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Proceedings Paper

Fluorescence correlation spectroscopy to study antibody binding and stoichiometry of complexes
Author(s): Kerry M. Swift; Edmund D. Matayoshi
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Paper Abstract

FCS (fluorescence correlation spectroscopy) was used to study the association at the single molecule level of tumor necrosis factor alpha (TNF-α) and two of its protein antagonists Humira(TM) (adalimumab), a fully humanized monoclonal antibody, and Enbrel(TM) (etanercept), a soluble form of the TNF receptor. Single molecule approaches potentially have the advantage not only of enhanced sensitivity, but also of observing at equilibrium the details that would otherwise be lost in classical ensemble experiments where heterogeneity is averaged. We prepared fluorescent conjugates of the protein drugs and their biological target, the trimeric soluble form of TNF-α. The bivalency of adalimumab and the trimeric nature of TNF-α potentially allow several forms of associative complexes that may differ in stoichiometry. Detailed knowledge of this reaction may be relevant to understanding adalimumab's pharmacological properties. Our FCS data showed that a single trimeric TNF-α can bind up to three adalimumab molecules. Under some conditions even larger complexes are formed, apparently the result of cross-linking of TNF-α trimers by adalimumab. In addition, distinct differences between Humira and Enbrel were observed in their association with TNF-α.

Paper Details

Date Published: 21 February 2008
PDF: 5 pages
Proc. SPIE 6862, Single Molecule Spectroscopy and Imaging, 686208 (21 February 2008); doi: 10.1117/12.767807
Show Author Affiliations
Kerry M. Swift, Abbott Labs. (United States)
Edmund D. Matayoshi, Abbott Labs. (United States)

Published in SPIE Proceedings Vol. 6862:
Single Molecule Spectroscopy and Imaging
Jörg Enderlein; Zygmunt K. Gryczynski; Rainer Erdmann, Editor(s)

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