AQP4 independent staining of false NMO-IgG positive sera.

<p>WT (A) and AQP4 knockout (B) brain sections were stained with a serum showing NMO IgG-like staining at low magnification. Inset (A) shows that the serum staining (green) is restricted to endothelial cells of the brain capillaries and not to astrocyte processes stained red by AQP4 antibodies. (B) Staining of AQP4−/− mouse endothelial cells of the serum under analysis. Magnification bar 10 µm.</p

Follow-up of NMO patient sera analyzed using a commercially available NMO-IgG kit and the HSS-CBA test.

<p>Note the difference between NMO-IgG evaluated using the test on monkey cerebellum or the HSS-CBA test. 9/15 samples were negative with the cerebellum-based test, while all the samples were positive on cells. Note the good correlation between the cell based test and Cascade Plasma Filtration therapy (CPFT) treatment. Serum titer was measured by limiting dilution.</p>*<p>Yes: exacerbation phase of the disease; No: inactivity phase of the disease.</p>**<p>Samples 7–14 were collected during a 15 day cycle of CPFT (one therapeutic session every other day) planned because of a severe exacerbation. Serum samples were collected before and after each CPFT session.</p

NMO-IgG binding on AQP4-M23 with C- or N- terminal fluorescent tag.

<p>NMO-IgG binding on AQP4-M23 with C- or N- terminal fluorescent tag.</p

Summary of the results obtained in terms of sensitivity and specificity for testing sera by immunofluorescence on cells transfected with AQP4-M23 or AQP4-M1 (AQP4-M1>M23) or AQP4-M1 (M1M23I).

<p>Note that the sensitivity is 97.5% and specificity is 100%.</p

NMO-IgG detection using the monkey cerebellum based IF test.

<p>Note the very low sensitivity and specificity of this test.</p

Correlation between AQP4-M1 CDS cloning strategy, LS frequency, the AQP4-M1/M23 ratio and NMO-IgG binding.

<p>(A): Immunoblot experiment performed using commercial AQP4 antibodies on cells transfected with different AQP4 expressing constructs. From the left: lane 1, AQP4-M23 exclusively producing AQP4-M23, lane 2, AQP4-M1M23I (with the methionine in position 23 mutated into Isoleucin), exclusively producing AQP4-M1, lane 3–6: the four constructs under analysis with G or C or T or A, in the N⁻³position, as indicated. On the right: densitometric analysis of the AQP4-M1 and AQP4-M23 bands reported as the AQP4-M1/M23 ratio (n = 4), *P<0.01, (A⁻³/G⁻³) vs (C⁻³/T⁻³). (B): Illustration showing the cap-dependent translation and relative LS frequency on AQP4-M1 mRNA depending on TIS. (C) Left: immunofluorescence experiment, using AQP4 commercial antibody, performed on AQP4-M1 with TIS (G)⁻³gc<u>AUG</u>A⁺⁴, and AQP4-M1 with TIS (C)⁻³gc<u>AUG</u>A⁺⁴, transfected cells. Right: quantitative analysis of the fluorescence signal obtained by TIRF microscopy of transfected cells with vectors having a purine (A⁻³/G⁻³) or a pyrimidine (C⁻³/T⁻³) at the N⁻³, (n = 4). (D) Left: immunofluorescence experiment performed on transiently transfected cells using NMO sera (collection of 20 NMO sera). The level of NMO-IgG binding is very low in cells transfected with AQP4-M1 with TIS (G)⁻³gc<u>AUG</u>A⁺⁴, while it is high in cells transfected with AQP4-M1 with TIS (C)⁻³gc<u>AUG</u>A⁺⁴. Right: quantitative analysis of NMO-IgG binding on cells transfected with vectors having a purine (A⁻³/G⁻³) or a pyrimidine (C⁻³/T⁻³) at the N⁻³, analyzed by immunofluorescence (n = 4). *p<0.01. Magnification bar 10 µm.</p

Data are expressed as mean±SEM. *p<0.01, AQP4 KO vs WT.

<p>Data are expressed as mean±SEM. *p<0.01, AQP4 KO vs WT.</p