Analysis of bispecific monoclonal antibody binding to immobilized antigens using an optical biosensor.
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.
Dmitriev
D A
DA
Department of Chemical Enzymology, School of Chemistry, Lomonosov Moscow State University, Moscow, 119992 Russia. dmitrievda@mtu-net.ru
Massino
Y S
YS
Segal
O L
OL
Smirnova
M B
MB
Pavlova
E V
EV
Kolyaskina
G I
GI
Gurevich
K G
KG
Gnedenko
O V
OV
Ivanov
Y D
YD
Archakov
A I
AI
Osipov
A P
AP
Dmitriev
A D
AD
Egorov
A M
AM
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
D000911:Antibodies, Monoclonal; D000941:Antigens; D007074:Immunoglobulin G