Peroxidase conjugated mouse anti armenian hamster antibody

NoV VP1-MUC1, NoV VP1 and WT L. tarentolae cell lysates were loaded on 10–20% precast WedgeWell Gel (Thermo Fisher 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 a 5% semi-skimmed milk solution (5%milk/TBS/0,01%Tween20) and incubated overnight with primary antibodies solution: Armenian hamster anti-MUC1 antibody (MA5-11,202, Thermo Fisher Scientific; 1:100 in 5%milk/TBS/0,01%Tween20) or rabbit anti-N terminal capsid protein of NoV antibodies (ab92976, Abcam; 1:1000 in 5%milk/TBS/0,01%Tween20). The following day, the membranes were washed (TBS/0,01%Tween20) and incubated with solution of Peroxidase-conjugated mouse anti-Armenian hamster antibody (Santa Cruz Biotechnology; 1:4000 in 5%milk/TBS/0,01%Tween20) or AffiniPure goat anti-rabbit antibodies (Jackson Immuno Research; 1:4000 in 5%milk/TBS/0,01%Tween20). A reaction was developed with SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific).

A 96-well ELISA plate (Pierce Streptavidin High Binding Capacity, Clear) was coated with 100 µl/well of biotinylated PDTRPAPGSTAPPAHGVTSA MUC1 peptide (synthesized by JPT Peptide Technologies) adjusted to 10 µg/ml. The coated plate was incubated for 2 h with shaking at room temperature. Then the plate was washed 4 × 5 min with 200 µl/well of wash buffer (Tris buffered saline pH 7,2/0,1%BSA/0,05%Tween20). Next, 100 µl/well of L. tarentolae cell lysates (WT, NoV VP1 and NoV VP1-MUC1 lysates) were used to coat the plate. The plate was incubated for 2 h with shaking in room temperature. The plate was washed as previously. Next, 100 µl/well of Armenian hamster anti-MUC1 antibody (MA5-11,202, Thermo Fisher Scientific; 1:100 in wash buffer) was added and the plate was incubated for 1 h at room temperature, then washed as previously. Then 100 µl/well of Peroxidase-conjugated mouse anti-Armenian hamster antibody (Santa Cruz Biotechnology; 1:2000 in wash buffer) was added and incubated for 1 h at room temperature. The plate was washed as previously 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 (Epoch, Biotek).