Analysis of Bispecific Monoclonal Antibody Binding to Immobilized Antigens Using an Optical Biosensor

D. A. Dmitriev^{1See footnote 1}, Y. S. Massino², O. L. Segal², M. B. Smirnova², E. V. Pavlova³, G. I. Kolyaskina³, K. G. Gurevich¹, O. V. Gnedenko⁴, Y. D. Ivanov⁴, A. I. Archakov⁴, A. P. Osipov¹, A. D. Dmitriev², and A. M. Egorov¹

¹Department of Chemical Enzymology, School of Chemistry, Lomonosov Moscow State University, Moscow, 119992 Russia; fax: (095) 939-2727; E-mail: dmitrievda@mtu-net.ru

²Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences, ul. Butlerova 5a, Moscow, 117865 Russia; fax: (095) 338-8500; E-mail: dmitr@rcmh.msk.ru

³Center of Mental Health, Russian Academy of Medical Sciences, Zagorodnoe Shosse 2/2, Moscow, 113152 Russia; fax: (095) 952-8940

⁴Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, Pogodinskaya ul. 10, Moscow, 119832 Russia; fax: (095) 245-0857; E-mail: yuiv@ibmh.msk.su

^* To whom correspondence should be addressed.

Received September 3, 2001; Revision received November 19, 2001

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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.

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KEY WORDS: bispecific antibodies, kinetic constants, bivalent interaction, antigen-antibody interaction, biosensor, IAsys, affinity, avidity

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