Detection of antigens using a protein-DNA chimera developed by enzymatic covalent bonding with phiX gene A*

Analytical Chemistry
Farhima AkterEiry Kobatake

Abstract

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 φX174 gene A* protein and Z(mab25) (A*-Zmab). The φX174 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 γ1 (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 80% after a 15 min reaction. Finally, sensitive detection of human interferon-γ (IFN-γ) was achieved by immuno-RCA using our fusion protein in sandwich ELISA format. This new approach of the use of site-...Continue Reading

References

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Citations

Nov 10, 2015·Chemical Reviews·Yongxi ZhaoChunhai Fan
Jan 24, 2018·Biotechnology Letters·Yasumasa MashimoEiry Kobatake
Jan 15, 2014·Analytical Sciences : the International Journal of the Japan Society for Analytical Chemistry·Toshiro Kobori, Hirokazu Takahashi
Oct 15, 2019·Bioconjugate Chemistry·Christiane StillerAmelie Eriksson Karlström
Jun 5, 2014·ACS Nano·Diana A PippigHermann E Gaub

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