Secondary antibody solution

Sera from the immunized mice were collected and pooled on day 56 after immunization. A 96-well ELISA plate (Greiner Microlon High-Binding, clear) was coated with 100 µl/well of  L. tarentolae-derived NoV- VP1 cell lysates. The coated plate was incubated overnight at 4 °C. Next, the plates were washed 4 × 5 min with 200 µl/well of washing buffer (PBS/0.05%Tween20) and blocked for 2 h with 250 µl/well of blocking buffer (3%BSA/PBS/0.05%Tween20) at 37 °C. The plates were washed as previously and serial dilutions of mouse sera (in 3%BSA/PBS/0.05%Tween20) were added to the wells and incubated for 1 h at room temperature. Serial dilutions of rabbit anti-NoV antibodies (Abcam ab92976) (in 3%BSA/PBS/0.05%Tween20) served as a positive control. After incubation plates were washed as previously, and appropriate secondary antibody solution (Jackson Immuno Research) (in 3%BSA/PBS/0.05%Tween20) was used for detection. Finally, following the last plate-washing step (6 × 5 min with 200 µl/well), 100 µl/well of HRP-substrate solution was added (1-Step Turbo TMB-ELISA, Thermo Scientific), the plate was incubated in dark until the blue color developed, and the reaction was stopped by adding 50 µl of 0.5 M sulfuric acid to each well. Signal intensity at 450 nm was measured using a plate reader (NanoQuant Microplate Reader, TECAN).

Sera from immunized mice were collected and pooled on day 56 after immunization. A 96-well ELISA plate (Greiner Microlon High-Binding, clear) was coated with 100 µl/well of L. tarentolae cell lysates (WT and NoV VP1-MUC1 lysates). The coated plate was incubated overnight at 4 °C. Next, the plate was washed 4 × 5 min with 200 µl/well of washing buffer (PBS/0,05%Tween20) and blocked for 2 h with 250 µl/well of blocking buffer (3%BSA/PBS/0,05%Tween20) at 37 °C. The plate was washed as previously and serial dilutions of pooled mouse sera (in 3%BSA/PBS/0,05%Tween20) were added to the wells and incubated for 1 h at room temperature. Serial dilutions of Armenian hamster anti-MUC1 antibody (MA5-11,202, Thermo Fisher Scientific; in 3%BSA/PBS/0,05%Tween20) served as a positive control. After incubation the plate was washed as previously, and appropriate secondary antibody solution (Jackson Immuno Research; in 3%BSA/PBS/0,05%Tween20) was used for detection. Finally, following the last plate-washing step (6 × 5 min with 200 µl/well), 100 µl/well of HRP-substrate solution was added (1-Step Turbo TMB-ELISA, Thermo Scientific), the plate was incubated in dark until the blue color developed, and the reaction was stopped by adding 50 µL of 0,5 M sulfuric acid to each well. Signal intensity at 450 nm was measured using a plate reader (Epoch, Biotek).

A 96-well ELISA plate (Greiner Microlon High-Binding, clear) was coated with 100 µl/well of sequential dilutions purified L. tarentolae-derived VLPs (NoV VP1 and NoV VP1-MUC1). WT L. tarentolae cell lysate served as a negative control. The coated plate was incubated overnight at 4 °C. Next, the plate was washed 4 × 5 min with 200 µl/well of washing buffer (PBS/0,05%Tween20) and blocked for 2 h with 250 µl/well of blocking buffer (3%BSA/PBS/0,05%Tween20) at 37 °C. The plate was washed as previously and 100 µl/well of primary rabbit anti-NoV antibodies (Abcam ab92976; in 3%BSA/PBS/0,05%Tween20) and Armenian hamster anti-MUC1 antibody (MA5-11,202, Thermo Fisher Scientific) were added. After incubation the plate was washed as previously, and the appropriate secondary antibody solution (Jackson Immuno Research; in 3%BSA/PBS/0,05%Tween20) was used for detection. Finally, following the last plate-washing step (6 × 5 min with 200 µl/well), 100 µl/well of HRP-substrate solution was added (1-Step Turbo TMB-ELISA, Thermo Scientific), the plate was incubated in dark until the blue color developed, and the reaction was stopped by adding 50 µL of 0,5 M sulfuric acid to each well. Signal intensity at 450 nm was measured using a plate reader (Epoch, Biotek).