Horseradish peroxidase hrp conjugated goat anti mouse igg

Purified gp350^1-425-His was coated on 96-well microplates (Corning) (100 ng/well in PBS) for 2 h at 37°C. The plates were washed once and then blocked with blocking buffer (PBS pH 7.4, containing 2% gelatin, 0.5% casein and 0.1% ProClin 300) overnight at 4°C. Then, 5-fold serial dilutions of sera were added to the plates and incubated for 1 h at 37°C. The plates were washed 5 times and incubated for 30 min at 37°C with 100 μl of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Promega) (1:20,000 dilution). Signals were developed using EL-TMB kit (Sangon Biotech). Absorbance was measured at 450 nm using a microplate reader (Molecular Devices). The cutoff value was set to 0.1 which was determined by the OD₄₅₀ value of preimmune sera.

At 7, 14, 28 and 35 days after the first immunization, the blood samples from the tail vein of mice were collected, sera were separated and used as the primary antibody for an enzyme-linked immunosorbent assay (ELISA), with MntC as the coating antigen. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG, anti-IgG1, anti-IgG2a, anti-IgG2b and anti-IgG3 (Promega, San Luis Obispo, CA, USA) were used as the secondary antibodies respectively. The substrate was 3,3’,5,5’- tetramethylbenzidine (Sigma-Aldrich), and the stop solution was 2 M H₂SO₄ in this reaction. After terminating, the titres were measured at OD450 using a microplate reader (Bio-Rad, Hercules, CA, USA).