Merz H, Malisius R, Mannweiler S, Zhou R, Hartmann W, Orscheschek K, Moubayed P, Feller A C
Department of Pathology, Medical University of Lübeck, Germany.
Lab Invest. 1995 Jul;73(1):149-56.
Since the introduction of mAb, immunohistochemistry has become an important tool in research and in surgical pathology. The most widely used fixative in routine histopathology is formaldehyde, and it has become the gold standard for morphologic tissue preservation. Although the molecular mechanism underlying the tissue fixation is not well understood, it has become clear that available immunoreactive Ag are progressively lost during the fixation process. For a long time, it was thought that formalin-sensitive Ag might be irreversibly destroyed during the fixation process. Although monoclonal anti-Ig Ab frequently worked inadequately, polyclonal anti-Ig Ab were shown to produce reproducible staining results. It thus appeared possible that most cellular Ag might not be irreversibly destroyed but only masked.
Although some Ag may be retrieved under appropriate conditions, there might still be many for which available antigenic epitopes are still too sparse to be visualized, as observed for a large number of leukocyte differentiation Ag. One reliable approach to resolve this dilemma is the use of a combination of an optimized Ag retrieval system and a powerful immunohistochemical staining protocol introducing a biotin amplification step, in which signal amplification is accomplished by covalent deposition of biotin molecules.
Cryostat and paraffin sections were stained with the avidin-biotin complex technique and, for comparison, with the new maximized immunohistochemical staining protocol, termed the ImmunoMax method. Each step was monitored to establish how effectively it enhanced the overall sensitivity. Although pretreatment with detergent, protease, a chaotropic substance, or microwave heating resulted in only moderately improved immunostaining, the biotinylated tyramine enhancement step proved to be the most efficient one, although the latter is not sufficient for many Ag when used without pretreatment steps. The combination of an Ag retrieval step with the biotinylated tyramine enhancement step resulted in a 100 to 10,000-fold boost in sensitivity without loss of specificity.
With the ImmunoMax method, defined Ag can be reproducibly detected in formalin-fixed, paraffin-embedded tissues, and the sensitivity of the method is tremendously enhanced. Moreover, it also allows many previously unreactive or unsatisfactorily reactive Ag to be detected, as shown here for IgD, IgM, and CD7 with the use of mAb.
自从单克隆抗体（mAb）问世以来，免疫组织化学已成为研究和外科病理学中的一项重要工具。常规组织病理学中使用最广泛的固定剂是甲醛，它已成为形态学组织保存的金标准。尽管组织固定的分子机制尚未完全明了，但很明显，在固定过程中可用的免疫反应性抗原（Ag）会逐渐丢失。长期以来，人们认为对福尔马林敏感的抗原可能在固定过程中被不可逆地破坏。尽管单克隆抗Ig抗体常常效果不佳，但多克隆抗Ig抗体已显示能产生可重复的染色结果。因此，似乎大多数细胞抗原可能并非被不可逆地破坏，而只是被掩盖了。
尽管在适当条件下一些抗原可能被找回，但仍可能有许多抗原，其可用的抗原表位仍然过于稀少而无法显现，就像大量白细胞分化抗原的情况一样。解决这一困境的一个可靠方法是使用优化的抗原找回系统和引入生物素放大步骤的强大免疫组织化学染色方案相结合，其中信号放大是通过生物素分子的共价沉积来实现的。
用冰冻切片机切片和石蜡切片采用抗生物素蛋白-生物素复合物技术染色，并为作比较采用新的最大化免疫组织化学染色方案，即免疫增强法（ImmunoMax法）染色。对每个步骤进行监测，以确定其增强总体敏感性的效果如何。尽管用去污剂、蛋白酶、离液剂或微波加热进行预处理仅使免疫染色有适度改善，但生物素化酪胺增强步骤被证明是最有效的，不过在不使用预处理步骤时，对于许多抗原而言后者并不充分。抗原找回步骤与生物素化酪胺增强步骤相结合使敏感性提高了100至10000倍，且不失特异性。
采用免疫增强法（ImmunoMax法），可以在福尔马林固定、石蜡包埋的组织中可重复地检测特定抗原，且该方法的敏感性得到极大提高。此外，它还能检测许多以前无反应或反应不令人满意的抗原，如本文用单克隆抗体检测IgD、IgM和CD7所显示的那样。
