Viral Antibody Avidity ELISA

ELISA with the inclusion of urea wash procedure was performed to measure the avidity of vaccination-induced antibodies to viral surface antigens, as described previously (Bachmann et al., 1997 (link); Fleury et al., 1999 (link); Polack et al., 2003 (link); Delgado et al., 2009 (link)). 96-well plates were coated with 10⁴ PFU of PR8 virus, and the wells were blocked by 1% BSA for 1 h at RT. After washing, twofold serial dilutions of sera were added to the plates, followed by incubation for 1 h at RT. After washing, 100 μL of distilled water (DW) or 7–9 M urea dissolved in DW were added to each well and incubated for 30 min for the dissociation of antibodies from the viruses. The urea was removed by washings and HRP-conjugated goat anti-mouse IgG antibody (Bethyl Laboratories) was added to the wells and incubated for 1 h. After washing, TMB substrate solution was added to the wells and incubated for 30 min. The colorimetric reaction was stopped by the addition of 1 M H₂SO₄, and the absorbance was read at 450 nm on an ELISA reader.