Detection of Antigens Using a Protein-DNA Chimera Developed by Enzymatic Covalent Bonding with phiX Gene A~(*)

摘要

The chemical reactions used to make antibody--DNA conjugates in many immunoassays diminish antigen-binding activity and yield heterogeneous products. Here, we address these issues by developing an antibody-based rolling circle amplification (RCA) strategy using a fusion of (phi)X174 gene A~(*) protein and Z_(mab25) (A~(*)-Zmab). The (phi)X174 gene A~(*) protein is an enzyme that can covalently link with DNA, while the Z_(mab25) protein moiety can bind to specific species of antibodies. The DNA in an A~(*)-Zmab conjugate was attached to the A~(*) protein at a site chosen to not interfere with protein function, as determined by enzyme-linked immunosorbent assay (ELISA) and gel mobility shift analysis. The novel A~(*)-Zmab-DNA conjugate retained its binding capabilities to a specific class of murine immunoglobulin gamma1 (IgG1) but not to rabbit IgG. This indicates the generality of the A~(*)-Zmab-based immuno-RCA assay that can be used in-sandwich ELISA format. Moreover, the enzymatic covalent method dramatically increased the yields of A~(*)-Zmab-DNA conjugates up to 80percent after a 15 min reaction. Finally, sensitive detection of human interferon-gamma (IFN-gamma) was achieved by immuno-RCA using our fusion protein in sandwich ELISA format. This new approach of the use of site-specific enzymatic DNA conjugation to proteins should be applicable to fabrication of novel immunoassays for biosensing.