Development of an indirect ELISA method for detection of porcine acute diarrhea syndrome coronavirus IgG antibody based on recombinant spike protein

Porcine Acute Diarrhea Syndrome Coronavirus (SADS-CoV) is a newly discovered porcine enteric pathogenic coronavirus that can cause watery diarrhea in newborn piglets and cause significant economic losses to the pig industry. At present, there is no suitable serological method to evaluate the effectiveness of SADS-CoV infection and vaccine, so there is an urgent need to use an effective enzyme-linked immunosorbent assay (ELISA) to make up for this deficiency. Here, a recombinant plasmid expressing SADS-CoV spike (S) protein fused with human IgG Fc domain was constructed to produce recombinant baculovirus and expressed in HEK 293F cells. The S-Fc protein is purified with protein G resin and retains the reactivity with anti-human Fc and anti-SADS-CoV antibodies. Then S-Fc protein was used to develop indirect ELISA (S-iELISA) and optimize the reaction conditions of S-iELISA. As a result, by analyzing the OD450nm value of 40 SADS-CoV negative sera confirmed by immunofluorescence assay (IFA) and Western blotting, the cut-off value was determined to be 0.3711. The coefficient of variation (CV) of the 6 SADS-CoV positive sera within and between runs of S-iELISA were all less than 10%. The cross-reactivity test showed that S-iELISA has no cross-reactivity with other porcine virus sera. In addition, based on the detection of 111 clinical serum samples, the overall coincidence rate of IFA and S-iELISA was 97.3%. The virus neutralization test with 7 different OD450nm values ​​of serum showed that the OD450nm value detected by S-iELISA was positively correlated with the virus neutralization test. Finally, S-iELISA was performed on 300 pig farm serum samples. Commercial kits of other porcine enteroviruses showed that the IgG positive rates of SADS-CoV, TGEV, PDCoV and PEDV were 81.7%, 54%, and 65.3%, respectively. , 6%, respectively. The results show that the S-iELISA is specific, sensitive, and reproducible, and can be used to detect SADS-CoV infection in the pig industry. This article is protected by copyright. all rights reserved.