ELISA plates were coated with 10 μg/ml of purified LTA from S. aureus (Invivogen) by incubation for 1 h at 37°C, and blocked by incubation with 5% milk in PBS for 1 h at room temperature. Plasma isolated from fresh blood collected into heparin was diluted 1/20, 1/100 and 1/500 and incubated on the plate for 1 h at room temperature. Anti-LTA antibodies were detected with peroxidase-conjugated goat anti-human IgG antibodies and the TMB microwell peroxidase substrate (50-76-00 KPL). The reaction was stopped by adding H2SO4 (1.8 M). Optical density was determined with an MRX microplate reader (Thermolab Systems).
For IVIgs a starting dilution at 10 mg/ml was made to get closer from human plasma concentration of Igs. Then, OD was assessed at the following dilutions 1/20, 1/100, 1/500, 1/2500, 1/7500, 1/22500.
For IVIgs a starting dilution at 10 mg/ml was made to get closer from human plasma concentration of Igs. Then, OD was assessed at the following dilutions 1/20, 1/100, 1/500, 1/2500, 1/7500, 1/22500.