Härtig W, Kirazov L, Brückner G, Holzer M, Gärtner U, Bigl V
Paul Flechsig Institute for Brain Research, Department of Neurochemistry, University of Leipzig, Germany.
Brain Res Brain Res Protoc. 1997 Dec 1;2(1):35-43. doi: 10.1016/s1385-299x(97)00026-3.
While the digoxigenin-anti-digoxigenin (DIG) method is currently the preferred tool for non-radioactive in situ hybridization this study extends its application field to Western blotting of proteins and summarizes advantageous properties of digoxigenylated antibodies in immunocytochemistry. An established protocol for the preparation of digoxigenylated primary antibodies is complemented by dot blot analyses confirming the high sensitivity of hapten-anti-hapten techniques based on primary digoxigenylated antibodies. The comparative Western blot analysis of calcium-binding proteins in nervous tissue is used as an example to show the highly specific detection of relevant antigens with unmodified primary antibodies, digoxigenylated secondary antibodies and anti-digoxigenin-peroxidase conjugates. The application of the DIG technology seems to be especially indicated in tissues containing high amounts of endogenous biotin-bearing proteins which might induce false-positive staining in conventional streptavidin/biotin techniques. Finally, the previously shown suitability of digoxigenylated antibodies for different immunocytochemical procedures is completed here by examples for sensitive single immunoperoxidase staining of neural markers in rat brain and for carbocyanine double immunofluorescence labelling of senile plaques in old rhesus monkeys.
虽然地高辛配体 - 抗地高辛配体（DIG）方法目前是非放射性原位杂交的首选工具，但本研究将其应用领域扩展到蛋白质的免疫印迹，并总结了地高辛标记抗体在免疫细胞化学中的优势特性。制备地高辛标记一抗的既定方案通过斑点印迹分析得到补充，该分析证实了基于地高辛标记一抗的半抗原 - 抗半抗原技术的高灵敏度。以神经组织中钙结合蛋白的比较免疫印迹分析为例，展示了使用未修饰的一抗、地高辛标记的二抗和抗地高辛 - 过氧化物酶结合物对相关抗原进行高度特异性检测。DIG技术似乎特别适用于含有大量内源性生物素结合蛋白的组织，这些蛋白可能在传统的链霉亲和素/生物素技术中诱导假阳性染色。最后，此前显示的地高辛标记抗体适用于不同免疫细胞化学程序的结论，在此通过大鼠脑中神经标记物的灵敏单免疫过氧化物酶染色以及老年恒河猴脑中老年斑的碳青染料双免疫荧光标记的实例得到完善。
