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. 2011 Jul 6;6(7):e21962. doi: 10.1371/journal.pone.0021962

Figure 6. Recombinant proteins binding to human plasminogen.

Figure 6

(A) Binding of leptospiral recombinant proteins to human plasminogen. Human purified plasminogen (10 µg/ml) was coated to ELISA plates and allowed to interact with the recombinant proteins Lsa66 and Lp30 (10 µg/ml). BSA and fetuin were used as a negative control for nonspecific binding. The binding was detected by specific antibodies. Bars represent the mean of absorbance at 492 nm ± the standard deviation of three replicates for each protein and are representative of three independent experiments. For statistical analyses, the binding of Lsa66 and Lp30 to human PLG was compared to its binding to BSA as well as fetuin by two-tailed t test (*P<0.005 and **P<0.0005). (B) Similar as described in (A) but the binding of the recombinant proteins was detected by specific anti-polyhistidine monoclonal antibodies. (C)Ten µg/ml pf PLG was immobilized into 96-wells ELISA plates and 0 to 2,000 nM of each recombinant protein was added for interaction. The binding was detected using antiserum raised in mice against each protein in appropriate dilutions (1∶500 for Lsa66 and 1∶500 for Lp30) followed by horseradish peroxidase-conjugated anti-mouse IgG. Data represent the mean absorbance values ± the standard deviation of three replicates for each experimental group. The dissociation constant (K D) was calculated based on ELISA data for the recombinant proteins that have reached the equilibrium concentration. (D) Plasmin generation by PLG bound to recombinant proteins was assayed by modified ELISA as immobilized proteins received the following treatment: PLG + uPA + specific plasmin substrate (PLG + uPA + S), or controls lacking one of the three components (PLG + uPA; PLG + S; uPA + S). BSA was employed as negative control. Bars represent mean absorbance at 405 nm, as a measure of relative substrate degradation ± the standard deviation of four replicates for each experimental group and are representative of two independent experiments. Statistically significant binding in comparison to the negative control (BSA) are show: *P<0.001 and **P<0.02.