baseline, with co-incubation of cells with both Rotenone and Nicardipine similarly resulting in a significant increase in ROS (grey bars). *Significance refers to P < 0.05 by two-way ANOVA, experimental condition verses baseline. .
[0037] Figure 8 shows anti-VGCC Ab-mediated ROS generation in RinAl2 cells is inhibited by the DHP antagonist, Nicardipine. Pooled means of ROS generation in DCF-DA primed Rin A12 cells incubated with 1 mg/ml IgG derived from healthy donors (Control, n = 2) or anti-VGCC positive IgG from patients with T1D (T1D, n = 2), incubated in the presence (+ nicardipine) or absence of 10 mM Nicardipine. A significant increase compared to baseline is achieved in ROS generation incubated with T1D IgG in the absence of Nicardipine. All other incubations do not significantly alter ROS generation. *Significance refers to P < .05 by two-way ANOVA
[0038] Figure 9 shows neutralization of anti-VGCC Abs by anti -idiotypic antibodies in IgG from healthy individuals or functional antibody negative patients. ROS generation in DCF-DA primed Rin A12 cells incubated with 1 mg/ml IgG derived from healthy donor (CTR) or preincubated with an equimolar equivalent of IgG from a separate healthy individual (CTR + CTR) is not significantly different to baseline (cells without IgG incubation). In contrast, IgG from a patient with anti-VGCC Abs (T1D) induces a significant increase in ROS expression compared to baseline, which is reduced by pre-incubation with IgG either from a healthy donor (T1D + CTR) or a patient negative for anti-VGCC Abs (T!D + T1D neg), resulting in ROS levels not significantly different compared to baseline. As such, Ig pre-incubation of anti-VGCC Abs can neutralize the functional effect of the channel antibodies on ROS generation in RinAl2 cells. Values are the mean and SEM from at least 2 independent assays incorporating duplicate samples. * Significance refers to P < .05 by two-way ANOVA.
[0039] Figure 10 shows ROS stress generation as a percenatge of untreated baseline. Time indicates incubation period. Assay conducted using caprylic acid precipitated IgG. Cell line: Rin A12. [0040] Figure 11 shows PI positivity as a percentage of untreated baseline. Time indicates incubation period. Assay conducted using caprylic acid precipitated IgG. Cell line: Rin A12.
DETAILED DESCRIPTION
[0041] The present disclosure relates to methods for detecting functional autoantibodies, methods for identifying subjects suffering from, or susceptible to, diseases, conditions or states associated with functional autoantibodies, methods for identifying functional autoantibodies and their antigenic targets, and methods for identifying agents that modulate binding of a functional autoantibody to an antigenic target.
[0042] The present disclosure is based, at least in part, on the use of a neuroendocrine cell line, RinAl2, which is a derivative of the rat insulinoma 5F line. This cell line has glucose sensitivity, and is capable of producing low levels of insulin release. Features of beta cell lines such as Rin cells include low free radical scavenger production, and ROS generation under stimulation, factors which benefit their use in an assay based on measuring ROS generation. The time of the assay duration described herein is based largely on a requirement for the build up of detectible ROS in response to antibody- mediated intracellular ion disruption, which may not occur in other cell types less susceptible to accumulation of ROS. In addition to the RinAl2 cell line, the assay has successfully been conducted with a second insulin cell line, that being the murine BTC3 cell.
[0043] The present disclosure provides the ability to detect different anti-channel autoantibodies. For example, studies conducted as part of this study demonstrate that the anti-VGCC antibody associated with type 1 diabetes of the present disclosure is phenotypically distinguished from an anti-neuronal ion channel antibody in human narcolepsy with cataplexy.
[0044] The assay of the present disclosure is a pan-anti-ion channel (or G-Coupled Protein Receptor) assay, in that it will detect functional (target modifying) antibodies where the functional modification results in intracellular ion derangement resulting in stress generation. In one embodiment, the successful detection of an anti-ion channel antibody in a patient with idiotypic dysautonomia resulted in a phenotypic assay presentation distinct from that produced by the anti-VGCC antibody associated with T1D.
[0045] Certain embodiments of the present disclosure provide a method of detecting a functional autoantibody against an antigenic target.
[0046] The method permits the detection of functionally active autoantibodies, for example as present in the IgG fraction of patient serum. The method can be used in some embodiments as a real time assay for detecting autoantibody-mediated stress generation, and for assessing the degree of potential autoantibody pathogenicity, and in some embodiments has clinical utility in diagnosis or prognosis of type 1 diabetes and/or in autonomic dysfunction.
[0047] In certain embodiments, the present disclosure provides a method of detecting a functional autoantibody against an antigenic target, the method comprising exposing a cell expressing the antigenic target to the autoantibody, wherein the antigenic target has an activity to alter the level of reactive oxygen or nitrogen species in the cell and binding of the autoantibody to the antigenic target alters the activity of the antigenic target, and detecting the functional antibody on the basis of a change in the level of the reactive oxygen or nitrogen species in the cell.
[0048] The term“functional autoantibody” as used herein refers to an auto-reactive antibody against an antigenic target in a host, the presence of which has some form of pathophysiologic effect in the host. The present disclosure permits the detection of a variety of different autoantibodies, as demonstrated by the studies described herein.
[0049] In certain embodiments, the functional autoantibody is an IgG antibody. In certain embodiments, the functional autoantibody is an IgM antibody.
[0050] In certain embodiments, the functional autoantibody is present in a biological sample from a subject.
[0051] In certain embodiments, the functional antibody is present in a mix of other biological factors. In certain embodiments, the functional antibody is a semi-purified or a purified antibody. In certain embodiments, the functional autoantibody is an isolated antibody. In certain embodiments, the functional antibody is present in a sample enriched for immunoglobulins.
[0052] The present method contemplates detecting autoantibodies in crude samples, in processed samples, in enriched samples and/or as semi-purified or purified antibodies.
[0053] Methods for enriching and purifying antibodies are known in the art.
[0054] Various antigenic targets are contemplated. The term“antigenic target” as used herein refers to a target for an autoantibody.
[0055] In certain embodiments, the antigenic target is an extracellular target. In certain embodiments, the antigenic target is a membrane associated target.
[0056] In certain embodiments, the antigenic target comprises an endogenous target.
[0057] In certain embodiments, the antigenic target comprises an exogenous target introduced into a cell, for example for the purposes of establishing a cell line for detecting functional autoantibodies. In certain embodiments, the antigenic target is a cloned antigenic target.
[0058] In certain embodiments, the antigenic target comprises a membrane bound ion channel. In certain embodiments, the antigenic target comprises a membrane bound receptor.
[0059] In certain embodiments, the antigenic target comprises a calcium ion channel, a potassium ion channel, a sodium ion channel or a chloride channel.
[0060] In certain embodiments, the antigenic target comprises a L-type voltage gated calcium channel (VGCC). In the human, the L-type voltage gated calcium channel is the ion channel that is defined by the genes for the subunits CACNA1C, CACNA1D , CACNA1S and CACNA1F. The equivalent ion channel in other species may be readily identified by a method known in the art. [0061] In certain embodiments, the antigenic target comprises a G protein-coupled receptor, such as GPR40, GLP-l and a2 adrenoreceptor. In certain embodiments, the antigenic target comprises a tyrosine kinase receptor, such as an EGF receptor.
[0062] In certain embodiments, the identity of the antigenic target is known. In certain embodiments, the identity of the antigenic target is not known. Indeed, the present disclosure does not rely on needing to know the identity of the antigenic target.
[0063] Methods for assessing the level of reactive oxygen or nitrogen species in a cell are known in the art. In this regard, the term“reactive oxygen species” refers to reactive species and free radical containing species derived from oxygen or containing oxygen. The term “reactive nitrogen species” refers to reactive species and free radical nitrogenous containing species.
[0064] In certain embodiments, the level of reactive oxygen or nitrogen species in the cell increases upon binding of the autoantibody to the antigenic target.
[0065] In certain embodiments, the level of reactive oxygen or nitrogen species decreases upon binding of the autoantibody to the antigenic target.
[0066] Examples of reactive oxygen species and reagents for their detection are shown in Table 1. Some of these reagents may also be used for detection of reactive nitrogen species.
Figure imgf000017_0001
[0067] In certain embodiments, the level of the reactive oxygen or reactive nitrogen species is determined using a fluorescent marker or probe to determine the level.
[0068] Fluorescent markers or probes for detecting reactive oxygen species are described, for example, in Gomes et al. (2005). Journal of Biochemical and Biophysical Methods 65(2-3): 45-80, hereby incorporated by reference. Fluorescent probes for detecting reactive nitrogen species are also described in Gomes et al. (2006). Journal of Fluorescence 16(1): 119-139, hereby incorporated by reference.
[0069] In certain embodiments, the level of the reactive oxygen species is determined using the marker 2',7'-dichlorofluorescein diacetate.
[0070] Other methods for detecting reactive oxygen species or reactive nitrogen species are known in the art, such as the fluorescent protein-based redox sensors (eg roGFP), and agents for detecting glutathione, all of which are known in the art and commercially available.
[0071] In certain embodiments, the method of detecting a functional autoantibody against an antigenic target comprises use of flow cytometry. Other methods are also contemplated, such as fluorescent microscopy or microplate analysis, which are known in the art.
[0072] In certain embodiments, the method comprises determining or assessing the level of reactive oxygen species or reactive nitrogen species in the cell. Qualitative and quantitative methods for determining or assessing the level of reactive oxygen species or reactive nitrogen species in a cell are known in the art.
[0073] In certain embodiments, the method of detecting a functional autoantibody against an antigenic target comprises comparison of the level of reactive oxygen species or nitrogen species with a control and/or reference level. For example, the level of reactive oxygen species or nitrogen species known to be associated with a basal level of the species, or when induced by the particular autoantibody or other agent may be used for comparison.
[0074] In certain embodiments, the method is used to identify the presence of functional autoantibodies in a subject. In certain embodiments, the method is used to detect the presence or level of functional autoantibodies in a subject. In certain embodiments, the method is used to assess the presence or level of functional autoantibodies in a subject. In certain embodiments, the method is used to confirm the presence of functional autoantibodies in the subject. In certain embodiments, the method is used to confirm the absence of functional autoantibodies in the subject.
[0075] In certain embodiments, the method comprises detecting the functional autoantibody in a biological sample from a subject.
[0076] Methods for obtaining biological samples containing antibodies are known in the art. In certain embodiments, the biological sample is a biological fluid. In certain embodiments, the biological fluid comprises urine, saliva or blood, plasma or serum.
[0077] In certain embodiments, the biological sample comprises saliva or blood.
[0078] In certain embodiments, the method comprises processing the biological sample to allow detection of autoantibodies in the biological sample. In certain embodiments, the method comprises obtaining a biological sample from a subject and processing the sample to detect functional autoantibodies. In certain embodiments, the biological sample comprises a processed biological sample.
[0079] In certain embodiments, the processed biological sample is enriched for IgG and/or IgM. In certain embodiments, the method comprises enriching for immunoglobulins. Methods for enriching for immunoglobulins are known in the art.
[0080] In certain embodiments, the method comprises exposing the cells to at least 0.3 mg/ml IgG, at least 0.6 mg/ml IgG, or at least 1 mg/ml IgG.
[0081] In certain embodiments, the subject is a human subject.
[0082] In certain embodiment, the subject is suffering from, or susceptible to, a disease, condition or state, as described herein. In certain embodiments, the functional autoantibody is associated with a disease, condition or state.
[0083] In certain embodiments, the subject is a human subject suffering from, or susceptible to, type 1 diabetes. In certain embodiments, the functional autoantibody is associated with type 1 diabetes [0084] In certain embodiments, the subject is a human subject suffering from, or susceptible to, an autonomic dysfunction. In certain embodiments, the autonomic dysfunction comprises gastrointestinal and/or bladder autonomic neuropathy, or a cardiovascular autonomic disorder. In certain embodiments, the functional autoantibody is associated with an autonomic disorder.
[0085] In certain embodiments, the subject is a mammalian subject. In certain embodiments the subject is an animal, such as a livestock animal (such as a horse, a cow, a sheep, a goat, a pig), a domestic animal (such as a dog or a cat) and other types of animals, such as monkeys, rabbits, mice, rats and laboratory animals. Veterinary applications of the present disclosure are contemplated.
[0086] In certain embodiments, the cell expressing the antigenic target is a cell naturally expressing the antigenic target. In certain embodiments, the cell expressing the antigenic target is a cell engineered to express the antigenic target. Methods of introducing and expressing exogenous proteins in cells are known in the art, for example as described in Sambrook et al. Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)) and Ausubel et al. Current Protocols in Molecular Biology (2003) John Wiley & Sons, both of which are herein incorporated by reference.
[0087] For example, the cell may express one or more subunits of an exogenous VGCC introduced by transformation or transfection into the cell, such as by viral transfection.
[0088] In certain embodiments, the cell is one or more cells. In certain embodiments, the cell is a plurality of cells. In certain embodiments, the cell is an isolated cell, or a cell present in a population of other cells, which may be substantially similar or different to the cell. Methods for isolating and culturing cells are known in the art.
[0089] Suitable cells expressing the antigenic target may be selected.
[0090] In certain embodiments, the cell expressing the antigenic target to the autoantibody is a cell of neuroendocrine origin. In this regard, a cell of neuroendocrine origin expresses a large range of neuro-receptors and neuroendocrine associated ion channels also found in nerve cells and other excitable cells.
[0091] In certain embodiment, the cell is an insulinoma cell.
[0092] In certain embodiments, the cell is a Rin A12 cell, or a variant or derivative thereof. In certain embodiments, the cell is a murine BTC cell.
[0093] Other types of cells are contemplated.
[0094] The cell may be exposed to the autoantibody for a suitable amount of time.
[0095] In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of at least 2 hours. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of 2 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of at least 3 hours. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of 3 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of at least 4 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 4 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of at least 5 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 5 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of at least 6 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 6 hours or greater. Other time periods are contemplated.
[0096] In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of time in one of the following ranges: 2 to 6 hours, 2 to 5 hours, 2 to 4 hours, 2 to 3 hours, 3 to 6 hours, 3 to 5 hours, 3 to 4 hours, 4 to 6 hours, 4 to 5 hours and 5 to 6 hours. Other time periods are contemplated.
[0097] In certain embodiments, a level of cells positive for changed reactive oxygen species and/or changed reactive nitrogen species one standard deviation above the mean value for a control is indicative of the presence of a disease, condition or state associated with the functional autoantibodies. In certain embodiments, a level of cells positive for changed reactive oxygen species or changed reactive nitrogen species two standard deviations above the mean value for a control is indicative of the presence of a disease, condition or state associated with the functional autoantibodies.
[0098] In certain embodiments, the method is used to identify a subject suffering from an autoimmune disease, condition or state, to identify a subject suffering from, or being susceptible to, type 1 diabetes, for diagnosis or prognosis of type 1 diabetes, for assessing the risk or likelihood of a subject suffering from, or being susceptible to, type 1 diabetes, to identify an antigenic target on a cell associated with the binding of a functional autoantibody, or to identify a therapeutic agent.
[0099] In certain embodiments, the method is used to screen for functional autoantibodies, to identify a subject suffering from an autoimmune disease, condition or state, to identify a subject suffering from, or being susceptible to, an autonomic disorder, for diagnosis or prognosis of an autoimmune disease, condition or state, for diagnosis or prognosis of an autonomic disorder, for assessing the risk or likelihood of a subject suffering from, or being susceptible to, an autoimmune disease, condition or state, for assessing the risk or likelihood of a subject suffering from, or being susceptible to, an autonomic disorder.
[00100] In certain embodiments, the present disclosure provides a method of detecting a functional autoantibody against a L-voltage gated calcium channel in a subject. In certain embodiments, the present disclosure provides a method of detecting a functional autoantibody against a L-voltage gated calcium channel in a subject suffering from, or susceptible to, a disease condition or state.
[00101] In certain embodiments, the present disclosure provides a method of detecting a functional autoantibody against a L-voltage gated calcium channel in a subject suspected of suffering from, or being susceptible to, type 1 diabetes.
[00102] In certain embodiments, the present disclosure provides a method of detecting a functional autoantibody against a L-voltage gated calcium channel in a subject suspected of suffering from, or being susceptible to, type 1 diabetes, the method comprising exposing a cell expressing a L-voltage gated calcium channel to a sample containing antibodies from the subject and detecting the functional antibody in the subject on the basis of a change in the level of the reactive oxygen species in the cell.
[00103] Methods for assessing the level of reactive oxygen species in a cell are as described herein.
[00104] In certain embodiments, the level of reactive oxygen species in the cell increases upon binding of the autoantibody to the L-voltage gated calcium channel.
[00105] Cells expressing antigenic targets and their use are as described herein.
[00106] In certain embodiments, the cell expressing the L-voltage gated calcium channel is a cell of neuroendocrine origin.
[00107] In certain embodiments, the cell is an insulinoma cell.
[00108] In certain embodiments, the cell is a Rin A12 cell, or a variant or derivative thereof. Other cells are as described herein.
[00109] The cell may be exposed to the autoantibody for a suitable amount of time.
[00110] In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of at least 2 hours. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of 2 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of at least 3 hours. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of 3 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of at least 4 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 4 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of at least 5 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 5 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of at least 6 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 6 hours or greater. Other time periods are contemplated.
[00111] In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of time in one of the following ranges: 2 to 6 hours, 2 to 5 hours, 2 to 4 hours, 2 to 3 hours, 3 to 6 hours, 3 to 5 hours, 3 to 4 hours, 4 to 6 hours, 4 to 5 hours and 5 to 6 hours. Other time periods are contemplated.
[00112] In certain embodiments, a level of cells positive for changed reactive oxygen species one standard deviation above the mean value for a control is indicative of the subject suffering from, or susceptible to type 1 diabetes. In certain embodiments, a level of cells positive for changed reactive oxygen species two standard deviations above the mean value for a control is indicative of the subject suffering from, or susceptible to type 1 diabetes.
[00113] In certain embodiments, the method is used for the diagnosis or prognosis of type 1 diabetes.
[00114] In certain embodiments, the present disclosure provides a method of detecting a functional autoantibody against a L-voltage gated calcium channel in a subject suspected of suffering from, or being susceptible to, an autonomic disorder.
[00115] In certain embodiments, the present disclosure provides a method of detecting a functional autoantibody against a L-voltage gated calcium channel in a subject suspected of suffering from, or being susceptible to, an autonomic disorder, the method comprising exposing a cell expressing a L-voltage gated calcium channel to a sample containing antibodies from the subject and detecting the functional antibody in the subject on the basis of a change in the level of the reactive oxygen species in the cell.
[00116] In certain embodiments, the method is used for diagnosis or prognosis of an autonomic disorder. [00117] In certain embodiments, the autonomic disorder comprises a gastrointestinal or bladder autonomic neuropathy, or cardiovascular autonomic complications.
[00118] Methods for assessing the level of reactive oxygen species in a cell are as described herein.
[00119] In certain embodiments, the level of reactive oxygen species in the cell increases upon binding of the autoantibody to the L-voltage gated calcium channel.
[00120] Cells expressing antigenic targets and their use are as described herein.
[00121] In certain embodiments, the cell expressing the L-voltage gated calcium channel is a cell of neuroendocrine origin. In certain embodiments, the cell is an insulinoma cell. In certain embodiments, the cell is a Rin A12 cell, or a variant thereof.
[00122] The cell may be exposed to the autoantibody for a suitable amount of time.
[00123] In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of at least 2 hours. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of 2 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of at least 3 hours. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of 3 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of at least 4 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 4 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of at least 5 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 5 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of at least 6 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 6 hours or greater. Other time periods are contemplated.
[00124] In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of time in one of the following ranges: 2 to 6 hours, 2 to 5 hours, 2 to 4 hours, 2 to 3 hours, 3 to 6 hours, 3 to 5 hours, 3 to 4 hours, 4 to 6 hours, 4 to 5 hours and 5 to 6 hours. Other time periods are contemplated.
[00125] In certain embodiments, a level of cells positive for changed reactive oxygen species one standard deviation above the mean value for a control is indicative of the subject suffering from, or susceptible to an autonomic disorder. In certain embodiments, a level of cells positive for changed reactive oxygen species two standard deviations above the mean value for a control is indicative of the subject suffering from, or susceptible to an autonomic disorder.
[00126] Certain embodiments of the present disclosure provide a method of identifying a subject suffering from, or susceptible to type 1 diabetes.
[00127] In certain embodiments, the present disclosure provides a method of identifying a subject suffering from, or susceptible to type 1 diabetes, the method comprising exposing a cell expressing a L-voltage gated calcium channel to a sample containing antibodies from the subject, and identifying the subject as suffering from, or being susceptible to, type 1 diabetes on the basis of an increase in the level of reactive oxygen species detected in the cell.
[00128] Methods for assessing the level of reactive oxygen species in a cell are as described herein.
[00129] Cells expressing antigenic targets and their use are as described herein.
[00130] In certain embodiments, the cell expressing the L-voltage gated calcium channel is a cell of neuroendocrine origin. In certain embodiments, the cell is an insulinoma cell. In certain embodiments, the cell is a Rin A12 cell, or a variant thereof.
[00131] The cell may be exposed to the autoantibody for a suitable amount of time.
[00132] In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of at least 2 hours. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of 2 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of at least 3 hours. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of 3 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of at least 4 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 4 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of at least 5 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 5 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of at least 6 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 6 hours or greater. Other time periods are contemplated.
[00133] In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of time in one of the following ranges: 2 to 6 hours, 2 to 5 hours, 2 to 4 hours, 2 to 3 hours, 3 to 6 hours, 3 to 5 hours, 3 to 4 hours, 4 to 6 hours, 4 to 5 hours and 5 to 6 hours. Other time periods are contemplated.
[00134] In certain embodiments, a level of cells positive for changed reactive oxygen species one standard deviation above the mean value for a control is indicative of the subject suffering from, or susceptible to type 1 diabetes. In certain embodiments, a level of cells positive for changed reactive oxygen species two standard deviations above the mean value for a control is indicative of the subject suffering from, or susceptible to type 1 diabetes.
[00135] Certain embodiments of the present disclosure provide a method of identifying a subject suffering from, or susceptible to an autonomic disorder associated with functional autoantibodies to a L-voltage gated calcium channel.
[00136] In certain embodiments, the present disclosure provides a method of identifying a subject suffering from, or susceptible to an autonomic disorder associated with functional autoantibodies to a L-voltage gated calcium channel, the method comprising exposing a cell expressing a L-voltage gated calcium channel to a sample containing antibodies from the subject and identifying the subject as suffering from, or being susceptible to the autonomic disorder on the basis of an increase in the level of reactive oxygen species detected in the cell.
[00137] Methods for assessing the level of reactive oxygen species in a cell are as described herein.
[00138] Cells expressing antigenic targets and their use are as described herein.
[00139] In certain embodiments, the cell expressing the L-voltage gated calcium channel is a cell of neuroendocrine origin. In certain embodiments, the cell is an insulinoma cell. In certain embodiments, the cell is a Rin A12 cell, or a variant thereof.
[00140] The cell may be exposed to the autoantibody for a suitable amount of time.
[00141] In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of at least 2 hours. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of 2 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of at least 3 hours. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of 3 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of at least 4 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 4 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of at least 5 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 5 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of at least 6 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 6 hours or greater. Other time periods are contemplated.
[00142] In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of time in one of the following ranges: 2 to 6 hours, 2 to 5 hours, 2 to 4 hours, 2 to 3 hours, 3 to 6 hours, 3 to 5 hours, 3 to 4 hours, 4 to 6 hours, 4 to 5 hours and 5 to 6 hours. Other time periods are contemplated.
[00143] In certain embodiments, a level of cells positive for changed reactive oxygen species one standard deviation above the mean value for a control is indicative of the subject suffering from, or susceptible to an autonomic disorder. In certain embodiments, a level of cells positive for changed reactive oxygen species two standard deviations above the mean value for a control is indicative of the subject suffering from, or susceptible to an autonomic disorder. Other levels are contemplated.
[00144] The methods of the present disclosure as described herein may also be used in screens to identify antigenic targets, functional autoantibodies and various types of agents.
[00145] Certain embodiments of the present disclosure provide a method of identifying an antigenic target for a selected functional autoantibody.
[00146] In certain embodiments, the present disclosure provides a method of identifying an antigenic target for a selected functional autoantibody, the method comprising:
exposing a cell expressing a candidate antigenic target to the selected functional autoantibody, wherein the antigenic target has an activity to alter the level of reactive oxygen or nitrogen species in the cell;
determining the ability of the selected functional antibody to alter the level of reactive oxygen or nitrogen species in the cell; and
identifying the candidate antigenic target as an antigenic target for the selected functional autoantibody.
[00147] In certain embodiments, the antigenic target comprises a target for a functional autoantibody associated with a disease, condition or state, as described herein.
[00148] In certain embodiments, the antigenic target comprises a target for a functional autoantibody associated with type 1 diabetes. [00149] In embodiments, the antigenic target comprises a target for a functional autoantibody associated with an autonomic disorder.
[00150] Functional autoantibodies are as described herein. In certain embodiments, the functional autoantibody is an IgG antibody. In certain embodiments, the functional autoantibody is an IgM antibody.
[00151] In certain embodiments, the functional autoantibody is present in a biological sample from a subject. In certain embodiments, the functional antibody is present in a biological sample from a subject suffering a disease, condition or state as described herein.
[00152] In certain embodiments, the functional antibody is present in a mix of other biological factors. In certain embodiments, the functional antibody is a semi-purified or a purified antibody. In certain embodiments, the functional autoantibody is an isolated antibody. In certain embodiments, the functional antibody is present in a sample enriched for immunoglobulins.
[00153] Methods for enriching and purifying antibodies are known in the art.
[00154] In certain embodiments, the identity of the functional antibody is unknown. In certain embodiments, the identity of the functional antibody is known. In this case, the method does not relay on needing to know the identity of the functional antibody.
[00155] Antigenic targets are as described herein.
[00156] In certain embodiments, the antigenic target is an extracellular target. In certain embodiments, the antigenic target is a membrane associated target.
[00157] In certain embodiments, the antigenic target comprises an endogenous target.
[00158] In certain embodiments, the antigenic target comprises an exogenous target introduced into a cell. In certain embodiments, the antigenic target is a cloned antigenic target. [00159] In certain embodiments, the antigenic target comprises a membrane bound ion channel. In certain embodiments, the antigenic target comprises a membrane bound receptor.
[00160] In certain embodiments, the antigenic target comprises a calcium ion channel, a potassium ion channel or a sodium ion channel. In certain embodiments, the antigenic target comprises a L-voltage gated calcium channel.
[00161] In certain embodiments, the antigenic target comprises a G protein-coupled receptor.
[00162] Methods for assessing the level of reactive oxygen or nitrogen species in a cell are as described herein.
[00163] In certain embodiments, the level of reactive oxygen or nitrogen species in the cell increases upon binding of the autoantibody to the antigenic target.
[00164] In certain embodiments, the level of reactive oxygen or nitrogen species decreases upon binding of the autoantibody to the antigenic target.
[00165] In certain embodiments, the method of identifying an antigenic target comprises use of flow cytometry. Other methods are contemplated, such as fluorescent microscopy or microplate analysis.
[00166] In certain embodiments, the method of identifying an antigenic target comprises determining or assessing the level of reactive oxygen species or reactive nitrogen species in the cell. Qualitative and quantitative methods for determining or assessing the level of reactive oxygen species or reactive nitrogen species in a cell are known in the art.
[00167] In certain embodiments, the method of identifying an antigenic target comprises comparison of the level of reactive oxygen species or nitrogen species with a control and/or reference level.
[00168] In certain embodiments, the method comprises using the functional autoantibody in a biological sample from a subject. Biological samples are as described herein. In certain embodiments, the method comprises processing the biological sample.
[00169] In certain embodiments, the method comprises use of a processed biological sample enriched for IgG and/or IgM. In certain embodiments, the method comprises enriching for immunoglobulins.
[00170] In certain embodiments, the method comprises exposing the cells to at least 0.3 mg/ml IgG, at least 0.6 mg/ml IgG, or at least 1 mg/ml IgG.
[00171] In certain embodiments, the cell expressing the candidate antigenic target is a cell naturally expressing the candidate antigenic target. In certain embodiments, the cell expressing the candidate antigenic target is a cell engineered to express the candidate antigenic target.
[00172] In certain embodiments, the cell is one or more cells. In certain embodiments, the cell is a plurality of cells. In certain embodiments, the cell is an isolated cell, or a cell present in a population of other cells, which may be substantially similar or different to the cell.
[00173] In certain embodiments, the cell is a population of cells expressing one or more candidate antigenic targets. For example, a library of possible antigenic targets may be introduced into cells, and the antigenic target for the selected functional autoantibody identified by identifying cells showing a change in the level of reactive oxygen or nitrogen species. The particular cells may then be enriched or purified, and the identity of the antigenic target determined.
[00174] Suitable cells expressing candidate antigenic targets may be selected.
[00175] In certain embodiments, the cell expressing the candidate antigenic target is a cell of neuroendocrine origin. In certain embodiment, the cell is an insulinoma cell. In certain embodiments, the cell is a Rin A12 cell, or a variant thereof. Other cells are as described herein.
[00176] The cell may be exposed to the selected functional autoantibody for a suitable amount of time.
[00177] In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of at least 2 hours. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of 2 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of at least 3 hours. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of 3 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of at least 4 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 4 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of at least 5 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 5 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of at least 6 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 6 hours or greater. Other time periods are contemplated.
[00178] In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of time in one of the following ranges: 2 to 6 hours, 2 to 5 hours, 2 to 4 hours, 2 to 3 hours, 3 to 6 hours, 3 to 5 hours, 3 to 4 hours, 4 to 6 hours, 4 to 5 hours and 5 to 6 hours. Other time periods are contemplated.
[00179] The method of identifying the antigenic target may further comprises a variety of other in vitro or in vivo techniques known in the art to assist with the identification of the target, such as immunoprecipitation, Western blotting, functional cloning, and blocking agents.
[00180] Certain embodiments of the present disclosure provide a method of identifying an antigenic target for a functional autoantibody associated with an autonomic disorder.
[00181] In certain embodiments, the present disclosure provides a method of identifying an antigenic target for a functional autoantibody associated with an autonomic disorder, the method comprising:
exposing a cell expressing a candidate antigenic target to the functional autoantibody associated with an autonomic disorder, wherein the antigenic target has an activity to alter the level of reactive oxygen or nitrogen species in the cell;
determining the ability of the functional antibody to alter the level of reactive oxygen or nitrogen species in the cell; and
identifying the candidate antigenic target as an antigenic target for the functional autoantibody associated with the autonomic disorder.
[00182] Methods for identifying antigenic targets are as described herein.
[00183] In certain embodiments, the identity of the functional antibody is known. In certain embodiments, the identity of the functional antibody is unknown.
[00184] In certain embodiments, the cell expressing the candidate antigenic target is a cell in a population of cells having different possible antigenic targets.
[00185] In certain embodiments, exposing the cell to the functional autoantibody comprises exposing the cell to a sample from a subject known to be suffering from the autonomic disorder.
[00186] Certain embodiments of the present disclosure provide a method of identifying an antigenic target for a functional autoantibody associated with type 1 diabetes.
[00187] In certain embodiments, the present disclosure provides a method of identifying an antigenic target for a functional autoantibody associated with type 1 diabetes, the method comprising:
exposing a cell expressing a candidate antigenic target to the functional autoantibody associated with type 1 diabetes, wherein the antigenic target has an activity to alter the level of reactive oxygen or nitrogen species in the cell; determining the ability of the functional antibody to alter the level of reactive oxygen or nitrogen species in the cell; and
identifying the candidate antigenic target as an antigenic target for the functional autoantibody associated with type 1 diabetes.
[00188] Methods for identifying antigenic targets are as described herein.
[00189] In certain embodiments, the identity of the functional antibody is known. In certain embodiments, the identity of the functional antibody is unknown.
[00190] In certain embodiments, the cell expressing the candidate antigenic target is a cell in a population of cells having different possible antigenic targets.
[00191] In certain embodiments, exposing the cell to the functional autoantibody comprises exposing the cell to a sample from a subject known to be suffering from type 1 diabetes.
[00192] Certain embodiments of the present disclosure provide a method of identifying a functional autoantibody for a selected antigenic target.
[00193] In certain embodiments, the present disclosure provides a method of identifying a functional autoantibody for a selected antigenic target, the method comprising:
exposing a cell expressing the selected antigenic target to a candidate antibody, wherein the selected antigenic target has an activity to alter the level of reactive oxygen or nitrogen species in the cell;
determining the ability of the candidate antibody to alter the level of reactive oxygen or nitrogen species in the cell; and
identifying the candidate antibody as a functional autoantibody to the selected antigenic target.
[00194] In certain embodiments, the cell expresses an antigenic target able to bind to bind to a functional autoantibody associated with a disease, condition or state as described herein.
[00195] In certain embodiments, the cell expresses an antigenic target able to bind a functional autoantibody associated with type 1 diabetes. In certain embodiments, the functional antibody is a functional antibody associated with type 1 diabetes. In certain embodiments, the cell expresses an antigen target able to bind a functional autoantibody associated with type 1 diabetes.
[00196] In certain embodiments, the cell expresses an antigenic target able to bind a functional autoantibody associated with an autonomic disorder. In certain embodiments, the functional antibody is a functional antibody associated with an autonomic disorder. In certain embodiments, the cell expresses an antigenic target able to bind a functional autoantibody associated with an autonomic disorder.
[00197] Functional antibodies are as described herein. In certain embodiments, the functional autoantibody is an IgG antibody. In certain embodiments, the functional autoantibody is an IgM antibody.
[00198] In certain embodiments, the functional autoantibody is present in a biological sample from a subject.
[00199] In certain embodiments, the functional autoantibody is present in a mix of other biological factors. In certain embodiments, the functional autoantibody is a semi- purified or a purified antibody. In certain embodiments, the functional autoantibody is an isolated antibody. In certain embodiments, the functional autoantibody is present in a sample enriched for immunoglobulins.
[00200] In certain embodiments, the functional autoantibody is present in a subject with a disease, condition or state associated with the presence of the autoantibody. In certain embodiments, the functional autoantibody is present in a subject with type 1 diabetes. In certain embodiments, the functional autoantibody is present in a subject with an autonomic disorder.
[00201] Methods for enriching and purifying antibodies are known in the art.
[00202] In certain embodiments, the identity of the selected antigenic target is known. In certain embodiments, the identity of the selected antigenic target is unknown. In this case, the method does not rely on needing to know the identity of the selected antigenic target. [00203] In certain embodiments, the selected antigenic target is an extracellular target. In certain embodiments, the selected antigenic target is a membrane associated target.
[00204] In certain embodiments, the selected antigenic target comprises an endogenous target.
[00205] In certain embodiments, the selected antigenic target comprises an exogenous target introduced into a cell. In certain embodiments, the selected antigenic target is a cloned antigenic target.
[00206] In certain embodiments, the selected antigenic target comprises a membrane bound ion channel. In certain embodiments, the selected antigenic target comprises a membrane bound receptor.
[00207] In certain embodiments, the selected antigenic target comprises a calcium ion channel, a potassium ion channel or a sodium ion channel. In certain embodiments, the selected antigenic target comprises a L-voltage gated calcium channel.
[00208] In certain embodiments, the selected antigenic target comprises a G protein- coupled receptor.
[00209] In certain embodiments, the selected antigenic target is associated with a disease, condition or state. In certain embodiments, the selected antigenic target is associated with type 1 diabetes. In certain embodiments, the selected antigenic target is associated with an autonomic disorder.
[00210] Methods for assessing the level of reactive oxygen or nitrogen species in a cell are as described herein.
[00211] In certain embodiments, the level of reactive oxygen or nitrogen species in the cell increases upon binding of the functional autoantibody to the selected antigenic target.
[00212] In certain embodiments, the level of reactive oxygen or nitrogen species decreases upon binding of the functional autoantibody to the selected antigenic target. [00213] In certain embodiments, the method of identifying the functional autoantibody comprises use of flow cytometry. Other methods are contemplated, such as fluorescent microscopy or microplate analysis.
[00214] In certain embodiments, the method of identifying the functional autoantibody comprises determining or assessing the level of reactive oxygen species or reactive nitrogen species in the cell. Qualitative and quantitative methods for determining or assessing the level of reactive oxygen species or reactive nitrogen species in a cell are known in the art.
[00215] In certain embodiments, the method of identifying the functional autoantibody comprises comparison of the level of reactive oxygen species or nitrogen species with a control and/or reference level.
[00216] In certain embodiments, the method comprises using the functional autoantibody in a biological sample from a subject. Biological samples are as described herein. In certain embodiments, method comprises processing the biological sample.
[00217] In certain embodiments, the method comprises use of a processed biological sample enriched for IgG and/or IgM. In certain embodiments, the method comprises enriching for immunoglobulins.
[00218] In certain embodiments, the method comprises exposing the cells to at least 0.3 mg/ml IgG, at least 0.6 mg/ml IgG, or at least 1 mg/ml IgG.
[00219] In certain embodiments, the cell expressing the selected antigenic target is a cell naturally expressing the selected antigenic target. In certain embodiments, the cell expressing the selected antigenic target is a cell engineered to express the selected antigenic target.
[00220] Cells are as described herein.
[00221] In certain embodiments, the cell expressing the selected antigenic target is a cell of neuroendocrine origin. In certain embodiment, the cell is an insulinoma cell. In certain embodiments, the cell is a Rin A12 cell, or a variant thereof. Other cells are contemplated. Other cells are as described herein.
[00222] The cell may be exposed to the candidate antibody for a suitable amount of time.
[00223] In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of at least 2 hours. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of 2 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of at least 3 hours. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of 3 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of at least 4 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 4 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of at least 5 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 5 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of at least 6 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 6 hours or greater. Other time periods are contemplated.
[00224] In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of time in one of the following ranges: 2 to 6 hours, 2 to 5 hours, 2 to 4 hours, 2 to 3 hours, 3 to 6 hours, 3 to 5 hours, 3 to 4 hours, 4 to 6 hours, 4 to 5 hours and 5 to 6 hours. Other time periods are contemplated.
[00225] A variety of methods for identify a candidate antibody binding to the selected antigenic target are known in the art. For example, the selected antigenic target can be used as a target for affinity purification of the functional autoantibody. Alternatively, blocking peptides may be used to identify the antibody.
[00226] In certain embodiments, the present disclosure provides a method of identifying a functional autoantibody associated with type 1 diabetes, the method comprising:
exposing a cell expressing a selected antigenic target able to bind to a functional antibody associated with type 1 diabetes to a candidate antibody, wherein the antigenic target has an activity to alter the level of reactive oxygen or nitrogen species in the cell;
determining the ability of the candidate antibody to alter the level of reactive oxygen or nitrogen species in the cell; and
identifying the candidate antibody as a functional autoantibody associated with type 1 diabetes.
[00227] In certain embodiments, the present disclosure provides a method of identifying a functional autoantibody associated with an autonomic disorder, the method comprising:
exposing a cell expressing a selected antigenic target able to bind to a functional antibody associated with an autonomic disorder to a candidate antibody, wherein the antigenic target has an activity to alter the level of reactive oxygen or nitrogen species in the cell;
determining the ability of the candidate antibody to alter the level of reactive oxygen or nitrogen species in the cell; and
identifying the candidate antibody as a functional autoantibody associated with an autonomic disorder.
[00228] Certain embodiments of the present disclosure provide a method of identifying an agent that modulates the binding of a functional autoantibody to an antigenic target. Such agents may be potential therapeutic agents and/or agents suitable for research purposes.
[00229] In certain embodiments, the present disclosure provides a method of identifying an agent that modulates the binding of a functional autoantibody to an antigenic target, the antigenic target having an activity to alter the level of reactive oxygen or nitrogen species in a cell, the method comprising:
exposing a cell expressing the antigenic target to the functional autoantibody and a candidate agent; determining the ability of the candidate agent to modulate the change in reactive oxygen or nitrogen species in the cell caused by the functional autoantibody ; and
identifying the candidate agent as an agent that modulates the binding of the functional autoantibody to antigenic target.
[00230] In certain embodiments, the method is used to identify a therapeutic candidate for a disease, condition or state, or to identify an agent for use in a method for detecting the autoantibody.
[00231] In certain embodiments, the method may be used to identify an agent that modulates the binding of a functional autoantibody associated with type 1 diabetes to an antigenic target. In certain embodiments, the functional antibody is a functional autoantibody associated with type 1 diabetes.
[00232] In certain embodiments, the method may be used to identify an agent that modulates the binding of a functional autoantibody associated an autonomic disorder to an antigenic target. In certain embodiments, the functional antibody is a functional autoantibody associated with an autonomic disorder.
[00233] Functional autoantibodies are as described herein.
[00234] In certain embodiments, the functional autoantibody is an IgG antibody. In certain embodiments, the functional autoantibody is an IgM antibody.
[00235] In certain embodiments, the functional autoantibody is present in a biological sample from a subject.
[00236] In certain embodiments, the functional autoantibody is present in a mix of other biological factors. In certain embodiments, the functional autoantibody is a semi- purified or a purified autoantibody. In certain embodiments, the functional autoantibody is an isolated antibody. In certain embodiments, the functional autoantibody is present in a sample enriched for immunoglobulins. [00237] Methods for enriching and purifying antibodies are known in the art.
[00238] In certain embodiments, the identity of the functional antibody is known. In certain embodiments, the identity of the functional antibody is not known. In this case, the method does not rely on needing to know the identity of the selected functional antibody.
[00239] Antigenic targets are as described herein.
[00240] In certain embodiments, the antigenic target is an extracellular target. In certain embodiments, the antigenic target is a membrane associated target.
[00241] In certain embodiments, the antigenic target comprises an endogenous target.
[00242] In certain embodiments, the antigenic target comprises an exogenous target introduced into a cell. In certain embodiments, the antigenic target is a cloned antigenic target.
[00243] In certain embodiments, the antigenic target comprises a membrane bound ion channel. In certain embodiments, the antigenic target comprises a membrane bound receptor.
[00244] In certain embodiments, the antigenic target comprises a calcium ion channel, a potassium ion channel or a sodium ion channel.
[00245] In certain embodiments, the antigenic target comprises a L-type voltage gated calcium channel (VGCC).
[00246] In certain embodiments, the antigenic target comprises a G protein-coupled receptor.
[00247] In certain embodiments, the identity of the antigenic target is known. In certain embodiments, the identity of the antigenic target is not known. In this case, the method does not rely on need to know the identity of the antigenic target. [00248] Methods for assessing the level of reactive oxygen or nitrogen species in a cell are as described herein.
[00249] In certain embodiments, the agent comprises a drug, a small molecule, a protein, a polypeptide, a nucleic acid, a lipid, a ligand, a lipid, a carbohydrate, a nucleic acid, an oligonucleotide, a ribozyme, a biologic, an aptamer, a peptide, a cofactor, a ligand, a ligand mimetic, a receptor, an enzyme, a metal ion, a chelate, a nucleic acid, an antibody, and an amino acid. Other types of molecules are contemplated.
[00250] In certain embodiments, the method of identifying the agent comprises use of flow cytometry. Other methods are contemplated, such as fluorescent microscopy or microplate analysis.
[00251] In certain embodiments, the method comprises determining or assessing the level of reactive oxygen species or reactive nitrogen species in the cell. Qualitative and quantitative methods for determining or assessing the level of reactive oxygen species or reactive nitrogen species in a cell are as described herein and known in the art.
[00252] In certain embodiments, the method of identify an agent that modulates the binding of a functional autoantibody to an antigenic target comprises comparison of the level of reactive oxygen species or nitrogen species with a control and/or reference level.
[00253] In certain embodiments, the method comprises using functional autoantibody in a biological sample from a subject. Biological samples are as described herein.
[00254] In certain embodiments, the processed biological sample is enriched for IgG and/or IgM. In certain embodiments, the method comprises enriching for immunoglobulins. Methods for enriching for immunoglobulins are known in the art.
[00255] In certain embodiments, the method comprises exposing the cells to at least 0.3 mg/ml IgG, at least 0.6 mg/ml IgG, or at least 1 mg/ml IgG. [00256] In certain embodiments, the functional autoantibody is from a subject suffering from, or susceptible to, a disease, condition or state associated with functional autoantibodies.
[00257] In certain embodiments, the functional autoantibody is from a subject suffering from, or susceptible to, type 1 diabetes.
[00258] In certain embodiments, the functional autoantibody is from a subject suffering from, or susceptible to, an autonomic dysfunction. In certain embodiments, the autonomic dysfunction comprises gastrointestinal and/or bladder autonomic neuropathy, or a cardiovascular autonomic disorder.
[00259] Cells expressing antigenic targets are as described herein. Suitable cells expressing the antigenic target may be selected.
[00260] In certain embodiments, the cell expressing the antigenic target is a cell naturally expressing the antigenic target. In certain embodiments, the cell expressing the antigenic target is a cell engineered to express the antigenic target.
[00261] In certain embodiments, the cell expressing the antigenic target to the autoantibody is a cell of neuroendocrine origin. In certain embodiment, the cell is an insulinoma cell. In certain embodiments, the cell is a Rin A12 cell, or a variant or derivative thereof.
[00262] The cell may be exposed to the functional autoantibody for a suitable amount of time.
[00263] In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of at least 2 hours. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of 2 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of at least 3 hours. In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of 3 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of at least 4 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 4 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of at least 5 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 5 hours or greater. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of at least 6 hours. In certain embodiments, the method comprises exposing the cells to the functional antibody for a period of 6 hours or greater. Other time periods are contemplated.
[00264] In certain embodiments, the method comprises exposing the cells to the functional autoantibody for a period of time in one of the following ranges: 2 to 6 hours, 2 to 5 hours, 2 to 4 hours, 2 to 3 hours, 3 to 6 hours, 3 to 5 hours, 3 to 4 hours, 4 to 6 hours, 4 to 5 hours and 5 to 6 hours. Other time periods are contemplated.
[00265] The method of identifying an agent that modulates the binding of a functional autoantibody to an antigenic target may further utilise a variety of other in vitro or in vivo techniques known in the art to assist with the identification of the agent, such as immunoassay (eg ELISA), immunoprecipitation, Western blotting, affinity purification, binding assays, and competition assays.
[00266] Certain embodiments of the present disclosure provide an agent identified according to a method as described herein.
[00267] Examples of agents include a drug, a small molecule, a protein, a polypeptide, a nucleic acid, a lipid, a ligand, a lipid, a carbohydrate, a nucleic acid, an oligonucleotide, a ribozyme, a biologic, an aptamer, a peptide, a cofactor, a ligand, a ligand mimetic, a receptor, an enzyme, a metal ion, a chelate, a nucleic acid, an antibody, and an amino acid. Other types of molecules are contemplated.
[00268] In certain embodiments, the agent is a candidate therapeutic agent. In certain embodiments, the agent may be used to detect the functional autoantibody in other assays using binding of the agent to the antibody. Such assays are known in the art, and include for example, enzyme-linked immunosorbent assay ELISA (eg direct ELISA, indirect ELISA, and sandwich ELISA). [00269] Certain embodiments of the present disclosure provide a method of detecting a functional autoantibody, the method comprising using an identified agent to detect the functional autoantibody. Methods of detecting a functional autoantibody using an agent are known in the art.
[00270] Certain embodiments of the present invention provide a method of identifying a therapeutic agent for a disease, condition or state associated with binding of a functional autoantibody to an antigenic target.
[00271] In certain embodiments, the present disclosure provides a method of identifying a therapeutic agent for a disease, condition or state associated with binding of a functional autoantibody to an antigenic target, the antigenic target having an activity to alter the level of reactive oxygen or nitrogen species in a cell, the method comprising:
exposing a cell expressing the antigenic target to the functional autoantibody and a candidate agent;
determining the ability of the candidate agent to modulate the change in reactive oxygen or nitrogen species in the cell caused by the functional antibody ; and
identifying the candidate agent as a therapeutic agent for the disease, condition or state associated with binding of a functional autoantibody to an antigenic target.
[00272] Agents, and methods for identifying agents, are as described herein.
[00273] In certain embodiments, the disease, condition or state is type 1 diabetes.
[00274] In certain embodiments, the disease, condition or state is an autonomic disorder. In certain embodiments, the autonomic disorder comprises a gastrointestinal or bladder autonomic neuropathy, or cardiovascular autonomic complications.
[00275] Methods for determining the ability of an agent to have a therapeutic ability are known in the art, and include for example use of in vitro assays for determining the effect of an agent on cells and/or one or more animal models for a disease, condition or state.
[00276] Certain embodiments of the present disclosure provide a kit for performing a method as described herein.
[00277] In certain embodiments, the kit comprises one or more reagents, cells and instructions as described herein.
[00278] Certain embodiments of the present disclosure provide use of a cell expressing an antigenic target for a functional autoantibody for identifying an agent that modulates the binding of the functional autoantibody to the antigenic target.
[00279] Cells, functional autoantibodies and antigenic targets, are as described herein. Methods for using cells expressing an antigenic target are as described herein.
[00280] In certain embodiments, the present disclosure provides use of a cell expressing an antigenic target for a functional autoantibody for identifying an agent that modulates the binding of the functional autoantibody to the antigenic target, wherein the antigenic target has an activity to alter the level of reactive oxygen or nitrogen species in the cell and binding of the autoantibody to the antigenic target alters the activity of the antigenic target.
[00281] Certain embodiments of the present disclosure provide use of a cell expressing an antigenic target for a functional autoantibody for identifying a therapeutic agent for a disease, condition or state associated with the binding of the functional autoantibody to the target.
[00282] In certain embodiments, the present disclosure provides use of a cell expressing an antigenic target for a functional autoantibody for identifying a therapeutic agent for a disease, condition or state associated with the binding of the functional autoantibody to the target, wherein the antigenic target has an activity to alter the level of reactive oxygen or nitrogen species in the cell and binding of the autoantibody to the antigenic target alters the activity of the antigenic target. [00283] Cells, and antigenic targets, are as described herein. Methods for using cells expressing an antigenic target are as described herein.
[00284] Certain embodiments of the present disclosure provide a kit for detecting a functional autoantibody.
[00285] In certain embodiments, the present disclosure provides a kit for detecting a functional autoantibody, the kit comprising:
a cell expressing an antigenic target to the functional autoantibody, wherein the antigenic target has an activity to alter the level of reactive oxygen or nitrogen species in the cell; and
a marker to determine the level of reactive oxygen or nitrogen species in the cell; and
optionally positive and/or negative control reagents for the level of reactive oxygen or nitrogen species in the cell.
[00286] Examples of cells, markers and reagents for detecting a functional autoantibody are as described herein.
[00287] Certain embodiments of the present disclosure provide isolated complexes.
[00288] In certain embodiments, the present disclosure provide an isolated complex comprising a cell expressing an antigenic target that has an activity to alter the level of reactive oxygen or nitrogen species in the cell, a functional autoantibody bound to the antigenic target on the cell, and a marker in the cell for detecting the level of reactive oxygen or nitrogen species.
[00289] Methods for forming complexes between a functional autoantibody bound to the antigenic target on the cell are described herein.
[00290] Antigenic targets are as described herein.
[00291] Methods for detecting antibody-antigen complexes are known in the art.
[00292] Markers for detecting the level of reactive oxygen or nitrogen species in cells are as described herein.
[00293] Certain embodiments of the present disclosure provide a method of detecting a functional autoantibody to an antigenic target on a cell, the method comprising using a complex as described herein to detect the functional autoantibody.
[00294] Standard techniques and equipment may be used for recombinant DNA technology, DNA sequencing, DNA arrays, oligonucleotide synthesis, molecular biology, cell biology and enzymatic reactions. The foregoing techniques and procedures may be generally performed according to methods known in the art and/or as commercially available, and are as described for example in Sambrook et al. Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)) and Ausubel et al Current Protocols in Molecular Biology (2003) John Wiley & Sons, both of which are herein incorporated by reference.
[00295] The present disclosure is further described by the following examples. It is to be understood that the following description is for the purpose of describing particular embodiments only and is not intended to be limiting with respect to the above description.
EXAMPLE 1 - A cell-based assay for the detection of pathogenic anti -Voltage-Gated Calcium Channel autoantibodies in immunoglobulin G from patients with Type 1
Diabetes
[00296] An assay of reactive oxygen species (ROS) stress induction in the rat insulinoma cell line Rin A12, as determined by 2’, 7’ -Diehl orofluorescein diacetate (DCF-DA) fluorescence detection by flow cytometry has been developed. We demonstrate that incubation of Rin A12 cells with immunoglobulin G (IgG) containing anti-VGCC activity from patients with T1D mediates a significant increase in ROS, with subsequent induction of apoptosis, as determined by positivity for annexin V expression. Neither T1D patient-derived IgG lacking anti-VGCC activity or IgG from healthy donors altered ROS or annexin V expression, indicating the new assay is specific for the detection of functional anti-VGCC Abs. Subsequent screening of IgG samples derived from individual patients indicated a prevalence of approximately 75% in a cohort of 20 patients with T1D. The new cell-based assay provides, for the first time, experimental evidence supporting a plausible pathophysiological mechanism underlying anti-VGCC Ab-mediated apoptosis induction in b cells. Additionally, the assay is a considerable advance on previously published methods for detecting and characterising the functional activity of anti-VGCC Abs in patient-derived samples.
[00297] 1. Introduction
[00298] Type 1 diabetes mellitus (T1D) is a chronic autoimmune disease characterised by insulin deficiency arising from immune-mediated destruction of insulin-producing beta (b) cells of the pancreas. Typically arising in childhood or early adolescence, the prevalence of T1D is highest in developed regions such as Europe and North America, with increasing incidence observed in these countries. While the aetiology of autoimmune-mediated destruction of b cells remains a topic of investigation, it is believed that T cells play a central role, as studies using post mortem pancreatic samples obtained from patients with recent or established disease indicate onset of symptoms is concurrent with predominantly CD8+ T cell-mediated insulitis. Furthermore, autoreactive CD8+ and CD4+ T cells specific to islet-derived epitopes have been demonstrated in isolates obtained from patient islets.
[00299] To date, considerations of humeral responses in T1D have focused on the role of antibodies as diagnostic and/or prognostic markers of disease. For example, since the publication over 20 years ago of seminal studies linking risk for T1D with the presence of serum antibodies against islet cell cytoplasmic antigens, a considerable body of research has confirmed the predictive value of serotyping autoantibody responses in at risk individuals. To date, fine specification of the autoantibody responses in T1D has identified a number of b cell autoantigens. What remains to be established, however, is a relationship between the generation in individuals of autoantibodies to cytoplasmic b cell autoantigens and immune-mediated pathogenesis resulting in b cell loss.
[00300] In this stud, we have used a cell-based assay of reactive oxygen species (ROS) generation to demonstrate that anti-VGCC Abs from patients with T1D induce stress and apoptosis in the rat insulinoma cell line, Rin A12, findings that support a pathogenic role for these antibodies in the immune processes associated with the disease. Furthermore, the cell-based stress induction assay is suitable for screening patients for the presence of these functional autoantibodies, thereby offering for the first time, a diagnostic assay capable of demonstrating both the presence and activity of these functional antibodies in larger patient cohorts, potentially improving the understanding of the role these antibodies play in the pathogenesis of T1D.
[00301] 2. Materials and methods
[00302] 2.1 Patient and control samples
[00303] Blood samples were obtained with informed consent from patients with T1D (n=20) and healthy individuals (h=10) (Table 1.). Eight of 20 T1D patients had previously been assessed for the presence of serum-derived anti-VGCC immunoglobulin G (IgG) autoantibodies, as defined using a whole colon migrating motor complex assay (Jackson, M.W. & Gordon, T.P. (2010) A novel impedance-based cellular assay for the detection of anti-calcium channel autoantibodies in type 1 diabetes. J Immunol Methods , 361, 31-36), with anti-VGCC activity detected in IgG derived from 6 of 8 patients with T1D (Table 2.).
Table 2. Codes and demographic information for patients and controls.
Figure imgf000052_0001
Footnote: Anti-VGCC Ab positive or negative patients indicate patients where anti-VGCC Abs have been detected in the IgG fraction of serum using a colon assay of colonic migrating motor activity and pharmacological confirmation, as reported in Jackson, M.W. & Gordon, T.P. (2010) A novel impedance-based cellular assay for the detection of anti-calcium channel autoantibodies in type 1 diabetes. J Immunol Methods, 361, 31-36. Uncharacterised patients refers to patients recruited to the study where anti-VGCC Ab status is unknown. [00304] 2.2 Preparation of IgG
[00305] IgG was prepared from individual serum samples using the caprylic acid precipitation technique (Steinbuch and Audran, Arch Biochem Biophys. 1969 134(2): 279-84.) as described previously (Jackson et al. J Neurosci. 2008 28(49): 13303-9). Briefly, serum was lowered to a pH of 4.8 using a solution of acetic acid (1M) and sodium hydroxide (0.5M), and stirred vigorously for 30 mins at room temperature. Caprylic acid was added while stirring the solution, to achieve a final caprylic acid to serum ratio of 0.74: 10. Samples were then centrifuged at 12000 rpm for 20 mins, and supernatants recovered and filtered through a Millipore 0.20 pm filter (Sartorius Stedim Biotech, Goettingen, Germany). Filtered samples were then combined with Hanks’ Balanced Salt Solution (HBSS, cat H6648, Sigma- Aldrich, Pty. Ltd, Australia) to a final volume of 10 mL. Samples were then spun down (4700 x g) for 25 mins at l5°C using a Millipore B15 concentrator (10 kDa, Merck Millipore Ltd, Darmstadt, Germany). This step was repeated 3 times. Recovered samples were suspended in HBSS at a final volume of 500 uL. The purified IgG was then dialysed against HBSS at pH 7.4 to reduce residual caprylic acid contamination using 7 kDa dialysis tubing (Medical International Ltd, London).
[00306] 2.3 Cell culture
[00307] Rat insulinoma cells (Rin A12) were cultured at 37 °C in an atmosphere of 5% C02 in air, in RPMI 1640 (Sigma- Aldrich, Australia) containing penicillin/streptomycin, 5% L-glutamine and supplemented with 10% fetal calf serum (Sigma-Aldrich, Australia). The cells were harvested at confluences of between 60-70 % by trypsin digestion, washed twice and re-suspended in HBSS.
[00308] 2.4 Detection of Reactive Oxygen Species generation by 2’, 7’ -
Dichlorofluorescein diacetate (DCF-DA)
[00309] For determining levels of intracellular reactive oxygen species (ROS), Rin A12 cells were primed by incubation with 40 mM DCF-DA for 5 mins at 37°C, using a modification of previously reported methodology (Tampo et al. (2003) Circulation Research , 92: 56-63). DCF-DA primed cells were then washed and re-suspended in individual vials in HBSS and incubated as follows: i) for establishment of positive controls for ROS induction, cells were incubated in HBSS for 4 hrs, followed by 5 mins of incubation in the presence or absence of Rotenone (Sigma-Aldrich, Australia) at between 0.1 and 2 mM, or for 4 hours in HBSS either with or without 30 pM Bay K8644 (Alomone Labs, Hadassah Ein Kerem, Israel), with addition or not of between 1 to 30 pM of nicardipine (Sigma-Aldrich, Australia); ii) in HBSS in the presence or absence of patient or control IgG at final concentrations of between 0.3 and 5 mg/mL. Experiments conducted with IgG also included duplicate vials of cells incubated in HBSS alone for 4 hours, followed by incubated for 5 mins in the presence of 1 pM Rotenone for confirmation of stress induction in each individual experimental run. Following incubations, cells were washed three times, and resuspended in HBSS (200 pl) at room temperature. Propidium iodide (PI) (Sigma-Aldrich) (50 pg/ml) was added just prior to flow cytometric analysis. All samples were assessed in triplicate and on at least 2 separate occasions. DCF-DA incubation, and all subsequent incubations were performed in the dark.
[00310] 2.5 Assessment of apoptosis using Annexin V antibody.
[00311] For assessment of IgG-mediated apoptosis, cells (2.5 X l05/ml) were incubated in HBSS in the presence or absence of 1 mg/ml of patient or control IgG for 4 or 16 hrs. Following incubation, cells were washed in binding buffer (eBioscience, Invitrogen, Australia) and re-suspended in 100 pl of the same buffer, and a FITC-conjugated antibody against annexin V (BD Biosciences, Maryland) added at a ratio of 5 parts per 100 of binding buffer, and the cells incubated at room temperature in the dark for 10 mins. Controls included cells incubated for 4 or 16 hrs in binding buffer alone, either with or without annexin V labelling, and cells incubated in the presence of 0.1 pM staurosporine (Sigma-Aldrich, Australia) as a positive control for induction of apoptosis. Following incubation with the annexin V antibody, cells were washed 3 times in binding buffer, and PI (50 pg/ml) was added just prior to flow cytometric analysis. [00312] 2.6 Flow cytometric analysis
[00313] Analysis of ROS levels (as determined by DCF-DA) or Annexin V expression in cells exposed to the various treatments was determined by flow cytometry using an Accuri C6 (Becton Dickinson, USA). Briefly, just prior to acquisition, cells within individual vials were mixed by finger tap, and acquired at a fluidic flow rate of 5xl03 cells per min with a medium flow core size of 25 mM. DCF-DA positivity was determined for each experimental run by assessment of DCF-DA primed but untreated cells, where cells were gated to exclude debris, and the scatter plot for DCF-DA fluorescence used to determine a line of positivity. For each individual experimental run, a line of positivity was placed to the right of the captured cells such that approximately 98% of cells were present in the left quadrant of the scatter plot. Subsequently, cells captured following incubation with the various experimental conditions were assessed as DCF-DA positive if they appeared in the right quadrant of the scatter plot. To correct for inter experimental variation, treated cells captured within individual experimental runs were normalised to the values obtained from untreated cells within the same experiment. To ensure that only viable cells were assessed for DCF-DA positivity, cells positive for PI were excluded from analysis.
[00314] Similarly, for determination of positivity for Annexin V, untreated but Annexin V antibody-labelled cells were acquired and used as the baseline for subsequent comparisons with treated cells from the same experimental run. As with DCF-DA primed cells, PI positive cells were excluded from analysis for Annexin V positivity.
[00315] 3. Results
[00316] 3.1 DCF-DA is a suitable marker of reactive species generation, and the L-type VGCC agonist, Bay K8466, mediates reversible stress generation in Bin A 12 cells.
[00317] To establish a reliable positive control for the stress induction assays, DCF-DA primed Rin A12 cells were incubated in media for 4 hrs, followed by 5 mins incubation with Rotenone at between 0.1 and 2 mM. Flow cytometric analysis of PI negative cells indicated that incubation with Rotenone resulted in a concentration dependent increase in DCF-DA positive cells compared to baseline values, with significance by 2-way ANOVA achieved at Rotenone concentrations of 1 and 2 mM (Fig. 1 A). In all subsequent experiments, 1 mM of Rotenone was used as a positive control to confirm reactive species generation. The cells were then assessed for reactive species generation in response to the L-type VGCC agonist, Bay K8644. Incubation of cells for 4 hours in the presence of 30 nM Bay K8644 resulted in a significant increase of approximately 350% in DCF-DA positive cells compared to baseline. Co-incubation of cells with both Bay K8644 and nicardipine reduced the number of DCF-DA positive cells compared to Bay K8644 alone, with a significant reduction in DCF-DA positive cells achieved at 10 nM nicardipine (Fig. 1 B).
[00318] 3.2 Diabetic IgG with anti-VGCC activity induces reactive species generation in Rin A12 Beta cells as determined by flow cytometric analysis of DCF-DA.
[00319] DCF-DA primed cells were first incubated with IgG from healthy individuals (h=10) to establish background DCF-DA positivity values. In these experiments, incubation of cells for 4 hrs with concentrations of IgG ranging from 0.3-2 mg/mL produced no significant change in DCF-DA positivity, however, both 3 and 5 mg/ml of IgG resulted in significantly increased values when compared to no IgG addition (by 2- way ANOVA) (Figure 1 C), likely resulting from non-specific effects of high IgG concentrations. Hence, IgG incubation of cells using patient-derived samples was performed using IgG concentration of between 0.3-2 mg/ml. To optimise the time- course for the cell-based stress assay, IgG at between 0.3-2 mg/ml from control subjects was incubated with primed Rin A12 cells for between 1 and 7 hrs, and DCF-DA positivity assessed. At 1, 2 and 4 hrs of incubation, DCF-DA positivity values were not significantly altered by the presence of IgG, but were significantly higher following 7 hrs incubation with IgG at all concentrations when compared to cells incubated without IgG (Figure 1 D). Therefore, incubation with patient IgG was performed for 4 hrs.
[00320] To establish whether anti-VGCC Abs mediate increased stress in Rin A12 cells, IgG samples from T1D patients previously characterized for the presence of anti- VGCC activity (designated TlD+) were then assessed by incubation for 4 hrs with DCF- DA primed cells. Across the range of IgG concentrations between 0.3-2 mg/ml, TlD+ IgG from 6 of 6 patients mediated a significant increase in stress generation, as measured by DCF-DA positivity, in Rin A12 cells compared to the pooled mean baseline values (IgG from healthy individuals by 2way ANOVA) (Figure 2, Table 3).
Table 3. Stress induction in Rin A12 cells by patient or control IgG as determined by DCF-DA positivity.
Figure imgf000057_0001
Footnotes: Rin A12 cells were incubated with corresponding IgG concentration for 4 hrs. Values in bold are above the mean + 2 SD of corresponding pooled means derived from IgG samples from healthy individuals. DCF-DA positivity is expressed as a percentage of baseline (values for cells incubated without IgG). CTRs refers to IgG from healthy individuals. T1D+ indicates individual patients previously characterised as positive for the presence of anti-VGCC Abs in the IgG fraction (see Table 1). T1D- refers to individual patients previously characterised as negative for the presence of anti-VGCC Abs in the IgG fraction (see Table 1).
[00321] By contrast, incubating DCF-DA primed Rin A12 cells with IgG from patients previously characterised to be negative for anti-VGCC Abs (designated T1D-) did not result in a significant increase in DCF-DA positivity (Figure 2. C&D, Table 2). Hence, the cell-based DCF-DA stress assay has the ability to detect the presence in IgG of functional antibodies to L-type VGCCs. [00322] 3.3 The DCF-DA cell-based stress assay is a suitable screening assay for determining the presence in patient IgG of functional anti-VGCC Abs.
[00323] After first establishing the utility of the DCF-DA cell-based assay for detecting anti-VGCC Ab-mediated stress induction in cultured b cells, we then analysed the DCF- DA positivity results from the previously characterised patients, and from controls, to determine an optimum IgG concentration for screening for the presence of anti-VGCC Abs. To achieve this, results at each IgG incubation concentration for patients were plotted against the mean + 2 standard deviations (SDs) of results from controls at the equivalent IgG incubation concentration (Table 2). Analysis of the DCF-DA positivity results from the 6 previously characterised T1D+ patients indicated that incubation of Rin A12 cells at 0.3 and 0.6 mg/ml of IgG resulted in mean values above the mean plus 2 SDs of comparable control values in 4 of 6 patients. However, mean values arising from incubation with 1 mg/ml IgG from 6 of 6 T1D+ patients were above the mean plus 2 SDs of DCF-DA positivity values produced by control IgG (Figure 3 A) (Table 2). By contrast, DCF-DA positivity values obtained using IgG from previously characterised T1D- patients were below corresponding control means plus 2 SDs in all cases and at all IgG incubation concentrations (Figure 3 A) (Table 2). As such, 1 mg/ml appears to be a suitable IgG incubation concentration for detecting the presence of anti- VGCC Abs in IgG derived from T1D patients. Indeed, analysis of results obtained using 2 mg/ml of IgG from T1D+ patients resulted in mean values above corresponding control means plus 2 SDs in only 2 of 6 cases. We then assessed optimisation of the incubation time. For this, primed cells were incubated for 1, 2 or 4 hrs with 1 mg/ml IgG from T1D+ patient P2 (Table 2) and a control donor. Significant induction of stress, as determined by DCF-DA positivity values compared to controls, was observed only in cells incubated with T1D+ IgG at 4 hrs (Figure 2 E). Thus, the optimum incubation time for the cell-based stress assay is 4 hrs.
[00324] After establishing 1 mg/ml IgG and 4 hrs incubation as optimal for the detection of anti-VGCC Abs as determined by induction of stress in DCF-DA-primed Rin A12 cells, a further 12 patients with T1D (not previously characterised for the presence of anti-VGCC Abs by the CMMC assay) were assessed for the presence of functional Abs using the DCF-DA cell-based stress induction assay. The criteria for determining the presence of anti-VGCC Abs in individual IgG samples was a DCF-DA value above the mean + 2 SDs of equivalent pooled control IgG values (Table 4).
Table 4. Determination of the presence of anti-VGCC Abs in patient IgG using DCF- DA primed Rin A12 cells.
SCREENED SAMPLES % DCF-DA Positivity Positivity for Anti- values VGCC Abs
Pooled Healthy Donor 79 ±39
Controls (h=10)
Figure imgf000059_0001
Footnotes: Values are derived from Rin A12 cells incubated with 1 mg/ml IgG, and are expressed as percentage of baseline (cells incubated without IgG). Values above the mean + 2 SD of pooled values derived from control IgG indicate positivity for stress induction
[00325] Of the 12 additional patients screened, individual IgG samples from 9 patients mediated a mean increase in DCF-DA positivity values above the criteria for the presence of anti-VGCC Abs (Figure 3 B) (Table 3). Thus, in the cohort investigated in the current study, anti-VGCC Abs were present in the IgG fraction of serum from 15 of 20 patients with T1D. Some patients recruited for the study had been assessed for the presence of other T1D autoantibodies, including anti-GAD, IA-2 or ZnT8 (data not shown). There was not apparent correlation between the presence in patient serum of anti-VGCC Abs and other T1D autoantibodies.
[00326] 3.4 Anti-VGCC Abs induce apoptosis in Rin A12 cells as determined by assessment of Annexin V
[00327] Anti-VGCC Ab-mediated apoptosis has been reported in cultured cell lines derived from pancreatic b cells. To establish whether incubation with anti-VGCC Abs induced apoptosis in the current study, Rin A12 cells incubated with patient or control IgG were assessed using antibody labelling of annexin V. For these experiments, Rin A12 cells were incubated for 4 or 16 hrs in the presence or absence of IgG (1 mg/ml) from either T1D+ (n=2, patients P2 & P7: Table 3) and T1D- (n=2, patients P17 & P19: Table 3) patients, or control donors (n=2). Annexin positivity, as determined by flow cytometric analysis of annexin V-antibody labelled cells, was significantly higher in cells incubated with T1D+ IgG compared to controls following 16 hrs of incubation (by 2-way ANOVA, pooled means of annexin V positivity values; T1D+ verses control), but not at 4 hrs, while T1D- IgG did not significantly alter annexin positivity at either time point (Fig. 4).
[00328] 4. Discussion
[00329] In an attempt to investigate the pathophysiological mechanism of anti-VGCC antibodies in T1D, a cell-based assay of intracellular RS generation was established using DCF-DA primed Rin A12 cells. In the current study, IgG from patients previously tested positive for anti-VGCC Abs using the CMMC assay mediated increased stress in the DCF-DA cell-based assay. Similarly, IgG from patients previously characterised as negative for anti-VGCC Ab had no effect on the cells. The use of previously characterised patients allows a direct comparison between the gold standard CMMC assay and the current assay for the ability to discriminate the presence of anti-VGCC Abs in patient samples. In the current study, a 100% correlation in results was achieved between the two assays, hence the current assay is a suitable substitute for the CMMC assay. This represents a considerable advancement, as the new cell-based assay is considerably less complex to perform than the tissue-based CMMC assay, and can be scaled to provide increased diagnostic capacity. Indeed, once validated, the RS cell assay was used to screen IgG from 12 previously untested patients with T1D. Within this cohort, IgG samples from 9 individual patients were identified as having the ability to induce ROS stress induction. Hence, in the total cohort used for the current study, 15 of 20 individual patient IgG samples screened contained functional anti-VGCC Abs capable of mediating RS stress induction in pancreatic b cells.
[00330] In addition to an ability to detect the presence in immunoglobulin samples of anti-VGCC Abs, the new assay incorporating a functional assessment of stress induction has the ability to directly investigate the pathogenic effect of these Abs on b cells, the main target of autoimmune responses in T1D. Induction of RS-associated stress is particularly harmful for pancreatic b cells due to a low anti -oxidative capacity including low expression of superoxide dismutase (SOD), catalase, and glutathione peroxidas, leaving these cells vulnerable to dysfunction and apoptosis. The current study, which assesses RS generation, provides for the first time a potential pathogenic mechanism underlying anti-VGCC Ab-mediated b cell apoptosis. Given the site of antibody activity is the L-type VGCC, it is highly likely that increased Ca2+ flux mediated by Ab binding results in the stress response reported in the current study. Indeed, increased Ca2+ is a well described mediator of stress in a number of cell types, including b cells, and preliminary studies using the L type VGCC agonist, Bay K8644, demonstrated that direct activation of these channels results in stress induction in the current assay. Further to this, the effects of the channel agonist were blocked by addition of the DHP antagonist, nicardipine. Thus, the effect of anti-VGCC Abs was mimicked by an agonist acting at the DHP site. It is likely that the majority of the increased stress observed in the IgG-incubated cells is ROS, arising from the tricarboxylic acidic cycle in mitochondria, which has been reported to result from Ca2+ mediated activation of b cells, with excess exogenous Ca2+ flux via VGCCs reported to drive increases in ROS. The effect of the Abs was apparent, in terms of RS generation, after about 4 hours which is consistent with other studies assessing Ca2+ mediated stress. Other forms of RS include reactive nitrogen species, which generally arise from upregulation of inducible nitric oxide synthase, a feature of cytokine-mediated stress responses in pancreatic b cells. The current assay does not utilize cytokines in the incubation media, thus it is proposed that a sustained elevation in exogenous Ca2+ flux mediated by anti-VGCC Abs drives an increase in ROS, secondary to elevated mitochondrial activity.
[00331] In excess, the toxic effects of increased free radical generation via ROS has the potential to induce apoptosis. Indeed, in the current study, induction of annexin V expression was detected in Rin A12 cells incubated with IgG containing anti- VGCC activity. While numerous studies have demonstrated that increased Ca2+ flux resulting from over-activity of VGCCs is an inducer of apoptosis in various human, rat, and mouse cell types, the current study is the first to hypothesize a link in b cells between autoantibody-mediated stress induction and apoptosis. It is therefore contended that the apoptotic effects of anti-VGCC antibodies on pancreatic b cells results from Ca2+-mediated generation of ROS. Assessment of colonic migrating motor complex activity and bladder cystometry following passive transfer of IgG from anti-VGCC Ab positive patients to mice has demonstrated disrupted VGCC channel activity in vivo , indicating these antibodies are potentially pathogenic in vivo. The current study suggests further research is needed to address the direct effects of these pathogenic antibodies on b cell function and survival in patients, particularly in the preclinical phase when intervention of specific autoimmune processes by targeting b cells may alter disease outcomes. To date, a pathogenic role for autoantibodies in T1D remains unproven, which is perplexing given observations that humoral autoimmunity appears early in disease and is prognostic for disease progression, features shared with a number of other autoimmune diseases, including Sjogren’s syndrome and rheumatoid arthritis, where the pathogenic role of autoantibodies are well described (reviewed in (Rowley & Whittingham, 2015). Interestingly, in vivo neutralisation using immune-modulation by pooled IgG has been reported for anti-VGCC Abs, attributed to the blocking action of anti -idiotypic antibodies. To date however, clinical outcomes arising from humoral immune-modulation have been disappointing, likely due to application of these therapies after significant b cell loss has already occurred. It is intriguing to speculate that early identification of autoantibody responses coupled with immunomodulation in‘at risk’ individuals may provide clinically beneficial disease outcomes. [00332] In conclusion, we have developed a cell-based assay of stress induction in Rin A12 cells to detect the presence in patient IgG of autoantibodies to L-type VGCCs. The new cell assay provides both a method of detection and an indication of pathogenicity arising from the functional activity of the antibodies, which is a considerable benefit compared to assays assessing only antibody binding. By providing a cell-based platform for screening larger patient cohorts, the new assay should facilitate a greatly improved understanding of the prevalence, timing and consequences of anti-VGCC Ab responses in patients with type 1 diabetes.
EXAMPLE 2 - Pharmacological characterization of VGCC-mediated ROS generation in Rin A12 cells
[00333] Introduction
[00334] We next sought to investigate the role of VGCCs and the effect of the anti- VGCC Ab in relation to ROS generation using pharmacological characterization, and to examine whether the action of the anti-VGCC Ab in mediating ROS generation can be blocked by pre-incubation with IgG from healthy individuals or T1D-ROS patients.
[00335] Methods
[00336] 2.1 IgG samples
[00337] IgG preparation from T1D patients characterized as a positive for ROS stress induction in Rin A12 (designated T1D+ROS i.e., P010, P014, P015 and P016), or inability to induce ROS generation in Rin A12 (designated T1D-ROS i.e., P018, P019, and P020), were used as positive and negative controls. In addition, IgG preparations from healthy donors (i.e., C007, C008, C009 and C010) were used as controls (n=4).
[00338] 2.2 Pharmacological characterization of ROS generation
[00339] 2.2.1 Exogenous Ca2+ chelation study
[00340] To assess the role of exogenous Ca2+ in VGCC-mediated ROS stress induction, cells were prepared and primed with DCF-DA as described earlier and incubated with or without 1 uM of the Ca2+ chelator, ethylene glycol tetra-acetic acid (EGTA) (Sigma Aldrich, AUSTRALIA). ROS generation was assessed in cells following 4 hrs of incubation with IgG at a concentration of 1 mg/ml, from either T1D+ROS patients, T1D-ROS patients, or healthy donors, or following acute addition of rotenone.
[00341] 2.2.2 DHP Analysis
[00342] DHP analysis
[00343] To assess the role of DHP agonists and antagonists in VGCC-mediated ROS stress induction, cells were prepared and primed with DCF-DA as described earlier with or without the DHP agonist, Bay K8644 (1-100 nM) (Alomone Lab, Israel), and/or the antagonist, nicardipine (1-100 nM) (Sigma-Aldrich Pty. Ltd, Australia). ROS generation was assessed in cells incubated for 4 hours following the addition of drugs, and/or IgG at a concentration of 1 mg/ml, or following acute addition of rotenone.
[00344] Immunological analysis
[00345] To assess neutralization of functional autoantibody, 1 mg/ml of IgG from T1D+ROS patients was combined and pre-incubated for 30 mins at room temperature with 1 mg/ml of IgG from either T1D-ROS patients or healthy individuals. For ROS assessment, cells were prepared and primed with DCF-DA, and incubated with either individual or pre-incubated IgG preparation for 4 hrs/
[00346] Statistical analysis
[00347] Statistical comparisons of DCF-DA positivity for cells incubated with IgG or drugs including rotenone, EGTA, nicardipine or Bay K8644 were performed on individual sample means using 2-way or l-way Analysis of Variance (ANOVA) at 0.05 level of confidence.
[00348] Results
[00349] Exogenous Ca2+ is required for rotenone-mediated ROS generation [00350] To investigate the involvement of exogenous Ca2+ in the b cell stress response, rotenone was used to stimulate ROS generation in cells following incubation with media containing either free Ca2+ or the chelating agent, EGTA. As indicated in Figure 5, assessment of DCF-DA positivity for cells incubated in chelator-containing media resulted in unchanged values compared to cells incubated without EGTA (baseline DCF-DA positivity). In contrast, assessment of DCF-DA positivity for cells incubated in Ca2+-containing media with acute addition of rotenone resulted in a significant increase of 538% compared to baseline. The rotenone-mediated increase in DCF-DA positivity was reversed in cells incubated with EGTA, with positivity values not significantly different to baseline.
[00351] Characterization by pharmacological manipulation of the role of the DHP site of L-type VGCCs in ROS generation
[00352] Bay K8644 stimulates ROS generation in Rin A12 cells
[00353] To assess the effect of DHP drugs in DCF-DA primed Rin A12 cells, DCF- DA positivity was assessed following treatment by Bay K8644 and nicardipine, at concentrations ranging from 1-100 nM. Assessment of DCF-DA positivity for cells incubated with Bay K8644 at concentrations of between 1-10 nM or 100 nM resulted in no significant increase compared to untreated cells (baseline DCF-DA positivity), however incubation with 30 nM resulted in a significant increase of DCF-DA positivity of 477% of baseline (Figure 6A). In contrast, assessment of DCF-DA positivity for cells incubated with nicardipine (1-100 nM) displayed no statistical change across compared to baseline the concentration range (Figure 6B).
[00354] Nicardipine reverses Bay K8644-mediated ROS generation
[00355] To assess the ability of nicardipine to block the agonistic effect of Bay K8644-mediated ROS generation, DCF-DA primed Rin A12 cells were incubated without or with nicardipine at concentrations between 3-30 nM following addition of 30 nM Bay K8644. As indicated in Figure 6C, assessment of DCF-DA positivity for cells incubated without nicardipine treatments following the addition of Bay K8644 resulted in a significant DCF-DA positivity increase of 307% compared to baseline. In contrast, assessment of DCF-DA positivity for cells incubated with nicardipine at a concentration of 10 nM and Bay K8644 resulted in a value of 46% of baseline, representing an 85% reduction compared to Bay K8644 alone. While complete reversal was observed at a nicardipine concentration of 10 nM, partial reversal was achieved using 3 and 30 nM, with DCF-DA positivity values of 173 and 214%, respectively, representing a reduction of 44% and 29%, respectively, compared to DCF-DA positivity in cells incubated with Bay K8644 alone. Thus this data indicates that the DHP agonist and antagonist drugs mediate ROS generation in b cells.
[00356] Nicardipine does not reverse rotenone -mediated ROS generation
[00357] To investigate the involvement of DHP-expressing VGCCs in the rotenone- mediated ROS generation, nicardipine was used at a concentration of 10 nM to assess the b cell stress response to rotenone. As indicated in Figure 7, assessment of DCF-DA positivity in unstimulated cells incubated with nicardipine resulted in unchanged values compared to untreated cells (baseline DCF-DA positivity). By contrast, assessment of DCF-DA positivity for cells incubated without nicardipine and stimulated with rotenone resulted in a significant increase of 891% compared to the baseline. Assessment of DCF-DA positivity for cells incubated with nicardipine and stimulated with rotenone indicated that nicardipine did not reverse the rotenone- mediated ROS generation, with a value of 1091% of baseline (Figure 7).
[00358] Chelation of exogenous Ca2+ does not reverse anti-VGCC Ab-mediated ROS generation
[00359] To investigate the role of exogenous Ca2+ in anti-VGCC Ab-mediated ROS generation, DCF-DA positivity was assessed in cells incubated with IgG from T1D+ROS patients, or healthy controls, in media containing free Ca2+ or I mM EGTA.
[00360] Anti-VGCC Abs-mediated ROS generation is reversed by nicardipine [00361] To assess the role of DHP-expressing VGCCs in anti-VGCC Ab-mediated ROS generation, DCF-DA positivity was assessed in cells incubated with IgG from T1D+ROS patients, or healthy donor controls, either alone or with 10 nM of nicardipine.
[00362] As demonstrated in Figure 9, incubation of cells with anti-VGCC Ab- containing IgG from 2 individual T1D patients resulted in a statistically significant increase in ROS generation compared to baseline values. Addition of nicardipine to the same IgG reversed the ROS generation, such that ROS values were not significantly different to baseline. By contrast, for cells incubated with IgG from healthy controls, neither IgG alone or in combination with nicarpidine resulted in a significant chance in ROS generation compared to baseline.
[00363] Functional anti-VGCC Abs are blocked by pre-incubation with IgG from healthy donors or T1D-ROS patients
[00364] To investigate the neutralization of anti-VGCC Abs-mediated ROS generation by IgG from healthy donors or T1D-ROS patients, DCF-DA positivity was assessed in cells incubated with IgG containing anti-VGCC Abs from individual T1D patients, either alone or following pre-incubated with an equimolar concentration of IgG from healthy donor controls or T1D patients negative for anti- VGCC antibodies.
[00365] As indicated in figure 10, pooled ROS generation values in cells incubated with T1D IgG containing anti-VGCC Abs resulted in a significant increase in ROS generation compared to baseline. Significance was lost following pre-incuabtion of the IgG with equimolar IgG from healthy controls or T1D patient IgG lacking anti- VGCC Abs. By contrast, healthy donor IgG, either alone or following pre-incubation with T1D patient IgG lacking anti-VGCC Abs, resulted in no significant change in ROS generation compared to baseline. [00366] Discussion
[00367] Over the last 30-40 years a number of studies investigating the role of functional autoantibodies in autoimmunity have identified autoantibodies against various ion channels, including Ca, K and Na channels (Majoie, H. J. M., de Baets, M., Renier, W., Lang, B., & Vincent, A. (2006). Epilepsy Research , 71(2): 135- 141). In most cases the presence of these anti-channels Abs is associated with activation or inhibition of cellular ionic exchange, leading to physiologically deleterious effects and subsequently driving cellular or tissue dysfunction or cell death (Hajnoczky, G., Davies, E., & Madesh, M. (2003). Biochem Biophys Res Commun , 304(3): 445-454; Jackson, M. W., Gordon, T. P., & Waterman, S. A. (2004). Gastroenterology, 126(3): 819-828; Koneczny, I. (2018). Front Immunol, 9” 97; Vincent, A., Lang, B., & Kleopa, K. A. (2006). Neuron, 52(1): 123-138; Waterman, S. A., Jackson, M. W., & Gordon, T. P. (2006). CHAPTER 17 - Functional Effects of Autoantibodies A2 - Mackay, Noel R. Roselan R The Autoimmune Diseases (Fourth Edition) (pp. 217-236). St. Louis: Academic Press.). Because these targets are available on the cell surface, the autoantibodies are able to bind and exert a functional effect depend on the cell types and membrane potential. For example, there are inhibitory autoantibodies described in Lambert-Eaton myasthenic syndrome (LEMS) targeting structures such as nicotinic acetylcholinergic receptors, which inhibit skeletal muscle contraction (Kleopa, K. A. (2011). Curr Neuropharmacol , 9(3): 458-467; Takamori, M. (2008). J
Neuroimmunol, 201-202, 145-152.). Other structures targeted by Abs in LEMS are voltage gated Ca2+ channels (VGCC), where the Abs exert an inhibitory effect resulting in reduced Ca2+ flux (Roberts, A., Perera, S., Lang, B., Vincent, A., & Newsom-Davis, J. (1985). Nature, 317(6039): 737.).
[00368] The detection and characterization of functional autoantibodies has been slow compared to traditional autoantibodies. For example, autoantibodies against anti-nuclear antigens (ANA), first discovered over 60 years ago have been extensively studied in systemic autoimmune diseases such as lupus and Sjogren and form part of the diagnostic criteria for these diseases. Assays for the presence of anti-ANA Abs such as Ro and La utilize traditional immunological methods including ELISA or Immunofluorescence. Similarly, in T1D, conventional immunological approaches have facilitated the identification of autoantibodies targeting intracellular structures in pancreatic b cells including GAD, IA-2 and ZnT8 which currently are used as markers for the disease.
[00369] In contrast, detection and characterization of functional autoantibodies require more complex approaches including live tissue or cell assays associated with pharmacological and/or physiological analysis. Such an approach was used in the current study to further characterize the anti-VGCC Ab-mediated ROS generation. In these studies the role of exogenous Ca2+ was first assessed using chelation. Surprisingly, addition of the chelating agent to cells incubated with patient derived anti-VGCC Abs did not significantly reduce ROS generation. This result is somewhat counter intuitive, as increased Ca2+ flux through the L-type VGCCs is a proposed mechanism for anti-VGCC Ab activity.
[00370] Interestingly, chelation significantly reversed rotenone-mediated ROS generation. A close interpretation of the methodology used for this study may explain these results, as the b cells were incubated with both the anti-VGCC Abs and the chelating agent at the same time over 4 hrs. In contrast, in the rotenone experiment, rotenone is added for only 5 mins of the last 4 hrs incubation, thus the cells had been incubated with the chelating agent for 3.55 hrs prior the addition of the rotenone. Therefore, it is possible that the chelating agent takes time to work, during which the anti-VGCC Ab could mediate ROS generation. It is possible that during the early period of 4 hours of incubation, there is sufficient available Ca2+ flux to cause a partial depolarization of cells and allow internal Ca2+ release, and therefore stress generation to occur. An alternative possibility is that binding of anti-VGCC Ab to the VGCC has a secondary effect capable of modifying ROS generation and internal Ca2+ release independent of exogenous Ca2+. There are well described pathways of ROS generation that do not rely on external calcium, most notably the iNOS, which can be driven by a number of cellular physiological responses, including to various ligands. While the mechanism where by anti-VGCC Abs might achieve this remain unknown, internalization of antibody bound surface targets are well described, which is one possible driver of cellular responses independent of external calcium. [00371] More encouraging is the result using the DHP antagonist nicardipine, where reversal of the anti-VGCC Ab-mediated ROS generation confirms the crucial involvement of the DHP site in mediating the Abs effects. Interestingly, the involvement of the DHP site in ROS generation was supported by Bay K8644 which mimicked the Ab effect. These observations matched with the data demonstrating pharmacological approaches used in other assay systems, such as the CMMC assay where anti-VGCC Ab-mediated effects on the visceral smooth muscle is also mimicked by Bay K8644, and blocked by nicardipine. Therefore, this study establishes the crucial role of L-type VGCCs in Ab-mediated ROS generation in b cells.
[00372] While these studies do not confirm the specificity of the target 100%, given the body of work now done, including previous studies demonstrating that anti- VGCC Ab mediated apoptosis in b cell is Ca2+ dependent, the pharmacological characterization conducted here further supports that L-type VGCCs are a crucial target of the functional Ab in T1D. Currently, autonomic dysfunction is the only clinically relevant symptoms associated with the presence of anti-VGCC Abs.
[00373] In the current study, IgG from healthy individuals neutralized the functional Ab-mediated ROS generation, a finding that was further extended by demonstrating Ab neutralization using IgG from T1D patients lacking anti-VGCC Ab activity (T1D-ROS).
[00374] These current studies also provide a basis for screening methods that allow the identification of antigenic targets, functional autoantibodies and various agents.
EXAMPLE 3 - Autoimmune Primary Dysautonomia
[00375] Primary dysautonomia in young patients often poses a diagnostic challenge as no standard testing is available to aid in differential diagnoses. Autoimmune hypothesis has been postulated as a possible pathophysiological mechanism especially where hereditary and secondary causes have been excluded. However, the tests for auto-antibodies may only be done in specialized labs only, and are not available in many countries. [00376] Herein, we describe a young female with severe dysautonomia manifesting with gut and bladder dysmotility in addition to POTS where a novel functional antibody assay assisted with the diagnosis.
[00377] The l9-year-old woman had an 8-year history of severe progressive gut dysmotility without structural causes. She has a venting PEG, a PEJ for medication administration, and is fully dependent on TPN for her severe malnutrition. She had Mitrofanoff procedure for her neurogenic bladder. She was on glucocorticoid and dexamphetamine replacement for her recurrent hypoglycaemia, which was thought to be part of her dysautonomia. POTS was diagnosed by a cardiologist, and she was commenced on ivabradine for management. She had 6 months of severe sicca symptoms resulting in dental decay, necessitating full dental extraction. An extensive routine autoimmune and genetic work up had been non-revealing.
[00378] Figure 10 shows ROS the stress generation as a % of untreated baseline. Figure 11 shows the PI positivity as a percentage of untreated baseline. The assays were conducted using caprylic acid precipitated IgG with the Rin A12 cell line.
[00379] As can be seen, the female patient tested positive on the cell based research assay designed for detection of pathogenic functional auto-antibodies directed against voltage gated calcium channels. The data was generated from at least 2 separate assays, on different days.
[00380] The successful detection of an anti-ion channel antibody in the patient with idiotypic dysautonomia resulted in a phenotypic assay presentation distinct from that produced by the anti-VGCC antibody associated with T1D.
[00381] The results produced by the functional antibody in this instance resulted in increased apoptosis at 2, 4 and 6 hours incubation, and an increased stress response across both 4 and 6 hours. The typical profile for anti-VGCC antibodies is an increased stress response at 4 hours with no increase in apoptosis at this time point.
[00382] Further, the autonomic profile of the dysautonomia patient is substantially more pronounced than that associated with the anti-VGCC antibody. For example, the patient has severe gut dysmotility, atonic/neurogenic bladder, and postural othostatic tachycardia syndrome. The autonomic dysfunction associated with the anti-VGCC antibody in type 1 diabetes appears limited to mild to medium gastrointestinal dysfunction, particularly gastroparesis.
[00383] Table 5 shows the correlations between the presence in T1D patients of anti- VGCC antibodies and symptoms of gastrointestinal dysfunction (gastroparesis). The table shows the correlation between presence of anti-VGCC Ab and autonomic gastrointestinal tract dysfunction symptoms in 15 patients with T1D screened for the presence of anti-VGCC Abs and for the presence of symptoms of gastrointestinal tract dysfunction (gastroparesis) as determined by treating physician.
Table 5
Figure imgf000072_0001
[00384] Footnote: The presence of symptoms is positively correlated with positivity for anti-VGCC Abs (P<0.05 by 2-tailed Chi Square).
[00385] These results indicates a pathophysiological role of some form of channelopathy to explain the myriad clinical manifestations in the patient. Therapeutic interventions to remove and/or reduce this antibody may be considered to improve the prognosis of this morbid disease.
[00386] The results also support the use of the live cell-based bioassay for anti channel antibodies. As discussed earlier, this assay is based on an excitable neuroendocrine pancreatic beta cell line (Rin A12), and is capable of detecting functional antibodies disrupting ion channels (or GPCRs) present on the cell surface and modifying cellular ion transport.
[00387] The basic readout of the assay is reactive oxygen stress (ROS) generation (as determined by DCFDA) resulting from altered ion channel activity, with an additional assessment of cell viability (as determined by PI). As described previously herein, the assay has been validated for the detection of anti-VGCC autoantibodies in type 1 diabetes.
[00388] The results indicate the presence in the patient IgG fraction of an anti-ion channel antibody. It is apparent that over multiple time points, IgG from the patient induces increased ROS stress in the cell bioassay, with a corresponding decrease in cell viability (as demonstrated by the increase in PI) up to 6 hours. The data indicates an antibody-mediated stress response leading to the induction of apoptosis in the incubated cells.
[00389] The data indicate that at least some of the autonomic issues observed in the patient are attributable to the presence of an anti-ion channel antibody. There is significant literature supporting a role for functional antibodies in several human channelopathies, and as we have also demonstrated a correlation between autonomic symptoms and the presence in patients of anti-VGCC autoantibodies in type 1 diabetes. We have also passively transferred these antibodies to mice and reproduced autonomic dysfunctions similar to some of those described in the current patient.
[00390] These current studies also provide a basis for screening methods that allow the identification of antigenic targets, functional autoantibodies and various agents. EXAMPLE 4 - Kits and product for detecting functional autoantibodies
[00391] A kit or combination product for detecting functional autoantibodies may include one or more of the following components:
(i) Reagents for processing serum samples from a patient. For example solutions for adjusting pH, reagents for enriching immunoglobulins (eg caprylic acid), and Hanks’ Balanced Salt Solution.
(ii) Reagents for cell culture and handling.
(iii) Reagents for cell labelling to detect reactive oxygen species. For example, 2’, 7’ -Dichlorofluorescein diacetate (DCF-DA).
(iv) Control/Reference Reagents. For example, rotenone, nicardipine, control antibodies.
(iv) Flow cytometric reagents.
[00392] A kit or product may also include instructions for processing samples, cell culture, use of controls and reference standards, antibody-antigen binding conditions, and analysis.
[00393] Although the present disclosure has been described with reference to particular embodiments, it will be appreciated that the disclosure may be embodied in many other forms. It will also be appreciated that the disclosure described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to, or indicated in this specification, individually or collectively, and any and all combinations of any two or more of the steps or features.
[00394] Also, it is to be noted that, as used herein, the singular forms“a”,“an” and “the” include plural aspects unless the context already dictates otherwise. [00395] Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as“comprises” or“comprising”, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
[00396] The term“about” or“approximately” means an acceptable error for a particular value, which depends in part on how the value is measured or determined. In certain embodiments,“about” can mean one or more standard deviations. When the antecedent term "about" is applied to a recited range or value it denotes an approximation within the deviation in the range or value known or expected in the art from the measurements method. For removal of doubt, it shall be understood that any range stated herein that does not specifically recite the term“about” before the range or before any value within the stated range inherently includes such term to encompass the approximation within the deviation noted above.
[00397] Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.
[00398] The subject headings used herein are included only for the ease of reference of the reader and should not be used to limit the subject matter found throughout the disclosure or the claims. The subject headings should not be used in construing the scope of the claims or the claim limitations.
[00399] The description provided herein is in relation to several embodiments which may share common characteristics and features. It is to be understood that one or more features of one embodiment may be combinable with one or more features of the other embodiments. In addition, a single feature or combination of features of the embodiments may constitute additional embodiments.
[00400] All methods described herein can be performed in any suitable order unless indicated otherwise herein or clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the example embodiments and does not pose a limitation on the scope of the claimed invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential.
[00401] Future patent applications may be filed on the basis of the present application, for example by claiming priority from the present application, by claiming a divisional status and/or by claiming a continuation status. It is to be understood that the following claims are provided by way of example only, and are not intended to limit the scope of what may be claimed in any such future application. Nor should the claims be considered to limit the understanding of (or exclude other understandings of) the present disclosure. Features may be added to or omitted from the example claims at a later date.

Claims

1. A method of detecting a functional autoantibody against an antigenic target, method comprising exposing a cell expressing the antigenic target to the autoantibody, wherein the antigenic target has an activity to alter the level of reactive oxygen or nitrogen species in the cell and binding of the autoantibody to the antigenic target alters the activity of the antigenic target, and detecting the functional autoantibody on the basis of a change in the level of the reactive oxygen or nitrogen species in the cell.
2. The method according to claim 1, wherein the antigenic target comprises a membrane bound cell ion channel.
3. The method according to claims 1 or 2, wherein the antigenic target comprises a calcium ion channel, a potassium ion channel or a sodium ion channel.
4. The method according to claim 1, wherein the antigenic target comprises a membrane bound receptor.
5. The method according to claim 1 or 4, wherein the antigenic target comprises a G protein-coupled receptor.
6. The method according to any one of claims 1 to 5, wherein the level of reactive oxygen or nitrogen species in the cell increases upon binding of the autoantibody to the antigenic target.
7. The method according to any one of claims 1 to 6, wherein the level of the reactive oxygen or nitrogen species is determined using a fluorescent marker to determine the level.
8. The method according to claim 7, wherein the level of the reactive oxygen species is determined using the marker 2,,7’-dichlorofluorescein diacetate.
9. The method according to any one of claims 1 to 8, wherein the method comprises detecting the functional autoantibody in a biological sample from a subject.
10. The method according to claim 9, wherein the biological sample comprises a processed biological sample.
11. The method according to claims 9 or 10, wherein the biological sample comprises blood, serum or plasma.
12. The method according to any one of claims 9 to 11, wherein the processed biological sample is enriched for IgG.
13. The method according to any one of claims 1 to 12, wherein the antigenic target comprises a L-type voltage gated calcium channel (VGCC).
14. The method according to any one of claims 1 to 14, wherein the cell is of neuroendocrine origin.
15. The method according to any one of claims 1 to 14, wherein the cell is an insulinoma cell.
16. The method according to any one of claims 1 to 15, wherein is cell is a Rin A12 cell.
18. The method according to any one of claims 10 to 17, wherein the subject is suffering from, or susceptible to, type 1 diabetes.
19. The method according to any one of claims 10 to 18, wherein the subject is suffering from, or susceptible to, autonomic dysfunction.
20. The method according to claim 19, wherein the autonomic dysfunction comprises gastrointestinal and/or bladder autonomic neuropathy, or a cardiovascular autonomic disorder.
21. The method according to any one of claims 1 to 21, wherein the method is used to screen for functional autoantibodies, to identify a subject suffering from an autoimmune disease, condition or state, to identify a subject suffering from, or being susceptible to, an autonomic disorder, for diagnosis or prognosis of an autoimmune disease, condition or state, for diagnosis or prognosis of an autonomic disorder, for assessing the risk or likelihood of a subject suffering from, or being susceptible to, an autoimmune disease, condition or state, for assessing the risk or likelihood of a subject suffering from, or being susceptible to, an autonomic disorder, to identify a specific functional autoantibody, to identify an antigenic target on a cell associated with the binding of a functional autoantibody, or to identify a therapeutic agent.
22. A method of detecting a functional autoantibody against a L-voltage gated calcium channel in a subject suspected of suffering from, or being susceptible to, an autonomic disorder, the method comprising exposing a cell expressing the L-voltage gated calcium channel to a sample containing antibodies from the subject and detecting the functional autoantibody in the subject on the basis of a change in the level of the reactive oxygen or nitrogen species in the cell.
23. The method according to claim 22, wherein the method is used for diagnosis or prognosis of the autonomic disorder.
24. The method according to claim 23, wherein the autonomic disorder comprises a gastrointestinal or bladder autonomic neuropathy, or cardiovascular autonomic complications.
25. A method of identifying a subject suffering from, or susceptible to an autonomic disorder associated with functional autoantibodies to a L-voltage gated calcium channel, the method comprising exposing a cell expressing the L-voltage gated calcium channel to a sample containing antibodies from the subject and identifying the subject as suffering from, or being susceptible to the autonomic disorder on the basis of an increase in the level of reactive oxygen species detected in the cell.
26. A method of identifying an antigenic target for a selected functional autoantibody, the method comprising:
exposing a cell expressing a candidate antigenic target to the selected functional autoantibody, wherein the antigenic target has an activity to alter the level of reactive oxygen or nitrogen species in the cell;
determining the ability of the selected functional autoantibody to alter the level of reactive oxygen or nitrogen species in the cell; and
identifying the candidate antigenic target as an antigenic target for the selected functional autoantibody.
27. The method according to claim 26, wherein the antigenic target comprises a target for a functional autoantibody associated with type 1 diabetes.
28. The method according to claim 26, wherein the antigenic target comprises a target for a functional autoantibody associated with an autonomic disorder.
29. A method of identifying a functional autoantibody for a selected antigenic target, the method comprising:
exposing a cell expressing the selected antigenic target to a candidate antibody, wherein the selected antigenic target has an activity to alter the level of reactive oxygen or nitrogen species in the cell;
determining the ability of the candidate antibody to alter the level of reactive oxygen or nitrogen species in the cell; and
identifying the candidate antibody as a functional autoantibody to the selected antigenic target.
30. The method according to claim 29, wherein the functional antibody is a functional autoantibody associated with type 1 diabetes.
31. The method according to claim 29, wherein the functional antibody is a functional autoantibody associated with an autonomic disorder.
32. A method of identifying an agent that modulates the binding of a functional autoantibody to an antigenic target, the antigenic target having an activity to alter the level of reactive oxygen or nitrogen species in a cell, the method comprising:
exposing a cell expressing the antigenic target to the functional autoantibody and a candidate agent;
determining the ability of the candidate agent to modulate the change in reactive oxygen or nitrogen species in the cell caused by the functional antibody ; and
identifying the candidate agent as an agent that modulates the binding of the functional autoantibody to antigenic target.
33. The method according to claim 32, wherein the functional antibody is a functional autoantibody associated with type 1 diabetes.
34. The method according to claim 32, wherein the functional antibody is a functional autoantibody associated with an autonomic disorder.
35. The method according to any one of claims 32 to 34, wherein the method is used to identify a therapeutic candidate for a disease, condition or state, or to identify an agent for use in a method for detecting the autoantibody.
36. An agent identified according to claim 35.
38. A method of detecting a functional autoantibody, the method comprising using an agent according to claim 36 to detecting the functional autoantibody.
39. A method of identifying a therapeutic agent for a disease, condition or state associated with binding of a functional autoantibody to an antigenic target, the antigenic target having an activity to alter the level of reactive oxygen or nitrogen species in a cell, the method comprising:
exposing a cell expressing the antigenic target to the functional autoantibody and a candidate agent;
determining the ability of the candidate agent to modulate the change in reactive oxygen or nitrogen species in the cell caused by the functional antibody ; and
identifying the candidate agent as a therapeutic agent for the disease, condition or state associated with binding of a functional autoantibody to an antigenic target.
40. The method according to claim 39, wherein the disease, condition or state is type 1 diabetes.
41. The method according to claim 39, wherein the disease, condition or state is an autonomic disorder.
42. The method according to claim 41, wherein the autonomic disorder comprises a gastrointestinal or bladder autonomic neuropathy, or cardiovascular autonomic complications.
43. A kit for performing a method according to any one of claims 1 to 25.
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Citations (5)

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WO2007013124A2 (en) * 2005-07-28 2007-02-01 Vittorio Enrico Avvedimento Stimulatory auto-antibodies to the pdgf receptor as pathology marker and therapeutic target
WO2012013813A1 (en) * 2010-07-30 2012-02-02 Gianluca Moroncini Epitopes of the human pdgf receptor able to bind human auto-antibodies, antibodies and uses thereof
WO2016146979A1 (en) * 2015-03-13 2016-09-22 Queen Mary University Of London Antibodies against modified insulin for use in treatment of and assays for diabetes
EP3109257A1 (en) * 2015-06-26 2016-12-28 Roberto Paterno' Diagnosis and therapy of multiple sclerosis

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004100951A1 (en) * 2003-05-13 2004-11-25 Flinders Medical Centre Diabetic neuropathy diagnostic and therapeutic
WO2007013124A2 (en) * 2005-07-28 2007-02-01 Vittorio Enrico Avvedimento Stimulatory auto-antibodies to the pdgf receptor as pathology marker and therapeutic target
WO2012013813A1 (en) * 2010-07-30 2012-02-02 Gianluca Moroncini Epitopes of the human pdgf receptor able to bind human auto-antibodies, antibodies and uses thereof
WO2016146979A1 (en) * 2015-03-13 2016-09-22 Queen Mary University Of London Antibodies against modified insulin for use in treatment of and assays for diabetes
EP3109257A1 (en) * 2015-06-26 2016-12-28 Roberto Paterno' Diagnosis and therapy of multiple sclerosis

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