Tructions (Thermo Scientific). Convalescent sera from 23 individuals with laboratory-confirmed leptospirosis were pooled and 800 ml was added to 3.7 ml of 10 mM phosphate buffered saline, pH 7.4 (PBS) followed by filtration through 0.45 mm filter. Two ml of filtered sera was added to the affinity column and mixed by rotation for 1 h at room temperature. One ml of PBS added to the column, the flow-through (FT) fraction was collected and the rest of filtered sera (2.2 ml) was added to the column repeating the process as described above. The column was washed four times with 2 ml of PBS and LipL32-specific antibodies were recovered by addition of IgG elution buffer (Thermo Scientific) to the affinity column.Materials and Methods Ethics statementThis study was conducted according to principles expressed in the Declaration of Helsinki. Informed written consent was obtained from participants and the study was approved by the Institutional Review Board A of the Research and Development Committee, VA Greater Los Angeles Healthcare System (PCC #2012 – 050702). Co-Author David A. Haake has a patent on leptospiral protein LipL32. This does not alter our adherence to all PLoS ONE policies on sharing data and materials.Membrane fractionationFor membrane affinity experiments, total membranes were isolated as described previously [26]. Briefly, 56109 leptospiral cells were washed twice with PBS, containing 5 mM MgCl2 and resuspended in 0.9 ml of lysis buffer (10 mM TrisHCl, pH 8.0, 5 mM EDTA, 0.5 protease inhibitor cocktail, Sigma-Aldrich) containing 1 mg/ml of lysozyme. The suspension was incubated for 5 min at 4uC and subjected to three cycles of freezing (280uC) and thawing (room temperature) with vigorous vortexing. Then DNase I (Sigma-Aldrich) was added to a final concentration of 5 mg/ml and the cell suspension was incubated on ice for 20 min. Membranes were recovered by centrifugation at 16,0006 g for 15 min at 4uC and resuspended in 0.5 ml of lysis buffer (without lysozyme). A 100 ml aliquot of the membrane suspension was mixed with 100 ml of either 0.2 M Na2CO3, 3.2 M urea, 1.2 M NaCl, or lysis buffer and incubated for 15 min at 4uC. The samples were pelleted at 16,0006 g for 15 min at 4uC and the supernatants were precipitated with acetone. Each membrane The assays. Thus, patients in the MC and NE groups had pellet and its supernatant precipitate were resuspended in 50 ml of Novex NuPage sample buffer (Invitrogen, Carlsbad, CA).Bacterial strains and growth conditionsLeptospira interrogans serovar Title Loaded From File Copenhageni strain Fiocruz L1-130 was isolated from a patient during a leptospirosis outbreak in Salvador, Brazil [5]. Leptospires were cultivated at 30uC in ProbuminTM Vaccine Grade Solution (84-066-5, Millipore, Billerica, MA) diluted five-fold into autoclaved distilled water [21]. Competent E. coli NEB 5-a (New England Biolabs, Ipswich, MA), and BLR(DE3)pLysS (Novagen, Madison, WI) were used for cloning and expression, respectively. E. coli were grown in LuriaBertani (LB) broth or on agar plates with 50 mg/ml carbenicillin, 12.5 mg/ml tetracycline, 34 mg/ml chloramphenicol, 40 mg/ml kanamycin or 40 mg/mlspectinomycin (Sigma-Aldrich, St. Louis, MO) when appropriate.Gel electrophoresis, antibodies and immunoblottingProtein samples were boiled for 5 min in Novex NuPage sample buffer (Life Technologies, Carlsbad, CA) in the presence of 2.5 b-mercapthoethanol and separated through Bis-Tris 4?2 polyacrylamide gradient NuPage gels using the Novex XCell Sure Lock electrophoresis cell (Life Technologies).Tructions (Thermo Scientific). Convalescent sera from 23 individuals with laboratory-confirmed leptospirosis were pooled and 800 ml was added to 3.7 ml of 10 mM phosphate buffered saline, pH 7.4 (PBS) followed by filtration through 0.45 mm filter. Two ml of filtered sera was added to the affinity column and mixed by rotation for 1 h at room temperature. One ml of PBS added to the column, the flow-through (FT) fraction was collected and the rest of filtered sera (2.2 ml) was added to the column repeating the process as described above. The column was washed four times with 2 ml of PBS and LipL32-specific antibodies were recovered by addition of IgG elution buffer (Thermo Scientific) to the affinity column.Materials and Methods Ethics statementThis study was conducted according to principles expressed in the Declaration of Helsinki. Informed written consent was obtained from participants and the study was approved by the Institutional Review Board A of the Research and Development Committee, VA Greater Los Angeles Healthcare System (PCC #2012 – 050702). Co-Author David A. Haake has a patent on leptospiral protein LipL32. This does not alter our adherence to all PLoS ONE policies on sharing data and materials.Membrane fractionationFor membrane affinity experiments, total membranes were isolated as described previously [26]. Briefly, 56109 leptospiral cells were washed twice with PBS, containing 5 mM MgCl2 and resuspended in 0.9 ml of lysis buffer (10 mM TrisHCl, pH 8.0, 5 mM EDTA, 0.5 protease inhibitor cocktail, Sigma-Aldrich) containing 1 mg/ml of lysozyme. The suspension was incubated for 5 min at 4uC and subjected to three cycles of freezing (280uC) and thawing (room temperature) with vigorous vortexing. Then DNase I (Sigma-Aldrich) was added to a final concentration of 5 mg/ml and the cell suspension was incubated on ice for 20 min. Membranes were recovered by centrifugation at 16,0006 g for 15 min at 4uC and resuspended in 0.5 ml of lysis buffer (without lysozyme). A 100 ml aliquot of the membrane suspension was mixed with 100 ml of either 0.2 M Na2CO3, 3.2 M urea, 1.2 M NaCl, or lysis buffer and incubated for 15 min at 4uC. The samples were pelleted at 16,0006 g for 15 min at 4uC and the supernatants were precipitated with acetone. Each membrane pellet and its supernatant precipitate were resuspended in 50 ml of Novex NuPage sample buffer (Invitrogen, Carlsbad, CA).Bacterial strains and growth conditionsLeptospira interrogans serovar Copenhageni strain Fiocruz L1-130 was isolated from a patient during a leptospirosis outbreak in Salvador, Brazil [5]. Leptospires were cultivated at 30uC in ProbuminTM Vaccine Grade Solution (84-066-5, Millipore, Billerica, MA) diluted five-fold into autoclaved distilled water [21]. Competent E. coli NEB 5-a (New England Biolabs, Ipswich, MA), and BLR(DE3)pLysS (Novagen, Madison, WI) were used for cloning and expression, respectively. E. coli were grown in LuriaBertani (LB) broth or on agar plates with 50 mg/ml carbenicillin, 12.5 mg/ml tetracycline, 34 mg/ml chloramphenicol, 40 mg/ml kanamycin or 40 mg/mlspectinomycin (Sigma-Aldrich, St. Louis, MO) when appropriate.Gel electrophoresis, antibodies and immunoblottingProtein samples were boiled for 5 min in Novex NuPage sample buffer (Life Technologies, Carlsbad, CA) in the presence of 2.5 b-mercapthoethanol and separated through Bis-Tris 4?2 polyacrylamide gradient NuPage gels using the Novex XCell Sure Lock electrophoresis cell (Life Technologies).
