Delfia enhancement buffer

Gradient fractions were diluted in 0.5 M GuHCl and coated on to Nunc 96-well polisorp plates (ThermoFisher Scientific, 475094) overnight at 4°C. The plates were washed 3 times with DELFIA washing solution (5 mM Tris-HCl, 0.15 M NaCl, 0.005% Tween 20, 0.01% NaN₃, pH 7.75). Periodate-oxidation was performed for 20 min with 25 mM sodium metaperiodate in 0.1 M sodium acetate buffer (pH 5.5). The plates were washed again 3 times with DELFIA washing solution and one time with PBS/0.05% Tween, and the wells were blocked for 1h with DELFIA blocking solution (50 mM Tris-HCl, 0.15 M NaCl, 90 µM CaCl₂, 4 µM EDTA, 0.02% NaN₃, and 0.1% BSA) at 22–24°C. After discarding the blocking buffer, the plates were incubated for 1 hour with 2.5 µM biotin-hydrazide solution in 0.1 M sodium acetate buffer (pH 5.5) at 22–24°C. The plates were washed again 3 times and then incubated for 1 hour at 22–24°C with europium labelled Streptavidin (PerkinElmer, 1244–360) 1:1,000 in DELFIA assay buffer (PerkinElmer, 1244-111). This was followed by washing the plates six times with DELFIA washing solution and incubated for 5 min on a shaker with DELFIA enhancement buffer (PerkinElmer, 1244-114). The fluorescence was measured using a Wallac 1420 VICTOR2 plate reader with Europium label protocol (PerkinElmer, Waltham, MA, USA).

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).