title: Immobilizing affinity proteins to nitrocellulose: a toolbox for paper-based assay developers.
abstract: To enable enhanced paper-based diagnostics with improved detection capabilities, new methods are needed to immobilize affinity reagents to porous substrates, especially for capture molecules other than IgG. To this end, we have developed and characterized three novel methods for immobilizing protein-based affinity reagents to nitrocellulose membranes. We have demonstrated these methods using recombinant affinity proteins for the influenza surface protein hemagglutinin, leveraging the customizability of these recombinant "flu binders" for the design of features for immobilization. The three approaches shown are: (1) covalent attachment of thiolated affinity protein to an epoxide-functionalized nitrocellulose membrane, (2) attachment of biotinylated affinity protein through a nitrocellulose-binding streptavidin anchor protein, and (3) fusion of affinity protein to a novel nitrocellulose-binding anchor protein for direct coupling and immobilization. We also characterized the use of direct adsorption for the flu binders, as a point of comparison and motivation for these novel methods. Finally, we demonstrated that these novel methods can provide improved performance to an influenza hemagglutinin assay, compared to a traditional antibody-based capture system. Taken together, this work advances the toolkit available for the development of next-generation paper-based diagnostics.
authors.affiliation: Department of Bioengineering, University of Washington, 3720 15th Ave NE, Seattle, WA, 98105, USA. cholst@uw.edu.
authors.forename: Carly A
authors.initials: CA
authors.lastname: Holstein
authors.affiliation: Department of Bioengineering, University of Washington, 3720 15th Ave NE, Seattle, WA, 98105, USA.|Department of Biochemistry, University of Washington, 1705 NE Pacific St., Seattle, WA, 98195-7350, USA.
authors.forename: Aaron
authors.initials: A
authors.lastname: Chevalier
authors.affiliation: Department of Bioengineering, University of Washington, 3720 15th Ave NE, Seattle, WA, 98105, USA.
authors.forename: Steven
authors.initials: S
authors.lastname: Bennett
authors.affiliation: Department of Bioengineering, University of Washington, 3720 15th Ave NE, Seattle, WA, 98105, USA.
authors.forename: Caitlin E
authors.initials: CE
authors.lastname: Anderson
authors.affiliation: Department of Bioengineering, University of Washington, 3720 15th Ave NE, Seattle, WA, 98105, USA.
authors.forename: Karen
authors.initials: K
authors.lastname: Keniston
authors.affiliation: General Electric Global Research Center, 1 Research Cir, Niskayuna, NY, 12309, USA.
authors.forename: Cathryn
authors.initials: C
authors.lastname: Olsen
authors.affiliation: General Electric Global Research Center, 1 Research Cir, Niskayuna, NY, 12309, USA.
authors.forename: Bing
authors.initials: B
authors.lastname: Li
authors.affiliation: General Electric Global Research Center, 1 Research Cir, Niskayuna, NY, 12309, USA.
authors.forename: Brian
authors.initials: B
authors.lastname: Bales
authors.affiliation: General Electric Global Research Center, 1 Research Cir, Niskayuna, NY, 12309, USA.
authors.forename: David R
authors.initials: DR
authors.lastname: Moore
authors.affiliation: School of Chemical, Biological, and Environmental Engineering, Oregon State University, 103 Gleeson Hall, Corvallis, OR, 97331, USA.
authors.forename: Elain
authors.initials: E
authors.lastname: Fu
authors.affiliation: Department of Biochemistry, University of Washington, 1705 NE Pacific St., Seattle, WA, 98195-7350, USA.
authors.forename: David
authors.initials: D
authors.lastname: Baker
authors.affiliation: Department of Bioengineering, University of Washington, 3720 15th Ave NE, Seattle, WA, 98105, USA.
authors.forename: Paul
authors.initials: P
authors.lastname: Yager
mesh_terms: D000906:Antibodies / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods*; D003101:Collodion / Q000737:chemistry / Q000378:metabolism*; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism*; D010209:Paper; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D019809:Streptavidin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000906:Antibodies; D001426:Bacterial Proteins; D002352:Carrier Proteins; D055767:Immobilized Proteins; D011994:Recombinant Proteins; D003101:Collodion; D019809:Streptavidin
keywords: Nitrocellulose; Paper-based assays; Protein engineering; Protein immobilization