Antibodies that neutralize human beta interferon biologic activity recognize a linear epitope: analysis by synthetic peptide mapping.
The location of biologically relevant epitopes on recombinant human beta interferon in which Ser-17 replaces Cys-17 (rh[Ser17]IFN-beta) was evaluated by testing the immunoreactivity of antibodies against 159 sequential, overlapping octamer peptides. Three monoclonal antibodies (mAbs) that neutralize rh[Ser17]IFN-beta biologic activity, designated A1, A5, and A7, bound to peptides spanning only residues 39-48, whereas nonneutralizing mAb bound less specifically at multiple sites near the amino terminus. The immunoreactivity of peptides spanning residues 40-47 that contained a series of single amino acid substitutions suggested that residues 41-43 (Pro-Glu-Glu) and 46 (Gln) are important for the binding of neutralizing mAbs. The reactivity of mAbs to larger synthetic peptides containing rh[Ser17]IFN-beta sequences from residue 32 through residue 56 was evaluated. All mAbs except A7 reacted with synthetic peptides representing rh[Ser17]IFN-beta residues 32-47, 40-56, and 32-56, but only mAbs A1 and A5 bound to the core peptide composed of residues 40-47. Peptide 32-56 effectively blocked the binding of mAbs A1 and A5 to rh[Ser17]IFN-beta and markedly inhibited their neutralizing activity. Biologic activity of the peptides was undetectable. Rabbit antisera raised against peptides 32-47 and 40-56 recognized rh[Ser17]IFN-beta but did not neutralize its antiviral activity. Thus, structure-function analysis by peptide mapping has permitted the identification of a linear epitope recognized by neutralizing antibody on a biologically active cytokine. We conclude that the region spanning residues 32-56 is of major importance in the expression of the biologic activity of human IFN-beta.
Redlich
P N
PN
Department of Microbiology, Medical College of Wisconsin, Milwaukee 53226.
Hoeprich
P D
PD
Colby
C B
CB
Grossberg
S E
SE
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000939:Epitopes; D066298:In Vitro Techniques; D007370:Interferon Type I / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010455:Peptides / Q000276:immunology; D011487:Protein Conformation; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007370:Interferon Type I; D010455:Peptides