Tmb substrate

Edmonston strain-derived MV antigens (Jena Bioscience) or recombinant S protein (ABIN6952426, Antibodies Online) were coated on NUNC MAXISORP 96-well immuno-plates (Thermo Fisher) at 1 µg/ml. Coated plates were incubated overnight at 4 °C. After washing and blocking, sera from immunized mice were serially diluted in the binding buffer and incubated on plates for 1 h at 37 °C. After washing steps, an HRP-conjugated goat anti-mouse IgG (H + L) antibody (Jackson ImmunoResearch, 115-035-146, 1:5000 dilution) was added for 1 h at 37 °C. Antibody binding was detected by the addition of the TMB substrate (Eurobio) and the reaction was stopped with 100 µl of 30% H₂SO₄. The optical densities were recorded at 450 and 620 nm wavelengths using the EnSpire 2300 Multilabel Plate Reader (Perkin Elmer). Endpoint titers for each individual serum sample were calculated as the reciprocal of the last dilution giving twice the absorbance of the negative control sera. Isotype determination of the antibody responses was performed using HRP-conjugated isotype-specific (IgG1 or IgG2a) goat anti-mouse antibodies (AB97240 and AB97245, Abcam, 1:5000).

MV antigen (Edmonston strain, #PR-BA102-S-L, Jena Bioscience) at 1 µg/ml in PBS, and CSPb or CSPf recombinant proteins (produced at the Recombinant Protein and Antibodies Production Core Facility of the Institut Pasteur by J. Bellalou and V. Bondet, using the BioPod F800 microfermentor battery) at 1 µg/ml in carbonate buffer were coated overnight at 4 °C onto 96-well plates (#439454, Thermo Scientific) and then blocked for 1 h at 37 °C with a saturation buffer (PBS, 0.05% Tween, 3% BSA). Sera samples from immunized mice were serially diluted (PBS, 0.05% Tween, 1% BSA) and incubated on plates for 1 h at 37 °C. After washing steps (0.05% Tween in PBS), a secondary HRP-conjugated goat anti-mouse Ig antibody (#115-035-146, Jackson ImmunoResearch) was added at a dilution of 1/1000 for 1 h at 37 °C. Antibody binding was revealed by addition of the TMB substrate (#5120-0047, Eurobio) and the reaction was stopped by addition of H₂SO₄ 1 M. The optical densities (O.D.) were recorded at 450 nm. The endpoint titers for each individual serum were calculated as the reciprocal of the last dilution giving twice the absorbance of negative control sera.