Multiskan go elisa plate reader

Wells selected for testing were incubated with 50 uL/well of blocking buffer (Thermo Fisher Scientific, MA, USA) for 30 min at 37 °C. After discarding the buffer, 100 µL/well of serum samples and positive and negative controls previously diluted 1 in 100 in blocking buffer were incubated on the plates at 37 °C for 90 min. Then, the wells were washed 3 × 250 µL/well with 1× PBS plus 0.05% Tween 20 (PBS-T) and incubated for 45 min at room temperature with 100 uL/well of secondary antibody (Merck KGaA, Darmstadt, Germany) previously diluted 1 in 30,000 in blocking buffer. The wells were washed with PBS-T as described above, and 100 µL/well of TMB substrate (Mabtech, Nacka Strand, Sweden) were added. After 12 min in the dark, 100 µL/well of 1 M phosphoric acid (Merck KGaA, Darmstadt, Germany) was added to stop the reaction. Plates were read at 450 nm with the Multiskan Go ELISA plate reader (Thermo Fisher Scientific, MA, USA). The absorbance was calculated by subtracting the optical density (OD) measured on wells without sera from the values obtained from the sera-containing wells for each sample.

The mouse serum and HaCaT cell culture media of each group were further analysed to determine the concentrations of interferon gamma‐induced protein 10 (CXCL10) and IFN‐β using ELISA kits, according to the manufacturer's instructions (Elabscience) (Mouse IFN‐β ELISA Kit: E‐EL‐M0033c, Mouse CXCL10 ELISA Kit: E‐EL‐M0021c, Human CXCL10 ELISA Kit: E‐EL‐H0050c). Absorbance was measured at 450 nm using an Multiskan™ GO ELISA plate reader (Thermo Fisher Scientific) and the relative protein concentrations were calculated.

Soluble S. mansoni adult worm antigens (SoSmAWA) were prepared for ELISA (55 (link)). Twenty adult worms per milliliter were suspended in sterile PBS with a protease inhibitor cocktail (Complete Mini EDTA-Free, Roche 04 693 159 001). The mixture was homogenized, frozen and thawed, sonicated, and then centrifuged at 30,000 g for 30 min at 4°C. Supernatant protein concentration was determined using a Micro BCA Protein Assay Kit. Blood samples were collected from mice before immunization and infection, and at the necropsy, and analyzed by indirect ELISA to detect specific IgG, IgG1, and IgG2a antibodies anti-SmKT, anti-SmKB, and anti-SoSmAWA. A Corning Costar 96-well microplate (Cambridge, MA) was coated with 1 μg/ml of each peptide and SoSmAWA. The plates were then blocked with 2% of bovine serum albumin (Sigma) in PBS with 0.05% Tween 20 (PBST) for 1 h at 37°C. Sera samples diluted at 1:100 in PBST were added in duplicate wells and incubated for 1 h at 37°C. Goat anti-mouse IgG-HRP, IgG1-HRP, or IgG2a-HRP conjugates (Sigma) were used at 1:1000 in PBST and incubated for 1 h at 37°C. The plates were washed and developed adding H₂O₂ (0.012%) and orthophenylenediamine substrate (0.04%) in 0.1 M citrate/phosphate buffer, pH 5.0. The reaction was stopped with 3 N H₂SO₄ and read at 492 nm on a MultiSkan GO ELISA plate reader (Thermo Fisher Scientific, Vantaa).