Chemical modification
of BSA. (A) Schematic of BSA modification
with FAM and/or Ad–PEG–amine via EDC-catalyzed amidation.
(B) FAM–BSA excitation and emission scans (λmax ex/em = 480/525 nm, left). Dependence of the FAM-per-BSA modification
ratio on the molar feed ratio of FAM to BSA (right). Differences between
all reaction conditions were highly significant, ****P < 0.0001. (C) The affinity-based thermodynamic dissociation constant
of individual GH interactions (KD, bottom
left) and extent of Ad–BSA modification (N, bottom right),
determined by ITC (see Figures S5 and S6). (D) Avidity-controlled Ad–BSA complex half-life (bottom
left) and dissociation rate constant (bottom right), determined by
SPR. Data represent mean ± SD; n = 3; ns = not
significant, *P < 0.05, **P <
0.01, ***P < 0.001, ****P <
0.0001; ANOVA, Tukey HSD.