ELISA and Direct Immunofluorescence Test to Detect Equine Arteritis Virus (EAV) using a Monoclonal Antibody Directed to the EAV-N Protein

Abstract

A monoclonal antibody (mAb) directed against the equine arteritis virus (EAV) nucleocapsid (N) protein was used for indirect enzyme-linked immunosorbent assays (ELISAs) using viral antigen from different sources. The same mAb was labelled with fluorescein isothiocyanate for direct immunofluorescence tests (DIFTs). The N-specific mAb appeared to be suitable for the detection in both ELISA and DIFT of different EAV strains and field isolates from semen and tissue samples after passage in lines of RK-13, Vero and fetal equine kidney cells. The ELISA described is an easy and fast method which can be used in most cases to replace the microneutralization test to prove the EAV specificity of the cytopathic effect of cell cultures. The DIFT, however, is more sensitive than both the ELISA and the microneutralization test because EAV antigen can be detected even in cell cultures without or with very weak cytopathic effect.

Equine Viral Arteritis in a Newborn Foal: Parallel Detection of the Virus by Immunohistochemistry, Polymerase Chain Reaction and Virus Isolation

“…For example, nucleotide sequence comparison of EAV‐nucleocapsid (N) protein from different isolates revealed only 3–7% differences, rendering the N protein ideal for diagnostic purposes (Chirnside et al., 1994). Additionally N protein‐specific Mabs proved to be able to detect several different EAV strains (Weiland et al., 2000; Starick et al., 2001), which further supports the use of such Mabs in diagnostic work.…”

“…For example, nucleotide sequence comparison of EAV-nucleocapsid (N) protein from different isolates revealed only 3-7% differences, rendering the N protein ideal for diagnostic purposes (Chirnside et al, 1994). Additionally N protein-specific Mabs proved to be able to detect several different EAV strains (Weiland et al, 2000;Starick et al, 2001), which further supports the use of such Mabs in diagnostic work. This is the first report on the detection of EAV in formaldehyde-fixed and paraffin-embedded tissue sections using N protein-specific Mab with horseradish peroxidase (HRP)-labelled streptavidin-biotin.…”

“…In this study, the virus was detected using a HRP‐labelled streptavidin–biotin method in formaldehyde‐fixed and paraffin‐embedded tissue sections. The distribution of the EAV‐antigen in cell culture and in tissue sections was similar to previous studies (Crawford and Henson, 1973; Little et al., 1995; Mac Lachlan et al., 1996; Del Piero et al., 1997; Starick et al., 2001). The reaction with the N‐specific Mab was specific, which was supported by the use of EAV‐specific equine IgG, VI and PCR.…”

“…With N protein-specific Mab it was possible to detect EAV using direct immunofluorescence (IF) in cell cultures (Starick et al, 2001), indirect IF in formaldehyde-fixed and paraffinembedded tissue samples (Wada et al, 1994), and a streptavidin-biotin method in cell cultures or cryostat sections (Little et al, 1995;Mac Lachlan et al, 1996). In this study, the virus was detected using a HRP-labelled streptavidin-biotin method in formaldehyde-fixed and paraffin-embedded tissue sections.…”

Equine arteritis virus

“…Several laboratories have developed and evaluated enzyme-linked immunosorbent assays (ELISAs) to detect antibodies to EAV using whole virus, synthetic peptides, or recombinant viral proteins (e.g. GP5, M, and N) as antigens (Castillo-Olivares et al, 2003;Cook et al, 1989;Duthie et al, 2008;Hedges et al, 1998;Kondo et al, 1998;Nugent et al, 2000;Starik et al, 2001;Wagner et al, 2003). Various studies have shown that the source of antigen as well as the sera evaluated can markedly influence the results obtained with EAV protein specific ELISAs and competitive ELISA.…”