Figure 3.

Assessment of thermal stability of monomeric and assembled E2 antigens. DSF of monomeric Δ123 (A) and monomeric Δ123A7 (B). BN-PAGE of Δ123r (C) and Δ123A7r (D) under nondenaturing conditions either with no heating (RT) or with pretreatment at the indicated temperature for 5 min in the presence or absence of the 0.2 mm DTT. M, monomer prior to refolding. Signal intensity of HMW bands analyzed by BN-PAGE in the presence of 0.2 mm DTT at the indicated temperatures relative to RT + 0.2 mm DTT, which was assigned a value of 1.0 (E). ELISA reactivity of MAb14 (F) and HC84.27 (G) after pretreatment of E2 antigens at the indicated temperatures for 30 min prior to coating. Titers were obtained by interpolating fitted curves at 25-fold above background (given by reactivity to BSA) and were expressed relative to that of RT, which was assigned a value of 1.0. Error bars in E represent the S.D. of two independent experiments, and error bars in F and G represent the S.D. of three independent experiments. Note that in one case each for Δ123 monomers and Δ123A7 monomers, the threshold for titration to HC84.27 after treatment at 100 °C was not met, in which case they were assigned a titer of the highest concentration of antibody used (3,160 ng/ml).