title: Genetic fusion of single-chain variable fragments to partial spider silk improves target detection in micro- and nanoarrays.
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 were 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.
authors.affiliation: Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden.
authors.forename: Naresh
authors.initials: N
authors.lastname: Thatikonda
authors.affiliation: Department of Immunotechnology and CREATE Health, Lund University, Lund, Sweden.
authors.forename: Payam
authors.initials: P
authors.lastname: Delfani
authors.affiliation: Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden.
authors.forename: Ronnie
authors.initials: R
authors.lastname: Jansson
authors.affiliation: Department of Immunotechnology and CREATE Health, Lund University, Lund, Sweden.
authors.forename: Linn
authors.initials: L
authors.lastname: Petersson
authors.affiliation: Barentzymes AS, Tromsoe, Norway.
authors.forename: Diana
authors.initials: D
authors.lastname: Lindberg
authors.affiliation: Department of Immunotechnology and CREATE Health, Lund University, Lund, Sweden. christer.wingren@immun.lth.se.
authors.forename: Christer
authors.initials: C
authors.lastname: Wingren
authors.affiliation: Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden. myh@kth.se.|Division of Protein Technology, School of Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden. myh@kth.se.
authors.forename: My
authors.initials: M
authors.lastname: Hedhammar
mesh_terms: D000818:Animals; D055910:Antibodies, Immobilized / Q000235:genetics / Q000378:metabolism; D000915:Antibody Affinity; D006801:Humans; D046228:Microarray Analysis; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D047011:Silk / Q000235:genetics* / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000235:genetics* / Q000378:metabolism; D013112:Spiders / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D055910:Antibodies, Immobilized; D011993:Recombinant Fusion Proteins; D047011:Silk; D057127:Single-Chain Antibodies
keywords: Antigen detection; Microarray; Nanoarray; Single-chain variable fragment; Spider silk