Micropreparative ligand fishing with a cuvette-based optical mirror resonance biosensor

We have previously demonstrated the role of an optical biosensor (BIAcore 2 000) as a specific detector to monitor chromatographic fractions during the purification and characterisation of ligands for orphan biomolecules. We h ave now extended this application to perform micropreparative ligand fishin g directly on the sensor surface using an automated cuvette-based optical b iosensor (Iasys Auto+) equipped with a high-capacity carboxymethyldextran s urface (surface area 16 mm(2)). Using a F(ab)(2)' fragment of the A33 monoc lonal antibody as bait, we have recovered microgram quantities of essential ly homogeneous A33 ligand from the sensor surface in a form suitable for su bsequent sensitive and specific down stream analysis (micropreparative HPLC , sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western bl otting). The design of the cuvette-based system facilitates recovery of des orbed material from the constrained workspace in small volumes at high conc entration. The use of on-surface detection allows the surface viability to be continuously monitored and permits direct quantitation of both bound and recovered material. (C) 2000 Elsevier Science BN. All rights reserved.