Analysis and optimization of nonequilibrium capillary electrophoresis of alpha-fetoprotein isoforms

The L3 isoform of alpha-fetoprotein (AFP) is a specific marker for hepatocellular carcinoma. The separation and quantitation of L3 isoform from the L1 isoform is facilitated by Lens culinaris agglutin (LCA) affinity of the L3 isoform. The affinity-based separation is characterized by nonequilibrium conditions since electrophoresis perturbs the species concentrations away from equilibrium. The design of such separations requires careful consideration of the interplay between the reaction, diffusion, and separation time scales. We performed experiments to investigate the effect of separation parameters such as LCA concentration and CE voltage on the L1-L3 separation dynamics. We also describe a comprehensive mathematical model to predict electropherograms for affinity-based separations. The model includes the effects of molecular diffusion, electromigration, nonequilibrium reaction, and detection process. Together, the results demonstrate a process by which to optimize the affinity-based separations of AFP isoforms. We also obtained the kinetic rate constants for LCA affinity (kon=1.6x10(3) mol(-1) s(-1) L, koff=1x10(-3) s(-1)) by comparing the model predictions with experimental data. This study provides insight into the physics of affinity-based separations and can be extended to describe and optimize other nonequilibrium CE systems.