MAPPING OF THE ANTIBODY-BINDING AND RECEPTOR-BINDING DOMAINS OF GRANULOCYTE-COLONY-STIMULATING FACTOR USING AN OPTICAL BIOSENSOR - COMPARISON WITH ENZYME-LINKED-IMMUNOSORBENT-ASSAY COMPETITION STUDIES

An automated optical biosensor instrument for measuring molecular inte ractions (Pharmacia BIAcore) has been used to characterise the epitope s recognised by 15 monoclonal antibodies raised against recombinant hu man granulocyte colony-stimulating factor (G-CSF). The BIAcore combine s an autosampler and integrated microfluidic cartridge for the introdu ction and transportation of samples to the sensor chip surface, with s urface plasmon resonance to detect binding events. A rabbit anti-mouse Fc antibody, coupled to the sensor surface in situ using conventional protein chemistry techniques, was used to capture an anti-G-CSF monoc lonal antibody. G-CSF was bound to this antibody by injection over the sensor surface. Multi-site binding experiments were then performed in which other anti-G-CSF monoclonal antibodies were injected sequential ly over the surface, and their ability to bind to the G-CSF in a multi molecular complex monitored in real time. Results obtained using the b iosensor have been compared with data obtained by cross competition st udies using biotinylated antibodies or antibody binding studies using chemically or enzymatically derived G-CSF peptide fragments or synthet ic peptides. The results of these studies are in excellent agreement w ith the data from the BIAcore, although modification of the antibody o r G-CSF occasionally altered the epitope affinity.