Nunc immuno maxisorp 96 well plate

Nunc-Immuno Maxisorp 96-well plates (Thermo Scientific) were coated with U1-70K at 2 μg/mL in PBS (autoantigen information is shown in Supplementary Table 1). A BSA-coated plate (10 mg/mL) was used to measure non-specific binding. For biotinylated H2B peptides, Streptavidin High Binding Capacity Coated 96-Well Plates (Pierce) were coated with the peptides at 2 μg/mL in PBS. The plates were washed 5 times in wash buffer (PBS with 0.05% Tween-20), before blocking with 10 mg/mL BSA in PBS with 0.05% Tween-20. After washing, patient sera were diluted 1/100 or commercial antibodies were diluted at the indicated concentrations in antibody buffer (10 mg/mL BSA in PBS with 0.05% Tween-20) and incubated overnight at 4 °C. All samples were measured in duplicate wells. After washing, plates were probed with Europium conjugated goat-anti rabbit, rabbit anti-mouse, or mouse anti-human IgG antibodies (Perkin Elmer AD0105, AD0124 and 1244-330) diluted 1/500 in Delfia Assay Buffer (Perkin Elmer). Plates were washed and incubated in Delfia enhancement buffer (Perkin Elmer) for 25 minutes at 37 °C before measuring the time-resolved fluorescence with a Wallac Victor model 1420 Multilabel Counter (Perkin Elmer).

ELISAs were performed against SUMO-amphSH3. Nunc-Immuno MaxiSorp 96-well plates (ThermoScientific) were coated overnight at 4 °C with 100 µL of a 30 nM SUMO-amphSH3 solution in NaHCO₃ (50 mM, pH 9.6). Wells were then blocked with 200 µL of blocking buffer (PBS, 0.5% (wt/vol) biotin-free BSA) for 1 h at room temperature. After removing the blocking buffer, wells were washed four times with PT buffer (PBS with 0.05% tween-20). Biotinylated peptide dilutions were performed in PT buffer and 100 µL of each dilution was added to the ELISA plates and incubated on a rocking shaker for 1 h at room temperature. Following 16 washes with PT, 100 µL of horseradish-peroxidase-conjugated streptavidin (Jackson ImmunoReaserch) diluted at 1:3000 in PT) was added to each well. Samples were incubated on a rocking shaker for 30 min, and were then washed 6 times with PT and 4 times with PBS. Next, 100 µL of 1-Step Ultra (3,3′,5,5′-tetramethylbenzidine) TMB-ELISA was added and the reaction was stopped with 100 µL 1 M H₃PO₄. Absorbance was read at 450 nm on a Multiskan GO plate reader and corrected for absorbance at 800 nm as well as for no-specific binding of the peptides by subtracting the response in absence of SH3 domain. Data from independent run were normalized to the maximum values of the D54-dyn2 peptide and fitted using the One site binding hyperbola formula from GraphPad Prism v7.02.

Lysate-loaded APCs were cultured for 24–72 h. IL-12 levels were
assayed by anti-IL-12 ELISA using Nunc-Immuno Maxisorp 96 well plates
(Thermo Fisher Scientific, Roskilde, Denmark), purified anti-mouse IL-12,
recombinant mouse IL-12 and biotinylated anti-mouse IL-12 (all from
BioLegend).

Sera from mice were collected by tail-bleed prior to boost and challenge. Nunc-Immuno Maxisorp 96-well plates (NUNC) were coated with 100 ng recombinant protein (Pvs25FhCMB or r-Pvs25) overnight. Test sera was incubated for 2 h followed by goat anti-mouse IgG-AP secondary (Sigma), 1:3000. Plates were developed using pNPP and read at OD₄₀₅ until end-point detection set for each antigen. For r-Pvs25 ELISA, control regime titres were subtracted from mean end-point titres. The cut-off for determining end-point titer was the mean OD value for pre-immune sera + three times the standard deviation.

Nunc-Immuno Maxisorp 96-well plates (NUNC) were coated with 100 ng recombinant protein per well (r-Pfs25, described in^{12 (link),17 (link)}). After blocking with 5% skimmed-milk, sera were incubated for 2 hours followed by goat anti-mouse IgG-AP (Sigma), 1:5000. Plates were developed using pNPP and read at OD₄₀₅ until set end-point detection. Control regime titers were subtracted from mean end-point titers. Endpoint titer was defined as the x-axis intercept of the dilution curve at an absorbance value three standard deviations greater than the mean OD of pre-immune sera.