Enzymatic antigen retrieval

Other significant improvements were the development of the enzymatic antigen retrieval methods by Huang et al. [18] and the improvement of systems for secondary antibody detection with the introduction of the avidin-biotin-peroxidase complex (ABC) and the labeled streptavidin-biotin complex (LSAB) by Hsu etal. [19-22]. 

The majority of keratin immunostaining is performed on FFPE tissues. The duration of formalin fixation is a key factor when trying to optimize the technical performance of keratin immunoperoxidase stains. The fixation time is closely related to the time required for enzymatic predigestion.212 Generally, tissue fixed in 10% formalin for more than 2 days requires greater antigen retrieval, with less time required for tissues fixed briefly (hours) in 10% formalin. Most, if not all, keratin antibodies require epitope retrieval (depending on antibody and fixation duration) for optimal keratin antibody performance. 

The staining protocol and differences in pretreatment methods are an important factor when formalin-fixed and paraffin-embedded material is nsed, as not all antibodies work satisfactorily under these conditions. Several different approaches can be found in the literature. Heat-induced antigen retrieval by the use of microwave heat treatment has been demonstrated to be more effective in labeling intraneuronal Ap compared to no or enzymatic pretreatment [29]. Information on the use of FA is inconsistent. It is widely used to increase the immunoreactiv-ity of amyloid plaques in AD however, it has been shown that the effect on intraneuronal Ap is low and that use of FA sometimes results in a loss of cellular detail [30]. A recent study tried to quantify the effect of FA pretreatment and analyzed plaque load and intraneuronal Ap immunoreactivity using an APi, antibody in 6-month-old APP/PSIKI mice (Fig. 3 reproduced from [25] with permission from Elsevier). 

The development of primary antibodies reacting with fixation-resistant epitopes has somewhat improved this situation. However, the introduction of enzymatic pre-treatments for tissue sections — and particularly the introduction of methods for the heat retrieval of tissue antigenicity—were aimed at restoring the affinity and avidity of the immune reaction and have become very important pre-treatment tools in immunohistochemistry (IHC) (3). 

RL Ward, MA Clark, J Lees, NJ Hawkins. Retrieval of human antibodies from phage-display libraries using enzymatic cleavage. J Immunol Meth 189 73-82, 1996. EV Meulemans, R Slobbe, P Wasterval, FC Ramaekers, GJ van Eys. Selection of phage-displayed antibodies specific for a cytoskeletal antigen by competitive elution with a monoclonal antibody. J Mol Biol 244 353-360, 1994. 

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