Characterization of the interaction between human complement protein C4 and a single-chain variable fragment antibody by capillary electrophoresis and surface plasmon resonance

Immunoaffinity capillary electrophoresis and surface plasmon resonance have been used for the characterization of the interaction between two large-sized proteins, the human complement protein C4 and the single-chain variable fragment C43. The rather high kinetic rate constants as determined by surface plasmon resonance pointed out that a capillary electrophoresis method had to be applied, in which the labeled C4 is preincubated with C43 before injection and the same concentration of C43 is included in the running buffer. Analysis of the concentration dependence of the small mobility shift of the fluorescent C4 signal upon binding of C43 resulted in a dissociation constant that was comparable to the one obtained with surface plasmon resonance. This study is one of the few examples where capillary electrophoresis is successfully used to characterize the interaction between large proteins.