Positive reactions on Western blots do not necessarily indicate the epitopes on antigens are continuous

Abstract

Epitope mapping (identification of an antigenic site recognized by an antibody) is an important component of vaccine development and immunological assays. It is widely accepted that in Western blots, antibodies react exclusively with continuous epitopes: discontinuous epitopes are assumed to be irreversibly destroyed by electrophoresis under the denaturing conditions used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Here, we demonstrate that the epitopes recognized by four different monoclonal antibodies were identified as discontinuous epitopes when characterized by radioimmunoprecipitation assays and enzyme-linked immunosorbent assays, yet each of these antibodies reacted with the corresponding antigen on Western blots. Reaction on Western blots may be due to epitope renaturation during or after the transfer of the protein to a membrane. Therefore, positive reactions on Western blots do not necessarily indicate that epitopes are continuous and this caveat should be kept in mind while characterizing them.

Inhibition of β-Secretase in Vivo via Antibody Binding to Unique Loops (D and F) of BACE1

“…All data together suggested that the epitope of mAb 1A11 was not linear. It has been previously reported that conformational epitopes can be detected by Western blot, probably because of epitope renaturation during or after the transfer of the protein to the blotting membrane (51). Thus, we speculate that mAb 1A11 may bind to a conformational epitope of BACE1.…”

Characterization of Conformation-dependent Prion Protein Epitopes

“…However, our findings indicate that, subsequent to denaturing treatments, PrP readily refolds during or after Western blotting to a structure amenable to detection by conformation-dependent antibodies. Although it is widely assumed that discontinuous epitopes are generally irreversibly destroyed under the denaturing conditions used for SDS-PAGE, positive reactions on Western blots with antibodies recognizing discontinuous epitopes are not without precedent (44).…”

Cross-Reactive Anti-Avian H5N1 Influenza Neutralizing Antibodies in a Normal ‘Exposure-Naive’ Australian Blood Donor Population

“…However, a caveat needs to be raised regarding the long held perception that antibodies bind in western blots to only linear epitopes, in the light of recent findings by Zhou et al that challenge this view, with evidence that Ab-binding to discontinuous regions can occur. They have suggested that this is possible as a result of protein refolding of secondary structures during gel-blot processing [21]. …”