Protease accessibility assay of LIC11834 and LIC12253 encoded proteins of L. interrogans. Viable leptospires were incubated with 25 μg/ml of proteinase K at the indicated times. The suspensions were sedimented, washed, ressuspended in PBS and coated in a microplate. Antibodies against recombinant proteins Lsa33, Lsa25, LipL32 and DnaK were added. After incubation, anti-IgG peroxidase conjugated was added and the reaction was developed with OPD peroxidase substrate. Blanks were run in parallels but antibodies against the proteins were omitted. Readings were taken at 492 nm. Bars represent the mean of absorbance ± the standard deviation of three replicates for each protein and are representative of three independent experiments. For statistical analyses, the signal was compared between 0 hour and hours of treatment with PK by two-tailed t test (*P < 0.05).