Affinity probe capillary electrophoresis: analysis of recombinant human growth hormone with a fluorescent labeled antibody fragment.
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.
Shimura
K
K
Barnett Institute, Northeastern University, Boston, Massachusetts 02115.
Karger
B L
BL
D004586:Electrophoresis / Q000379:methods; D005455:Fluorescent Antibody Technique; D013006:Growth Hormone / Q000032:analysis* / Q000276:immunology; D006801:Humans; D007128:Immunoglobulin Fragments / Q000032:analysis / Q000276:immunology; D011994:Recombinant Proteins / Q000032:analysis / Q000276:immunology
D007128:Immunoglobulin Fragments; D011994:Recombinant Proteins; D013006:Growth Hormone