Patients

The index patient was diagnosed and treated at the Asklepios Klinik St. Georg, Hamburg, Germany. He gave written informed consent for evaluation of his medical records.

Control groups included 38 patients with therapy-naïve relapsing-remitting multiple sclerosis who fulfilled the revised McDonald criteria (1) and of who 19 presented with transverse myelitis, 33 patients with neurological symptoms and defined anti-neural autoantibodies (5x anti-NMDAR, 5x anti-Hu, 2x anti-Hu/anti-Ri, 3x anti-Yo, 2x anti-Yo/anti-Ri, 3x anti-Ri, 5x anti-AQP4, 5x anti-LGI1, 3x anti-CASPR2), 52 patients with stiff person syndrome or progressive encephalomyeltis with rigidity and myoclonus with antibodies against glutamic acid decarboxylase (GAD), and 205 normal healthy participants.

Methods

Reagents were obtained from Merck, Darmstadt, Germany or Sigma-Aldrich, Heidelberg, Germany if not specified otherwise.

Indirect immunofluorescence assay (IFA)

IFA was conducted as in Scharf et al. (2): briefly, slides with a biochip array of brain tissue cryosections (hippocampus of rat, cerebellum of rat, monkey and pig) combined with 28 recombinant HEK293 cells separately expressing brain antigens were used in conjunction with a biochip mosaic containing frozen sections of 30 different human, monkey and rat organs and tissues. Live-cell IFA with primary neurons was conducted as described by Dalmau et al. (3). In some experiments, a polyclonal rabbit antibody against GluRd2 (Sigma-Aldrich, dilution 1:500) was used in the first step followed by incubation with anti-rabbit IgG-Cy3 (Jackson Research, Suffolk, United Kingdom).

Histo-immunoprecipitation (HIP) and identification of the antigen

HIP, SDS-PAGE, MALDI-TOF analysis and Western blotting were conducted as in Scharf et al. (2). Some Western blots were incubated with the polyclonal rabbit antibody against GluRd2 (dilution 1:2000) in the first step followed by an incubation with anti-rabbit-IgG-alkaline phosphatase (Jackson Research) in the second step.

Cloning and expression of GluRd2 in HEK293

The cDNA for human GluRd2 (UNIPROT Acc. # O43424) was obtained by performing PCR on a commercially available cDNA (Source Biosciences, Germany, Genbank BC099652) with primers ATA GAA GAC GGC ATG GAA GTT TTC CCC TTC CTC TTG GTT TTG TCC GTC and TAT GAA GAC GCT CGA TTA TGG AGG TGC CTC GGT C. The amplification product was digested with BbsI and ligated with pTriEx-1 (Merck Biosciences, Germany). Expression of GluRd2 in HEK293, preparation of substrates for RC-IFA and generation of cell lysates were done as in Scharf et al. (2). For live-cell IFA with transfected HEK293 cells, they were seeded in Lab-Tek II Chamber Slides (ThermoFisher Scientific, Schwerte, Germany) and transfected identically.

Reference List

(1) Polman CH, Reingold SC, Banwell B, et al. Diagnostic criteria for multiple sclerosis: 2010 revisions to the McDonald criteria. Ann Neurol 2011 Feb;69:292-302.

(2) Scharf M, Miske R, Heidenreich F, et al. Neuronal Na+/K+ ATPase is an autoantibody target in paraneoplastic neurologic syndrome. Neurology 2015 Mar 25;84:1673-1684.

(3) Dalmau J, Gleichman AJ, Hughes EG, et al. Anti-NMDA-receptor encephalitis: case series and analysis of the effects of antibodies. Lancet Neurol 2008 Oct 11.

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