Elisa plates

Haemolytic assays were carried out as previously described [30 (link)]. Briefly, whole blood was washed twice with phosphate-buffered saline (PBS) and resuspended in PBS to obtain 8% (wt/vol.). Blood was used within the first hour. To prepare the supernatants, five isolated colonies of each strain (ST121: Jwt, CEU268, CEU890 and ST96: DL9, CEU852, CEU886) were sown in 5 mL of TSB and kept at 37 °C for 6 h with shaking. All the cultures were centrifuged for 10 min at 3000 RPM and the supernatant was filtered. Then 100 μL of 8% red blood cells (RBC) were mixed with 100 μL of the different dilutions of the culture supernatants (100%, 50%, 25% and 10%) in 1.5 mL tubes, which resulted in 4% of the final RBC. The mixture was incubated at 37 °C for 30 min and centrifuged for 2 min at 7 K RPM in a minispin centrifuge (Eppendorf, Hamburg, Germany). Next 150 μL of supernatants were transferred to ELISA plates (Sigma-Aldrich, Missouri, USA) without disturbing the pellet. OD 450 nm was read to determine the haemolysis capacity of strains ST121 and ST96. For the positive control, RBC were mixed with 20% Triton X-100. For negative control, RBC were mixed with PBS. This protocol was carried out using three strains of each ST type to compare the virulence between strains ST121 and ST96.

The immune response to a T cell‐dependent antigen was studied by intraperitoneal injection of 100 μg of NP‐KLH (Biosearch Technologies) in alum. The frequencies of NP‐specific IgM antibody‐secreting cells (ASCs) were determined in the spleen by enzyme‐linked immunospot (ELISPOT) assay, as described (Smith et al, 1997).
The serum titer of NP‐specific IgM, IgG1, and IgG2b antibodies was determined by enzyme‐linked immunosorbent assay (ELISA; Smith et al, 1997) by using ELISA plates (Sigma‐Aldrich), which were coated with 25 μg/ml of NP₇‐BSA or NP₂₄‐BSA to capture high‐affinity IgG1 or total NP‐specific IgM, IgG1, and IgG2b antibodies, respectively.

Serum samples obtained from immunised mice were serially diluted with PBS containing 0.02% sodium azide and 2% bovine serum albumin (BSA), and then applied to ELISA plates (Sigma-Aldrich) coated with mouse recombinant tumour necrosis factor ligand superfamily member 11 (TNFSF11; 10 μg/mL, R&D Systems). Reactivity of serum antibodies to the target protein was determined using an HRP-conjugated goat anti-mouse IgG secondary antibody (Thermo Fisher Scientific) at a dilution of 1/1000 in PBS/0.02% sodium azide/2% BSA. After development with 1,2- phenylenediamine dihydrochloride (0.4 mg/mL in 0.066 M disodium phosphate, 0.035 M citric acid and 0.01% hydrogen peroxide), absorbance was measured in an ELISA plate reader at 450 nm.