Sheep anti human plasminogen igg

In order to determine whether the FhES extract has the ability to bind plasminogen, an enzyme-linked immunosorbent assay (ELISA) was performed (González-Miguel et al., 2012 (link)). In brief, multiwell microplates (Costar) were coated with 1 µg per well of FhES extract diluted in carbonate buffer, pH 9.6, overnight at 4 °C. The wells were blocked with 1% BSA in PBS and incubated successively with increasing amounts (from 0 µg to 3 µg) of human plasminogen (Acris Antibodies), with a sheep anti-human plasminogen IgG (Acris Antibodies) at 1:2000 dilution and then with a peroxidase-conjugated donkey anti-sheep IgG (Sigma) at 1:4000 dilution. All incubations were performed for 1 h at 37 °C and between each step washed three times with PBS wash buffer (PBS containing 0.05% Tween₂₀). Ortho-phenylene-diamine was used as a chromogen. Optical densities (OD) were measured at 492 nm in an Easy Reader (Bio-Rad). In parallel, competition assays were performed by including 50 mm of the lysine analogue ε-aminocaproic acid (εACA) during plasminogen incubation. Some wells coated with BSA only were used as negative controls.

To determine which proteins of FhES extract bind plasminogen, they were electrotransferred from 2D gels to nitrocellulose membranes at 20 V for 30 min using a Trans-Blot SD Semi-Dry Transfer cell (Bio-Rad). Blots were blocked with 2% BSA in PBS wash buffer, for 1 h at room temperature. FhES membranes were incubated overnight at 4 °C with 10 µg mL⁻¹ of human plasminogen. Then, the blots were incubated with a sheep anti-human plasminogen IgG (Acris Antibodies) at 1:1000 dilution and with a peroxidase-conjugated donkey anti-sheep IgG (Sigma) at 1:2000 dilution. These incubations were performed at 37° C for 90 min with shaking and between each step washed three times with washing buffer for 5 min per wash. Protein bands were revealed with 4-chloro naphthol. Negative controls were also used in which the plasminogen had been omitted. Membranes were digitized with the scanner GS-800 Densitometer (Bio-Rad) using the Quantity One Software v.4.6.5 (Bio-Rad). Matching of 2-D gels with the homologous Western blot to identify plasminogen-binding proteins, the assignment of molecular weights (MW) and isoelectric points (pI) of each protein were analysed using the PDQuest Software v.8.0.1 (Bio-Rad). All assays were performed in triplicate to assess the reproducibility of the spot pattern.