Rabbit anti mouse igg1

Levels of RV-specific total IgG, IgG1, and IgG2c in serum samples from immunised and control mice were determined by direct ELISA as described previously [36 (link)]. In brief, maxisorp plates were coated with Rh452 viral antigen diluted in bicarbonate coating buffer (Na₂CO₃, NaHCO₃, in water, pH 9.6) and incubated overnight at room temperature. Non-specific protein binding sites were then blocked with PBS containing 2% skim milk powder for 2 h at room temperature. 50μL of serially diluted serum samples were added to the appropriate wells for 2 h at room temperature, followed by the addition of horseradish peroxidase conjugated goat anti-mouse IgG (Thermo Scientific), rabbit anti-mouse IgG1 (Invitrogen USA), or goat anti-mouse IgG2c (Southern Biotech USA). Plates were then incubated at room temperature for 2 h, and developed using TMB peroxidase substrate in the dark for 30 mins. The reaction was stopped with 2 mol H₂SO₄, and absorbance measured at 450/620 nm using a Microplate ELISA reader (Biotrack II plate reader). End-point titres expressed as the reciprocal of the last dilution where OD value ≥ cut-off value. Cut-off determined as mean + (3 × S.D.) of OD values of samples from control mice.

Serum levels of anti-CII Abs were measured by enzyme-linked immunosorbent assay (ELISA). Briefly, microtiter plates were coated with chicken CII (10 μg/ml) overnight at 4 °C. After washing and blocking, serum samples were added in serial dilutions and incubated for 2 h at room temperature. After four washes, peroxidase-conjugated goat anti-mouse IgG (KPL, Baltimore, MD, USA), rabbit anti-mouse IgG1 (Invitrogen, Carlsbad, CA, USA), IgG2c (Invitrogen), or biotin-conjugated anti-mouse IgM (II/41; eBioscience, San Diego, CA, USA) was added and incubated for 2 h at room temperature. For the anti-mouse IgM, streptavidine–HRP (R&D System, Minneapolis, MN, USA) was added after four washes and incubated for 30 min at room temperature. Ab binding was visualized using TMBS (eBioscience).