Antigen-Binding Activity of Monoclonal Antibodies after Incubation with Organic Solvents

Ya. I. Melnikova, S. G. Odintsov, Z. I. Kravchuk, and S. P. Martsev^*

Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, ul. Akademika Kuprevicha 5/2, Minsk, 220141 Belarus; fax: +375-17-263-7274; E-mail: martsev@ns.iboch.ac.by

^* To whom correspondence should be addressed.

Received May 17, 2000

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Effects of four organic solvents--methanol, trifluoroethanol, dimethylsulfoxide, and dimethylformamide (DMF)--on the ferritin-binding activity of three monoclonal mouse antibodies of IgG2a and IgG1 subclasses were studied. The ferritin-binding constants of monoclonal antibodies G10 and F11 (the IgG2a subclass) were increased 2-6-fold after incubation with DMF and removal of the organic solvent by gel filtration. The maximum effect on the F11 antibodies was found in the presence of 5-13% DMF and on the G10 antibodies at 11-40% DMF. The effect remained after the removal of DMF from the incubation medium, and this suggests that the incubation with DMF resulted in irreversible conformational changes of the antibodies and in production of active conformers of the G10 and F11 antibodies. These conformations occurred within 15-60 min. The long-term stability and the fluorescence of the antibodies exposed to DMF suggest that the conformational changes were not global, but involved small and relatively independent structural elements of the antibodies, either of hypervariable CDR loops in variable domains or of the hinge region of the antibodies. The affinity of the C5 antibodies of the mouse IgG1 subclass was decreased after incubation with DMF. The activation was a solvent-specific effect because incubation of the G10 antibodies with methanol and dimethylsulfoxide decreased the affinity for the antigen, and incubation with trifluoroethanol virtually did not affect it. Relatively small changes in the antigen-binding activity of the antibodies were found even after the incubation with 5% organic solvent.

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KEY WORDS: immunoglobulin, IgG2a, IgG1, antibody conformers, antibody modification, affinity of antibodies, intrinsic fluorescence, dimethylformamide

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