Figure 5.

The human IgG extracted from brain of mice infused with patients’ CSF is specific for NMDARs. (A and B) HEK293 cells expressing the GluN1 subunit of the NMDAR immunolabelled with acid-extracted IgG fractions (top row in A) or pre-extraction fractions (top row in B) from hippocampus of mice infused with patients’ CSF and sacrificed on the indicated days. The maximal reactivity with GluN1-expressing cells was noted in acid-extracted IgG fractions from Days 13 and 18 (A); none of the pre-extraction fractions showed GluN1 reactivity (B) indicating that the reactivity of acid-extracted fractions corresponds to IgG antibodies that were bound to brain NMDAR receptors. The second row in A and B shows the reactivity with a monoclonal GluN1 antibody, and the third row the colocalization of immunolabelling. Scale bars = 10 µm. (C) Quantification of NMDAR antibody titre in IgG-extracted fractions from hippocampus of animals treated with patients’ CSF (n = 5 mice per each time point, except four mice for Day 5). Solid line = median. Significance was tested by Kruskal-Wallis with an α-error of 0.05 (asterisks) and post hoc testing with Dunn’s test ($). **^{, $$}P < 0.01, ***^{, $$$}P < 0.001. See Supplementary Table 2 for detailed statistics. (D and E) HEK293 cells expressing the GluN1 subunit of the NMDAR immunolabelled with acid-extracted IgG fractions (D) and pre-extraction fractions (E) from hippocampus (Hippo), cerebral cortex (Ctx) and cerebellum (Cb) of mice infused with patients’ CSF (Day 18). The acid-extracted IgG fraction from hippocampus showed higher level of NMDAR antibodies than those extracted from cerebral cortex (Ctx) and cerebellum (Cb). Scale bars = 10 µm. n.s = not significant.