title: Significance of antibody orientation unraveled: well-oriented antibodies recorded high binding affinity.
abstract: To investigate the effect of antibody orientation on its immunological activities, we developed a novel and versatile platform consisting of a well-defined phospholipid polymer surface on which staphylococcal protein A (SpA) was site-selectively immobilized. The application of a biocompatible phospholipid-based platform ensured minimal denaturation of immobilized antibodies, and the site-selective immobilization of SpA clarified the effect of antibody orientation on immunological activities. The phospholipid polymer platform was prepared on silicon substrates using the surface-initiated atom transfer radical polymerization (SI-ATRP) technique. An enzymatic reaction was performed for orientation-selective coupling of SpA molecules to the polymer brush surface. Orientation-controlled antibodies were achieved using enzymatic reactions, and these antibodies captured 1.8 ± 0.1 antigens on average, implying that at least 80% of immobilized antibodies reacted with two antigens. Theoretical multivalent binding analysis further revealed that orientation-controlled antibodies had antigen-antibody reaction equilibrium dissociation constants (K(d)) as low as 8.6 × 10(-10) mol/L, whereas randomly oriented and partially oriented antibodies showed K(d) values of 2.0 × 10(-7) and 1.2 × 10(-7) mol/L, respectively. Strict control of antibody orientation not only formed an approximately 100-fold stronger antigen-antibody complex than the controls but also sustained the native antibody K(d) (10(-10)-10(-9) mol/L). These findings support the significance of antibody orientation because controlling the orientation resulted in high reactivity and theoretical binding capacity.
authors.affiliation: Department of Materials Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan.
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Tajima
authors.forename: Madoka
authors.initials: M
authors.lastname: Takai
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Ishihara
mesh_terms: D000818:Animals; D055910:Antibodies, Immobilized / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D000915:Antibody Affinity; D000941:Antigens / Q000737:chemistry / Q000276:immunology; D006801:Humans; D007118:Immunoassay; D008689:Methacrylates / Q000737:chemistry; D008970:Molecular Weight; D014442:Monophenol Monooxygenase / Q000378:metabolism; D010084:Oxidation-Reduction; D010743:Phospholipids / Q000378:metabolism; D010767:Phosphorylcholine / Q000031:analogs & derivatives / Q000737:chemistry; D011109:Polymethacrylic Acids; D011489:Protein Denaturation; D013205:Staphylococcal Protein A / Q000737:chemistry / Q000378:metabolism; D013499:Surface Properties; D014443:Tyrosine / Q000378:metabolism
chemical_list: D055910:Antibodies, Immobilized; D000941:Antigens; D008689:Methacrylates; D010743:Phospholipids; D011109:Polymethacrylic Acids; D013205:Staphylococcal Protein A; C115766:poly(2-methacryloyloxyethyl-phosphorylcholine); D010767:Phosphorylcholine; D014443:Tyrosine; D014442:Monophenol Monooxygenase