The binding of bispecific monoclonal antibodies to the solid phase-adsorbed antigens

• Published in: Bioorganicheskaia khimiia Vol. 27; no. 4; p. 265
• Main Authors: Dmitriev, D A, Massino, Iu S, Smirnova, M B, Segal, O L, Pavlova, E B, Koliaskina, G I, Osipov, A P, Egorov, A M, Dmitriev, A D
• Format: Journal Article
• Language: Russian
• Published: Russia (Federation) 01-07-2001
• Subjects: Animals; Antibodies, Bispecific - immunology; Antigen-Antibody Complex - immunology; Binding Sites, Antibody - immunology; Enzyme-Linked Immunosorbent Assay - methods; Horseradish Peroxidase - immunology; Humans; Immunoglobulin G - immunology; Mice; Myoglobin - immunology; Radioimmunoassay - methods; Sensitivity and Specificity
• ISSN: 0132-3423

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-hIgG/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 (Ka) of antiperoxidase parental Mabs to immobilized HRP is 21 and 38 times higher than Ka for 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.