Antibody arrays hold considerable potential in a variety of applications including proteomics research,
drug discovery, and diagnostics. Many of the schemes used to fabricate the arrays fail to immobilize the
antibodies at a uniform density or in a single orientation; consequently, the
immobilized antibodies recognize their
antigens with variable efficiency. This paper describes a strategy to immobilize
antibodies in a single orientation, with a controlled density, using the covalent interaction between
cutinase and its suicide substrate.
Protein fusions between
cutinase and five
antibodies of three different types (scFv, V(HH), and FN3) were prepared and immobilized upon self-assembled monolayers (
SAMs) presenting a
phosphonate capture
ligand. The
immobilized antibodies exhibit high affinity and selectivity for their target
antigens, as monitored by surface plasmon resonance and fluorescence scanning. Furthermore, by changing the density of capture
ligand on the SAM the density of the immobilized antibody could be controlled. The monolayers, which also present a tri(
ethylene glycol) group, are inert to nonspecific adsorption of
proteins and allow the detection of a specific
antigen in a
complex mixture. The demonstration of
cutinase-directed antibody immobilization with insert
SAMs provides a straightforward and robust method for preparing antibody chips.