The Binding of Bispecific Monoclonal Antibodies to the Solid Phase-Adsorbed Antigens |
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Authors: | Dmitriev D A Massino Yu S Smirnova M B Segal O L Pavlova E V Kolyaskina G I Osipov A P Egorov A M Dmitriev A D |
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Institution: | (1) Chemical Faculty, Moscow State University, Vorob'evy gory, GSP-3, Moscow, 119899, Russia;(2) Institute of High Nervous Activity and Neurophysiology, Russian Academy of Sciences, ul. Butlerova 5a, Moscow, 117865, Russia;(3) Center of Mental Health, Russian Academy of Medical Sciences, Moscow, Russia |
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Abstract: | The ability of bispecific antibodies (Babs) formed by fusion of hybridomas and parent monoclonal antibodies (Mabs) to interact with the solid phase-adsorbed antigens was studied. Mabs specific to the three different antigens horseradish peroxidase (HRP), human IgG (hIgG), and human myoglobin (Mb)] as well as Babs with the double specificity antimyoglobin/antiperoxidase (anti-Mb/HRP) and anti-human IgG/antiperoxidase (anti-hIgG/HRP)] were used. It was shown by radioimmunological and immunoenzyme assays that parent Mabs bind to solid phase-adsorbed antigens considerably more effectively than Babs. The observed equilibrium binding constant (K
a) of antiperoxidase parental Mabs to immobilized HRP is 21 and 38 times higher than K
afor Babs binding sites (anti-Mb/HRP and anti-hIgG/HRP, respectively) to peroxidase. It was calculated that about 90–95% of all bound parental antiperoxidase Mabs were associated with immobilized HRP bivalently, and only about 5–10% were bound monovalently. On the contrary, parental Mabs against hIgG bind to the sorbed antigen essentially only monovalently. It was also shown that the avidity of anti-Mb/HRP Babs significantly increased when two antigens, Mb and HRP, were simultaneously adsorbed on the solid phase. These data imply that Babs bearing an enzyme-binding site (for example, binding to HRP) cannot be more effective than standard conjugates (e.g., enzyme-conjugated antibodies) in heterogeneous noncompetitive immunoassays. |
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Keywords: | affinity avidity bispecific antibodies bivalent interaction ELISA |
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