Ab105460

Equal amount of 20 μg salivary proteins from different groups were applied to the 10% SDS-PAGE and transferred from gels to the PVDF membrane as described in 2.4. After being blocked with 5% skimmed milk in TBST for 1 h, the membrane was incubated with primary antibody of mouse monoclonal anti-human MUC5B (1:1000 in TBST, Abcam, Ab105460) or rabbit monoclonal anti-human immunoglobulin A (IgA) (1:1000 in TBST, Abcam, Ab124716) at 4 °C overnight. The membrane was then washed three times with TBST and incubated with the horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. The goat anti-mouse Immunoglobulin G heavy and light chains (IgG H&L) for MUC5B (1:5000 in TBST; Abcam, Ab205719) and the goat anti-rabbit IgG H&L for IgA (1:5000 in TBST, Abcam, Ab205718) were used, respectively. After being washed three times with TBST, the membranes were finally detected by using the chemiluminescent HRP substrate (Beyotime, Haimen, China) and scanned with the Tannon 5200 Imaging System (Tanon, Shanghai, China).

For immobilization, 100 μL of 5 μg/mL MUC5B antibody (Abcam, Ab105460) or IgA antibody (Abcam, Ab124716) diluted in the coating buffer (0.1 M carbonate/bicarbonate, pH 9.4) was added to each well of the 96-well plate and incubated at 4 °C overnight. The wells used as the blank control were added with 100 μL of coating buffer without any antibody. The next day, the plate was washed three times with PBST and blocked with carbo-free solution at 37 °C for 1 h on the shaker. Then, the plate was washed again and 50 μg of salivary proteins (1 μg/μL) from each group was added to the well in triplicate and incubated at 37 °C for 1 h. After the plate being washed, 100 μL of the biotinylated SNA or MAL-II (Vector Laboratories Inc, Burlingame, CA, USA), which was 1:3000 or 1:200 diluted in carbon-free solution, was applied to each well and incubated at 37 °C for another 1 h. Next, the plate was washed again and 100 μL of streptavidin-HRP (Vector Laboratories) with 1:2000 dilution in carbon-free was added to the well and incubated at 37 °C for 50 min. The HRP signal was finally detected by 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (Beyotime, Haimen, China) and the absorbance of 450 nm for each well was read by the microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).