METHODS IN LABORATORY INVESTIGATION IMMUNOMAX - A MAXIMIZED IMMUNOHISTOCHEMICAL METHOD FOR THE RETRIEVAL AND ENHANCEMENT OF HIDDEN ANTIGENS

BACKGROUND: Since the introduction of mAb, immunohistochemistry has be come an important tool in research and in surgical pathology. The most widely used fixative in routine histopathology is formaldehyde, and i t has become the gold standard for morphologic tissue preservation. Al though the molecular mechanism underlying the tissue fixation is not w ell understood, it has become clear that available immunoreactive Ag a re progressively lost during the fixation process. For a long time, it was thought that formalin-sensitive Ag might be irreversibly destroye d during the fixation process. Although monoclonal anti- Ig Ab frequen tly worked inadequately, polyclonal anti-Ig Ab were shown to produce r eproducible staining results. It thus appeared possible that most cell ular Ag might not be irreversibly destroyed but only masked. EXPERIMEN TAL DESIGN: Although some Ag may be retrieved under appropriate condit ions, 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 thi s dilemma is the use of a combination of an optimized Ag retrieval sys tem and a powerful immunohistochemical staining protocol introducing a biotin amplification step, in which signal amplification is accomplis hed by covalent deposition of biotin molecules.RESULTS: Cryostat and p araffin sections were stained with the avidin-biotin complex technique and, for comparison, with the new maximized immunohistochemical stain ing protocol, termed the ImmunoMax method. Each step was monitored to establish how effectively it enhanced the overall sensitivity. Althoug h pretreatment with detergent, protease, a chaotropic substance, or mi crowave heating resulted in only moderately improved immunostaining, t he biotinylated tyramine enhancement step proved to be the most effici ent one, although the latter is not sufficient for many Ag when used w ithout pretreatment steps. The combination of an Ag retrieval step wit h the biotinylated tyramine enhancement step resulted in a 100 to 10,0 00-fold boost in sensitivity without loss of specificity.CONCLUSIONS: 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 previou sly unreactive or unsatisfactorily reactive Ag to be detected, as show n here for IgD, IgM, and CD7 with the use of mAb.