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Genetic fusion of single-chain variable fragments to partial spider silk improves target detection in micro- and nanoarrays.

Thatikonda, Naresh; Delfani, Payam LU ; Jansson, Ronnie; Petersson, Linn LU ; Lindberg, Diana; Wingren, Christer LU and Hedhammar, My (2015) In Biotechnology Journal 11(3). p.437-448
Abstract
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... (More)
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. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Biotechnology Journal
volume
11
issue
3
pages
437 - 448
publisher
John Wiley & Sons
external identifiers
  • pmid:26470853
  • scopus:84959232625
  • wos:000372141400016
ISSN
1860-6768
DOI
10.1002/biot.201500297
language
English
LU publication?
yes
id
a51e8dd6-262e-41fe-a003-f6f01c66a938 (old id 8152161)
date added to LUP
2015-11-13 13:16:24
date last changed
2017-10-01 03:15:52
@article{a51e8dd6-262e-41fe-a003-f6f01c66a938,
  abstract     = {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.},
  author       = {Thatikonda, Naresh and Delfani, Payam and Jansson, Ronnie and Petersson, Linn and Lindberg, Diana and Wingren, Christer and Hedhammar, My},
  issn         = {1860-6768},
  language     = {eng},
  month        = {10},
  number       = {3},
  pages        = {437--448},
  publisher    = {John Wiley & Sons},
  series       = {Biotechnology Journal},
  title        = {Genetic fusion of single-chain variable fragments to partial spider silk improves target detection in micro- and nanoarrays.},
  url          = {http://dx.doi.org/10.1002/biot.201500297},
  volume       = {11},
  year         = {2015},
}