Zellbasierte Testverfahren zur Untersuchung von NMDA-Rezeptor-Antikörpern

• Main Author: Leubner, Jonas
• Format: Doctoral or Postdoctoral Thesis
• Language: German
• Published: Charité - Universitätsmedizin Berlin 2017
• Subjects: NMDA-receptor-encephalitis; NMDAR antibodies; 600 Technik; Medizin; angewandte Wissenschaften::610 Medizin und Gesundheit; Ursus maritimus
• Online Access: https://pub.dzne.de/record/144863; https://pub.dzne.de/search?p=id:%22DZNE-2020-00288%22

Discovered in 2007, anti-N-Methyl-D-Aspartat receptor (NMDAR) encephalitis is one of the most commonly identified causes for encephalitis. The disease often runs a severe and rapid course showing reduced state of consciousness, epileptic seizures, autonomic dysregulation and hypopnea leading to intensive care unit treatment and even death. Clinical diagnosis is made by detection of pathogenic NMDAR antibodies from a patient’s cerebrospinal fluid (CSF) or serum. Little is known about prognosis factors for the disease severity, but would be helpful for providing early and appropriate treatment. We suppose the affinity of NMDAR antibodies might be an important predicting factor. We developed highly sensitive methods to detect low concentrations of NMDAR antibodies, which can be used to analyze antibody affinity of different patients in future. Therefore, cDNA of the NR1 subunit of the NMDA receptors was amplified by polymerase chain reaction, cloned into different vectors and purified from Escheria coli for transient and stable transfection. Immunofluorescence staining on transfected HEK-cells detected lower concentrations of human NMDAR antibodies than commercial assays and flow cytometry showed exact quantification of the antibody titer, which can be used to monitor clinical progress and evaluate therapeutic success in follow-up examinations. Further CSF was conjugated with Alexa Fluor 594 to detect human and non-human NMDA receptor antibodies without secondary antibodies. The post mortem CSF analysis of a young polar bear (Ursus maritimus) suffering epileptic seizures showed strong binding to HEK cells expressing NMDA receptors. Tissue immunohistochemistry exploration demonstrated a typical neuropil signal in hippocampus and cerebellum as in human patients with NMDAR encephalitis and histopathological examination showed an encephalitis with infiltration of plasma cells. We conclude, death was caused by NMDAR encephalitis. This is the first reported non-human case and suggests, that other mammals might suffer ...