Anti mouse igg1

The assessment of total serum IgG levels (anti-Toxocara) was performed by indirect immunoenzymatic assay (ELISA) using TES as the antigen at a concentration of 1 μg/ml and serum dilution of the primary antibody (1:50), as described by Avila et al. (2012) (link). For the secondary serum, horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibody produced in goat (Sigma-Aldrich, St. Louis, Missouri, USA), a 1:5000 dilution in PBS-T, was used. Serum samples were examined individually and in duplicate, and readings were performed at a wavelength of 492 nm. The evaluated isotypes were IgG1, IgG2a, IgG2b and IgG3, following the recommendations of Monoclonal Mouse Antibody Isotyping Reagents (Sigma-Aldrich, St. Louis, Missouri, USA). The concentration of antigen used in sensitization and the dilution of the primary serum were the same as those used for the evaluation of total IgG. Each sample (pool of sera) was examined in triplicate to obtain the mean absorbance. The secondary antibodies anti-mouse IgG1, IgG2a, IgG2b and IgG3 (Sigma-Aldrich, St. Louis, Missouri, USA) produced in goat were diluted 1:2000 in PBS-T and applied to the plates. After, the peroxidase-conjugated anti-goat antibody produced in rabbit (Sigma-Aldrich, St. Louis, Missouri, USA) was diluted 1:4000 in PBS-T was added.

On day 28 after primary immunization, mice were exsanguinated, and serum samples were collected for an enzyme-linked immunosorbent assay (ELISA). Microtiter plate wells (Corning Incorporated, United States) were coated with PA0833_26-237 (200 ng per well) in 0.05 M carbonate buffer (pH 9.5) overnight at 4°C. Diluted serum samples were used as the primary antibodies, and the secondary antibodies were horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG, anti-mouse IgG1, anti-mouse IgG2a or anti-mouse IgG2b (Sigma). The optical density at 450 nm was measured, and the titers were defined as the highest dilution that yielded an absorbance value of more than twice the value of the pre-immune serum.

Mouse serum samples were collected for an enzyme-linked immunosorbent assay (ELISA), which was performed as previously described [23 (link)]. In brief, microtiter plate wells (Corning Incorporated, New York City, NY, USA) were coated with mHI_N2, MntC, mSEB or SpA5 (200 ng per well) in 0.05 M carbonate buffer (pH 9.5) overnight at 4 °C. The primary antibodies were diluted serum samples, and the secondary antibodies were HRP-conjugated goat anti-mouse IgG, anti-mouse IgG1, anti-mouse IgG2a or anti-mouse IgG2b (Sigma). The optical density was measured at 450 nm, and the titres were defined as the highest dilution that yielded an absorbance value of more than twice the value of the pre-immune serum.