Hou H S, Su Y P, Shieh H K, Lee L H
Department of Veterinary Medicine, National Chung Hsing University, Taichung, 403, Taiwan.
Virology. 2001 Mar 30;282(1):168-75. doi: 10.1006/viro.2001.0814.
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.
制备了十种针对禽呼肠孤病毒S1133非结构蛋白sigmaNS的单克隆抗体（MAb）。其中八种在与辣根过氧化物酶偶联后用于双向竞争结合试验。结果确定了三个表位，分别命名为A、B和C。用MAb对sigmaNS的聚（A）-琼脂糖结合活性进行封闭试验表明，识别表位B的MAb能够阻断聚（A）寡聚物结合，这表明表位B参与了sigmaNS与单链RNA的结合。采用免疫斑点结合试验分析变性对表位抗体识别的影响。所有MAb均能与天然形式的蛋白sigmaNS结合。在SDS和2-巯基乙醇中煮沸变性后，识别表位B和C的MAb的结合不受影响。识别表位A的MAb的反应性因变性而完全丧失。这些结果表明，针对表位A的MAb的结合依赖于构象；然而，MAb对表位B和C的识别不依赖于构象。此外，MAb对异源禽呼肠孤病毒株的交叉反应结果表明，这三个表位在这些病毒株中高度保守。
