Identification of a linear epitope of interferon-alpha2b recognized by neutralizing monoclonal antibodies.
Four monoclonal antibodies (mAbs) directed against the recombinant human interferon-alpha2b (IFN-alpha2b) were used as probes to study the interaction of the IFN molecule to its receptors. The [125I]IFN-alpha2b binding to immobilized mAbs was completely inhibited by IFN-alpha2b and IFN-alpha2a but neither IFNbeta nor IFNgamma showed any effect. Gel-filtration HPLC of the immune complexes formed by incubating [125I]IFN-alpha2b 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-alpha2b anti-viral and anti-proliferative activities as well as [125I]IFN-alpha2b 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-alpha2b 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.
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Instituto de Química y Fisicoquímica Biológicas (UBA-CONICET), Facultad de Farmacia y Bioquímica, Buenos Aires, Argentina.
Sterin-Prync
A
A
Retegui
L
L
Vidal
A
A
Criscuolo
M
M
Roguin
L P
LP
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001665:Binding Sites; D001667:Binding, Competitive; D018604:Epitope Mapping; D000939:Epitopes; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000276:immunology* / Q000378:metabolism*; D016899:Interferon-beta / Q000378:metabolism; D007371:Interferon-gamma / Q000378:metabolism; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D011485:Protein Binding; D053633:Receptor, Interferon alpha-beta; D017471:Receptors, Interferon / Q000378:metabolism; D011994:Recombinant Proteins
D000911:Antibodies, Monoclonal; D000939:Epitopes; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D017471:Receptors, Interferon; D011994:Recombinant Proteins; D053633:Receptor, Interferon alpha-beta; D016899:Interferon-beta; D007371:Interferon-gamma