Figure 4.

RhuMab SSM5 targets the same restricted epitope of the GluN1 subunit of the NMDAR as patient's CSF antibodies. (A–C) Patient‐derived rhuMab shows the same staining pattern as CSF from a patient with anti‐NMDAR encephalitis in immunohistochemistry with HEK cells expressing full‐length or mutant GluN1/GluN2B. Immunofluorescence with HEK293 cells expressing full‐length GluN1/GluN2B (A) or mutant GluN1 with full‐length GluN2B (B, C) and stained for human IgG (green) and with a commercial GluN1‐specific antibody (red); nuclear counterstaining with DAPI (blue). Cells were treated with human CSF (diluted 1:16), patient‐derived rhuMab (SSM5, 4.3 μg/mL) or control rhuMab (isotype, rhuMab 12D7, 4.3 μg/mL). The epitope‐disrupting G369I mutant (B) does not show staining by patients' CSF or rhuMab, while a mutant with less epitope disruption G369S (C) does not affect staining. Scale bar represents 10 μm. (D) Confirmation of antigen specificity of patient‐derived rhuMab by immunoprecipitation (IP). Proteins precipitated from brain membrane lysates were separated by PAGE electrophoresis and blotted onto PVDF membranes. Western blot detection was performed with a commercial GluN1‐specific antibody. A band of around 120 kDa corresponding to the GluN1 subunit of the NMDA receptor was identified using SSM5 rhuMab (5 μg, covalently coupled to Dynabeads) in the membrane fraction of brain tissue. Commercially available antibodies to the respective target antigen were used as positive control in IPs and in western blot detections. A rhuMab specific for an irrelevant target (12D7) was included as a negative control (NC) for IP experiments.