pneumoniae-negative by the CFT and an IgM ELISA test (Platelia, Bio-Rad). Furthermore, the specificity of the rAtpD protein-based ELISAs was assessed using 55 additional serum samples, 18 that were positive for a C. pneumoniae infection (National Reference Center for Chlamydiae, Université Victor Segalen Bordeaux 2, France), 10 that were positive for a L. pneumophila infection (National Reference Center for Legionella, Université Lyon 1, France), 10 that were positive for a C. burnetii infection (Pellegrin hospital, Bordeaux,

France), 8 that were from patients harboring a S. pneumoniae RTI (Raymond Poincaré hospital, Garches, MK-4827 France), 8 that were positive for a B. pertussis infection (Marcel Merieux Laboratory, Lyon, France), and 1 that was positive for a C. psittaci infection (National Reference Center for Chlamydiae, Université Victor Segalen Bordeaux 2, France). The present project is in compliance with the GDC-0941 chemical structure Helsinki Declaration (Ethical Principles for Medical Research Involving Human Subjects). BIBW2992 molecular weight The study was done in accordance with the guidelines of the ethical committees of the participating hospitals. In each hospital, specimens were collected as part

of the routine management of patients without any additional sampling, and patients provided no objection for their samples to be used. According to the French legacy, this study did not need to be examined by the French “”Comité pour la Protection des Personnes”" and allowed the exemption of patient’s written informed consent. All patient data shown in the present work were anonymously reported, without offering

any possibility Thymidylate synthase to trace the actual patients. 2D-E The bacterial pellet were suspended in rehydratation solution (Ready-Prep 2-D Rehydratation/Sample Buffer 1, Bio-Rad) composed of 7 M urea, 2 M thiourea, 1% (wt/vol) ASB-14 detergent, 40 mM Tris, 4% 3[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfate (CHAPS), 0.2% (vol/vol) immobilised pH gradient (IPG) buffer, pH 3-10, 20 mM dithiothreitol (DTT) and 0.002% bromophenol blue. Cell lysis was performed by sonication three times 20 s (Branson Sonifier), and the un-disrupted cells were removed by centrifugation (20,817 × g; 45 min; 21°C). Total protein concentration was determined using a 2-D Quant kit (GE Healthcare) according to the manufacturer’s instructions. The protein concentration was calculated using bovine serum albumin (BSA) as a standard. Isoelectric focusing was performed using the Protean IEF Cell system and Immobilised pH gradient (IPG) strips with a pH range of 5-8 (Bio-Rad). Two hundred and fifty μg of the protein samples in 150 μl of rehydratation solution was used to rehydrate the IPG strips (7 cm, pH 5-8) overnight at 20°C under mineral oil. The proteins were focused for 10 kVh with a maximum voltage of 4,000 V at 20°C.