A molecular model of antigen retrieval using a peptide array.
Even though antigen retrieval is highly denaturing, it paradoxically restores immunoreactivity after formalin fixation. It is unclear how this happens. We address this question using a peptide array to model formalin fixation and antigen retrieval. The peptides are linear stretches based on the native protein sequence, containing antibody epitopes of HER-2, estrogen receptor, progesterone receptor, and Ki-67. Of the 7 peptides, 6 retain their immunoreactivity after formalin fixation. However, if formalin fixation is performed in the presence of an irrelevant protein, immunoreactivity is abrogated, regardless of the peptides' amino acid composition. Fixation of an external protein around the antibody epitope prevents antibody binding. Antigen retrieval restores immunoreactivity. These findings demonstrate that native protein conformation is not relevant during antigen retrieval. Moreover, the loss and recovery of immunoreactivity associated with fixation and antigen retrieval, respectively, can be accounted for completely with a model of steric interference by adjacent proteins.
Medical Discovery Partners, Boston, MA 02118, USA.
Seshi R
SR
Sompuram
Kodela
K
Vani
Steven A
SA
Bogen
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000937:Antigen-Antibody Reactions / Q000187:drug effects; D000941:Antigens / Q000032:analysis / Q000302:isolation & purification*; D000939:Epitopes; D005404:Fixatives; D005557:Formaldehyde; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D019394:Ki-67 Antigen / Q000276:immunology; D051379:Mice; D008958:Models, Molecular; D019151:Peptide Library; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology; D011960:Receptors, Estrogen / Q000276:immunology; D011980:Receptors, Progesterone / Q000276:immunology
D000911:Antibodies, Monoclonal; D000941:Antigens; D000939:Epitopes; D005404:Fixatives; D005557:Formaldehyde; D019394:Ki-67 Antigen; D019151:Peptide Library; D018719:Erb-b2 Receptor Tyrosine Kinases; D011960:Receptors, Estrogen; D011980:Receptors, Progesterone