Streptavidin peroxidase

LAD-2 cells (National Institute of Allergy and Infectious Diseases, Bethesda, MD, USA) were maintained in StemPro-34 medium (Life Technologies, Grand Island, NY, USA) supplemented with 2 mM l-glutamine, 100 U/mL penicillin, 50 μg/mL streptomycin, and 100 ng/mL recombinant human stem cell factor (R&D Systems). LAD-2 cells were sensitized using 100 ng/mL biotinylated-IgE (BioPorto Diagnostics, Hellerup, Denmark) with or without 5 µg/mL EETs. Cells were stimulated with 100 ng/mL streptavidin peroxidase (Sigma-Aldrich) in Tyrode’s buffer containing 0.1% BSA. Total β-hexosaminidase was obtained via lysing of LAD-2 cells in 0.1% Triton X-100 in PBS. The supernatants were collected and incubated with an equal volume of 4 mM p-nitrophenyl N-acetyl-β-d-glucosamide (Sigma-Aldrich) in citrate buffer for 1 h. The reactions were stopped via the addition of 0.4 M glycine buffer, and signals were read at a wavelength of 405 nm. Levels of TNF-α and MCP-1 (R&D Systems) were measured using ELISA kits according to the manufacturer’s protocol.

Levels of T. gondii-specific total IgG, IgG1, and IgG2a antibodies were measured by ELISA. High-affinity microtiter plates were coated with Stag (10 μg/ml), washed with PBS plus 0.05% Tween 20 (PBS-T) and blocked with 5% skim milk in PBS-T for 1 h at room temperature. Serum samples were diluted 1:25 in 1% skim milk-PBS-T and incubated for 1 h (for IgG detection) or 2 h (for IgG1 and IgG2a detection) at 37°C. After washing, peroxidase-labeled goat anti-mouse IgG (1:1,000; Sigma Chemical Co., St Louis, MO) or biotin-labeled goat anti-mouse IgG1 (1:4,000) or anti-mouse IgG2a (1:2,000) antibodies (Caltag Lab. Inc., South San Francisco, CA) were added and incubated for 1 h at 37°C. Next, streptavidin-peroxidase (1:1,000; Sigma) was added for IgG1 and IgG2a detection assays. The assays were developed with 0.01 M 2,2-azino-bis-3-ethyl-benzthiazoline sulfonic acid (ABTS; Sigma) and 0.03% H₂O₂. Optical density (OD) values were determined in a plate reader (M2e, Molecular Devices) at 405 nm. Results were expressed in ELISA index (EI) to the formula: EI = OD sample/OD cut off, where cut off was calculated as the mean OD for negative control sera plus three standard deviations.

Levels of T. gondii-specific total IgG, IgG1 and IgG2a antibodies were measured by ELISA as described elsewhere [18 (link)], with modifications. Samples were collected at 10, 20 and 30 dpi, and the sera stored at −20°C for further analysis. High-affinity microtiter plates were coated with soluble tachyzoite antigens (STAg, 10 μg/ml), washed with PBS plus 0.05% Tween 20 (PBS-T) and blocked with 5% skim milk in PBS-T for 1 h at room temperature. Serum samples were diluted 1:25 in 1% skim milk-PBS-T and incubated for 1 h (for IgG detection) or 2 h (for IgG1 and IgG2a detection) at 37°C. After washing, peroxidase-labeled goat anti-mouse IgG (1:1000; Sigma Chemical Co., St Louis, MO) or biotin-labeled goat anti-mouse IgG1 (1:4000) or anti-mouse IgG2a (1:2000) antibodies (Caltag Lab. Inc., South San Francisco, CA) were added and incubated for 1 h at 37°C. Next, streptavidin-peroxidase (1:1000; Sigma) was added for IgG1 and IgG2a detection assays. The assays were developed with 0.01 M 2,2-azino-bis-3-ethyl-benzthiazoline sulfonic acid (ABTS; Sigma) and 0.03% H₂O₂. Optical density (OD) values were determined in a plate reader at 405 nm. Results were expressed in ELISA index (EI), according to the formula: EI = OD sample/OD cut off, where cut off was calculated as the mean OD for negative control sera plus three standard deviations.