Evaluation of serological assays available in a biosafety level 2 laboratory and their application for survey of Middle East respiratory syndrome coronavirus among livestock in Ethiopia

TERAMICHI, Takurou; FUKUSHI, Shuetsu; HACHIYA, Yuma; MELAKU, Simenew Keskes; OGUMA, Keisuke; SENTSUI, Hiroshi

The MERS-CoV RBD was used as the antigen of the cELISA. For the preparation of recombinant RBD, the mammalian expression plasmid pCAGGS-RBD, which encodes histidine-tagged MERS-CoV RBD (amino acid 358-588), was transfected to 293T cells. At 2 days after transfection, the recombinant RBD was purified from the supernatant of transfected cells using His-Bind Purification Kit (Merck, Damastadt, Germany). The cELISA was performed as described by Fukushi et al. [9] . Briefly, MERS-CoV recombinant RBD with pre-determined optimal quantity was coated on a 96-well ELISA plate at 4°C for overnight, following which the wells were incubated with PBS containing 2% bovine serum albumin and 0.05% tween 20 (blocking reagent) for 2 hr at 37°C. Following the removal of the blocking reagent, 100 µl of a biotin-labeled monoclonal antibody mixed with serially diluted serum samples was added and incubated for 1 hr at 37°C. One of camel serum that showed a high antibody titer in