2.6 ELISA
Ninety-six well plates were coated with 500 ng/well of purified
recombinant G1-NTD and G2-NTD overnight at 4 ºC. The coated plates were
washed three times with PBST, and blocked with 5% non-fat dry milk in
PBST
at
37 ºC for 1 h. The plates were washed three times, and then incubated
with G1- and G2-NTD antisera (1:200 dilution in PBST). The plates were
incubated at 37 ºC for 1.5 h and washed three times with PBST, and
HRP-conjugated
goat anti-mouse IgG (1:5000 dilution in PBST) was added to each well.
The plates were incubated t 37 ºC for 1.5 h and washed as above.
Finally, 100 μl 3, 3’, 5, 5’- tetramethylbenzidine (TMB) was added to
each well, and the plates were incubated for additional 15 min at 37 ºC
in dark. The reaction was stopped by the addition of 1 N sulfuric acid.
The absorbance was read at a wavelength of 450 nm (OD450) using
Multiskan GO (Thermo Fisher Scientific) ELISA plate reader.