title: Intracellular and non-neuronal targets of voltage-gated potassium channel complex antibodies.
abstract: Autoantibodies against the extracellular domains of the voltage-gated potassium channel (VGKC) complex proteins, leucine-rich glioma-inactivated 1 (LGI1) and contactin-associated protein-2 (CASPR2), are found in patients with limbic encephalitis, faciobrachial dystonic seizures, Morvan's syndrome and neuromyotonia. However, in routine testing, VGKC complex antibodies without LGI1 or CASPR2 reactivities (double-negative) are more common than LGI1 or CASPR2 specificities. Therefore, the target(s) and clinical associations of double-negative antibodies need to be determined.
  
  
  
  Sera (n=1131) from several clinically defined cohorts were tested for IgG radioimmunoprecipitation of radioiodinated α-dendrotoxin (125I-αDTX)-labelled VGKC complexes from mammalian brain extracts. Positive samples were systematically tested for live hippocampal neuron reactivity, IgG precipitation of125I-αDTX and125I-αDTX-labelled Kv1 subunits, and by cell-based assays which expressed Kv1 subunits, LGI1 and CASPR2.
  
  
  
  VGKC complex antibodies were found in 162 of 1131 (14%) sera. 90 of these (56%) had antibodies targeting the extracellular domains of LGI1 or CASPR2. Of the remaining 72 double-negative sera, 10 (14%) immunoprecipitated125I-αDTX itself, and 27 (38%) bound to solubilised co-expressed Kv1.1/1.2/1.6 subunits and/or Kv1.2 subunits alone, at levels proportionate to VGKC complex antibody levels (r=0.57, p=0.0017). The sera with LGI1 and CASPR2 antibodies immunoprecipitated neither preparation. None of the 27 Kv1-precipitating samples bound live hippocampal neurons or Kv1 extracellular domains, but 16 (59%) bound to permeabilised Kv1-expressing human embryonic kidney 293T cells. These intracellular Kv1 antibodies mainly associated with non-immune disease aetiologies, poor longitudinal clinical-serological correlations and a limited immunotherapy response.
  
  
  
  Double-negative VGKC complex antibodies are often directed against cytosolic epitopes of Kv1 subunits and occasionally against non-mammalian αDTX. These antibodies should no longer be classified as neuronal-surface antibodies. They consequently lack pathogenic potential and do not in themselves support the use of immunotherapies.
authors.affiliation: Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK.
authors.forename: Bethan
authors.initials: B
authors.lastname: Lang
authors.affiliation: Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK.
authors.forename: Mateusz
authors.initials: M
authors.lastname: Makuch
authors.affiliation: Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK.
authors.forename: Teresa
authors.initials: T
authors.lastname: Moloney
authors.affiliation: Institute for Experimental Immunology, Lubeck, Germany.
authors.forename: Inga
authors.initials: I
authors.lastname: Dettmann
authors.affiliation: Institute for Experimental Immunology, Lubeck, Germany.
authors.forename: Swantje
authors.initials: S
authors.lastname: Mindorf
authors.affiliation: Institute for Experimental Immunology, Lubeck, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Probst
authors.affiliation: Institute for Experimental Immunology, Lubeck, Germany.
authors.forename: Winfried
authors.initials: W
authors.lastname: Stoecker
authors.affiliation: Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK.
authors.forename: Camilla
authors.initials: C
authors.lastname: Buckley
authors.affiliation: Department of Psychiatry, University of Oxford, Oxford, UK.
authors.forename: Charles R
authors.initials: CR
authors.lastname: Newton
authors.affiliation: Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK.
authors.forename: M Isabel
authors.initials: MI
authors.lastname: Leite
authors.affiliation: Department of Neurology, Queen's Medical Centre, Nottingham, UK.
authors.forename: Paul
authors.initials: P
authors.lastname: Maddison
authors.affiliation: Institute for Experimental Immunology, Lubeck, Germany.
authors.forename: Lars
authors.initials: L
authors.lastname: Komorowski
authors.affiliation: Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK.
authors.forename: Jane
authors.initials: J
authors.lastname: Adcock
authors.affiliation: Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK.
authors.forename: Angela
authors.initials: A
authors.lastname: Vincent
authors.affiliation: Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK.
authors.forename: Patrick
authors.initials: P
authors.lastname: Waters
authors.affiliation: Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK.
authors.forename: Sarosh R
authors.initials: SR
authors.lastname: Irani
mesh_terms: D001323:Autoantibodies / Q000097:blood*; D020274:Autoimmune Diseases of the Nervous System / Q000276:immunology*; D001921:Brain / Q000276:immunology; D001927:Brain Diseases / Q000175:diagnosis / Q000276:immunology*; D015331:Cohort Studies; D003600:Cytosol / Q000276:immunology; D004546:Elapid Venoms / Q000276:immunology; D000939:Epitopes / Q000276:immunology; D057809:HEK293 Cells / Q000276:immunology; D006624:Hippocampus / Q000276:immunology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D042541:Intracellular Space / Q000276:immunology; D007457:Iodine Radioisotopes; D008565:Membrane Proteins / Q000276:immunology; D009419:Nerve Tissue Proteins / Q000276:immunology; D009468:Neuromuscular Diseases / Q000276:immunology*; D009474:Neurons / Q000276:immunology; D024642:Potassium Channels, Voltage-Gated / Q000276:immunology*; D011506:Proteins / Q000276:immunology; D051637:Shaker Superfamily of Potassium Channels / Q000276:immunology
chemical_list: D001323:Autoantibodies; C403049:CNTNAP2 protein, human; D004546:Elapid Venoms; D000939:Epitopes; D047908:Intracellular Signaling Peptides and Proteins; D007457:Iodine Radioisotopes; C116499:LGI1 protein, human; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D024642:Potassium Channels, Voltage-Gated; D011506:Proteins; D051637:Shaker Superfamily of Potassium Channels; C020273:dendrotoxin