Agglutination was examined by dark-field microscopy and titres were measured as the last dilution where at least 50% of the leptospires were agglutinated (Cole et al., 1973). MAT results were read blind by two expert operators in two different centres; the reactions with LaiWT and LaiMut were tested at the same time. Twenty millilitres of 14-day cultures of LaiWT MG-132 ic50 or LaiMut were centrifuged (3500 g at 4 °C) for

6 min. The pellet was washed twice with sterile distilled water and then resuspended in 700 μL of distilled water, to which 350 μL of 3 × treatment buffer [0.125 M Tris-HCl (pH 6.8); SDS (sodium dodecyl sulphate, 4%); glycerol (20%); 2, β-mercaptoethanol (15%); bromophenol blue (0.1%)] was added. The mixture was heated to 100 °C for 5 min, 1 μg proteinase K was added, and incubated overnight at 60 °C (Hitchcock & Brown, 1983), and the solution was stored frozen until analysis. A discontinuous SDS-PAGE (gradient 6–15%) was used to analyse lipopolysaccharide molecules from LaiWT and LaiMut (Laemmli, 1970). For the

visualization of lipopolysaccharide, polyacrylamide gels were stained using the procedure described by Tsai & Frasch (1982) as modified by Hitchcock & Brown (1983). Following electrophoresis, lipopolysaccharide was transferred to Immobilon-P membranes (Millipore, St. Louis, MO) (Towbin et al., 1979) and probed with mAb F70C7 at a 1 : 100 dilution as the primary antibody and www.selleckchem.com/products/SB-203580.html alkaline phosphatase-labelled goat anti-mouse immunoglobulin G (Kirkegaard and Perry, MD) at a 1 : 5000 dilution as the secondary antibody. Reactions were visualized colourimetrically with a solution containing 90 μL of NBT (75 mg mL−1 of nitroblue tetrazolium in 70% dimethylformamide), 70 μL of BCIP Glutamate dehydrogenase (50 mg mL−1 of 5-bromo-4-chloro-3-indolyl phosphate), and 20 mL of alkaline buffer (100 mM Tris, 100 mM NaCl, 50 mM MgCl2, pH 9.5). Each sequence read was trimmed for quality and mapped to a region of the reference genome sequence of serovar Lai (Ren et al.,

2003) representing the lipopolysaccharide biosynthesis locus using sequencher 3.1 (Gene Codes, Ann Arbor, MI). An escape mutant strain of LaiWT was obtained after serial subculture in the presence of mAb F70C7. Strains LaiWT and LaiMut had identical RFLP patterns using either EcoRI or BamHI (data not shown). This near genetic identity was further confirmed by sequencing of the secY gene, which was identical in both strains. The MAT titre of F70C7 against LaiWT was 1280, whereas LaiMut was not agglutinated by F70C7. Additional MAT testing with a set of mAbs and polyclonal sera revealed that the agglutination of LaiMut was decreased by all reactive mAbs and polyclonal sera against serovar Lai, except for mAb F20C4-1, which showed an increased titre.