Monoclonal antibodies against different epitopes of nonstructural protein sigma NS of avian reovirus S1133

Ten monoclonal antibodies (MAbs) were prepared against the nonstructural pr otein sigma NS of avian reovirus S1133. Eight of them were selected for two -way competitive binding assay after coupling with horseradish peroxidase. The results allowed the definition of three epitopes, designated A, B, and C. Blocking assay of poly(A)-Sepharose binding activity of sigma NS with MA bs indicated that MAb recognizing epitope B was able to block poly(A) oligo mer binding, suggesting that epitope B is involved in ssRNA binding of sigm a NS. An immune-dot binding assay was used to analyze the effect of denatur ation on antibody recognition of the epitopes. All MAbs bound to protein si gma NS in its native form. After denaturation by boiling in SDS and 2-merca ptaethanol, the binding of MAbs recognizing epitopes B and C was not affect ed. The reactivity of MAbs recognizing epitope A was fully abolished by den aturation. These results suggest that the binding of MAbs directed against epitope A is conformation-dependent however, the recognition by MAbs of epi topes B and C is not conformation-dependent. In addition, the results from the cross-reactivity of MAbs to heterologous avian reovirus strains suggest that the three epitopes are highly conserved among these virus strains. (C ) 2001 Academic Press.