Kinetic analysis of interactions between bispecific monoclonal antibodies and immobilized antigens using a resonant mirror biosensor.
A resonant mirror biosensor (IAsys) protocol is described for the comparative kinetic analysis of the ability of monoclonal antibodies (Mabs) and bispecific antibodies (Babs) to bind immobilized antigens. The protocol has been optimized and validated using the panel of affinity-purified antibodies, including two parental Mabs, one specific to human immunoglobulin G (hIgG) and another specific to horseradish peroxidase (HRP), and a Bab derived thereof by cell fusion (anti-hIgG/HRP Bab). The real-time kinetic analysis of antigen-antibody interactions using this protocol allows to demonstrate the differences in the avidity of bivalently binding Mabs and monovalent Babs. As shown in our previous study [J. Immunol. Methods 261 (2002) 103], the observed equilibrium association constants (Kass) determined by IAsys using this protocol yield figures almost overlapping with those obtained by solid-phase radioimmunoassay (RIA). The described protocol is suited for the investigation of the effects of valency on the binding properties of antibodies. It also may be applied for the selection of Mabs and Babs with desired features, for different fields of application.
Dmitriev
Dmitriy A
DA
Department of Chemistry, Division of Chemical Enzymology, Moscow State University, Moscow, Russian Federation. dmitrievda@mail.ru
Massino
Yulia S
YS
Segal
Olga L
OL
D000818:Animals; D018033:Antibodies, Bispecific / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D000937:Antigen-Antibody Reactions / Q000502:physiology*; D015374:Biosensing Techniques / Q000379:methods*; D006735:Horseradish Peroxidase / Q000276:immunology; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D051379:Mice; D018448:Models, Immunological
D018033:Antibodies, Bispecific; D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D006735:Horseradish Peroxidase