Detection of Anti-PCV2 Antibodies
- Serum-virus neutralization (SVN) assay
- Indirect immunoperoxidase monolayer assay (IPMA)
- Indirect immunofluorescence (IFA) assay
- Enzyme-linked immunosorbent assay (ELISA)
Serum-virus neutralization (SVN) assay
The basis of the SVN assay is that it detects the presence of antibodies that have the ability to prevent virus from attaching to and/or infecting cells. Neutralizing antibody assays for PCV2 have been described in the literature (Meerts et al., 2006, 2005c; Pogranichnyy et al., 2000). Neutralizing antibodies were detected between 15 (Meerts et al., 2005c) and 28(Pogranichnyy et al., 2000) days post PCV2-infection and were correlated with protection or clearance of PCV2 infection in gnotobiotic pigs (Meerts et al., 2005c).
The IPMA is similar to the IFA with the exception that the antibody used is a peroxidase-conjugated anti-swine IgG. IPMA for PCV2 is widely used (Ellis et al., 1998; Balasch et al., 1999). An interlaboratory testing comparing IFA and IPMA results on the same 20 serum samples performed in different laboratories in Europe and Canada found a wide variation in detected titers between laboratories (McNair et al., 2004). In general, IPMA gave higher titers than IFA, and paraformaldehyde used as fixative gave higher titers than did acetone or ethyl alcohol (McNair et al., 2004).
This assay detects the ability of the antibody in the serum to bind to a fixed monolayer of virus-infected cells. The specific antibodies are labeled with fluorescein-conjugated anti-swine-IgG. IFA assays for PCV2 have been described in the literature (Allan et al., 1998; Pogranichnyy et al., 2000; Tischer et al, 1995). Recently, an IFA assay based on an ORF2 protein has been described(Racine et al., 2004) and it was found that the regular whole PCV2-based IFA had only a 57.1% relative sensitivity compared to the ORF-2 protein based IFA. IFAs have also been described for antibodies against non-pathogenic PCV1 (Fenaux et al., 2003) and appear to be specific. Studies have shown that there is a low level of cross-reactivity between PCV1 and PCV2 on IFA test (Allan et al., 1998, Pogranichnyy et al., 2000).
There are several peer-reviewed articles describing PCV2 ELISA assays (Blanchard et al., 2003; Liu et al., 2004; Nawagitgul et al., 2002). Recently, commercially available IgG and IgM PCV2-ELISA assays have been introduced in Europe (Ingezim PCV IgG® and Ingezim PCV IgM®, Ingenasa, Madrid, Spain). Combined IgG and IgM values might be useful to determine the timing of PCV2-infection: IgM values ≥ IgG values: early active infection (within the first 21 DPI); IgM values < IgG values: active infection (approximately between 20 to 50 DPI); High IgG values and negative IgM values: late or resolving infection of convalescent (approximately 2 months after infection) (Segalés et al., 2005). A variant of the regular ELISA assay is the competitive (blocking) ELISA (Walker et al., 2000). A competitive ELISA specific for PCV2-antibodies is available in Europe (Synbiotics, Europe, Lyon, France). An antibody-detection blocking ELISA to detect PCV2-specific antibodies on feces is also described(Lopez et al., 2005) and is commercially available in Europe (Synbiotics Europe, Lyon, France).
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