Fabrication of cysteine-modified antibodies with Fc-specific conjugation for covalent and oriented immobilization of native antibodies.

ABSTRACT

Covalent and oriented immobilization of antibodies (Abs) can substantially improve the sensitivity and stability of solid-phase immunoassays. By modifying the natural Abs with functional groups that provide unique handles for further conjugation, Abs could be immobilized onto the solid matrices with uniform orientation. Herein, an effective approach for Fc-specific modification of Abs was developed for the oriented and covalent immobilization of Abs. Twelve photoreactive Z-domain variants, incorporated with a photoactivable probe (p-benzoyl-L-phenylalanine, Bpa) at different positions and carrying a C-terminal Cys-tag (i.e. ZBpa-Cys variants), were individually constructed and produced in Escherichia coli and tested for photo-cross-linking to various IgGs. The different ZBpa-Cys variants demonstrated large differences in photo-conjugation efficiency for the tested IgGs. The conjugation efficiencies of 17thZBpa-Cys ranged from 90 % to nearly 100 % for rabbit IgG and mouse IgG2a, IgG2b and IgG3. Other variants, including 5thZBpa-Cys, 18thZBpa-Cys, 32thZBpa-Cys, and 35thZBpa-Cys, also displayed conjugation efficiencies of 61 %-83 % for mouse IgG1, IgG2a and IgG3. Subsequently, the photo-modified Abs, namely IgG-Cys conjugates, were covalently immobilized onto a maleimide group-functionalized solid-phase carrier on the basis of the reaction of sulfhydryl and maleimide. Thus, a generic platform for the controlled and oriented immobilization of Abs was developed, and the efficacy and potential of the proposed approach for sensitive immunoassays was demonstrated by detecting human α-fetoprotein.