Marriott S J, Roeder D J, Consigli R A
J Virol. 1987 Sep;61(9):2747-53. doi: 10.1128/JVI.61.9.2747-2753.1987.
Anti-idiotypic antibodies have been successfully used to identify and isolate the receptor for several cell ligands. To prepare an immunologic probe for identification of the polyomavirus receptor on mouse kidney cells, polyclonal antisera against antipolyomavirus antibodies were prepared in rabbits. Fab fragments of the previously characterized monoclonal antibody E7, which neutralizes polyomavirus infection, were used for immunization (S. J. Marriott and R. A. Consigli, J. Virol. 56:365-372, 1985). Sera containing the greatest anti-idiotype activity were identified by enzyme-linked immunosorbent assay (ELISA) and purified by a series of affinity columns. The anti-idiotypic antibodies recognized the E7 idiotope in an ELISA, and anti-idiotype binding could be inhibited by preincubation of E7 monoclonal antibody with polyomavirus virions. When mixed with anti-idiotype immunoglobulin G (IgG), E7 was no longer capable of binding or immunoprecipitating polyomavirus virions or neutralizing polyomavirus infection. Direct immunofluorescence showed anti-idiotype IgG reactivity with a cell surface component of mouse kidney cells. Anti-idiotype F(ab')2 effectively competed with polyomavirus for binding to mouse kidney cells and displayed binding kinetics similar to those of polyomavirus. Virus infection of mouse kidney cells was blocked in a dose-dependent manner following treatment of the cells with anti-idiotype IgG. The anti-idiotype identified several proteins (95, 50, and 24 to 31 kilodaltons) in an immunoblot of mouse kidney cell membrane proteins but reacted predominantly with a single 50-kilodalton protein in a radioimmunoassay. The anti-idiotype failed to react with virus proteins in three assays, including ELISA, immunoprecipitation, and immunoblotting. The implications of this work for future identification and characterization of the polyomavirus receptor on mouse kidney cells are discussed.
抗独特型抗体已成功用于鉴定和分离几种细胞配体的受体。为制备用于鉴定小鼠肾细胞上多瘤病毒受体的免疫探针，在兔体内制备了针对抗多瘤病毒抗体的多克隆抗血清。用先前鉴定的可中和多瘤病毒感染的单克隆抗体E7的Fab片段进行免疫（S. J. 马里奥特和R. A. 康西格利，《病毒学杂志》56:365 - 372, 1985）。通过酶联免疫吸附测定（ELISA）鉴定出具有最大抗独特型活性的血清，并通过一系列亲和柱进行纯化。抗独特型抗体在ELISA中识别E7独特型表位，并且E7单克隆抗体与多瘤病毒颗粒预孵育可抑制抗独特型结合。当与抗独特型免疫球蛋白G（IgG）混合时，E7不再能够结合或免疫沉淀多瘤病毒颗粒或中和多瘤病毒感染。直接免疫荧光显示抗独特型IgG与小鼠肾细胞的细胞表面成分发生反应。抗独特型F(ab')2有效地与多瘤病毒竞争结合小鼠肾细胞，并表现出与多瘤病毒相似的结合动力学。用抗独特型IgG处理细胞后，小鼠肾细胞的病毒感染以剂量依赖方式被阻断。抗独特型在小鼠肾细胞膜蛋白的免疫印迹中鉴定出几种蛋白质（95、50以及24至31千道尔顿），但在放射免疫测定中主要与单一的50千道尔顿蛋白质发生反应。在包括ELISA、免疫沉淀和免疫印迹在内的三种测定中，抗独特型未能与病毒蛋白发生反应。讨论了这项工作对未来鉴定和表征小鼠肾细胞上多瘤病毒受体的意义。
