Immunograde 96 well elisa plates

Immunograde 96-well ELISA plates (Midsci) were coated with 100 ng S1 in 50 μL PBS per well overnight at 4°C, and then blocked in 200 μL of 5% non-fat dry milk/PBS for 1 h at 37°C. Serum samples from immunized mice were serially diluted in PBS/0.1% BSA starting at either a dilution of 1:50 or 1:100. Blocking buffer was removed, and diluted sera were added to the wells and incubated for 2 h at 37°C followed by overnight incubation at 4°C. Plates were washed three times with PBST (0.05% Tween20), and alkaline phosphatase conjugated secondary antibodies diluted in PBS/0.1% BSA were added (goat-anti-mouse IgG, IgG1, IgG2b, or IgG2c Jackson Immuno Research Laboratories). Plates were incubated at 37°C for 1h, washed with PBST, and then developed at RT by addition of 100 μL of p-nitrophenyl phosphate (pNPP) substrate (Sigma-Aldrich) per well. Absorbance was measured at 405 nm on a microplate spectrophotometer. Titers were calculated against naïve sera, using a cutoff value defined by the sum of the average absorbance at the lowest dilution and two times the standard deviation.

Immunograde 96-well ELISA plates (Midsci) were coated with 100 ng RBD in 50 μL PBS/well overnight at 4°C, and then blocked in 200 μL of 5% non-fat dry milk/PBS for 1 h at 37°C. Sera from immunized mice were serially diluted in PBS/0.1% BSA. Blocking buffer was removed, and diluted sera were added to the wells and incubated for 2 h at 37°C followed by overnight incubation at 4°C. Plates were washed with PBST (0.05% Tween20), and alkaline phosphatase conjugated secondary antibodies diluted in PBS/0.1% BSA were added (goat-anti-mouse IgG, IgG1, IgG2b, or IgG2c Jackson Immuno Research Laboratories). Plates were incubated at 37°C for 1h, washed with PBST, and then developed at RT by addition of p-nitrophenyl phosphate (pNPP) substrate (Sigma-Aldrich). Absorbance was measured at 405 nm, and titers were determined using a cutoff value defined by the sum of the average absorbance at the lowest dilution of naïve serum and two times the standard deviation.

Immunograde 96-well ELISA plates (Midsci) were coated with 100 ng RBD in 50 μL PBS/well overnight at 4 °C, and then blocked in 200 μL of 5% non-fat dry milk/PBS for 1 h at 37 °C. Sera or BAL from immunized mice were serially diluted in PBS/0.1% BSA. Blocking buffer was removed, and diluted sera were added to the wells and incubated for 2 h at 37 °C followed by overnight incubation at 4 °C. Plates were washed with PBST (0.05% Tween20), and respective alkaline phosphatase conjugated secondary antibodies diluted in PBS/0.1% BSA were added (1:5,000 goat-anti-mouse IgG H + L (cat. 115-055-003), 1:10,000 goat-anti-mouse IgG1 (cat. 115-055-205), 1:5,000 goat-anti-mouse IgG2b (cat.115-055-207), or 1:5,000 goat-anti-mouse IgG2c (at.115-055-208) from Jackson ImmunoResearch Laboratories, or 1:2,000 rabbit-anti-mouse IgA (cat. 610-4506) from Rockland Immunochemicals). Plates were incubated at 37 °C for 1 h, washed with PBST, and then developed at RT by the addition of p-nitrophenyl phosphate (pNPP) substrate (Sigma-Aldrich). Absorbance was measured at 405 nm, and titers were determined using a cutoff value defined by the sum of the average absorbance at the lowest dilution of naïve serum and two times the standard deviation.