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

Anti-MERS-CoV-rNP MAbs were produced using a protocol as described previously. 19 Briefly, six-week-old female BALB/c mice were immunized with 100 mg purified MERS-CoV-rNP emulsified in an equal volume of Freund's complete adjuvant (Sigma-Aldrich, Saint Louis, MO, USA) subcutaneously. Two booster immunizations using the same antigen emulsified in incomplete Freund's adjuvant were carried out on days 14 and 28. Blood samples were obtained from the tail veins of the immunized mice and tested for titers against purified MERS-CoV-rNP by ELISA on MERS-CoV-rNP-coated microplates. After the final booster dose, the spleens of the mice were removed, and splenocytes were fused with the mouse myeloma cell line Sp2/0-Ag-14. Culture supernatant from individual hybridoma clones was then screened against purified MERS-CoV-rNP by ELISA. After subcloning three times by limiting dilution, stable hybridoma cells were injected into the abdominal cavity of a mouse to produce ascitic fluid. The MAbs were purified first by precipitating with 50% ammonium sulfate, then with diethylaminoethyl column by high performance liquid chromatography. The concentrations of purified IgG were determined by their absorbance at OD 280 . All animal procedures were approved officially by the Xiamen University Institutional Committee for the Use and Care of Laboratory Animal.