Responders were determined by ELISA with the recombinant proteins and serum samples from patients of both phases of the disease. The reactivity was evaluated as IgG antibodies. Serum was considered MAT positive or MAT negative if agglutination was detected when the sera were tested for their reactivity’s with isolates of the 22 serovars (see Methods). The cutoff values are depicted as horizontal bars and were defined as the mean plus
3 standard deviations obtained for sera from 12 healthy individuals. (A) shows the data for Lsa33, (B) for Lsa25, and (C) depicts the Ilomastat data when both proteins were employed (Lsa33 plus Lsa25). Recombinant proteins adhesion to ECM components The ability of Lsa33 and Lsa25 proteins to mediate host colonization by adhering to extracellular matrix proteins was examined by ELISA. Laminin, collagen Type I, collagen Type IV, cellular fibronectin, plasma fibronectin, and the control proteins fetuin and gelatin were BIIB057 mouse immobilized on microdilution wells and recombinant protein attachment was A-1155463 concentration assessed by ELISA using antibodies against the proteins. As shown in Figure 5A, both recombinant proteins exhibited adhesiveness to laminin, while no statistically significant
binding was observed with these proteins when wells were coated with collagen Type I and IV, cellular and plasma fibronectin, gelatin or with the highly glycosylated control
protein fetuin. The interaction of recombinant proteins with laminin was also observed when anti – polyhistidine monoclonal antibodies were employed to probe the ligands (Figure 5B). The binding between Lsa33 and Lsa25 with laminin was also evaluated on a quantitative basis as depicted in Figure 5C. A dose – dependent and saturable binding was observed Sclareol when increasing concentrations of the recombinant proteins (0–6 μM) were allowed to adhere to a fixed laminin concentration (1 μg) (Figure 5C). Binding saturation level was achieved by protein concentration of ~4 and 5 μM for Lsa33 and Lsa25, respectively. Based on ELISA data, the calculated dissociation equilibrium constants (K D) for the recombinant protein Lsa33 and Lsa25 with laminin is 367.5 and 415.4 nM, respectively. The role of laminin sugar moieties in the binding with Lsa33 and Lsa25 was assessed with laminin previously oxidized by increasing concentrations of sodium metaperiodate, ranging from 5 to 100 mM. The effect of metaperiodate concentration on the interaction is displayed in Figure 5D. Laminin oxidation had some effect on the interaction with Lsa25, being the reduction of 40% achieved at the highest metaperiodate concentration employed (100 mM). However, the attachment of Lsa33 to metaperiodate – treated laminin had no interference on the binding.