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.