title: Analysis of bispecific monoclonal antibody binding to immobilized antigens using an optical biosensor.
abstract: The interaction between two different monoclonal antibodies (Mabs) and their corresponding bispecific antibodies (Babs) with immobilized antigens was investigated using an optical biosensor (IAsys). The analyzed panel of affinity-purified antibodies included two parental Mabs (one of which was specific to human IgG (hIgG), and another one to horseradish peroxidase (HRP)), as well as Babs derived thereof (anti-hIgG/HRP). Babs resulting from the fusion of parental hybridomas bear two antigen-binding sites toward two different antigens and thus may interact with immobilized antigen through only one antigen-binding site (monovalently). Using an IAsys biosensor this study shows that the bivalent binding of Mabs predominates over the monovalent binding with immobilized HRP, whereas anti-hIgG parental Mabs were bound monovalently to the immobilized hIgG. The observed equilibrium association constant (K(ass)) values obtained in our last work [1] by solid-phase radioimmunoassay are consistent with those constants obtained by IAsys. The K(ass) of anti-HRP Mabs was about 50 times higher than that of anti-HRP shoulder of Babs. The dissociation rate constant (k(diss)) for anti-HRP shoulder of Babs was 21 times higher than k(diss) for anti-HRP Mabs. The comparison of the kinetic parameters for bivalent anti-HRP Mabs and Babs derived from anti-Mb/HRP and anti-hIgG/HRP, allowed to calculate that 95% of bound anti-HRP Mabs are bivalently linked with immobilized HRP, whereas only 5% of bound anti-HRP Mabs are monovalently linked. In general, the data obtained indicate that Babs bearing an enzyme-binding site may not be efficiently used instead of traditional antibody-enzyme conjugates in the case of binding of bivalent Mabs.
authors.affiliation: Department of Chemical Enzymology, School of Chemistry, Lomonosov Moscow State University, Moscow, 119992 Russia. dmitrievda@mtu-net.ru
authors.forename: D A
authors.initials: DA
authors.lastname: Dmitriev
authors.forename: Y S
authors.initials: YS
authors.lastname: Massino
authors.forename: O L
authors.initials: OL
authors.lastname: Segal
authors.forename: M B
authors.initials: MB
authors.lastname: Smirnova
authors.forename: E V
authors.initials: EV
authors.lastname: Pavlova
authors.forename: G I
authors.initials: GI
authors.lastname: Kolyaskina
authors.forename: K G
authors.initials: KG
authors.lastname: Gurevich
authors.forename: O V
authors.initials: OV
authors.lastname: Gnedenko
authors.forename: Y D
authors.initials: YD
authors.lastname: Ivanov
authors.forename: A I
authors.initials: AI
authors.lastname: Archakov
authors.forename: A P
authors.initials: AP
authors.lastname: Osipov
authors.forename: A D
authors.initials: AD
authors.lastname: Dmitriev
authors.forename: A M
authors.initials: AM
authors.lastname: Egorov
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000937:Antigen-Antibody Reactions; D000941:Antigens / Q000737:chemistry; D001665:Binding Sites; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007074:Immunoglobulin G / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D013997:Time Factors
chemical_list: D000911:Antibodies, Monoclonal; D000941:Antigens; D007074:Immunoglobulin G