Post-capillary affinity detection of protein microheterogeneity in capillary zone electrophoresis

Post-capillary affinity detection is developed to selectively evaluate protein microheterogeneity in complex sample matrices. In this scheme, separation of protein variants is accomplished prior to affinity detection. A post-capillary reactor is constructed to allow the transfer of separated variants as well as the addition of a fluorescein-labeled analyte-binding protein into the reaction capillary. Affinity complexes are formed within the reaction capillary and monitored by laser-induced fluorescence detection. Fluorescence enhancement, upon affinity complex formation, allows selective detection of protein variants. Successful resolution of IgG Fc variants is presented and compared with affinity capillary electrophoresis techniques reported in the literature. Additionally, the effects of analyte transfer and affinity complex formation on the resolution and detection limits of IgG Fc variants are discussed.