Tmb one

An ELISA was used to detect antigen-specific antibody responses in milk from 5 independent S. aureus culture-positive cows (SCC >200,000) and compared with 5 S. aureus culture-negative cows (SCC <200,000). Microtiter plates (Nunc, Rochester, NY) were coated with 10 μg of purified protein (EsxA and IsdC, described above) per well in 1× PBS, blocked with buffer (1% skim milk + 1× PBS), and incubated with 2-fold dilutions of bovine milk, starting at a 1:10 dilution, for 12 h at 4°C. Plates were then washed with PBS-T (1× PBS + 0.05% Tween 20), incubated with horseradish peroxide-conjugated anti-bovine IgG (1:10,000 dilution, A10–102P, as above) in blocking buffer for 1 h at 37°C, washed again, and developed with tetramethylbenzidine (TMB One, Promega, Madison, WI). Antibody titers were calculated after background values (protocol minus samples) were subtracted. Endpoint titers were defined as the reciprocal of the dilution giving an OD of 0.2. For the ovalbumin control, 10 μg of purified chicken ovalbumin (Thermo Fisher Scientific) was coated onto Microtiter plates and assays performed as above. Ovalbumin ELISA endpoint titers were defined as the reciprocal of the dilution, giving an OD of 0.02, and adjusted by a factor of 10 for comparison. The ELISA results are representative of the assay performed independently 3 times.