Mendelson E, Bustin M
Biochemistry. 1984 Jul 17;23(15):3459-66. doi: 10.1021/bi00310a012.
A series of monoclonal antibodies specific for distinguishable epitopes in chromosomal protein histone H5 were obtained from mice immunized with either free H5 or H5 . RNA complexes. The antibodies elicited by H5 could be distinguished from those elicited by H5 . RNA by their binding to native or acid-denatured H5, by their interaction with the globular region of H5, and by their cross-reactivity with H1o. The specificity of the antibodies was assessed by enzyme-linked immunosorbent assay (ELISA) and immunoblotting experiments. The antibodies could distinguish between H5 and the closely related histones H1 and H1o. The binding of some of the antibodies to the antigens was dependent on the type of assay used, suggesting nonrandom binding of the antigen to the solid supports used in ELISA and immunoblotting. Competitive ELISA experiments indicate that 8 of the 11 antibodies characterized bind to distinct epitopes. Three monoclonal antibodies bind to epitopes which are in close spatial proximity, causing mutual steric hindrance. The monoclonal antibodies bind to nuclei of fixed cells and to isolated chromatin, indicating that the epitopes are present both in the purified protein and in chromatin-complexed H5. These monoclonal antibodies can be used to study the organization of distinct regions of histones H5 and H1o in chromatin and chromosomes.
通过用游离的H5或H5 - RNA复合物免疫小鼠，获得了一系列针对染色体蛋白组蛋白H5中可区分表位的单克隆抗体。由H5引发的抗体与由H5 - RNA引发的抗体可通过它们与天然或酸变性H5的结合、与H5球状区域的相互作用以及与H1o的交叉反应性来区分。通过酶联免疫吸附测定（ELISA）和免疫印迹实验评估了抗体的特异性。这些抗体能够区分H5与密切相关的组蛋白H1和H1o。一些抗体与抗原的结合取决于所使用的测定类型，这表明抗原与ELISA和免疫印迹中使用的固相支持物存在非随机结合。竞争性ELISA实验表明，所鉴定的11种抗体中有8种与不同的表位结合。三种单克隆抗体与空间上紧密相邻的表位结合，导致相互的空间位阻。这些单克隆抗体与固定细胞的细胞核以及分离的染色质结合，表明这些表位在纯化的蛋白质和与染色质复合的H5中均存在。这些单克隆抗体可用于研究组蛋白H5和H1o在染色质和染色体中不同区域的组织情况。
