Antibodies Immunoreactive With Formalin-Fixed Tissue Antigens Recognize Linear Protein Epitopes

Seshi R. Sompuram, Kodela Vani, Laurie J. Hafer, Steven A. Bogen
2006 American Journal of Clinical Pathology  
A b s t r a c t It is not clearly understood why some monoclonal antibodies bind to their antigens in formalin-fixed, paraffin-embedded tissue sections but others do not. To address this question, we analyzed the protein epitopes of 9 monoclonal antibodies that are immunoreactive after formalin fixation and antigen retrieval. We identified the antibody contact sites by using phage display and synthesized corresponding peptides derived from the GenBank database sequence that contain the
more » ... ontain the predicted antibody binding sites. Our data indicate that all 9 antibodies bind to linear epitopes, ie, composed of contiguous amino acids. In addition, the amino acids proline, tyrosine, glutamine, and leucine are highly represented in these antibody contact sites. The epitopes tend to be mildly to moderately hydrophilic. These findings are the first detailed studies of antibody epitopes associated with antigen retrieval and suggest that antibodies must recognize linear sequences to bind after formalin fixation and antigen retrieval. Restoration of immunoreactivity to tissue antigens following antigen retrieval is a cornerstone technology for modern histopathologic diagnosis. Although the antigen-retrieval technique has been practiced for slightly more than a decade, the protein chemistry explaining it is poorly understood. A variety of potential mechanisms have been forwarded as explanations for the restoration of immunoreactivity after antigen retrieval. These include extraction of diffusible blocking proteins, precipitation of proteins, and rehydration of the tissue section, thereby allowing better penetration of antibody 1 ; removal of cage-like calcium complexes 2 ; and heat mobilization of trace remaining amounts of paraffin. 3 Based on the chemistry of formalin reactions, recently reviewed by Shi et al, 4 the most likely mechanisms are associated with protein cross-linking. Recently, formalin fixation and antigen retrieval were analyzed using model systems by biophysical techniques. 5,6 Those studies suggested that the formaldehyde reaction creates intramolecular and intermolecular cross-links, many of which are broken on antigen retrieval. Our own recent study using peptides that mimic antibody binding sites further suggested that these cross-links may be formed, at least in part, by the Mannich reaction. 7 A variety of monoclonal antibodies (mAbs) have gained popularity because of their usefulness in conjunction with formalin-fixed tissue samples after antigen retrieval. These mAbs represent a select subset, based on their clinical usefulness and ability to stain formalin-fixed, paraffin-embedded tissue sections after antigen retrieval. Antibodies that are not immunoreactive after antigen retrieval generally are not used for routine clinical immunopathology. It is not known why some antibodies are immunoreactive after antigen retrieval and others are not. The ability of an antibody to stain tissue proteins after
doi:10.1309/6h0arqf7k3y608eh pmid:16482995 fatcat:wow2ltovavfhrnlis74hithtki