NHBA Antigen Fragment Immunoreactivity

The immunoreactivity of NHBA antigenic fragments against the 31E10/E7 mAb was tested using a previously described ELISA inhibition assay [26 (link)]. Briefly, the 31E10/E7 mAb (1 μg/ml) was preincubated with micromolar concentrations of competitors (represented by the indicated NHBA fragments or by the entire antigen fused to an histidine tail) prior to the addition to microtiter wells sensitized with the whole fusion antigen (NHBA-NUbp fused to an histidine tail or NHBA-NUbp-His, 5 μg/ml). Alkaline phosphatase-conjugated goat anti-mouse IgG (Sigma) was then added at a 1:5,000 dilution followed by p-nitrophenyl phosphate disodium salt (Sigma). Percent inhibition was calculated by comparing the absorbance value of wells with and without the inhibitors.