BACKGROUND: Autoantibodies (Abs) targeting the central nervous system (CNS) can cause various neuropsychiatric autoimmune diseases. The potential response to immunotherapy necessitates the continuous expansion of Ab testing strategies including non-antigen-specific screening assays. This study investigated whether tissue-based screening using unfixed murine CNS sections can help to identify patients with immunotherapy-responsive neuropsychiatric diseases after routine Ab panels yielded negative results. METHODS: This retrospective single-center study screened cerebrospinal fluid (CSF) of 279 patients for immunoglobulin G (IgG) anti-CNS Abs using unfixed mouse brain. Patients had a variety of neuropsychiatric conditions, in which an autoimmune contribution was considered. Previous testing for a panel of established autoantibodies using cell-based assays remained negative. Of 238 patients, paired serum samples were available. RESULTS: A subgroup of 55 patients (20%) showed novel anti-CNS autoantibody patterns in CSF, consisting of anti-myelin (n=13), anti-neuropil (n=14), anti-vessel (n=8), anti-tight junction (n=5), anti-cellular (n=8), and anti-astroglial (n=7) autoantibodies. Thirty-six patients (65%) fulfilled criteria for possible, probable, or definite autoimmune encephalitis or paraneoplastic neurological syndrome. Memory impairment (73%) and psychiatric abnormalities (64%) were the most frequent symptoms. Antibody subtypes were not significantly associated with clinical parameters at this sample size, however, there was a trend towards better response to immunotherapy with antibodies against myelin, neuropil, and neuronal cells, while patients with anti-vessel antibodies did not improve. CNS autoantibodies mainly disappeared parallel to clinical improvement. In 46% of treated patients, physicians would not have started immunotherapy without detection of anti-CNS autoantibodies, and the vast majority of patients stabilized or improved. CONCLUSION: Novel CNS Abs were common in patients with suspected 'seronegative' autoimmune neuropsychiatric disorders. Detection facilitated identification of immunotherapy-responsive cases and enabled treatment initiation without increasing unnecessary treatments. Thus, tissue screening using unfixed mouse brain applied in patients with suspected neuropsychiatric autoimmune diseases parallel to established cell-based assays. Future studies should identify the underlying antigens, demonstrate the pathogenic role in animal models, and implement promising Abs into diagnostic routine panels.