GENETIC-ENGINEERING OF A SINGLE-CHAIN ANTIBODY FRAGMENT FOR SURFACE IMMOBILIZATION IN AN OPTICAL BIOSENSOR

The development of a biosensor based on a genetically engineered biomo lecule offers many potential advantages to sensors that rely on natura l proteins only. Here we present how protein engineering techniques ca n be used to introduce a functional unit for surface immobilization in to a single-chain antibody fragment (scF(v)). A peptide known to mimic the binding properties of biotin was fused to the carboxyterminus of the phosphorylcholine-binding scF(v) fragment of IgA McPC603. This fus ion protein could be immobilized on a streptavidin monolayer. The resu lting scF(v) monolayer was capable of binding a fluorescently labeled phosphorylcholine analog, as detected by total internal reflection flu orescence. In contrast, an scF(v) monolayer formed by introducing biot in through chemical modification was not capable of binding phosphoryl choline. These results demonstrate the utility of site-specific, orien ted attachment strategies in the formation of protein monolayers in op tical sensors, made possible by the use of protein engineering techniq ues. (C) 1998 Elsevier Science S.A. All rights reserved.