title: Capture molecules preconditioned for kinetic analysis of high-affinity antigen-antibody complex in Biacore A100.
abstract: Surface plasmon resonance (SPR) is routinely applied on determining association or dissociation constant rates of antigen-antibody complexes. In a SPR system such as Biacore, the capture method is a widely accepted procedure in kinetic analysis for association or dissociation of soluble antigen analytes with antibody ligands initially captured by anti-Fc molecules immobilized on the sensor chip. Appropriate preparations of anti-immunoglobulin G (IgG)-Fc molecules on sensor chips have not been examined yet for stable kinetic analysis of antibodies with several affinities to soluble antigens. Here, we constructed murine monoclonal antibodies (MoAbs) with various affinities to hen egg lysozyme (HEL) and performed kinetic analysis of these MoAbs captured by rat MoAbs against mouse IgG-Fc immobilized on the sensor chip. When capture molecules maximally immobilized on the sensor chip, we observed no apparent dissociation of MoAbs with extremely high affinity to soluble HEL antigens. In contrast, on the limited amount (1000-2000 response units) of capture molecule immobilized on the sensor chip, we could perform stable kinetic analysis of MoAbs with highest affinities to the antigen as well as those with lower or moderate binding affinities. Thus, in some cases, accurate kinetic analysis of high-affinity antibodies can be performed by minimization of capture molecule densities on the sensor chip in SPR.
authors.affiliation: Tsukuba Discovery Laboratory of KAN unit, Eisai Company Ltd, Tsukuba, Ibaraki, Japan. m-katayama@hhc.eisai.co.jp
authors.forename: Masahiko
authors.initials: M
authors.lastname: Katayama
authors.forename: Toshitaka
authors.initials: T
authors.lastname: Sato
authors.forename: Junro
authors.initials: J
authors.lastname: Kuromitsu
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D000915:Antibody Affinity / Q000276:immunology; D000936:Antigen-Antibody Complex / Q000032:analysis* / Q000276:immunology; D000937:Antigen-Antibody Reactions / Q000276:immunology; D000941:Antigens / Q000737:chemistry* / Q000276:immunology; D057205:Automation, Laboratory; D015374:Biosensing Techniques / Q000379:methods*; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry / Q000276:immunology; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000276:immunology; D007700:Kinetics; D051379:Mice; D009113:Muramidase / Q000737:chemistry / Q000276:immunology; D051381:Rats; D012995:Solubility; D020349:Surface Plasmon Resonance
chemical_list: D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000941:Antigens; D055767:Immobilized Proteins; D007141:Immunoglobulin Fc Fragments; C421864:hen egg lysozyme; D009113:Muramidase