title: Bio-nanocapsule-based scaffold improves the sensitivity and ligand-binding capacity of mammalian receptors on the sensor chip.
abstract: Mammalian receptors are recognized as target molecules for drug discovery, and chemical libraries have been screened for both potential antagonists and agonists mainly by ligand-binding assays using immobilized receptors. A bio-nanocapsule (BNC) of approximately 30 nm that displays a tandem form of the protein A-derived immunoglobulin G (IgG) Fc-binding Z domains (denoted as ZZ-BNC) has been developed for both clustering and oriented immobilization of IgGs on the solid phase of immunosensors. In this study, human IgG1 Fc-fused vascular endothelial growth factor (VEGF) receptor was immobilized through ZZ-BNC on the sensor chip of quartz crystal microbalance (ZZ-BNC-coating). When compared with direct adsorption and protein A-coating, the sensor chip showed higher sensitivity (∽46- and ∽165-fold, respectively) and larger ligand-binding capacity (∽4- and ∽18-fold, respectively). Furthermore, the number of VEGF molecules bound to its receptor increased from 0.20 (direct adsorption) to 2.06 by ZZ-BNC-coating, strongly suggesting that ZZ-BNC reduced the steric hindrance near ligand recognition sites through oriented immobilization. Similarly, the sensitivity and ligand-binding capacity of leptin and prolactin receptors were both enhanced at a level comparable to that observed for the VEGF receptor. Thus, the combination of ZZ-BNC and Fc-fused receptors could significantly improve the function of ligand-binding assays.
authors.affiliation: The Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Japan.|Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya, Japan.
authors.forename: Masumi
authors.initials: M
authors.lastname: Iijima
authors.affiliation: The Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Japan.|Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya, Japan.
authors.forename: Nobuo
authors.initials: N
authors.lastname: Yoshimoto
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya, Japan.
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Niimi
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya, Japan.
authors.forename: Andrés D
authors.initials: AD
authors.lastname: Maturana
authors.affiliation: The Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Japan. skuroda@sanken.osaka-u.ac.jp.|Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya, Japan. skuroda@sanken.osaka-u.ac.jp.
authors.forename: Shun'ichi
authors.initials: S
authors.lastname: Kuroda
mesh_terms: D000327:Adsorption; D015374:Biosensing Techniques / Q000379:methods; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry*; D020738:Leptin / Q000737:chemistry; D008024:Ligands; D053769:Nanocapsules / Q000737:chemistry*; D011981:Receptors, Prolactin / Q000737:chemistry; D040262:Receptors, Vascular Endothelial Growth Factor / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000276:immunology; D042461:Vascular Endothelial Growth Factor A / Q000032:analysis / Q000378:metabolism*
chemical_list: D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D020738:Leptin; D008024:Ligands; D053769:Nanocapsules; D011981:Receptors, Prolactin; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D040262:Receptors, Vascular Endothelial Growth Factor
keywords: Bio-nanocapsules; Biosensors; IgG Fc-fused receptors; Oriented immobilization; Quartz crystal microbalance