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Detection of Antigens Using a Protein-DNA Chimera Developed by Enzymatic Covalent Bonding with phiX Gene A
KTH, School of Biotechnology (BIO), Molecular Biotechnology.
KTH, School of Biotechnology (BIO), Molecular Biotechnology.ORCID iD: 0000-0003-4214-6991
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2012 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 84, no 11, 5040-5046 p.Article in journal (Refereed) Published
Abstract [en]

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(mab2s) (A*-Zmab). The phi X174 gene A* protein is an enzyme that can covalently link with DNA, while the Z(mab2s) 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 gamma 1 (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-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.

Place, publisher, year, edition, pages
2012. Vol. 84, no 11, 5040-5046 p.
Keyword [en]
Antigen-binding activities, Binding capability, Biosensing, Covalent bonding, Enzyme linked immunosorbent assay, Fusion proteins, Gel mobility, Heterogeneous products, Protein functions, Protein moiety, Rolling circle amplifications, Sensitive detection, Site-specific, Antibodies, Antigens, Genes
National Category
Analytical Chemistry
URN: urn:nbn:se:kth:diva-98720DOI: 10.1021/ac300708rISI: 000304783100057ScopusID: 2-s2.0-84861904144OAI: diva2:539143
QC 20120703Available from: 2012-07-03 Created: 2012-07-02 Last updated: 2012-07-03Bibliographically approved

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Grimm, SebastianNygren, Per-Åke
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