A Molecular Mechanism of Formalin Fixation and Antigen Retrieval
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.
In 1991, Shi and coworkers described their finding that boiling tissue sections in heavy metal solutions reversed the formalin-fixation effect. Namely, the reactivity of many antibodies for tissue epitopes can be restored by boiling tissue sections, a process often referred to as antigen retrieval. This finding and subsequent refinement of the technique helped facilitate the dramatic growth in the use of immunohistochemical analysis for surgical pathology. During the ensuing decade, numerous procedural modifications were described. These modifications include the composition of the antigen-retrieval buffer, temperature (eg, using a pressure cooker or not), and the use (or not) of microwave irradiation. Despite these methodological improvements, previous studies of the technique have largely been empirical in nature. Namely, certain procedural modifications were correlated with better or worse immunostaining, without a mechanistic understanding of the underlying process. To date, it has not been possible to delineate the precise molecular and structural features that are responsible for the formalin-fixation and antigen-retrieval phenomenon.
Our understanding of the effects of formaldehyde on proteins traces back to the original work of Fraenkel-Conrat and colleagues, published during the 1940s. Formaldehyde is capable of a variety of cross-linking reactions, recently summarized by Shi et al. In solution, formaldehyde is capable of binding to the following amino acids: lysine (K), arginine (R), tyrosine (Y), asparagine (N), histidine (H), glutamine (Q), and serine (S). It is not clear, however, which (if any) of these reactions might be relevant in the context of antigen retrieval. For example, it is not even clearly established whether antigen retrieval actually breaks formaldehyde cross-links. Other proposed hypotheses include extraction of diffusible blocking proteins, precipitation of proteins, and rehydration of the tissue section, thereby allowing better penetration of antibody; removal of cage-like calcium complexes; and heat mobilization of trace remaining amounts of paraffin. Thus, formaldehyde has a variety of effects on tissue, only some of which are likely to be associated with the antigen-retrieval phenomenon. It was our goal in the present study to identify the formaldehyde-induced effects associated with the loss of immunoreactivity and subsequent recovery associated with antigen retrieval.
To do this, we created a model immunostaining system using synthetic peptides. Each peptide mimics the antibody-combining site of a particular antigen, such as estrogen receptor (ER), progesterone receptor (PR), Ki-67, p53, or HER-2. We identified the peptides using phage display, a combinatorial display technique in which an antibody (or other binder) selects from billions of possible peptide combinations. The selected peptides represent essentially 1 epitope of a larger protein. The peptides are relatively short (approximately 20 mer) and conformationally constrained in a cyclic orientation. For this reason, we initially thought it unlikely that such peptides could undergo formalin fixation, leading to loss of antibody reactivity. It was, therefore, with surprise that we discovered that some of the peptides undergo fixation and antigen retrieval, just as the native proteins do. Other peptides do not. Since we know the precise amino acid composition of the peptides, we were able to precisely correlate the presence of specific amino acids with the formalin-fixation and antigen-retrieval phenomenon. We describe the model system for fixation and antigen retrieval, the correlation of the peptides' fixation characteristics with their amino acid sequence, and 2 potential applications of these findings.
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.
In 1991, Shi and coworkers described their finding that boiling tissue sections in heavy metal solutions reversed the formalin-fixation effect. Namely, the reactivity of many antibodies for tissue epitopes can be restored by boiling tissue sections, a process often referred to as antigen retrieval. This finding and subsequent refinement of the technique helped facilitate the dramatic growth in the use of immunohistochemical analysis for surgical pathology. During the ensuing decade, numerous procedural modifications were described. These modifications include the composition of the antigen-retrieval buffer, temperature (eg, using a pressure cooker or not), and the use (or not) of microwave irradiation. Despite these methodological improvements, previous studies of the technique have largely been empirical in nature. Namely, certain procedural modifications were correlated with better or worse immunostaining, without a mechanistic understanding of the underlying process. To date, it has not been possible to delineate the precise molecular and structural features that are responsible for the formalin-fixation and antigen-retrieval phenomenon.
Our understanding of the effects of formaldehyde on proteins traces back to the original work of Fraenkel-Conrat and colleagues, published during the 1940s. Formaldehyde is capable of a variety of cross-linking reactions, recently summarized by Shi et al. In solution, formaldehyde is capable of binding to the following amino acids: lysine (K), arginine (R), tyrosine (Y), asparagine (N), histidine (H), glutamine (Q), and serine (S). It is not clear, however, which (if any) of these reactions might be relevant in the context of antigen retrieval. For example, it is not even clearly established whether antigen retrieval actually breaks formaldehyde cross-links. Other proposed hypotheses include extraction of diffusible blocking proteins, precipitation of proteins, and rehydration of the tissue section, thereby allowing better penetration of antibody; removal of cage-like calcium complexes; and heat mobilization of trace remaining amounts of paraffin. Thus, formaldehyde has a variety of effects on tissue, only some of which are likely to be associated with the antigen-retrieval phenomenon. It was our goal in the present study to identify the formaldehyde-induced effects associated with the loss of immunoreactivity and subsequent recovery associated with antigen retrieval.
To do this, we created a model immunostaining system using synthetic peptides. Each peptide mimics the antibody-combining site of a particular antigen, such as estrogen receptor (ER), progesterone receptor (PR), Ki-67, p53, or HER-2. We identified the peptides using phage display, a combinatorial display technique in which an antibody (or other binder) selects from billions of possible peptide combinations. The selected peptides represent essentially 1 epitope of a larger protein. The peptides are relatively short (approximately 20 mer) and conformationally constrained in a cyclic orientation. For this reason, we initially thought it unlikely that such peptides could undergo formalin fixation, leading to loss of antibody reactivity. It was, therefore, with surprise that we discovered that some of the peptides undergo fixation and antigen retrieval, just as the native proteins do. Other peptides do not. Since we know the precise amino acid composition of the peptides, we were able to precisely correlate the presence of specific amino acids with the formalin-fixation and antigen-retrieval phenomenon. We describe the model system for fixation and antigen retrieval, the correlation of the peptides' fixation characteristics with their amino acid sequence, and 2 potential applications of these findings.
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