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The structural orientation of antibody layers bound to engineered biosensor surfaces

Lookup NU author(s): Anton Le Brun, Dr Deepan Shah, Professor Jeremy LakeyORCiD


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This paper describes a membrane protein array that binds immunoglobulin G at its constant regions whilst leaving the variable regions free to bind antigen. The scaffold of the array is the transmembrane domain of outer membrane protein A (tOmpA) from Escherichia colt engineered to assemble as an oriented monolayer on gold surfaces via a single cysteine residue. Other protein domains can be fused to the N and C termini of the scaffold. In this study we use circularly permuted ctOmpA fused to two Z domains of Staphylococcus aureus protein A (ZZctOmpA) to create the immunoglobulin G-binding array. The solution structure of the engineered proteins was assessed by circular dichroism spectroscopy. Assembly of the array, attachment of antibodies and antigen binding were measured using surface plasmon resonance and neutron reflection. Compared to mouse IgG2, polyclonal IgG from rabbit bound very strongly to ZZctOmpA and the dissociation of the immunoglobulin was slow enough to allow neutron reflection studies of the assembled layer with antigen. Using both magnetic and isotopic contrasts a complete layer by layer model was defined which revealed that the 223 A high layer contains antibodies in an upright orientation. (C) 2011 Elsevier Ltd. All rights reserved.

Publication metadata

Author(s): Le Brun AP, Holt SA, Shah DSH, Majkrzak CF, Lakey JH

Publication type: Article

Publication status: Published

Journal: Biomaterials

Year: 2011

Volume: 32

Issue: 12

Pages: 3303-3311

Print publication date: 08/02/2011

ISSN (print): 0142-9612

ISSN (electronic): 1878-5905

Publisher: Elsevier BV


DOI: 10.1016/j.biomaterials.2011.01.026


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