Bispecific antibody-mediated detection of the staphylococcus aureus thermonuclease

Wagstaffe, Sarah Jayne, Hill, Katja E. ORCID: https://orcid.org/0000-0002-8590-0117, Williams, David Wynne ORCID: https://orcid.org/0000-0002-7351-5131, Randle, Beverley J., Thomas, David William ORCID: https://orcid.org/0000-0001-7319-5820, Stephens, Philip ORCID: https://orcid.org/0000-0002-0840-4996 and Riley, D. Jason 2012. Bispecific antibody-mediated detection of the staphylococcus aureus thermonuclease. Analytical Chemistry 84 (14) , pp. 5876-5884. 10.1021/ac203403d

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Abstract

We report a novel fluorescence-based immunoassay which enables qualitative detection of the Staphylococcus aureus Thermonuclease (TNase) enzyme, thus providing confirmation of the presence of the S. aureus bacterium in vitro. The biomedical problem of chronic wound healing and the continuing emergence of antibiotic-resistant species is addressed in the development of a detection system capable of the rapid, real-time assessment of bacterial load and diversity. The use of bispecific antibodies (BsAb) provides integration of the molecular detection and signal response components of a standard immunoassay due to steric hindrance-mediated release of prebound fluorescent reporter molecules upon specific binding of TNase to adjacent sites. Rhodamine and fluorescein-labeled hemocyanin from Megathura crenulata (KLH) were prepared as effective immunoconjugates containing a sensitive fluorescent reporter moiety. BsAb that both specifically quenched the fluorescence of the reporter conjugate and bound the TNase target antigen were produced using cell fusion techniques. Assays were then performed to analyze the properties attributable to the steric hindrance-mediated release of the fluorescent reporter molecules upon adjacent TNase binding. This was performed by monitoring the intensity of fluorescence emission of the immunogenic reporter conjugate released into an aqueous environment at 578 and 520 nm, respectively.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Dentistry Systems Immunity Research Institute (SIURI)
Subjects:	Q Science > QR Microbiology R Medicine > R Medicine (General)
Publisher:	American Chemical Society
ISSN:	0003-2700
Funders:	EPSRC
Last Modified:	06 Nov 2024 22:25
URI:	https://orca.cardiff.ac.uk/id/eprint/31910

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