Peroxidase labeled goat anti mouse igg

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.

Twelve days after each immunization, blood was collected from the tail vein, and sera were analyzed for the presence of antibodies recognizing each recombinant protein. Antibodies were detected by enzyme-linked immunosorbent assay (ELISA), essentially as described in a previous study^{22 (link)}. The recombinant proteins NLP-CSP_CT, NLP-CSP_R^{23 (link)}, yPvCSP-VK210_CT, yPvCSP-VK247_CT, and yPvCSP-P. vivax-like _CT^{22 (link)} were employed as solid phase-bound antigens (200 ng/well). After an overnight incubation at RT, plates were washed with a solution of PBS containing 0.05% Tween-20 (PBS-T) and blocked with a blocking solution (PBS, 5% (w/v) skimmed milk) for 2 h at 37 °C. Serial dilutions of murine polyclonal sera were added to the wells and incubated for 1 h at RT; after washes with PBS-T, peroxidase-labeled goat anti-mouse IgG (Sigma, St. Louis, USA), diluted 1:3000, was added to each well. Reactions were developed with the OPD/acid stop system. Anti-IgG titers were determined based on the highest dilution of sera yielding an A₄₉₂ greater than 0.1.