Antibodies immunoreactive with formalin-fixed tissue antigens recognize linear protein epitopes.
It is not clearly understood why some monoclonal antibodies bind to their antigens informalin-fixed, paraffin-embedded tissue sections but others do not. To address this question, we analyzed the protein epitopes of 9 monoclonal antibodies that are immunoreactive after formalin fixation and antigen retrieval. We identified the antibody contact sites by using phage display and synthesized corresponding peptides derived from the GenBank database sequence that contain the predicted antibody binding sites. Our data indicate that all 9 antibodies bind to linear epitopes, ie, composed of contiguous amino acids. In addition, the amino acids proline, tyrosine, glutamine, and leucine are highly represented in these antibody contact sites. The epitopes tend to be mildly to moderately hydrophilic. These findings are the first detailed studies of antibody epitopes associated with antigen retrieval and suggest that antibodies must recognize linear sequences to bind after formalin fixation and antigen retrieval.
Sompuram
Seshi R
SR
Medical Discovery Partners LLC, Boston, MA 02118, USA.
Vani
Kodela
K
Hafer
Laurie J
LJ
Bogen
Steven A
SA
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D000941:Antigens / Q000032:analysis; D001666:Binding Sites, Antibody; D000939:Epitopes / Q000276:immunology*; D005404:Fixatives; D005557:Formaldehyde; D006801:Humans; D019394:Ki-67 Antigen / Q000276:immunology; D019151:Peptide Library; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology; D011960:Receptors, Estrogen / Q000276:immunology; D011980:Receptors, Progesterone / Q000276:immunology; D016159:Tumor Suppressor Protein p53 / 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; D016159:Tumor Suppressor Protein p53