The serum samples of 10 patients diagnosed with streptococcal pneumonia caused by Streptococcus pneumoniae and 25 healthy persons were obtained from the 307 Hospital of PLA (Beijing, China). These serum samples were all Q fever antibody negative (QAb-negative) tested as described previously [27]. The present project is in compliance with the Helsinki Declaration (Ethical Principles for Medical Research

Involving Human Subjects). This study was approved by the ethics committee of the Beijing Institute of Microbiology and Epidemiology. In each hospital, the serum samples of patients were collected as part of the routine management of patients without any additional sampling, and all patient data was deidentified. Two-dimensional (2-D) electrophoresis of C. burnetii proteins The BIX 1294 research buy purified C. burnetii organisms were GDC-0449 solubility dmso rinsed with cold PBS and centrifuged at 12,000 g for 30 min at 4°C

with an Allegra™ 21R centrifuge (Beckman, Fullerton, CA). see more The supernatant was discarded and the pellet resuspended in rehydration buffer (7 M urea, 2 M thiourea, 4% [wt/vol] CHAPS, 1% [wt/vol] DTT, 0.2% [vol/vol] Bio-lyte). The cell lysates were sonicated (300 W, 3 s on and 9 s off) for 30 min at 4°C using a ultrasonic processor (Sonics & Materials, Newtown, CT), then centrifuged at 20,000 g for 1 h at 17°C to remove any insoluble material prior to isoelectric focusing. The supernatant was collected and the proteins precipitated with a 2-D Clean-Up Kit (Amersham, Piscataway, NJ) according to the manufacture’s instruction. The pellets were resuspended in rehydration buffer and the protein concentration of the solution determined using the Bradford method [28]. The protein solution was aliquoted and stored

at −70°C until used. A 350 μl protein solution (800 μg of Coxiella protein) was loaded onto each 17-cm nonlinear Immobiline Protein kinase N1 DryStrips (pH 3 to 10, Bio-Rad, Hercules, CA). The isoelectric focusing was performed at 50v for 12 h, 200v for 1 h, 1000v for 1 h, 10, 000v for 11 h, and 500v for 8 h using a Protean IEF cell system (Bio-Rad, Hercules, CA). Following isoelectric focusing, the strips were equilibrated and placed on sodium dodecyl sulfate (SDS)-polyacrylamide gels for second-dimension electrophoresis as described previously [29]. The gels were then stained with modified Coomassie brilliant blue [30]. Immunoblotting of C. burnetii proteins Following 2-D electrophoresis, the Coxiella proteins in the gels were transferred onto a 0.45 μm polyvinylidene difluoride membranes (Millipore, Bedford, MA) at 0.8 mA/cm2 for 1 h with transfer buffer (48 mM Tris-base, 39 mM glycine, 0.04% [wt/vol] SDS, 20% [vol/vol] methanol) and then blocked overnight in blocking buffer (20 mmol/L Tris-base, 137 mmol/L NaCl supplemented with 0.05% [vol/vol] Tween 20, 5% [wt/vol] skimmed milk, pH 7.6) at 4°C.