Production of Monoclonal Antibodies to Tropheryma whipplei and Identification of Recognized Epitopes by Two-Dimensional Electrophoresis and Mass Spectrometry
Authors: Philippe Decloquement
Yuefei Yu
Malgorzata Kowalczewska
Claude Nappez
Bernard La Scola
Journal: Journal of Clinical Microbiology (2006)
Discipline: MICROBIOLOGY
Keywords: Tropheryma whipplei; monoclonal antibodies; epitope; 2-DE; mass spectrometry
Affiliations: Unit√© des Rickettsies, CNRS UMR 6020, Facult√© de M√©decine de Marseille, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 05, France., UniteÃÅ des Rickettsies, CNRS UMR 6020, FaculteÃÅ de MeÃÅdecine de Marseille, 13385 Marseille Cedex 05, France

Abstract:  Tropheryma whipplei, the agent of Whipple's disease, is a gram-positive rod-shaped bacterium that belongs to the group of actinobacteria. In order to produce monoclonal antibodies (MAbs) against this bacterium, we inoculated mice with two different strains, Slow2 and Endo5. We produced 13 and 10 MAbs against Slow2 and Endo5, respectively. Nine of the Slow2 MAbs and seven of the Endo5 MAbs recognized a 58-kDa epitope. In addition, three other Endo5 MAbs detected a unique 84-kDa epitope. These MAbs were species specific, as they did not react with a selection of 22 different bacterial species, but they were not strain specific, as they did react with six other strains of T. whipplei. Two-dimensional gel electrophoresis (2-DE) was combined with mass spectrometry (MS) to identify the 58-kDa and 84-kDa epitopes recognized by MAbs. After trypsin in-gel digestion of the spot, the 58-kDa protein was identified as an ATP synthase F1 complex beta chain, whereas the 84-kDa protein was identified as a polyribonucleotide nucleotidyltransferase by MS with matrix-assisted laser desorption ionization-time of flight. In an in vitro model, one of these MAbs allowed good detection of T. whipplei in stool samples, contrary to a rabbit polyclonal antibody, which led to high fluorescent background. In the prospective studies, the produced MAb will be tested for detection of T. whipplei in clinical samples, and the gene coding for identified 58-kDa and 84-kDa antigens will be tentatively cloned and then tested for its use in a diagnostic enzyme-linked immunosorbent assay for Whipple's disease. 

Retracted: No

PubPeer comments:
 Figure 3A and Figure 4A appear to look very similar. Legend Figure 3: *Two-dimensional Western blot showing the reactivity of MAbs with Slow2 proteins. The monoclonal antibodies against Slow2 were from the supernatant of hybridoma 7G2 (A), 7H3 (B), 6F5 (C), and 6C3 (D). The 7G2 antibody bound only one spot at 60 kDa and pI 5.1.* Legend Figure 4: *Two-dimensional Western blot showing the reactivity of MAbs with Endo5 proteins. The monoclonal antibodies against Endo5 were from the supernatant of hybridoma 11G10 (A) and 11F10 (B). The 11F10 antibody bound a unique spot at 84 kDa and pI 5.3. The MAb WE11G10 recognized the same spot as WS7G2.* Could the authors please clarify if the blot in Figure 3A (incubated with 7G2) was stripped and then incubated with WE11G10? The legend states the 2 monoclonals recognized the same spot, but the blots also look similar to each other. Shown with red boxes. Note a little dot on the top right corner, and the vertical striping of the membrane left of the 58 kDa spot. Image made a bit darker to bring out details.  I , 
 Surprisingly, one of the author names has been removed some time between the acceptance of the paper and the final publication. Here is a screenshot of the accepted paper, which lists Yuefei Yu , Malgorzata Kowalczewska , Philippe Decloquement , Claude Nappez , Didier Raoult, and Bernard La Scola:  In the published paper, however, the name of author Didier Raoult has disappeared.  In 2006, Raoult was banned from publishing in American Society for Microbiology journals for 1 year, which might have something to do with this author name removal (source: https: science.sciencemag.org content 335 6072 1033). ,