Generation of High‐Specificity Antibodies against Membrane Proteins Using DNA‐Gold Micronanoplexes for Gene Gun Immunization. (21st February 2018)

• Record Type: Journal Article
• Authors: Hansen, Debra T.; Craciunescu, Felicia M.; Fromme, Petra; Johnston, Stephen A.; Sykes, Kathryn F.
• Editors: Coligan, John E.; Dunn, Ben M.; Speicher, David W.; Wingfield, Paul T.
• Is Part Of: Current protocols in protein science. Volume 91(2018)
• Page Start: 29.20.1
• Page End: 29.20.22
• Publication Date: 2018-02-21
• Subjects: antibodies -- membrane protein -- genetic immunization -- DNA immunization -- conformational epitopes -- gene gun; Proteins -- Laboratory manuals; Proteins; Clinical Laboratory Techniques; Genetic Techniques; Immunologic Techniques; Proteins; Laboratory manuals; 572.6028
• DOI: 10.1002/cpps.50
• Languages: English
• ISSNs: 1934-3655

Abstract

Abstract: Membrane proteins are the molecular interface of the cell and its environs; however, studies of membrane proteins are highly technically challenging, mainly due to instability of the isolated protein. Towards the production of antibodies that recognize properly folded and stabilized forms of membrane protein antigen, we describe a DNA‐based immunization method for mice that expresses the antigen in the membranes of dendritic cells, thus allowing direct presentation to the immune system. This genetic immunization approach employs a highly efficient method of biolistic delivery based on DNA‐gold micronanoplexes, which are complexes of micron‐sized gold particles that allow dermal penetration and nanometer‐sized gold particles that provide a higher surface area for DNA binding than micron gold alone. In contrast to antibodies derived from immunizations with detergent‐solubilized protein or with protein fragments, antibodies from genetic immunization are expected to have a high capacity for binding conformational epitopes and for modulating membrane protein activity. © 2018 by John Wiley & Sons, Inc.