A sensitive and specific antigen detection assay for Middle East respiratory syndrome coronavirus

Chen, Yixin; Chan, Kwok-Hung; Kang, Yahong; Chen, Honglin; Luk, Hayes KH; Poon, Rosana WS; Chan, Jasper FW; Yuen, Kwok-Yung; Xia, Ningshao; Lau, Susanna KP; Woo, Patrick CY

The capture ELISA for MERS-CoV-NP detection was developed as described previously with minor modifications. 21 Microplates (Sigma Aldrich, Saint Louis, MO, USA) were pre-coated with the first anti-MERS-CoV-rNP MAb 1F6 and incubated at 37 6 C overnight with a blocking reagent (phosphate-buffered saline with 2% sucrose, 0.2% casein-Na, and 2% gelatin). Inactivated MERS-CoV, HCoV-229E, HCoV-OC43, two influenza virus A strains, one influenza virus B strain and one respiratory syncytial virus (RSV) strain, and purified rNPs of MERS-CoV, HCoV-229E, and HCoV-OC43 were diluted in phosphatebuffered saline with 2% skim milk. The inactivated viral lysates were diluted to the same virus titer of 10 6 TCID 50 and further serially diluted. Specifically, 50 mL of viral lysis buffer was added to the coated well and a 50 mL of sample (fixed concentrations of purified NP or viral cell culture lysates) was then added to the wells in duplicates. The plate was shaken for 2 min and incubated at 37 6 C for 30 min. After the wells were washed five times, 100 mL of the second anti-MERS-CoV-rNP MAb 7C4 conjugated with HRP was added and the plate was incubated at 37 6 C for 30 min. After five washes, detection was carried out by adding of 100 mL TMB per well with incubation for 10 min followed by the addition of 50 mL 0.2 M H 2 SO 4 . The OD 450/630 nm was measured with a microplate reader. The cutoff value was set as 0.151 mean value of 20 human nasopharyngeal aspirates (NPAs) negative for influenza A, B, and C viruses, adenovirus, RSV, parainfluenza viruses 1-4, human rhinoviruses, human metapneumovirus, HCoV-HKU1, HCoV-OC43, HCoV-229E, HCoV-NL63, and MERS-CoV.