Production and characterization of a genetically engineered anti-caffeine camelid antibody and its use in immunoaffinity chromatography

This work demonstrates the feasibility of using a camelid single domain antibody for immunoaffinity chromatographic seperation of small molecules An anti-caffeine VHH antibody was produced by grafting the complementanty determining sequences of a previously generated antibody onto an anti-RNase A antibody scaffold. followed by expression in E colt. Analysis of the binding properties of the antibody by ELISA and fluorescence-based thermal shift assays showed that it recognizes not only caffeine, but also theophylline. theobiomine. and paraxanthine, albeit with lower affinity Further investigation of the effect of environmental conditions. I e, temperature, pH. and ionic strength, on the antibody using these methods provided useful information about potential elation conditions to be used in chromatographic applications. Immobilization of the VHH onto a high flow-through synthetic support material resulted In a stationary phase capable of separating caffeine and its metabolites (C) 2009 Elsevier B.V. All rights reserved