(335b) Simple and Robust Antibody Fragment Immobilization for Detection of Antigens

Arrays of antibody fragments have great potential in diagnostics and screening of molecule libraries, but synthesis of arrays containing a large number of different antibody fragments requires a robust immobilization approach that does not depend on optimization for individual antibody fragments. We have developed a simple and robust immobilization method that takes advantage of the high-affinity and specific interaction of biotin and streptavidin to immobilize single-chain variable fragment (scFv) antibodies while maintaining their ability to bind their target antigens. scFvs are biotinylated in vivo during recombinant expression in Escherichia coli cells by genetically fusing the scFvs to biotinylation sequences. We efficiently immobilized a variety of biotinylated scFvs onto streptavidin-coated plates directly from cell lysates, with no need for purification prior to immobilization. The immobilized scFvs retain the ability to bind their target proteins and retain the specificity of the soluble scFvs. Antigens can be detected from a purified sample or from cell lysate. Furthermore, the immobilization of the scFvs is stable for at least 40 days after storage in buffer at 4 °C or -20 °C with no loss of binding activity. The simplicity and robustness of our method make it a promising approach for immobilizing a wide variety of antibody fragments and other proteins in an array format for diagnostics, screening, or any other application that necessitates immobilization of large number of functional antibody fragments.