title: Capture-stabilize approach for membrane protein SPR assays.
abstract: Measuring the binding kinetics of antibodies to intact membrane proteins by surface plasmon resonance has been challenging largely because of the inherent difficulties in capturing membrane proteins on chip surfaces while retaining their native conformation. Here we describe a method in which His-tagged CXCR5, a GPCR, was purified and captured on a Biacore chip surface via the affinity tag. The captured receptor protein was then stabilized on the chip surface by limited cross-linking. The resulting chip surface retained ligand binding activity and was used for monoclonal antibody kinetics assays by a standard Biacore kinetics assay method with a simple low pH regeneration step. We demonstrate the advantages of this whole receptor assay when compared to available peptide-based binding assays. We further extended the application of the capture-stabilize approach to virus-like particles and demonstrated its utility analyzing antibodies against CD52, a GPI-anchored protein, in its native membrane environment. The results are the first demonstration of chemically stabilized chip surfaces for membrane protein SPR assays.
authors.affiliation: Genzyme Corporation, 49 New York Avenue, Framingham, MA 01701, USA.
authors.forename: Ruiyin
authors.initials: R
authors.lastname: Chu
authors.affiliation: Genzyme Corporation, 49 New York Avenue, Framingham, MA 01701, USA.
authors.forename: David
authors.initials: D
authors.lastname: Reczek
authors.affiliation: Genzyme Corporation, 49 New York Avenue, Framingham, MA 01701, USA.
authors.forename: William
authors.initials: W
authors.lastname: Brondyk
mesh_terms: D000906:Antibodies / Q000378:metabolism; D000915:Antibody Affinity; D015703:Antigens, CD / Q000378:metabolism; D000951:Antigens, Neoplasm / Q000378:metabolism; D001681:Biological Assay / Q000379:methods; D000074301:CD52 Antigen; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D055550:Protein Stability; D054380:Receptors, CXCR5 / Q000737:chemistry / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism; D020349:Surface Plasmon Resonance / Q000379:methods
chemical_list: D000906:Antibodies; D015703:Antigens, CD; D000951:Antigens, Neoplasm; D000074301:CD52 Antigen; C000613862:CD52 protein, human; D006023:Glycoproteins; D008024:Ligands; D008565:Membrane Proteins; D010455:Peptides; D054380:Receptors, CXCR5; D011956:Receptors, Cell Surface