title: Detection of autoantibody against extracellular epitopes of N-methyl-D-aspartate receptor by cell-based assay.
abstract: The concept of anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis, a severe, potentially lethal, treatment-responsive disorder, mediated by autoantibodies against NMDAR was proposed. Because paraneoplastic anti-NMDAR encephalitis has a better prognosis after tumor resection and immunotherapy, rapid quantitative systems for detecting functional autoantibodies against extracellular epitopes of NMDAR are necessary. To detect autoantibodies recognizing extracellular epitopes of NMDAR, we stably expressed mutant NMDAR that decreases Ca(2+) permeability on a heterologous cell surface without any antagonist. Serum and CSF samples from patients were analysed using the cells expressing mutant NMDAR subunits by immunocytochemistry and on-cell Western analysis using live cells stably expressing mutant NMDAR. Furthermore, we were able to express mutant GluRζ1(NR1, GluN1) subunit of NMDAR alone on the cell surface and obtained direct evidence of the presence of autoantibodies recognizing extracellular epitopes of GluRζ1 and the induction of internalization by autoantibodies in serum and CSF from patients. The specificity of on-cell Western analysis was improved at 37°C. The combination of this rapid quantitative assay using our on-cell western analysis, detailed analysis of extracellular epitopes of NMDAR, and internalization assay of NMDAR will be valuable for the diagnosis, evaluation of clinical treatments, and follow-up of anti-NMDAR encephalitis.
authors.affiliation: Department of Molecular Neuroscience, Graduate School of Innovative Life Science, University of Toyama, Toyama 930-0194, Japan.
authors.forename: Shiho
authors.initials: S
authors.lastname: Takano
authors.forename: Yukitoshi
authors.initials: Y
authors.lastname: Takahashi
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kishi
authors.forename: Yoshiharu
authors.initials: Y
authors.lastname: Taguchi
authors.forename: Shutaro
authors.initials: S
authors.lastname: Takashima
authors.forename: Kortaro
authors.initials: K
authors.lastname: Tanaka
authors.forename: Atsushi
authors.initials: A
authors.lastname: Muraguchi
authors.forename: Hisashi
authors.initials: H
authors.lastname: Mori
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000818:Animals; D060426:Anti-N-Methyl-D-Aspartate Receptor Encephalitis / Q000175:diagnosis / Q000276:immunology; D001323:Autoantibodies / Q000096:biosynthesis / Q000097:blood* / Q000134:cerebrospinal fluid*; D015153:Blotting, Western / Q000379:methods; D016466:CHO Cells; D002648:Child; D002675:Child, Preschool; D006224:Cricetinae; D003412:Cricetulus; D000939:Epitopes / Q000097:blood* / Q000134:cerebrospinal fluid* / Q000276:immunology; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D007223:Infant; D008297:Male; D051379:Mice; D008875:Middle Aged; D016194:Receptors, N-Methyl-D-Aspartate / Q000097:blood / Q000276:immunology*; D055815:Young Adult
chemical_list: D001323:Autoantibodies; D000939:Epitopes; D016194:Receptors, N-Methyl-D-Aspartate