A Molecular Mechanism of Formalin Fixation and Antigen Retrieval

Despite the popularity of antigen-retrieval techniques, the precise molecular mechanism underlying the process remains enigmatic. We examined the molecular features underlying the loss of immunoreactivity following formalin fixation, with subsequent recovery by antigen retrieval. To do this, we first created a molecular model using short peptides that mimic the antibody-binding site of common clinical protein targets. The advantage of this model is that we know the amino acid sequence in and around the antibody-binding site. We observed that some, not all, of the peptides exhibited the formalin-fixation and antigen-retrieval phenomenon. Other peptides did not lose their ability to be recognized by antibody, even after prolonged incubation in formalin. A third, intermediate group exhibited the formalin-fixation and antigen-retrieval phenomenon only if another irrelevant protein was mixed with the peptide before fixation. Amino acid sequence analysis indicates that fixation and antigen retrieval are associated with a tyrosine in or near the antibody-binding site and with an arginine elsewhere, implicating the Mannich reaction as important in fixation and antigen retrieval.