Fig. 2

Evolution of the AMPA-reactivity profile in mice immunized with different PTM-antigens. A ELISA of the titrated murine sera showing the development of anti-modified protein antibody (AMPA) response to different post-translational modifications of fibrinogen, namely acetylated (AcFib) and carbamylated (CaFib) variants. Individual graphs depict titration ELISA results measured in the end of the experiment in mice groups with different immunization strategies. Medians and interquartile intervals per serum dilution are shown. Immunization experiments were performed twice with similar results. Data from one of the two representative immunization experiments are shown. B Timelines demonstrating median anti-AcFib and anti-CaFib reactivity of the immunized mice per immunization group. Arrows indicate the timepoints at which the mice were immunized (week 0 and week 5). Graphs depict ELISA OD-s measured with sera collected at different timepoints and diluted 1:50 for ELISA; medians and interquartile intervals are shown per timepoint. Data from one of the two representative immunization experiments are shown. p values (asterisk) refer to the change between two immunization groups within one timepoint according to Mann-Whitney U test (*p < 0.05). C Changes in AMPA titers of individual mice within 1 week after the booster (between timepoints 5 and 6), calculated by subtraction of the log-transformed timepoint 5 titer from the timepoint 6 titer; means ± SD are shown per immunization group. Pooled titer data from two immunization experiments are shown. p values (asterisk) refer to the change between two immunization groups according to Mann-Whitney U test (*p < 0.05, **p < 0.01, ***p < 0.001). All experiments were performed twice with similar results. The data obtained from one experiment is shown. D Relative evolution of the anti-CarP and AAPA responses: the titer evolution data were summarized per immunization group by depicting average titers. Due to the exponential nature of titer values, geometric mean was used to determine the average. Pooled titer data from the two immunization experiments are shown, measured with serum collected at week 5, week 6, and week 9. Example of performed calculations is shown in supplementary methods. E Model illustrating multireactive nature of AMPA is shown together with which antibodies are measured in AMPA assays applied in the study