Monoclonal antibodies against different epitopes of nonstructural protein sigmaNS of avian reovirus S1133.
Ten monoclonal antibodies (MAbs) were prepared against the nonstructural protein sigmaNS 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 sigmaNS with MAbs indicated that MAb recognizing epitope B was able to block poly(A) oligomer binding, suggesting that epitope B is involved in ssRNA binding of sigmaNS. An immuno-dot binding assay was used to analyze the effect of denaturation on antibody recognition of the epitopes. All MAbs bound to protein sigmaNS in its native form. After denaturation by boiling in SDS and 2-mercaptoethanol, the binding of MAbs recognizing epitopes B and C was not affected. The reactivity of MAbs recognizing epitope A was fully abolished by denaturation. These results suggest that the binding of MAbs directed against epitope A is conformation-dependent; however, the recognition by MAbs of epitopes 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.
Hou
H S
HS
Department of Veterinary Medicine, National Chung Hsing University, Taichung, 403, Taiwan.
Su
Y P
YP
Shieh
H K
HK
Lee
L H
LH
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000914:Antibodies, Viral / Q000276:immunology*; D000915:Antibody Affinity; D001667:Binding, Competitive; D001717:Birds; D003429:Cross Reactions; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology; D008623:Mercaptoethanol / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D011489:Protein Denaturation; D012367:RNA, Viral / Q000378:metabolism; D012087:Reoviridae / Q000235:genetics / Q000378:metabolism*; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000939:Epitopes; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D012967:Sodium Dodecyl Sulfate; D008623:Mercaptoethanol