Binding characteristics of Lsa33 and Lsa25 proteins to ECM components. (A) Wells were coated with 1 μg of laminin, collagen type I, collagen type IV, cellular fibronectin, plasma fibronectin and the control proteins gelatin and fetuin. One μg of the recombinant proteins Lsa33 and Lsa25 was added per well and the binding was measured by ELISA. In (A) the protein binding was detected by polyclonal antibodies against each protein, while in (B) protein binding was evaluated with monoclonal anti - polyhistidine serum. Data represent the mean ± the standard deviation from three independent experiments. For statistical analyses, the attachment of recombinant proteins to the ECM components was compared to its binding to gelatin by the two - tailed t test (*P < 0.05). (C) Dose - dependent binding experiments of recombinant proteins with laminin was performed by polyclonal antibodies against each protein; each point was performed in triplicate and expressed as the mean absorbance value at 492 nm ± standard error for each point. Gelatin was included as a negative control. The dissociation constants (KD) are depicted and were calculated based on ELISA data for the recombinant proteins that reached equilibrium. (D) Immobilized laminin was treated with sodium metaperiodate (5 to 100 mM) for 15 min at 4°C in the dark. Mean absorbance values at 492 nm (± the standard deviations of three independent experiments) were compared to those obtained with untreated laminin (0 mM).