title: Antigen retrieval causes protein unfolding: evidence for a linear epitope model of recovered immunoreactivity.
abstract: Antigen retrieval (AR), in which formalin-fixed paraffin-embedded tissue sections are briefly heated in buffers at high temperature, often greatly improves immunohistochemical staining. An important unresolved question regarding AR is how formalin treatment affects the conformation of protein epitopes and how heating unmasks these epitopes for subsequent antibody binding. The objective of the current study was to use model proteins to determine the effect of formalin treatment on protein conformation and thermal stability in relation to the mechanism of AR. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to identify the presence of protein formaldehyde cross-links, and circular dichroism spectropolarimetry was used to determine the effect of formalin treatment and high-temperature incubation on the secondary and tertiary structure of the model proteins. Results revealed that for some proteins, formalin treatment left the native protein conformation unaltered, whereas for others, formalin denatured tertiary structure, yielding a molten globule protein. In either case, heating to temperatures used in AR methods led to irreversible protein unfolding, which supports a linear epitope model of recovered protein immunoreactivity. Consequently, the core mechanism of AR likely centers on the restoration of normal protein chemical composition coupled with improved accessibility to linear epitopes through protein unfolding.
authors.affiliation: Department of Biophysics, Armed Forces Institute of Pathology, Rockville, Maryland 20850, USA.
authors.forename: Carol B
authors.initials: CB
authors.lastname: Fowler
authors.forename: David L
authors.initials: DL
authors.lastname: Evers
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: O'Leary
authors.forename: Jeffrey T
authors.initials: JT
authors.lastname: Mason
mesh_terms: D000941:Antigens / Q000737:chemistry*; D002942:Circular Dichroism; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes; D005404:Fixatives; D005557:Formaldehyde; D006358:Hot Temperature; D016612:Paraffin Embedding; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding; D011506:Proteins / Q000737:chemistry*
chemical_list: D000941:Antigens; D003432:Cross-Linking Reagents; D000939:Epitopes; D005404:Fixatives; D011506:Proteins; D005557:Formaldehyde