Micropreparative ligand fishing with a cuvette-based optical mirror resonance biosensor.
We have previously demonstrated the role of an optical biosensor (BIAcore 2000) as a specific detector to monitor chromatographic fractions during the purification and characterisation of ligands for orphan biomolecules. We have now extended this application to perform micropreparative ligand fishing directly on the sensor surface using an automated cuvette-based optical biosensor (Iasys Auto+) equipped with a high-capacity carboxymethyldextran surface (surface area 16 mm2). Using a F(ab)2' fragment of the A33 monoclonal antibody as bait, we have recovered microgram quantities of essentially homogeneous A33 ligand from the sensor surface in a form suitable for subsequent sensitive and specific down stream analysis (micropreparative HPLC, sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting). The design of the cuvette-based system facilitates recovery of desorbed material from the constrained workspace in small volumes at high concentration. The use of on-surface detection allows the surface viability to be continuously monitored and permits direct quantitation of both bound and recovered material.
Catimel
B
B
Ludwig Institute for Cancer Research, Melbourne Tumour Biology Branch, Royal Melbourne Hospital, Parkville, Victoria, Australia.
Weinstock
J
J
Nerrie
M
M
Domagala
T
T
Nice
E C
EC
D015374:Biosensing Techniques; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D008024:Ligands; D055095:Optics and Photonics; D014407:Tumor Cells, Cultured
D008024:Ligands