test, Fishers exact test and Chi-square test. magnification using a confocal microscope. It clearly shows the colocalization of membrane-associated NMDAR with serum antibodies of the patient but no colocalization with intracellular NMDAR probably residing within the endoplasmic reticulum (Fig 1B). Internalization of NMDAR in response to antibody binding observed in some but not all cells in the live CBA is 61422-45-5 usually shown in Fig 1C. Fig 1 Immunofluorescence CBA with 61422-45-5 HEK293A cells overexpressing functional NMDAR tagged with green neon protein transiently. With the CBA, in the breakthrough discovery group NMDAR antibodies had been discovered in 7/7 (100%) sufferers with NMDAR encephalitis, 0/37 (0%) neurological handles and 0/32 (0%) healthful handles (Desk 1). Awareness and specificity of the CBA had been 100% (95% self-confidence periods (CI) 59.0C100.0 and 94.8C100.0, respectively). Antibody titers in NMDAR encephalitis sufferers ranged from 1:640 to 1:20,480 (typical 1:1,280) (Fig 2A). Fig 2 NMDAR IgG antibody titers and MFI values in the finding group. For the FACS based assay, gating and analysis strategy for NMDAR-(Em)GFP and CD2-EmGFP conveying cells is usually shown in Fig 3. In the finding group the MFI was significantly higher in NMDAR patients (median 74,938, range 7,681 to 237,432) compared to neurological controls (median -401, range -16,158 to 16,646) and healthy controls (median 1,076, range -6,701 to 16,269; Fig 2B). Using ROC analysis a cut-off MFI value of 20,700 was decided (area under the contour 0.988, p<0.0001). NMDAR antibodies were detected in 6/7 (86%) NMDAR encephalitis 61422-45-5 patients, 0/37 (0%) neurological and 0/32 (0%) healthy controls (Table 1). Therefore, with a specificity of 100% (95% 61422-45-5 CI 94.8C100.0) the FACS based assay had a sensitivity of 86% (95% CI 42.1C99.6). Intra- and inter-assay variations (coefficient of variance) were 6% and 22C25%, respectively. Fig 3 Gating and analysis strategy for NMDAR and CD2 conveying HEK293A cells for FACS based analysis. Detection of NMDAR antibodies in the affirmation group In a next step the CBA was applied to 32 blinded samples of the affirmation group from Barcelona. All 16 patients with NMDAR encephalitis were positive for NMDAR antibodies and all 16 neurological controls were seronegative (Table 1). Antibody titers in NMDAR encephalitis patients ranged from 1:80 to 1:2,560 (median 1:640) (Fig 4A). Thus, the sensitivity and specificity of the CBA of 100% were confirmed in these blinded samples (95% CI 79.4C100.0). Fig 4 NMDAR IgG antibody titers and MFI values in the affirmation group. Similarly, the FACS assay was applied to 32 blinded samples of the affirmation group from Barcelona. 14/16 patients with NMDAR encephalitis (87%) were positive for NMDAR antibodies using the cut-off value decided in the finding group and all 16 neurological controls were seronegative (Table 1). The MFI was significantly higher in NMDAR patients (median 59,085, range 5,784 to 213,910) compared to neurological controls (median -1,239, range -3,751 to 2,169, Fig 4B). Thus, the sensitivity and specificity of the FACS 61422-45-5 assay were equally high in the affirmation group (95% CI 61.7C98.5 and 79.4C100.0, respectively) as in the finding group. Comparison of CBA and FACS The concordance kappa value between CBA and FACS was 0.943 (p<0.0001). 85 samples were seronegative and 20 samples were seropositive with both methods. Three samples were seropositive in the CBA, but seronegative in the FACS assay. Correlation of antibody titers of the CBA with MFI obtained by FACS based analysis was 0.697 (Spearmans ; p<0.0001; Fig 5). LAMA3 antibody Fig 5 Correlation of NMDAR IgG titers and MFI values decided.