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

We report the development of a MAbs-based antigen capture ELISA for MERS-CoV-NP. Our previous experience on SARS-CoV has shown that SARS-CoV-NP detection in SARS patients is highly feasible using an antigen capture ELISA-based method. 14, 15 Since NP is also an important, highly immunogenic and abundantly expressed structural protein in MERS-CoV, its detection using similar principles should also be feasible. The NP protein is a more preferable target than the S protein because the NP protein is present in much higher amount than the S protein. In this study, using hyperimmune sera obtained from mice immunized with three doses of MERS-CoV-rNP, we confirmed that MERS-CoV-NP is abundantly present in inactivated MERS-CoV cell cultures (Figure 2 ). The antigen capture ELISA was designed using two different MAbs (1F6 and 7C4) with high and specific antibody titers against MERS-CoV-rNP ( Figure 3 ). As half of the 24 MAbs generated showed different degrees of cross-reactivity against HCoV-229E and/or HCoV-OC43, suggesting the possibility of common epitopes across the different CoVs, this MAbs-based ELISA should be much more specific than a polyclonal antibodies-based ELISA using hyperimmune sera generated from two different kinds of animals immunized with MERS-CoV-rNP. In addition to the high specificity, we also observed that MERS-CoV-rNP and MERS-CoV-NP can be detected by the present assay with a protein and inactivated viral concentrations of as low as 1 ng/mL and 10 3 TCID 50 /mL respectively ( Figure 4) .