Hanham C A, Zhao F, Tignor G H
Department of Epidemiology and Public Health, Yale University School of Medicine, New Haven, Connecticut 06510.
J Virol. 1993 Jan;67(1):530-42. doi: 10.1128/JVI.67.1.530-542.1993.
We have developed idiotype-anti-idiotype monoclonal antibodies that provide evidence for rabies virus binding to the acetylcholine receptor (AChR). Hybridoma cell lines 7.12 and 7.25 resulted after fusion of NS-1 myeloma cells with spleen cells from a BALB/c mouse immunized with rabies virus strain CVS. Antibody 7.12 reacted with viral glycoprotein and neutralized virus infectivity in vivo. It also neutralized infectivity in vitro when PC12 cells, which express neuronal AChR, but not CER cells or neuroblastoma cells (clone N18), which have no AChR, were used. Antibody 7.25 reacted with nucleocapsid protein. Anti-idiotypic monoclonal antibody B9 was produced from fusion of NS-1 cells with spleen cells from a mouse immunized with 7.12 Fab. In an enzyme-linked immunosorbent assay and immunoprecipitation, B9 reacted with 7.12, polyclonal rabies virus immune dog serum, and purified AChR. The binding of B9 to 7.12 and immune dog serum was inhibited by AChR. B9 also inhibited the binding of 7.12 to rabies virus both in vitro and in vivo. Indirect immunofluorescence revealed that B9 reacted at neuromuscular junctions of mouse tissue. B9 also reacted in indirect immunofluorescence with distinct neurons in mouse and monkey brain tissue as well as with PC12 cells. B9 staining of neuronal elements in brain tissue of rabies virus-infected mice was greatly reduced. Rabies virus inhibited the binding of B9 to PC12 cells. Mice immunized with B9 developed low-titer rabies virus-neutralizing antibody. These mice were protected from lethal intramuscular rabies virus challenge. In contrast, anti-idiotypic antibody raised against nucleocapsid antibody 7.25 did not react with AChR.
我们已经研制出独特型-抗独特型单克隆抗体，这些抗体为狂犬病病毒与乙酰胆碱受体（AChR）结合提供了证据。NS-1骨髓瘤细胞与用狂犬病病毒株CVS免疫的BALB/c小鼠的脾细胞融合后，产生了杂交瘤细胞系7.12和7.25。抗体7.12与病毒糖蛋白反应，并在体内中和病毒感染性。当使用表达神经元AChR的PC12细胞，但不使用没有AChR的CER细胞或神经母细胞瘤细胞（克隆N18）时，它在体外也能中和感染性。抗体7.25与核衣壳蛋白反应。抗独特型单克隆抗体B9是由NS-1细胞与用7.12 Fab免疫的小鼠的脾细胞融合产生的。在酶联免疫吸附测定和免疫沉淀中，B9与7.12、狂犬病病毒免疫狗的多克隆血清以及纯化的AChR反应。AChR可抑制B9与7.12和免疫狗血清的结合。B9在体外和体内也抑制7.12与狂犬病病毒的结合。间接免疫荧光显示，B9在小鼠组织的神经肌肉接头处发生反应。B9在间接免疫荧光中也与小鼠和猴脑组织中的不同神经元以及PC12细胞发生反应。狂犬病病毒感染小鼠脑组织中神经元成分的B9染色大大减少。狂犬病病毒抑制B9与PC12细胞的结合。用B9免疫的小鼠产生了低滴度的狂犬病病毒中和抗体。这些小鼠在肌肉注射狂犬病病毒攻击时受到保护。相比之下，针对核衣壳抗体7.25产生的抗独特型抗体不与AChR反应。
