Identification of a linear epitope of interferon-α2b recognized by neutralizing monoclonal antibodies

Abstract

Four monoclonal antibodies (mAbs) directed against the recombinant human interferon-α2b (IFN-α2b) were used as probes to study the interaction of the IFN molecule to its receptors. The [125I]IFN-α2b binding to immobilized mAbs was completely inhibited by IFN-α2b and IFN-α2a but neither IFNβ nor IFNγ showed any effect. Gel-filtration HPLC of the immune complexes formed by incubating [125I]IFN-α2b with paired mAbs revealed the lack of simultaneous binding of two different antibodies to the tracer, suggesting that all mAbs recognize the same IFN antigenic domain. Furthermore, the mAbs were also able to neutralize the IFN-α2b anti-viral and anti-proliferative activities as well as [125I]IFN-α2b binding to WISH cell-membranes. As [125I]mAbs did not recognize IFN exposed epitopes in the IFN:receptor complexes, mAb induction of a conformational change in the IFN binding domain impairing its binding to receptors was considered unlikely. In order to identify the IFN region recognized by mAbs, IFN-α2b was digested with different proteolytic enzymes. Immunoreactivity of the resulting peptides was examined by Western blot and their sequences were established by Edman degradation after blotting to poly(vinylidene difluoride) membranes. Data obtained indicated that the smallest immunoreactive region recognized by mAbs consisted of residues 107-132 or 107-146. As this zone includes the sequence 123-140, which has been involved in the binding to receptors, and our mAbs did not show an allosteric behaviour, it is concluded that they are directed to overlapping epitopes located close to or even included in the IFN binding domain.