Affinity probe capillary electrophoresis: analysis of recombinant human growth hormone with a fluorescent labeled antibody fragment

Abstract

A new highly sensitive microscale analytical procedure called affinity probe capillary electrophoresis (APCE) is presented. One of the two species, which can form a biospecific complex, is labeled with a fluorescent dye. The affinity probe is used to detect the analyte as a complex after the separation of the excess free probe by capillary electrophoresis. As an example of this approach, an FAB' fragment of a mouse monoclonal antibody (anti-human growth hormone) was labeled with tetramethylrhodamine-iodoacetamide at a hinge region thiol group. Samples were mixed with the purified labeled antibody fragment, and the associated complex was separated by capillary isoelectric focusing with detection by laser-induced fluorescence. In these preliminary results, methionyl recombinant human growth hormone (met-rhGH) could be successfully determined down to detection levels of approximately 5 x 10(-12) M. Mono- and dideamidated variants of met-rhGH were detected simultaneously with the nondeamidated form of the antigen as separate peaks.