Development of rapid immunochromatographic strip test for the detection of porcine epidemic diarrhoea virus

Lyoo, Kwang-Soo; Yeom, Minjoo; Kim, Jungho; Kim, Donghyuk; Ha, Gunwoo; Na, Woonsung; Le, Van Phan; Song, Daesub

To produce monoclonal antibodies, BALB/c mice (six to eight weeks old) were intraperitoneally immunised with the purified N protein antigen emulsified in complete Freund's adjuvant. Three identical boosters were administrated with the N protein emulsified with incomplete Freund's adjuvant one-week intervals. The immunised mice were sacrificed to isolate spleen cells, which were washed in serum-free cell culture medium. Splenocytes (1×10 8 ) were mixed with myeloma cells line Sp2/0-Ag-14 (ATCC CRL 1581) (2×10 7 ) in a ratio of 5:1 in the presence of polyethylene glycol for cell fusion. The fused cells were seeded into 96-well tissue culture plates with hypoxanthine-aminopterin-thymidine (HAT) selection medium. Culture medium was exchanged with fresh HAT selection medium eight days after fusion. On day 11, the supernatants were screened by ELISA, and then mAb anti-PED 3B12-1A6 and mAb anti-PED 1H12-1C6 hybridomas, which strongly reacted with the PEDV protein, were selected. Each hybridoma cell was intraperitoneally injected into naïve BALB/C mice to generate ascetic fluid. The ascetic fluid was then purified on protein G Sepharose 4 Fast Flow (GE Healthcare, Uppsala, Sweden) according to the manufacturer's protocol. The antibody concentrations were measured using the Bradford protein assay (Bio-Rad Laboratories, Hercules, CA, USA).