Multivalent anchoring and oriented display of single-domain antibodies on cellulose

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DOIResolve DOI: http://doi.org/10.3390/s90705351
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TypeArticle
Journal titleSensors
ISSN1424-8220
Volume9
Issue7
Pages53515367; # of pages: 17
Subjectsingle-domain antibody; cellulose-binding module; bispecific; pentamer; Staphylococcus aureus; pathogen detection
AbstractAntibody 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 highavidity binding reagents on cellulosic filters for sensing of pathogens, biomarkers and environmental pollutants.
Publication date
LanguageEnglish
AffiliationNational Research Council Canada (NRC-CNRC); NRC Institute for Biological Sciences
Peer reviewedYes
NPARC number15329256
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Record identifier2abcdbb1-4cbe-4f7b-93c5-7c30b13cee94
Record created2010-05-21
Record modified2016-05-09
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