Horseradish peroxidase hrp conjugated goat anti human igg

Serum was first diluted 100-fold (40-fold for optimized conditions) with serum-dilution buffer (1% bovine serum albumin, 1% casein, 0.5% sucrose, 0.2% polyvinylpyrrolidone, 0.5% Tween20 in 0.01 M phosphate-buffered saline, Ph 7.4). Thereafter, 100 ΜL of the diluted sample was added to each microarray well and incubated for 30 min (or 2 h) on a shaker (500 rpm, 37 °C); the well incubated with serum-dilution buffer only was set as an experimental control. Thereafter, the microarray was rinsed three times with washing buffer (0.01 M PBST) and incubated with 100 ΜL of horseradish peroxidase (HRP) conjugated goat anti-human IgG (ZSGB-BIO, Beijing, China) for another 30 min on a shaker (500 rpm, 37 °C). Human HRP-IgG was diluted 10,000-fold with peroxidase conjugate stabilizer/diluent (Thermo Scientific, Waltham, MA, USA) for experimental use. Finally, 100 ΜL of one-step Ultra TMB-Blotting Solution (Thermo Scientific) was used to detect the informative signal of IgGs against probes using a microarray imager (Suzhou Epitope, Suzhou, China). The data were processed using IBT software, which was also developed by Suzhou Epitope (Suzhou, China). The signal for each dot was calculated using the following equation: Signal dot = Signal readout − Signal background.

For VP1- and VP2-specific IgG tests, 96-well ELISA plates (Thermo Fisher, USA) were coated with 2 μg/mL purified VP1 or VP2 protein at 4 °C overnight. Plates were blocked using 1% BSA in PBST with 0.05% Tween-20 for 2 h at 37 °C. Then, 1:100 diluted serum was added in PBST containing 0.5% BSA and incubated for 1 h at 37 °C, followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-human IgG (ZSGB-Bio, China) for 1 h at room temperature. PBST was used to wash between each step. Finally, 100 μL of TMB substrate solution (TIANGEN, China) was added for color development for 15 min, and then an equal volume stop solution (Sangon Biotech, China) was added to terminate the reactions. The absorbance was determined at 450 nm wavelength via a microplate spectrophotometer (Thermo Scientific Varioskan LUX, USA). For the peptide-specific IgG test, the procedure was similar to the one described above, except that the coated antigen was 5 M peptides, the serum dilution was 1:25, and the secondary antibody was diluted to 1:5000.