Figure 5. Maturation process of HBsAg (post DTT removal) at 37 °C as monitored with an “antigenicity ELISA” and a “mass-ELISA.” In the antigenicity assays, the HBsAg was captured using the disulfide insensitive 42B6 regardless of the disulfide status. Two different mAbs, same mAb 42B6 or disulfide sensitive 5F11, were employed as detection Ab in 2 different sandwich ELISAs to reveal the binding activity yielding unique information on different epiopes. (A) This assay (42B6: HBsAg: 5F11-HRP) is disulfide-dependent, detecting the HBsAg epitope development as a function oxidative maturation time. The red circles/line are the data obtained with facilitated maturation with cupric ion and the blue triangles or line are the data for the control (PBS only). Please note the expected promoting effect of cupric ion on the oxidative maturation of DTT-reduced HBsAg as indicated with the conformation-sensitive assays. (B) The other assay, with 42B6 on both sides (42B6: HBsAg: 42B6-HRP) is disulfide-independent. For this “mass-like” ELISA, it showed constant level during the whole oxidative maturation process. The standard curves used for antigenicity analysis were shown in the inset Figures in (A) and (B). The minimal detection level of HBsAg was for 2 assays shown in Figure (A) and (B) insets, ~0.98 ng/mL and ~3.9 ng/mL, respectively.