Elisa auto reader

To determine parasite-specific antibody titers in sera an indirect ELISA assay was performed. Briefly, 96-well microtiter plates (Nunc, Denmark) were coated with FhTE (2 μg/well) in 50 mM carbonate buffer (pH 9.6). After blocking with 1% gelatin in PBS, three washes with PBS containing 0.1% Tween-20 were performed. Serially diluted sera in PBS containing 0.1% Tween-20 and 0.5% gelatin were added to the wells for 1 h at 37°C. After three washes, wells were treated 1 h at 37°C using goat anti-mouse IgM, IgA, IgG IgG₁, IgG_2a, IgG_2b, or IgG₃ peroxidase-conjugates (Sigma-Aldrich, United States) followed by o-phenylenediamine (OPD) and H₂O₂ as substrates. Plates were read photometrically at 492 nm in an ELISA auto-reader (Thermo Fisher Scientific). Antibody titers were calculated to be the log₁₀ highest dilution, which gave twice the absorbance of control (mock) mouse sera with the minor dilution. Titers are shown as the arithmetic mean ± SEM.

Semiquantitative crystal violet assay for S. aureus biofilm was performed as previously described (19 (link)). Briefly, S. aureus strains were cultured in BHI at 37°C for 16 h. Then, the culture was 1:1,000 diluted with fresh BHI medium, inoculated into 24-well plates (1 mL per well), and incubated at 37°C for 24 h. After incubation, the culture supernatants were carefully aspirated, and the bacterial cells in each well were washed once with PBS and stained with 1% (mass/vol) crystal violet (Chongqing Boer Biotech Co., China) after drying. After staining for 20 min, the residual crystal violet was washed with slow water, dried, and dissolved in 100 μL of glacial acetic acid. The optical density value at 570 nm (OD₅₇₀) was read by micro-enzyme-linked immunosorbent assay (ELISA) autoreader (Thermo Scientific, USA).