Serum ELISA
The TeNT-specific antibody titre of each vaccinated animal was
determined in triplicate by serum ELISA. In brief, 96 well plates were
coated overnight with 0.5 µg of recombinant TeNT in carbonate coating
buffer pH 9.6. Plates were washed three times with PBS 0.05% tween and
blocked with PBS 5% skim milk for 1 h at 37°C. Plates were washed three
times with PBS 0.05% tween then coated with serial dilutions, in
triplicate, of the mouse serum in PBS 1% skim milk, starting at an
appropriate concentration for each sample determined by a pilot ELISA.
Each plate also had an internal control sample, derived from a pool of
sera determined to be suitable from the pilot ELISA. Plates were
incubated for 1 h at 37°C then washed three times with PBS 0.05% tween.
Wells were coated with 100 µL of Goat anti-mouse IgG HRP conjugated
antibodies (A4416 Sigma) diluted to 1:5000 in PBS 1% skim milk and
incubated for 1 h at 37°C. The plates were washed three times with PBS
0.05% tween, developed with TMB Chromogenic solution (Invitrogen.) in
the dark for 10–30 minutes, then stopped with 1 M HCl, according to the
manufacturer’s protocol. Absorbance at 450 nm was determined using a
Clariostar plus plate reader (BMG Labtech). Sample titres were
calculated as the antibody dilution that gave half the maximum OD (as
determined from the internal standards in each plate at a 1:16,000
dilution) and calculated using GraphPad Prism (version 8.3).