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.