title: Molecular mechanisms of antigen retrieval: antigen retrieval reverses steric interference caused by formalin-induced cross-links.
abstract: The overwhelming majority of antibodies useful for formalin fixed, paraffin embedded (FFPE) tissues require antigen retrieval to reverse the effect of formalin fixation and re-establish immunoreactivity. How this reversal happens is poorly understood. We developed a new experimental model for studying the mechanism of formalin fixation and antigen retrieval. Epitope mapping studies on nine antibodies useful for FFPE tissues revealed that each consisted of a contiguous stretch of amino acids in the native protein (linear epitope). Small peptides representing the epitopes of antibodies to human epidermal growth factor receptor type (HER2), estrogen, and progesterone receptors were attached covalently to glass microscope slides in a peptide array. Most peptides retained immunoreactivity after formalin fixation. Immunoreactivity was completely abrogated for all peptides, however, if an irrelevant large protein was present during formalin-induced cross-linking. We hypothesize that cross-linking the irrelevant protein to the peptide epitopes sterically blocked antibodies from binding. Antigen retrieval dissociates irrelevant proteins and restores immunoreactivity. Because the epitopes for clinical antibodies require only primary protein structure, the fact that antigen retrieval probably denatures the secondary and tertiary structure of the protein is irrelevant. The same mechanism may occur in tissue samples subjected to formalin fixation and antigen retrieval.
authors.affiliation: Medical Discovery Partners LLC, 715 Albany Street. sbogen@bu.edu
authors.forename: S A
authors.initials: SA
authors.lastname: Bogen
authors.forename: K
authors.initials: K
authors.lastname: Vani
authors.forename: S R
authors.initials: SR
authors.lastname: Sompuram
mesh_terms: D000937:Antigen-Antibody Reactions / Q000187:drug effects*; D003432:Cross-Linking Reagents; D018604:Epitope Mapping; D000939:Epitopes / Q000032:analysis* / Q000737:chemistry; D004967:Estrogens / Q000276:immunology; D005404:Fixatives; D005557:Formaldehyde / Q000494:pharmacology*; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology; D011980:Receptors, Progesterone / Q000276:immunology; D016707:Tissue Fixation / Q000379:methods*
chemical_list: D003432:Cross-Linking Reagents; D000939:Epitopes; D004967:Estrogens; D005404:Fixatives; D005557:Formaldehyde; D018719:Erb-b2 Receptor Tyrosine Kinases; D011980:Receptors, Progesterone; C508053:ERBB2 protein, human