Brown W Mark, Maxwell Perry, Graham Alastair N J, Yakkundi Anita, Dunlop Elaine A, Shi Zhanzhong, Johnston Patrick G, Lappin Terence R J
Department of Pathology, Institute of Pathology, Royal Group of Hospitals Trust, Grosvenor Road, Belfast BT12 6BA, United Kingdom.
Stem Cells. 2007 Mar;25(3):718-22. doi: 10.1634/stemcells.2006-0687. Epub 2006 Nov 16.
Immunohistochemical studies on formalin-fixed, paraffin-embedded (FFPE) tissue utilizing polyclonal antibodies form the cornerstone of many reports claiming to demonstrate erythropoietin receptor (EPOR) expression in malignant tissue. Recently, Elliott et al. (Blood 2006;107:1892-1895) reported that the antibodies commonly used to detect EPOR expression also detect non-EPOR proteins, and that their binding to EPOR was severely abrogated by two synthetic peptides based on the sequence of heat shock protein (HSP) 70, HSP70-2, and HSP70-5. We have investigated the specificity of the C20 antibody for detecting EPOR expression in non-small cell lung carcinoma (NSCLC) utilizing tissue microarrays. A total of 34 cases were available for study. Antibody absorbed with peptide resulted in marked suppression of cytoplasmic staining compared with nonabsorbed antibody. Four tumors that initially showed a membranous pattern of staining retained this pattern with absorbed antibody. Positive membranous immunoreactivity was also observed in 6 of 30 tumors that originally showed a predominantly cytoplasmic pattern of staining. Using the C20 antibody for Western blots, we detected three main bands, at 100, 66, and 59 kDa. Preincubation with either peptide caused abolition of the 66-kDa band, which contains non-EPOR sequences including heat shock peptides. These results call into question the significance of previous immunohistochemical studies of EPOR expression in malignancy and emphasize the need for more specific anti-EPOR antibodies to define the true extent of EPOR expression in neoplastic tissue.
利用多克隆抗体对福尔马林固定、石蜡包埋（FFPE）组织进行免疫组织化学研究，是许多声称在恶性组织中证明促红细胞生成素受体（EPOR）表达的报告的基石。最近，埃利奥特等人（《血液》2006年；107:1892 - 1895）报告称，常用于检测EPOR表达的抗体也能检测非EPOR蛋白，并且基于热休克蛋白（HSP）70、HSP70 - 2和HSP70 - 5序列的两种合成肽会严重抑制它们与EPOR的结合。我们利用组织微阵列研究了C20抗体在检测非小细胞肺癌（NSCLC）中EPOR表达的特异性。共有34例病例可供研究。与未吸收肽的抗体相比，用肽吸收的抗体导致细胞质染色明显抑制。最初显示膜性染色模式的4个肿瘤在用吸收肽的抗体处理后仍保留这种模式。在最初主要显示细胞质染色模式的30个肿瘤中的6个中也观察到阳性膜性免疫反应。使用C20抗体进行蛋白质印迹分析，我们检测到100、66和59 kDa的三条主要条带。与任何一种肽预孵育都会导致66 kDa条带消失，该条带包含包括热休克肽在内的非EPOR序列。这些结果使人对先前关于恶性肿瘤中EPOR表达的免疫组织化学研究的意义产生质疑，并强调需要更特异的抗EPOR抗体来确定肿瘤组织中EPOR表达的真实程度。
