Evaluation of the Efficacy of a Recombinant Adenovirus Expressing the Spike Protein of Porcine Epidemic Diarrhea Virus in Pigs

Liu, Xinsheng; Zhao, Donghong; Zhou, Peng; Zhang, Yongguang; Wang, Yonglu

ELISA. An indirect ELISA based on a PEDV S2 protein (approximately 57 kDa), which encodes the 790-1386 aa spike protein of the genotype GII PEDV strain and was expressed as a recombinant protein in Escherichia coli by our laboratory, was developed to assess IgG and IgA antibody responses in pigs immunized with rAd-PEDV-S. In brief, a concentration of 0.1 μg/mL purified PEDV S2 protein in 0.05 M NaHCO 3 was used to coat ELISA plates (Corning, USA) and then incubated with samples (1 : 200 dilution) at 37°C for 1 hour. After the plate was washed with PBST 3 times, an HRP-conjugated goat anti-pig IgG mAb (1 : 10000 dilution; Abcam, USA) or HRP-conjugated goat anti-pig IgA mAb (1 : 10000 dilution; Abcam, USA) was added and incubated at 37°C for 0.5 hours. en, the plate was washed with PBST 3 times, followed by the addition of the 3,3′,5,5′tetramethylbenzidine (TMB) substrate and incubation at 37°C for 15 minutes. en, 50 μL of stop solution was added, and the absorbance was determined at 450 nm.