Easy reader

The concentration of collagen in the supernatants was analyzed by ELISA. Treated and control cells were cultured with medium for 48 hours. Then the culture supernatants were collected, filtered and added to multi-well plates (Costar). After incubation overnight at 4°C, wells were blocked with 1% BSA in PBS and incubated with a rabbit anti-Type I Collagen antibody (1:2500) (Acris Antibodies), and then with a peroxidase-conjugated goat anti-rabbit IgG (Sigma) at 1:500 dilution. All incubations were performed for 1 h at 37°C and between each step washed three times with PBST. Ortho-phenylene-diamine was used as a chromogen. ODs were measured at 492 nm in an Easy Reader (Bio-Rad).

Sixty-six human serum samples from inhabitants of the Canary Islands, obtained from a previous study [6 (link)], were selected. Of them, 29 were positive to both anti-D. immitis IgG and anti-Wolbachia surface proteins (WSP) IgG (Group A), and 37 were seronegative (Group B). Sera were randomly selected from the group of positive samples and from the group of negative samples. Levels of total IgE were determined in all samples in the reference laboratory by using chemoluminescence-ELISA techniques (Service of Allergy of the Eurofins Megalab, Canary Islands, Spain). Seropositivity was considered when total IgE were >150 IU/mL.
All samples were further analyzed for the presence of specific anti-D. immitis IgE by non-commercial ELISA test, using adult D. immitis antigens. Then, 96-well microplates were coated with 0.8 μg of an extract of D. immitis adult worms. All serum samples were analyzed at a 1:100 dilution, respectively, and a goat anti-human IgE (H + L) conjugated to horseradish peroxidase (Thermo-Fisher, Barcelona, Spain) was used at 1:100 dilution. Optical densities (ODs) were measured at 492 nm in an Easy Reader (Bio-Rad Laboratories, Hercules, California, USA). The cut-off (OD = 0.5) was established by calculating the mean value + 3 standard deviations (3 SD) of 12 serum samples from clinically healthy blood donors (negative controls) living in a D. immitis-free area.

plasminogen activation and subsequent fibrinolysis was analysed in a test volume of 100 µL by measuring the amidolytic activity of generated plasmin (González-Miguel et al., 2012 (link)). In each well, 2 µg of human plasminogen (Acris Antibodies) were incubated in PBS with 3 µg of the chromogenic substrate D-Val-Leu-Lys 4-nitroanilide dihydrochloride (Sigma) in the presence of 1 µg of FhES. Activation of plasminogen was initiated by the addition of 15 ng of tPA (Sigma). In parallel, plasmin generation was also measured in the absence of t-PA, to observe the ability of the FhES extract proteins of activating plasminogen on their own. Plates were incubated at 37° C for 1 h and the hydrolysis of the chromogenic substrate was analysed by measuring absorbance at 405 nm in an Easy Reader (Bio-Rad).