Soluble S. mansoni Antigen ELISA Protocol

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).