Immunoprecipitation with two-dimensional pools as a hybridoma screening technique: production and characterization of monoclonal antibodies against adenovirus 2 proteins.
Immunoprecipitation with two-dimensional pools as a hybridoma screening technique: production and characterization of monoclonal antibodies against adenovirus 2 proteins.
复制标题
使用二维池的免疫沉淀作为杂交瘤筛选技术：针对腺病毒 2 蛋白的单克隆抗体的生产和表征。
DOI：
10.1016/0042-6822(81)90069-6
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发表时间：
1981
期刊：
影响因子：
3.7
通讯作者：
P. Sharp
中科院分区：
文献类型：
--
作者：
C. Cepko;P. Changelian;P. Sharp
建立了一种基于免疫沉淀的α-腺病毒单克隆抗体筛选技术。这种免疫沉淀过程可用作几乎任何抗体-抗原系统的快速初级筛选方法。该方法采用体内[35 S]甲硫氨酸标记的蛋白质作为抗原，采用杂交培养物上清液的二维池作为抗体，从而允许在第一次筛选中鉴定给定孔的特异性。迄今为止，已克隆的产生稳定的腹水产生肿瘤的α-腺病毒杂交体包括两个α-六邻体克隆、三个α-纤维克隆、一个α-蛋白IX克隆和一个沉淀六邻体加两种分子量为100，000和95，000道尔顿的蛋白的克隆。选择α-蛋白IX克隆以及α-六邻体克隆和α-五邻体碱基克隆，通过免疫沉淀分析表征其特异性。分泌针对六邻体（主要病毒体衣壳蛋白）的抗体的杂交细胞系被表征为与六邻体三聚体反应，所述六邻体三聚体在溶液中游离并且以九个一组（“九聚体”）组装。α-六邻体单克隆抗体不沉淀通过体外翻译制备的单体形式的六邻体或完整的成熟病毒粒子。这些特征以及沉淀从迄今为止测试的所有腺病毒血清型制备的六邻体的能力与组特异性六邻体血清所表现出的特征相同。五邻体碱基是存在于二十面体衣壳顶点的病毒体蛋白，其特异性单克隆抗体免疫沉淀通过体外翻译或变性成熟病毒体制备的单体形式的五邻体碱基。它不与与纤维组装的10 S完整五邻体形式的五邻体基底反应，也不沉淀成熟病毒体。α-蛋白IX抗体与蛋白IX的单体形式以及变性蛋白IX反应。还检测了所有三种抗体中和病毒感染性的能力，发现在本试验中为阴性。此外，使用这些抗体通过间接免疫荧光染色感染的细胞。α-六邻体和α-五邻体基础抗体产生了预期的明亮核荧光模式，而α-蛋白IX抗体产生了不寻常的明亮核火花模式。
A screening technique based on immunoprecipitation has been developed and used to select α-adenovirus monoclonal antibodies. This immunoprecipitation procedure can be used as a rapid primary screening method for virtually any antibody—antigen system. This method employsin vivo[35S]methionine-labeled proteins as antigens and two-dimensional pools of hybrid culture supernatants as antibody, thus allowing identification of the specificity of a given well in the first screen. The α-adenovirus hybrids that have been cloned to date to yield stable, ascites fluid-producing tumors include two α-hexon clones, three α-fiber clones, one α-protein IX clone, and one clone that precipitates hexon plus two proteins of molecular weights 100,000 and 95,000 daltons. The α-protein IX clone as well as an α-hexon clone and an α-penton base clone were chosen for characterization of their specificities by immunoprecipitation analysis. The hybrid cell line secreting antibody against hexon, the major virion capsid protein, was characterized as being reactive with hexon trimers, both free in solution and assembled in groups of nine (“ninemers”). The α-hexon monoclonal antibody did not precipitate the monomer form of hexon, prepared byin vitrotranslation, or intact mature virions. These characteristics, as well as the ability to precipitate hexons prepared from all adenovirus serotypes tested to date, are the same characteristics exhibited by group-specific hexon serum. The monoclonal antibody specific for penton base, the virion protein found at the vertices of the icosohedral capsid, immunoprecipitated the monomer form of penton base prepared byin vitrotranslation or denaturation of mature virions. It was not reactive with the 10 S complete penton form of penton base assembled with fiber, nor did it precipitate mature virions. The α-protein IX antibody was reactive with the monomer form of protein IX as well as denatured protein IX. All three of the antibodies were also tested for the ability to neutralize virus infectivity and were found to be negative in this assay. In addition, infected cells were stained by indirect immunofluorescence using these antibodies. The α-hexon and α-penton base antibodies gave the expected pattern of bright nuclear fluorescence, while the α-protein IX antibody gave rise to an unusual pattern of bright nuclear sparkles.