Fig. 1: Conception and development of heat-stable antibodies via chemical engineering for high-temperature deep immunostaining.
From: Antibody stabilization for thermally accelerated deep immunostaining
a, Schematic diagram of antibody (Ab) diffusion to reach the deep tissue antigen (Ag) target. Ka(T) is the association constant of the Ab–Ag binding reactions at a given temperature T, and Deff is the effective diffusion coefficient of free Ab as a function of the antibody spatial location (r) and T. ‡ denotes transition state. b, The general relationship between Deff, Ka, the percentage of active Abs and T. In a hypothetical heat-facilitated strategy, the Ab–Ag binding reaction is not favored at higher T (that is, it lowers Ka), but is also irreversibly denatured at sufficiently high T (brown solid line). Therefore, raising T to increase the free Ab proportion is viable only if the Abs can be protected from denaturation (brown dotted line). c, Reaction–diffusion simulation of Ab–Ag binding and Ab diffusion in a cylindrical arena. The time (t)-dependent concentration profiles of the Ab–Ag complex ([Ab–Ag]) along the diffusion distance (r) with different combinations of T-dependent Ab–Ag binding kinetics and Ab diffusivity are visualized (lower panels). d, [Ab–Ag] versus r at the end of the simulations in c. e, Strategies for stabilizing Abs against permanent heat denaturation: stage 1, complexation with a secondary Fab fragment to stabilize protein conformation; stage 2, multifunctional crosslinkers are used to crosslink the complex (insets). f, SDS–PAGE analysis on crosslinking primary Ab–Fab fragment complexes. AF594, Alexa Fluor 594; CH, heavy chain constant domain; CL, light chain constant domain; FITC, fluorescein isothiocyanate; MW, molecular weight; VH, heavy chain variable domain; VL, light chain variable domain. g, Gel filtration analysis of the optimized IgG–Fab complex crosslinking reaction mixture, including a pair of IgG and Fab with mismatched Fab host specificity. Gt, goat; Rb, rabbit. Albumin peaks have been removed for clarity. The full traces are shown in Extended Data Fig. 2.
