Polyethylene glycol 1450

Hybridoma cells secreting specific mAbs against the PEDV N protein were generated according to standard procedures, with some modifications [13 (link)]. Briefly, 6-week-old female BALB/c mice were immunized subcutaneously with the purified recombinant PEDV N protein in Freund’s complete adjuvant (Sigma-Aldrich Corporation, St. Louis, MO, USA). The mice received two booster immunizations with the N protein in Freund’s incomplete adjuvant at 2-week intervals. One additional intraperitoneal immunization without adjuvant was administered. Three days after the final vaccination, sera were collected from the mice and used for ELISA and western blot experiments, as described in this report. Spleen cells were harvested and fused with SP2/0 myeloma cells at a ratio of 5:1 using polyethylene glycol 1450 (Sigma-Aldrich Corporation). The supernatants of surviving hybridoma clones were screened for reactivity and specificity using an indirect ELISA as described by Liu et al. [17 (link)], with minor modifications. The supernatants of the hybridoma clones 15B12 and 4G7, which reacted strongly with the PEDV N protein in a western blot assay, were selected for further experiments. The protocols for all animal experiments in this study were approved by the Animal Care and Use Committee of our institute and performed in accordance with animal ethics guidelines.

LLC-PK1 cells, a porcine kidney cell line, were purchased from ATCC and subsequently cultured in Dulbecco’s modified Eagle’s medium (Gibco) containing 10% heat-inactivated fetal bovine serum (PAN-biotech) and a combination of penicillin and streptomycin (Solarbio), in a humidified incubator with an atmosphere of 5% CO₂ at 37 °C. PDCoV strain CH/JXJGS01/2016 (GenBank accession number KY293677.1) was isolated in our laboratory in 2016 from a newborn piglet with diarrhea. Mouse mAb against β-actin and hemagglutinin (HA) were bought from Medical and Biological Laboratories (Japan). Rabbit anti-B23 antibody was purchased from Proteintech (CHI, USA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H + L) was bought from antGene (China). FITC-conjugated goat anti-mouse IgG(H + L) and Alexa Fluor® 594 donkey anti-rabbit IgG(H + L) were bought from and TransGen Biotech (China). All enzymes used for the cloning procedures were purchased from Takara (Dalian, China). Freund’s complete/incomplete adjuvants, polyethylene glycol 1450 and HT/HAT medium were purchased from Sigma-Aldrich (MO, USA).