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Sensors 2009, 9(7), 5351-5367; doi:10.3390/s90705351

Multivalent Anchoring and Oriented Display of Single-Domain Antibodies on Cellulose

1,2, 1,#, 3, 3, 1,2,3 and 1,3,*
1 Institute for Biological Sciences, National Research Council of Canada, 100 Sussex Drive, Ottawa, Ontario, Canada, K1A 0R6 2 Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada, K1H 8M5 3 Department of Environmental Biology, University of Guelph, Guelph, Ontario, Canada, N1G 2W1 # Present address: Caprotec Bioanalytics GmbH, Volmerstr 5, 12489, Berlin, Germany
* Author to whom correspondence should be addressed.
Received: 31 May 2009 / Revised: 11 June 2009 / Accepted: 7 July 2009 / Published: 7 July 2009
(This article belongs to the Special Issue Pathogen Sensors)
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Antibody engineering has allowed for the rapid generation of binding agents against virtually any antigen of interest, predominantly for therapeutic applications. Considerably less attention has been given to the development of diagnostic reagents and biosensors using engineered antibodies. Recently, we produced a novel pentavalent bispecific antibody (i.e., decabody) by pentamerizing two single-domain antibodies (sdAbs) through the verotoxin B subunit (VTB) and found both fusion partners to be functional. Using a similar approach, we have engineered a bispecific pentameric fusion protein consisting of five sdAbs and five cellulose-binding modules (CBMs) linked via VTB. To find an optimal design format, we constructed six bispecific pentamers consisting of three different CBMs, fused to the Staphylococcus aureus-specific human sdAb HVHP428, in both orientations. One bispecific pentamer, containing an N-terminal CBM9 and C-terminal HVHP428, was soluble, non-aggregating, and did not degrade upon storage at 4 ºC for over six months. This molecule was dually functional as it bound to cellulose-based filters as well as S. aureus cells. When impregnated in cellulose filters, the bispecific pentamer recognized S. aureus cells in a flow-through detection assay. The ability of pentamerized CBMs to bind cellulose may form the basis of an immobilization platform for multivalent display of high-avidity binding reagents on cellulosic filters for sensing of pathogens, biomarkers and environmental pollutants.
Keywords: single-domain antibody; cellulose-binding module; bispecific; pentamer; Staphylococcus aureus; pathogen detection single-domain antibody; cellulose-binding module; bispecific; pentamer; Staphylococcus aureus; pathogen detection
This is an open access article distributed under the Creative Commons Attribution License (CC BY) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Hussack, G.; Luo, Y.; Veldhuis, L.; Hall, J.C.; Tanha, J.; MacKenzie, R. Multivalent Anchoring and Oriented Display of Single-Domain Antibodies on Cellulose. Sensors 2009, 9, 5351-5367.

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