Inhibition of L. interrogans attachment to immobilized laminin and PLG by recombinant proteins; The effect of laminin concentration on the binding of PLG to Lsa33. (A) Laminin or PLG (1 μg/well) was adsorbed onto microtiter plates followed by incubation with increasing concentrations of Lsa33 (0 to 10 μg) and in (B) laminin was adsorbed onto microtiter plates followed by incubation with increasing concentrations of Lsa25 (0 to 10 μg). In (A) and (B) the incubations were allowed to proceed for 90 min at 37°C. Live leptospires (100 μl/well of 4 X 107L. interrogans serovar Copenhageni strain M20 leptospires) were added and incubated for another 90 min at 37°C. The unbound leptospires were washed away, and the quantification of bound leptospires was performed indirectly by anti - LipL32 antibodies produced in mice (1: 4,000 dilution) followed by horseradish peroxidase - conjugated antimouse IgG antibodies. Each point represents the mean absorbance value at 492 nm ± standard deviation of three replicates. Data are representative of two independent experiments (*P < 0.05). (C) The effect of laminin on the binding of PLG (10 μg/ml) to immobilized rLIC11834 (10 μg/ml) was assessed with the addition of increasing concentrations of laminin (0 to 1.0 μg). The detection of rLIC11834-bound PLG was performed by use of specific antibodies anti - PLG. Bars represent the mean absorbance values ± standard deviation of four replicates for each condition and are representative of two independent experiments. Results of statistically significant interference on the binding in comparison with the control (no addition of laminin) are shown: *P < 0.05.