Wolf W P, Weis M, von Scheidt W
Medizinische Klinik I, University Hospital Grosshadern, Ludwig-Maximilians-University, Marchioninistrasse 15, 81377 Munich, Germany.
Histochem Cell Biol. 2001 Nov;116(5):411-26. doi: 10.1007/s00418-001-0334-6. Epub 2001 Oct 9.
Endothelin is an endothelium-derived peptide with potent vasoconstrictor and mitogenic properties. Since studies concerning the immunocytochemical localization of endothelin are often inconsistent we tried to clear up some of these discrepancies by comparing specificity and labeling patterns of different endothelin antibodies. Monoclonal and polyclonal endothelin antibodies ( n=7) were examined concerning their reactivity with endothelins and heart tissues by immunoblotting. Using immunofluorescence microscopy reactivities with human non-failing hearts, failing hearts, and cultured endothelial cells were examined. Specificity of labelings was assessed by absorption controls and functional controls using endothelial cells conditioned to produce different amounts of endothelin. As shown by immunoblotting five out of seven endothelin antibodies revealed both specific endothelin reactivity and negligible non-specific reactivities with heart proteins. Immunocytochemistry showed vascular reactivity of N-terminal endothelin antibodies to be associated with alpha-smooth muscle actin expression. One N-terminal antibody showed additional nuclear reactivity, and one C-terminal antibody vimentin-like labeling patterns. Although these reactivities were abolished in absorption controls these labelings had to be graded non-specific because functional specificity of endothelin antibodies could not be proven. The remaining antibodies revealed no endothelial reactivity even after tyramide signal amplification. Cellular endothelin concentrations ranged around 2,000-fold below the detection limit of immunocytochemical methods. Discrepancies in endothelin immunocytochemistry may originate from false-positive results and from expression levels of endothelin below the detection limit of immunocytochemical methods.
内皮素是一种由内皮细胞产生的具有强大血管收缩和促有丝分裂特性的肽。由于关于内皮素免疫细胞化学定位的研究结果常常不一致，我们试图通过比较不同内皮素抗体的特异性和标记模式来澄清其中一些差异。通过免疫印迹法检测了单克隆和多克隆内皮素抗体（n = 7）与内皮素和心脏组织的反应性。使用免疫荧光显微镜检查了它们与人正常心脏、衰竭心脏和培养的内皮细胞的反应性。通过使用条件性产生不同量内皮素的内皮细胞进行吸收对照和功能对照来评估标记的特异性。免疫印迹结果显示，七种内皮素抗体中有五种显示出特异性的内皮素反应性，并且与心脏蛋白的非特异性反应可忽略不计。免疫细胞化学显示，N端内皮素抗体的血管反应性与α-平滑肌肌动蛋白表达相关。一种N端抗体显示出额外的核反应性，一种C端抗体显示波形蛋白样标记模式。尽管这些反应性在吸收对照中被消除，但由于无法证明内皮素抗体的功能特异性，这些标记仍被判定为非特异性。其余抗体即使在酪胺信号放大后也未显示内皮反应性。细胞内内皮素浓度比免疫细胞化学方法的检测限低约2000倍。内皮素免疫细胞化学中的差异可能源于假阳性结果以及内皮素表达水平低于免疫细胞化学方法的检测限。
