(789e) Purification-Free Antibody Fragment Immobilization Using in Vivo Biotinylation

Authors: 
Ikonomova, S. P., University of Maryland
Karlsson, A. J., University of Maryland
Antibody fragments are attractive tools in research and diagnostics due to their strong antigen-binding ability and their ease of production in Escherichia coli. Such applications could benefit from an array of different antibody fragments immobilized on a surface, and constructing antibody arrays would necessitate an immobilization method that is amenable to many antibody fragments. We have established a simple immobilization method for single-chain variable fragment (scFv) antibodies that is based on the biotin-streptavidin interaction and does not require purifying scFvs prior to immobilization. We genetically fused two biotinylation tagsâ??the biotin carboxyl carrier protein (BCCP) or the AviTag minimal sequenceâ??to scFvs with different frameworks and antigens. scFvs were site-specifically biotinylated in vivo by endogenous E. coli biotin ligases during protein expression. All scFv fusions were successfully immobilized directly from cell lysates onto streptavidin-coated plates. We found that scFvs biotinylated via the BCCP tag tended to immobilize better than those biotinylated via the AviTag, even when biotinylation efficiency was improved with the biotin ligase BirA. The immobilized scFvs retained the ability to bind to their antigen, whether the antigen is a purified sample or it is detected in a cell lysate. The immobilization is stable, and the scFvs maintain their functionality to bind to their target for at least 100 days after immobilization and storage at 4 °C or -20 °C. The simplicity and robustness of our method make it a promising approach for future applications that require immobilization of multiple antibody fragments.