Production of monoclonal antibodies to Tropheryma whipplei and identification of recognized epitopes by two-dimensional electrophoresis and mass spectrometry.
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.
Yu
Yuefei
Y
Unité des Rickettsies, CNRS UMR 6020, Faculté de Médecine de Marseille, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 05, France.
Kowalczewska
Malgorzata
M
Decloquement
Philippe
P
Nappez
Claude
C
La Scola
Bernard
B
D039903:Actinobacteria / Q000276:immunology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D015153:Blotting, Western; D015180:Electrophoresis, Gel, Two-Dimensional; D000939:Epitopes; D005243:Feces / Q000382:microbiology; D006801:Humans; D013058:Mass Spectrometry; D051379:Mice; D008807:Mice, Inbred BALB C; D012680:Sensitivity and Specificity; D008061:Whipple Disease / Q000175:diagnosis* / Q000382:microbiology
D000911:Antibodies, Monoclonal; D000939:Epitopes