H. Merz et al., METHODS IN LABORATORY INVESTIGATION IMMUNOMAX - A MAXIMIZED IMMUNOHISTOCHEMICAL METHOD FOR THE RETRIEVAL AND ENHANCEMENT OF HIDDEN ANTIGENS, Laboratory investigation, 73(1), 1995, pp. 149-156
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.