Tmb substrate set

To assess the MPO peroxidation activity, reaction was developed by adding the TMB substrate set (BioLegend) to the collected supernatants. The reaction was stopped by adding 25% sulfuric acid (PanReac AppliChem). The optical densities (ODs) were read at 450 nm with a reference at 620 nm using the SUNRISE microplate reader (Tecan). To assess the MPO chlorination activity, we used the EnzChek™ Myeloperoxidase (MPO) Activity Assay Kit (ThermoFisher Scientific) according to manufacturer´s instructions. The fluorescence intensity was read in an Infinite F200 PRO plate reader (Tecan; ex. 485 nm, em. 530 nm). We used RIPA buffer (ThermoFisher Scientific) for complete cell lysis representing maximum MPO activity in the cell supernatant.

For the hamster model, each well of Nunc MaxiSorp ELISA Plates (Thermo Fisher Scientific, MA, USA) was coated with LigAc (500 ng), whole-cell lysate of L. interrogans serovar Pomona (1 × 10⁶ cells), or BSA (500 ng) in coating buffer (0.1 M Na₂CO₃, 0.1 M NaHCO₃, pH 9.5) at 4°C overnight. The coated wells were blocked with blocking buffer at 37°C for 1 h before incubation with serially diluted hamster sera (five-fold dilution of 1:500 to 1:312,500). The plates were incubated with either 1:5000 horseradish peroxidase (HRP)-labeled goat anti-hamster IgG antibody (KPL), mouse anti-hamster IgG1, IgG2/3, or IgG3 antibodies (Southern Biotech, Birmingham, AL, USA). All incubation steps were performed at 37°C for 1 h followed by three times washing with PBST. The reactivity of sera to antigens was detected using TMB Substrate Set (BioLegend, San Diego, CA, USA) and stopped with 2 M H₂SO₄. The absorbance was measured at 450 nm by the Varioskan Flash Spectral Scanning Multimode Reader (Thermo Scientific). Antibody titers were calculated using the midpoint titer from S-curve method with raw OD values (Supplementary File). For the mouse model, sera were tested for LigAc-specific antibodies using ELISA plates coated with LigAc (170 ng per well). Mouse serum IgG was detected using a goat anti-mouse IgG coupled to HRP (Southern Biotech).

96-well Immulon “U” Bottom plates were coated with 1 μg ml⁻¹ of ferritin proteins in Dulbecco’s Phosphate buffered saline, pH 7.4 (PBS). Plates were incubated at 4 °C overnight. After 30 min of blocking with blocking buffer (Dulbecco’s PBS containing 0.2% bovine serum albumin, pH 7.4), at RT, the plates were washed 3x with wash buffer (Dulbecco’s PBS containing 0.05% Tween 20, pH 7.4). Antibodies were serially diluted 5-fold in sample buffer (Dulbecco’s PBS containing 0.2% bovine serum albumin and 0.05% Tween 20, pH 7.4), or at a single concentration as indicated, and added to duplicate wells. The plates were incubated for 1 h at RT. The plates were then washed 4 times with wash buffer. Horseradish peroxidase (HRP)-conjugated goat anti-human IgG, gamma chain specific antibody (Sigma) was added and incubated at RT for 30 min, followed by 4 washes with wash buffer and one wash with PBS. For development, the substrate mixture from TMB Substrate set (Biolegend) was added and incubated for 10 min, before the addition of the Stop Solution for TMB Substrate (Biolegend). Absorbance (A) was measured at 450 nm or 650 nm as indicated, using an ELISA reader iD3 (Molecular Devices, San Jose, CA).

IL-10 levels in cell culture supernatants were measured using the ELISA MAX™ Standard Set Human IL-10 (BioLegend) or Human IL-10 ELISA Set (Diaclone), according to the manufacturer’s instructions, using ELISA Coating Buffer (BioLegend). IL-13 in cell culture supernatants was measured using the Human IL-13 ELISA Development Kit (HRP) (Mabtech) or Human IL-13 DuoSet ELISA (R&D Systems) according to the manufacturer’s instructions. For IL-17A measurement Human IL-17A ELISA Development Kit (HRP) (Mabtech) was used, according to the manufacturer’s instructions. Nunc-Immuno™ MicroWell™ 96 well plates (Sigma-Aldrich) were used. Plates were developed using the TMB Substrate Set (BioLegend), with H₂SO₄ added to stop the reaction. Absorbance was read at 450 nm and 570 nm wavelengths using an Epoch 2 Microplate Spectrophotometer (BioTek) or an Asys UVM340 microplate spectrophotometer (Biochrom). The absorbance at 570 nm was subtracted from that at 450 nm, and the concentrations were calculated based on standard curve equations.