Kuo C H, Gilon H, Blumenthal A B, Sedat J W
Exp Cell Res. 1982 Nov;142(1):141-54. doi: 10.1016/0014-4827(82)90418-9.
To study the structure and function of the cell nucleus, a library of 170 monoclonal antibodies was produced to nuclear antigens from 3-6 h old Drosophila embryos. In preparation for immunization, nuclei were separated, at neutral pH and in the presence of polyamines, into two fractions containing either urea-soluble non-histone nuclear proteins or histones plus small quantities of non-histone proteins complexed to DNA. The antibodies were characterized in a rapid, indirect immunofluorescent assay employing cultured Drosophila cells (Schneider's line 2). Low backgrounds and high specific fluorescence were achieved in this assay by purifying the rhodamine-labelled second antibody on a polystyrene resin and washing the cells with optimal concentrations of detergents. The assay categorized antigens according to their cellular locations: in nuclei, in nuclei plus cytoplasm, or primarily in cytoplasm. A subset of nuclear antigens reacted specifically with the nuclear envelope. In addition, some antibodies were characterized by their reactions with polytene chromosomes. The cultured cell assay provides a new, efficient method for expanding this antibody library. The monoclonal antibodies in the library now provide highly specific tools for investigating structural nuclear proteins and proteins that may be regulatory during embryonic development.
为了研究细胞核的结构和功能，制备了一个包含170种单克隆抗体的文库，这些抗体针对3至6小时大的果蝇胚胎中的核抗原。在免疫准备过程中，在中性pH值和多胺存在的条件下，将细胞核分离成两个部分，一部分含有尿素可溶性非组蛋白核蛋白，另一部分含有组蛋白以及与DNA结合的少量非组蛋白。这些抗体通过使用培养的果蝇细胞（施奈德2号线）进行的快速间接免疫荧光测定来表征。通过在聚苯乙烯树脂上纯化罗丹明标记的二抗并用最佳浓度的去污剂洗涤细胞，在该测定中实现了低背景和高特异性荧光。该测定根据抗原在细胞中的位置进行分类：在细胞核中、在细胞核加细胞质中或主要在细胞质中。一部分核抗原与核膜发生特异性反应。此外，一些抗体通过它们与多线染色体的反应来表征。培养细胞测定为扩展这个抗体文库提供了一种新的有效方法。文库中的单克隆抗体现在为研究核结构蛋白和可能在胚胎发育过程中起调节作用的蛋白提供了高度特异性的工具。
