Novel approach for immunoassay capture antibody immobilization – Click chemistry with expanded genetic code

Abstract

Antibodies are commonly used in biosensors for diagnostics and research due to their ability to specifically bind certain molecules. However, current manufacturing techniques leave biosensors to use randomly oriented capture antibodies. This results in decreased antibody function as some antibodies denature, capture sites are blocked or antibodies wear off. Site specific immobilization has been studied before, but not with expanded genetic code.

In this study E.coli C.321.deltaA.exp -strain was used to produce fragmented antibodies (Fab) with noncanonical p-azido-l-phenylalanine amino acid incorporated in protein. These fabs were immobilized covalently in DBCO magnetic beads via bio-orthogonal azide-alkyne cycloaddition reaction. Coated beads were compared to passively and randomly coated beads by saturating them with labeled antigen.

Results show that Fabs can be modified by adding p-azido-l-phenylalanine into C-terminal end of protein chain without any compromises to antibody functionality or stability.