Establishment of serological test to detect antibody against ferret coronavirus

MINAMI, Shohei; TERADA, Yutaka; SHIMODA, Hiroshi; TAKIZAWA, Masaki; ONUMA, Mamoru; OTA, Akihiko; OTA, Yuichi; AKABANE, Yoshihito; TAMUKAI, Kenichi; WATANABE, Keiichiro; NAGANUMA, Yumiko; KANAGAWA, Eiichi; NAKAMURA, Kaneichi; OHASHI, Masanari; TAKAMI, Yoshinori; MIWA, Yasutsugu; TANOUE, Tomoaki; OHWAKI, Masao; OHTA, Jouji; UNE, Yumi; MAEDA, Ken

ELISA: The concentration of purified recombinant proteins was adjusted to 5 µg ml −1 with adsorption buffer (0.05 M carbonate-bicarbonate buffer, pH 9.6). GST was used as a control at 5µg ml −1 . One hundred microliters of purified recombinant proteins and GST were added to 96well microplates (Maxisorp; Nunc, Roskilde, Denmark). After incubation at 37°c for 2 hr, plates were placed at 4°c overnight. The wells were washed three times with PBS containing 0.05% Tween 20 (PBS-T) and then incubated with 200 µl of 1% Block Ace (Dainippon Pharmaceutical, Osaka, Japan) in PBS at 37°c for 30 min. After washing three times with PBS-T, 100 µl of diluted sera or plasma were added to duplicate wells and incubated at 37°c for 30 min. Sera or plasma was diluted to 1:100 or 1:500 with PBS-T containing 0.4% Block Ace. Subsequently, wells were washed three times with PBS-T before 100 µl of peroxidase-conjugated anti-ferret immunoglobulin (ROcKLAND, Limerick, PA, U.S.A.) diluted with PBS-T containing 0.4% Block Ace was added and incubated at 37°c for 30 min. Following three washes with PBS-T, 100 µl of Horseradish Peroxidase Substrate (BIO-RAD) was added to each well. After incubation at room temperature for 30 min, the enzymatic reaction was stopped by adding 100 µl of 2% oxalic acid to each well. The absorbance was measured using a spectrophotometer (BIO-RAD) at 415 nm. All results were subtracted from the value for GST, and the cut-off value was arbitrarily set at 0.5. Phylogenetic analysis: A phylogenetic tree was constructed using the program MrBayes Ver. 3.2.2 [7] for MrModeltest analysis with a WAG substitution matrix [10] . We referred to the following sequences to construct the phylogenetic tree of N protein sequences; FREcV strain MSU-2 (GU338457), FREcV strain MSU-1 (DQ340562), FRScV strain MSU-1 (GU338456), mink coV strain WD1127 (HM245925), mink coV strain WD1133 (HM245926), ccoV type II strain fc1 (AB781790), FcoV type II strain M91-267 (AB781788), FcoV type I strain c3663 (AB535528), SARS-coV strain BJ182-12 (EU371564) and FRcoV strain Yamaguchi-1 (Lc029423). The tree was represented graphically using FigTree Ver. 1.4.2 [1] .