Formalin-fixed and heat-retrieved tissue antigens: A comparison of their immunoreactivity in experimental antibody diluents

Heat-induced antigen retrieval procedures have become an effective and therefore important part of immunohistochemistry. Although used with increasing frequency, it is not known by what mechanism or to what extent these procedures restore the molecular integrity of the antigen. In an attempt to explore these questions, the immune reactions of 14 tissue antigens with their corresponding monoclonal antibodies were compared both in the absence and after the application of two widely used heat-induced antigen retrieval procedures. The tissue antigens were selected either because of the increased or lack of response to heat retrieval. The nature of their immune reactions was appraised through the variable effects that some diluent solutes were previously shown to have on monoclonal antibodies at different pH. It was found that on heat retrieval, four antigens reacting with monoclonal antibodies of subclass IgG2a or IgG1 reversed their preference for an alkaline pH to that of an acid pH. Eight of nine antigens reacting with monoclonal antibodies of subclass IgG1 and one antigen reacting with an antibody of class IgM remained most reactive at pH 6.0. The retrieved epitope of a B-cell antigen reacting with an IgG3 subclass antibody retained its preference for the alkaline pH. These patterns of response by the antigens to heat-induced retrieval are compared with the previously reported modifications in the immune reactions of monoclonal antibodies when sodium chloride and sodium phosphates were included in the antibody diluent buffer. Use of antibody diluents containing these solutes in combination with heat-induced antigen retrieval resulted in the loss of most of the sensitivity gained through this technique. The possible common mechanisms underlying the causes for these results are discussed.