Peroxidase conjugated affinipure goat anti rabbit antibodies

Samples containing NoV VLPs were loaded on 10–20% precast WedgeWell Gel (Thermo Scientific) and run at the constant voltage of 165 V. After electrophoresis, semi-dry electrotransfer of proteins onto polyvinylidene difluoride membranes was performed. Membranes were then blocked for 1 h in 5% semi-skimmed milk solution (5%milk/TBS/0.01% Tween20) and incubated overnight at 8 °C with rabbit anti-N terminal capsid protein of NoV antibodies (Abcam ab92976) (1:1000 in 5%milk/TBS/0.01%Tween20). The following day, the membranes were washed 3 times with washing buffer (TBS/0.01%Tween20) and incubated for 1 h at room temperature in a solution of Peroxidase-conjugated AffiniPure Goat Anti-Rabbit antibodies (Jackson Immuno Research) (1:4000 in 5%milk/TBS/0.01%Tween20). After washing (same as above) the reaction was developed with SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Scientific).

A 96-well ELISA plate (Greiner Microlon High-Binding, clear) was coated with type III mucin from porcine stomach (Sigma-Aldrich; 10 µg/ml in PBS) for 4 h at room temperature. 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. After blocking the plate was washed as previously. Next, 100 µl/well of NoV VLPs produced in L. tarentolae cells were added to the wells and incubated for 1 h at room temperature. The NoV VLPs produced in S. frugiperda cells served as a positive control. After washing, 100 µl/well of rabbit anti-VP1 conformational antibodies (Immune Technology Corp.) (1:1000 in 3%BSA/PBS/0.05%Tween20) were added and the plate was incubated for 1 h at 37 °C followed by incubation with 100 µl/well of a Peroxidase-conjugated AffiniPure Goat Anti-Rabbit antibodies (Jackson Immuno Research) (1:2000 in 3% BSA/PBS/0.05%Tween20) for 1 h at room temperature. Finally, the plate was washed (6 × 5 min with 200 µl/well) and 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 was measured at 450 nm using a plate reader (NanoQuant Microplate Reader, TECAN).