Ono K, Jitsukawa T, Ishihara H, Fukuda A
J Biochem. 1986 Jan;99(1):269-79. doi: 10.1093/oxfordjournals.jbchem.a135469.
Three monoclonal antibodies against human liver ferritin were selected to study antigenic determinants (epitopes) of human isoferritins. These monoclonal antibodies were found to form immunoprecipitin lines with ferritin in double diffusion tests (Ouchterlony), indicating multiple epitopes on a single ferritin molecule. The antibodies revealed high species specificity as well. Monoclonal antibodies MA301 and MA311 appeared to recognize different epitopes, since they did not inhibit each other in competitive enzyme-linked immunosorbent assay (ELISA). However, MA309 recognized both epitopes for MA301 and MA311 with similar competitive inhibition. These epitopes were not detectable when ferritin was treated with 8M urea (pH 2.5) and were detectable upon reconstruction by dialysis against 2 M urea (pH 7.2), suggesting that these monoclonals recognize epitopes in the tertiary structure of the ferritin molecule. As a matter of fact, these monoclonals react preferentially with intact ferritin molecule and only negligibly with subunits. Isoelectric focusing patterns of human ferritins demonstrated that liver, spleen, placenta, and hepatoma cells (Li-7) transplanted in nude mice contained basic isoferritins, whereas HeLa cells (carcinoma), Wa cells (EB virus-transformed B cells), and Raji cells (Burkitt's lymphoma) contained acidic isoferritins. Human heart ferritin displayed a somewhat intermediate pattern between liver and HeLa ferritins. In spite of the heterogeneous population of human isoferritins, the dissociation constants (Kd) of the three monoclonal antibodies to liver, HeLa, and heart isoferritins were quite similar.
选择三种抗人肝铁蛋白的单克隆抗体来研究人异铁蛋白的抗原决定簇（表位）。在双向扩散试验（奥克特洛尼试验）中发现这些单克隆抗体与铁蛋白形成免疫沉淀线，表明单个铁蛋白分子上存在多个表位。这些抗体也显示出高度的种属特异性。单克隆抗体MA301和MA311似乎识别不同的表位，因为它们在竞争性酶联免疫吸附测定（ELISA）中互不抑制。然而，MA309以相似的竞争性抑制作用识别MA301和MA311的表位。当铁蛋白用8M尿素（pH 2.5）处理时，这些表位不可检测，而通过对2M尿素（pH 7.2）进行透析重建后则可检测到，这表明这些单克隆抗体识别铁蛋白分子三级结构中的表位。事实上，这些单克隆抗体优先与完整的铁蛋白分子反应，与亚基的反应可忽略不计。人铁蛋白的等电聚焦图谱表明，移植到裸鼠体内的肝、脾、胎盘和肝癌细胞（Li-7）含有碱性异铁蛋白，而人宫颈癌细胞（HeLa细胞）（癌）、Wa细胞（EB病毒转化的B细胞）和Raji细胞（伯基特淋巴瘤）含有酸性异铁蛋白。人心脏铁蛋白的图谱在肝脏和HeLa细胞铁蛋白之间呈现出某种中间模式。尽管人异铁蛋白群体具有异质性，但这三种单克隆抗体与人肝、HeLa细胞和心脏异铁蛋白的解离常数（Kd）相当相似。
