Fig. 2

Characterization of phage clones, phage-displayed peptides, and mimotopes. a-c For different NMO-IgG preparations, different experimental approaches were made to narrow down the number of phage-displayed peptides for further studies. a ELISA with single phage clones to evaluate interactions with the NMO-IgG preparation I. Single phage clones after 3 rounds of negative/positive selection (I-01 - > I-19) and 5 randomly picked phage clones without preceeding selection (L7 - > L11) were tested for their ability to react with the NMO-IgG preparation I, BSA, or control-IgG (Subcuvia). Each sample contained 10⁸ phages. Data represent three different experiments and are shown as mean + SEM (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, detected with one-way ANOVA followed by Sidak’s multiple comparisons test). b ELISA to verify that peptides, but not phage particles bind to the NMO-IgG preparation IV. IV was pre-incubated with specific, random, or no synthesized peptides prior to the incubation with peptide-displaying phages. Bound phages were then detected with horse radish peroxidase-conjugated anti-M13 antibodies and TMB substrate, and the absorbance was measured at 450 nm in an ELISA reader. Data represent triplicates of one experiment and are shown as mean + SEM. c Phage-displayed peptides identified with the NMO-IgG preparation III were mapped onto the 3D structure of human AQP4 (protein data base (pdb) accession number 3GD8) using PepSurf. Extracellular loops are displayed on top of the structure, intracellular parts of the molecule on the bottom of the 3D structure. The final alignment represents the best possible path in a defined surface graph. The probability obtaining the same alignment with a random sequence is given by the corresponding p-value. III-01 (P-value: 0.00017), III-04 (P-value: 0.00106), III-06 (P-value: 0.00007), and III-09 (P-value: 0.00028) mapped at least partially to the extracellular loops of AQP4 (red), while III-10 (P-value: 0.00008) and III-17 (P-value: 0.00047) mapped to intracellular or helical structures, respectively. d Selected peptides were tested for their ability to interfere with the binding of AQP4-reactive antibodies of NMO-IgG preparations to AQP4. Flow cytometry of AQP4 M23-transfected HEK293A cells reacting with NMO-IgG preparations V, VI, and III pre-incubated with the indicated peptides. Pre-incubation without peptide, or with a random peptide (SPRAISSYPLNEGGGS) served as negative controls. The data shown here are the mean values (+/−SEM) of the percentage of NMO-IgG binding after pre-incubation with peptides, obtained from 4 (V and III) or 5 (VI) independently performed experiments. Please note that the binding of the NMO-IgG preparations pre-incubated without peptide or with random peptides slightly differed from each other. Therefore, we referred to the binding of NMO-IgG without peptide as 100% (dashed red line) and always also show the percentage of binding of NMO-IgG pre-incubated with random peptide (solid red line). The lower one of these two different values was used as reference for the percentage of blocking achieved with the different peptides. Statistics was calculated using one-tailed, Welch-corrected t-tests. Blue arrows indicate mimotopes used for immunization