Characterization of an anti‐Borrelia burgdorferi OspA conformational epitope by limited proteolysis of monoclonal antibody‐bound antigen and mass spectrometricpeptide mapping

Abstract

Lyme borreliosis is a multisystem disorder caused by the spirochete Borrelia burgdorferi that is transmitted to humans by the tick Ixodes dammini. The immune response against the 31 kDa OspA, which is one of the most abundant B. burgdorferi proteins, appears to be critical in preventing infection and tissue inflammation. Detailed knowledge of the immunological and molecular characteristics of the OspA protein is important for the development of reliable diagnostic assays. In this study, we characterized a new conformational epitope present within the middle part of B. burgdorferi OspA. Our approach used enzymatic proteolyses of the immune complex followed by mass spectrometric identification of the peptides bound to the antibody. It appears to be one of the first reports on the characterization of a discontinuous epitope using mass spectrometry.

Rapid Identification of Novel Immunodominant Proteins and Characterization of a Specific Linear Epitope of Campylobacter jejuni

“…The state-of-the-art technique involves the cocrystallization of an antigen with a monoclonal antibody [50,51]. Besides, mutation analysis [52], mass-spectrometry [53] or CLIPS technology [54] are other methods to identify structural epitopes. However, the existing methods are extremely costly, time-consuming and demand high material inputs.…”

Involvement of the C‐terminal end of the prostrate‐specific antigen in a conformational epitope: characterization by proteolytic degradation of monoclonal antibody‐bound antigen and mass spectrometry

“…This methodology has already been shown to be efficient for the characterization of a conformational epitope (Legros et al, 2000). Binding of alternative PSA-related forms by mAbs 11E5C6 and 5D5A5, another anti-total PSA mAb that has been shown to be directed against a conformational epitope which is close to but different from that of 11E5C6 , allowed us to confirm the localization of the mAb 11E5C6 epitope and to emphasize the potential of this mAb for discriminating different PSArelated forms.…”

“…The combination of proteolytic degradation with mass spectrometry allowed this approach to become a powerful tool for epitope mapping applications (Zhao and Chait, 1994;Macht et al, 1996;Zhao et al, 1996;Yu et al, 1998;Kiselar and Downard, 1999;Suckau et al, 1990;Parker et al, 1996;Jeyarajah et al, 1998;Yi et al, 2000). However, although many studies describe the localization of linear epitopes, very few studies have identified discontinuous epitopes, permitting a conformational determination (Legros et al, 2000;Hochleitner et al, 2000a, b;Yi and Skalka, 2001).…”

“…We recently reported the characterization of an anti-Borrelia burgdorferi outer surface protein A (OspA) conformational epitope by limited proteolysis of mAb-bound antigen and MS analysis (Legros et al, 2000). This epitope was composed of two OspA parts, distant in the sequence, but close in the three-dimensional structure.…”

“…For each identified peptide, the intensity of selected ions was extracted, and the ratio of ion intensity of bound peptide contained in the eluate (E)/ion intensity of unbound peptide contained in the supernatant (S) was calculated for the most abundant ion, as previously described (Legros et al, 2000). It should be noted that the various ion signal intensities were peptide-dependent and could not be compared with each other, but in contrast the E/S ratios obtained for each peptide could be compared within the same analysis.…”