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Sensors 2014, 14(6), 10846-10863;

Isolation and Epitope Mapping of Staphylococcal Enterotoxin B Single-Domain Antibodies

American Society for Engineering Education, Postdoctoral Fellow at the Naval Research Laboratory, Washington, DC 20375, USA
Center for Biomolecular Science and Engineering, Naval Research Laboratory, Washington, DC 20375, USA
Author to whom correspondence should be addressed.
Received: 8 May 2014 / Revised: 9 June 2014 / Accepted: 10 June 2014 / Published: 19 June 2014
(This article belongs to the Special Issue Chemo- and Biosensors for Security and Defense)
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Single-domain antibodies (sdAbs), derived from the heavy chain only antibodies found in camelids such as llamas have the potential to provide rugged detection reagents with high affinities, and the ability to refold after denaturation. We have isolated and characterized sdAbs specific to staphylococcal enterotoxin B (SEB) which bind to two distinct epitopes and are able to function in a sandwich immunoassay for toxin detection. Characterization of these sdAbs revealed that each exhibited nanomolar binding affinities or better. Melting temperatures for the sdAbs ranged from approximately 60 °C to over 70 °C, with each demonstrating at least partial refolding after denaturation and several were able to completely refold. A first set of sdAbs was isolated by panning the library using adsorbed antigen, all of which recognized the same epitope on SEB. Epitope mapping suggested that these sdAbs bind to a particular fragment of SEB (VKSIDQFLYFDLIYSI) containing position L45 (underlined), which is involved in binding to the major histocompatibility complex (MHC). Differences in the binding affinities of the sdAbs to SEB and a less-toxic vaccine immunogen, SEBv (L45R/Y89A/Y94A) were also consistent with binding to this epitope. A sandwich panning strategy was utilized to isolate sdAbs which bind a second epitope. This epitope differed from the initial one obtained or from that recognized by previously isolated anti-SEB sdAb A3. Using SEB-toxin spiked milk we demonstrated that these newly isolated sdAbs could be utilized in sandwich-assays with each other, A3, and with various monoclonal antibodies. View Full-Text
Keywords: nanobodies; surface plasmon resonance; circular dichroism nanobodies; surface plasmon resonance; circular dichroism

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This is an open access article distributed under the Creative Commons Attribution License (CC BY 3.0).

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Turner, K.B.; Zabetakis, D.; Legler, P.; Goldman, E.R.; Anderson, G.P. Isolation and Epitope Mapping of Staphylococcal Enterotoxin B Single-Domain Antibodies. Sensors 2014, 14, 10846-10863.

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