Affinities of human NMDA receptor autoantibodies: implications for disease mechanisms and clinical diagnostics

Anti-N-methyl-d-aspartate receptor (NMDAR) encephalitis is a common autoimmune encephalitis presenting with psychosis, dyskinesias, autonomic dysfunction and seizures. The underlying autoantibodies against the NR1 subunit are directly pathogenic by disrupting synaptic NMDAR currents. However, antibody titers correlate only partially with the clinical outcome, suggesting the relevance of other factors such as antibody affinity. We thus determined the binding curves of human monoclonal autoantibodies and patients’ cerebrospinal fluid (CSF) against NR1-expressing HEK293 cells using flow cytometry. Antibody affinity was highly variable with binding constants (half-maximal concentration, c₅₀) ranging from 1 to 74 µg/ml for monoclonal antibodies. Comparing values of individual monoclonal antibodies with human CSF samples suggested that the CSF signal is predominantly represented by higher-affinity antibodies, potentially in a concentration range of NR1 antibodies between 0.1 and 5 µg/ml, roughly reflecting 1–10% of total CSF IgG in NMDAR encephalitis. Binding curves further depended on the CSF composition which must be considered when interpreting established clinical routine assays. Normalization of measurements using reference samples allowed high reproducibility. Accurate and reproducible measurement of NR1 antibody binding suggested that biophysical properties of the antibody might contribute to disease severity. Normalization of the data can be an elegant way to allow comparable inter-laboratory quantification of CSF NR1 antibody titers in autoimmune encephalitis patients, a prerequisite for use as surrogate markers in clinical trials. Based on our calculations, low-affinity antibodies can easily remain undetected in routine cell-based assays, indicating that their relation to clinical symptoms should be analyzed in future studies.