Absence of MERS-CoV antibodies in feral camels in Australia: Implications for the pathogen's origin and spread

Crameri, Gary; Durr, Peter A.; Barr, Jennifer; Yu, Meng; Graham, Kerryne; Williams, Owen J.; Kayali, Ghazi; Smith, David; Peiris, Malik; Mackenzie, John S.; Wang, Lin-Fa

A Luminex-based assay was developed using recombinant nucleocapsid (N) proteins of MERS-CoV and SARS-CoV using methodology established in our group for henipaviruses [22] . Recombinant CoV N proteins were produced in E. coli and purified directly from SDS-PAGE gels as previously described [23] . For coating onto Luminex beads, a total of 100 μg each of the two N proteins were coupled onto 100 μl of bead set 28 (SARS-CoV) and bead set 34 (MERS-CoV), respectively. Briefly, coupled microsphere sets were vortexed and sonicated prior to dilution in PBS-T containing 2% skim milk and transferred to 96well plate. The diluent was removed using an automated magnetic vacuum manifold followed by the addition of 100 μl of camel sera diluted 1:100 in PBS-T and incubated, shaking for 30 min at room temperature. Positive control camel sera used in this assay were derived from the natural infection of dromedary camels in Egypt during 2013 as part of a seroepidemiology study [24] . The serum was removed and the plate was washed twice with PBS-T followed by addition of Biotinylated Protein A (Pierce, Rockford, USA) and Protein G (Pierce, Rockford, USA) conjugates and incubated as described above. The conjugate was removed and the beads washed twice with PBS-T followed by addition of Streptavidin-phycoerythrin (Qiagen Pty Ltd, Australia) and a final incubation as described above. Assays were performed on a Bio-Plex Protein Array System integrated with Bio-Plex Manager Software (v 6.0) (Bio-Rad Laboratories, Inc., CA, USA). Results were recorded as median florescent intensity (MFI).