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Detection of Antige...
Detection of Antigens Using a Protein-DNA Chimera Developed by Enzymatic Covalent Bonding with phiX Gene A
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Akter, Farhima (author)
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Mie, Masayasu (author)
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- Grimm, Sebastian (author)
- KTH,Molekylär Bioteknologi
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- Nygren, Per-Åke (author)
- KTH,Molekylär Bioteknologi
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Kobatake, Eiry (author)
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(creator_code:org_t)
- American Chemical Society (ACS), 2012
- 2012
- English.
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In: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 84:11, s. 5040-5046
- Related links:
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https://urn.kb.se/re...
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https://doi.org/10.1...
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Abstract
Subject headings
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- 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.
Subject headings
- NATURVETENSKAP -- Kemi -- Analytisk kemi (hsv//swe)
- NATURAL SCIENCES -- Chemical Sciences -- Analytical Chemistry (hsv//eng)
Keyword
- 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
Publication and Content Type
- ref (subject category)
- art (subject category)
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