Genetic fusion of single-chain variable fragments to partial spider silk improves target detection in micro- and nanoarrays.
Author
Summary, in English
Immobilizing biomolecules with retained functionality and stability on solid supports is crucial for generation of sensitive immunoassays. However, upon use of conventional immobilization strategies, a major portion of the biomolecules (e.g. antibodies) frequently tends to lose their bioactivity. In this study, we describe a procedure to immobilize human single-chain variable fragment (scFv) via genetic fusion to partial spider silk, which have a high tendency to adhere to solid supports. Two scFvs, directed towards serum proteins, were genetically fused to partial spider silk proteins and expressed as silk fusion proteins in E. coli. Antigen binding ability of scFvs attached to a partial silk protein denoted RC was investigated using microarray analysis, whereas scFvs fused to the NC silk variant was examined using nanoarrays. Results from micro- and nanoarrays confirmed the functionality of scFvs attached to both RC and NC silk, and also for binding of targets in crude serum. Furthermore, the same amount of added scFv gives higher signal intensity when immobilized via partial spider silk compared to when immobilized alone. Together, the results suggest that usage of scFv-silk fusion proteins in immunoassays could improve target detection, in the long run enabling novel biomarkers to be detected in crude serum proteomes.
Department/s
Publishing year
2015-10-16
Language
English
Pages
437-448
Publication/Series
Biotechnology Journal
Volume
11
Issue
3
Document type
Journal article
Publisher
John Wiley & Sons Inc.
Topic
- Diagnostic Biotechnology
Status
Published
ISBN/ISSN/Other
- ISSN: 1860-6768