2.4 Enzyme-linked immunosorbent assay for detection of anti-BoHV-1 antibodies
Before each animal vaccination, sera were tested by anti BoHV-1 enzyme-linked immunosorbent assay (ELISA). Briefly, Immulon 1B (Dynatech, Laboratories) microtiter plates were coated with inactivated BoHV-1 (iBoHV) LA Strain (1/300) in 0.1 M carbonate-bicarbonate buffer, pH 9.6 and incubated overnight (ON) at 4 °C. Plates were blocked with PBS/0.05% Tween 20 (PBST) containing 1% ovalbumin (PBST-OVA). Serial dilutions of mice or cattle sera were prepared in PBST-OVA and dispensed in 50 µL/well. Plates were washed three times with PBST-OVA and incubated with anti-mouse or anti-bovine IgG peroxidase conjugate (KPL) respectively for 1 h at 37 °C. After extensive washing with PBST, ortho-phenylene-diamine (1,2-benzenediamine) dihydrochloride (OPD, SIGMA) and H2SO4 were added as substrate and absorbance was measured at 492 nm using MR 5000 microplate reader (Labsystems, MN, USA). Cut-off was established as the mean A492 of the negative sera +2 standard deviation (SD). Titers were expressed as the log10 of the reciprocal of the highest serum dilution giving an OD higher than the cut-off.