An ELISA for detection of antibodies against porcine epidemic diarrhoea virus (PEDV) based on the specific solubility of the viral surface glycoprotein

Abstract

Viral proteins of porcine epidemic diarrhoea virus (PEDV) were extracted from the cytoplasm of infected Vero cells using hypotonic conditions and a non-ionic detergent. Both the pH and the NaCl concentration of the extraction buffer were varied in attempts to increase the solubility of the virion spike glycoproteins (S-protein) and of the nucleocapsid proteins (N-protein). Monoclonal antibodies, hyperimmune sera and convalescent pig sera were used to identify and monitor these proteins by immunoprecipitation and Western blots. The solubility of the S-protein was optimal at pH 4, whereas that of the N-protein was optimal at pH 9. Consequently, it was possible to enrich for either S-protein or N-protein; increases in the NaCl concentration of the buffer were of no advantage in this respect. Enriched preparations of the S-protein and N-protein were used as ELISA antigen for the S-ELISA and N-ELISA, respectively. The S-ELISA proved to be the more effective of the two immunoassays. Antibodies against S-protein remained detectable for longer periods of time than anti-N-protein antibodies in the sera of PEDV-infected pigs. Using this ELISA of increased sensitivity, it was observed that only a small number of farms in Switzerland had been infected with PEDV.

Comparison of serum neutralization and enzyme-linked immunosorbent assay on sera from porcine epidemic diarrhea virus vaccinated pigs

“…SN is not useful for mass screening of swine sera for PED diagnosis. However, the serum neutralization test (SNT) has a high specificity and is useful for the determination of immunization status (Hofmann & Wyler 1990;Knuchel et al 1992;van Nieuwstadt & Zetstra 1991). A proteinbased ELISA can also be used to detect PEDV in which immunofluorescence analysis (IFA) with anti-PEDV-M antibody is used to distinguish PEDV-infected cells from cells infected with other enteric viruses (Ren et al 2011).…”

BAC-VAC, a novel generation of (DNA) vaccines: A bacterial artificial chromosome (BAC) containing a replication-competent, packaging-defective virus genome induces protective immunity against herpes simplex virus 1

“…ELISA was performed and antibody titers were determined as described (36) by using peroxidase-conjugated polyclonal anti-mouse IgG1, IgG2a, IgG2b, and IgG3 antibodies (Southern Biotechnology Associates). HSV-1 antigen was prepared from virus-infected Vero cells and titered for optimal performance in ELISA (40). Sera collected from immunized and control animals were analyzed for HSV-1 neutralization by using standard methods.…”

Cloning and further sequence analysis of the spike gene of attenuated porcine epidemic diarrhea virus DR13

“…Reading in the 5¢-3¢ direction, the PEDV genome contains genes for pol1 (P1) protein, spike (S) protein, an open reading frame (ORF3), envelope (E) protein, membrane (M) protein and nucleocapsid (N) protein [10,[17][18][19][20][21]. Among the proteins encoded by these genes, S protein, a glycoprotein peplomer (surface antigen) on the viral surface, plays an important role in binding to specific host cell receptor glycoproteins with subsequent penetration into the cells occurring via membrane fusion.…”