Development of an indirect ELISA for detecting swine acute diarrhoea syndrome coronavirus IgG antibodies based on a recombinant spike protein

Abstract Swine acute diarrhoea syndrome coronavirus (SADS‐CoV) is a newly identified swine enteropathogenic coronavirus that causes watery diarrhoea in neonatal piglets, leading to significant economic losses to the swine industry. Currently, there are no suitable serological methods to assess the infection of SADS‐CoV and effectiveness of vaccines, making an urgent need to exploit effective enzyme‐linked immunosorbent assay (ELISA) to compensate for this deficiency. Here, a recombinant plasmid that expresses the spike (S) protein of SADS‐CoV fused to the Fc domain of human IgG was constructed to generate recombinant baculovirus and expressed in HEK 293F cells. The S‐Fc protein was purified with protein G Resin, which retained reactivity with anti‐human Fc and anti‐SADS‐CoV antibodies. The S‐Fc protein was then used to develop an indirect ELISA (S‐iELISA) and the reaction conditions of S‐iELISA were optimized. As a result, the cut‐off value was determined as 0.3711 by analyzing OD 450nm values of 40 SADS‐CoV‐negative sera confirmed by immunofluorescence assay (IFA) and western blot. The coefficient of variation (CV) of 6 SADS‐CoV‐positive sera within and between runs of S‐iELISA were both less than 10%. The cross‐reactivity assays demonstrated that S‐iELISA was non‐cross‐reactive with other swine viruses' sera. Furthermore, the overall coincidence rate between IFA and S‐iELISA was 97.3% based on testing 111 clinical serum samples. Virus neutralization test with seven different OD 450nm values of the sera showed that the OD 450nm values tested by S‐iELISA are positively correlated with the virus neutralization assay. Finally, a total of 300 pig field serum samples were tested by S‐iELISA and commercial kits of other swine enteroviruses showed that the IgG‐positive for SADS‐CoV, TGEV, PDCoV and PEDV was 81.7, 54, 65.3 and 6%, respectively. The results suggest that this S‐iELISA is specific, sensitive, repeatable and can be applied for the detection of the SADS‐CoV infection in the swine industry.