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

The binding affinity of the mAbs was calculated following surface plasmon resonance (SPR) analysis using ProteOn XPR36 system (Bio-Rad Laboratories). Purified mAb 3B12-1A6 or mAb 1H12-1C6 was immobilised to a GLC chip (Bio-Rad Laboratories) using a standard Ethyl-3-(3-dimethylaminopropyl)-carbodiimide (ED-C)/N-hydroxysuccinimide (NHS) cross-linking reaction, and affinity values (ka, kd, KD) of each mAb for PEDV were determined by ProteOn Manager RM 2.1 software. Calculation of the binding affinity was performed in Gyeonggi Bio-Center (Suwon, Republic of Korea). To further characterise the mAbs for epitope recognition, we used a competition ELISA to test whether the mAbs recognise different epitopes of PEDV as in a previously described method. 11 Briefly, the recombinant N protein was coated onto a microtitre plate, and the plate was blocked with casein buffer. Biotinylated mAb and/or non-biotinylated mAb were added to the wells, and the plate was reacted with HRP-conjugated avidin (Thermo Fisher Scientific, Waltham, MA, USA). The colour reaction was stopped and read as optical density at 450 nm using an automated plate reader.