Microtiter plate

The antigen-specific IgY antibody titers were determined by Western blot and ELISA analysis. For Western blot, calf ADA protein (50 ng/lane) was resolved by SDS-PAGE and blotted as described above. After blocking (4 °C, overnight), the membrane was washed in PBST and cut into strips which were incubated with the cADA-specific IgY antibody solutions prepared in 0.5% skim milk in PBST at concentrations of 50, 10, 5, 1, and 0.1 μg/ml or with control IgY antibodies (50 μg/ml) for 1 h at 37 °C. Next, the membrane strips were washed with PBST and the secondary peroxidase-conjugated antibodies were used as previously described.
For indirect ELISA assay, a microtiter plate (MaxiSorp, Nunc, Gdańsk, Poland) was coated with cADA (0.5 μg/ml, 100 μl/well) in 50 mM sodium carbonate buffer, pH 9.6 (1 h, 37 °C). The plates were washed with PBST and blocked with 5% skim milk in PBST. The wells were then washed with PBST, and the antigen-specific or control IgY antibodies at different concentrations were added (50, 10, 5, 1, 0.1 μg/ml). After 1 h of incubation (37 °C), the plate was washed and secondary antibodies were applied (anti-IgY rabbit IgG-HRP antibodies, 1:5000 in 0.5% skim milk/PBST). The plate was developed with OPD solution as described above. The results were expressed as the ELISA index (EI), where EI = OD_sample/OD_control, with values of EI > 1.2 considered as positive [54 (link)].

All infected patients presented at least one palpable nodule [35] (link). For MF analysis, two skin biopsies of 1–3 mg were taken from the buttocks using a corneoscleral (Holth) punch (Koch, Hamburg, Germany). Each biopsy was immersed in 100 µl of 0.9% NaCl solution in a well of a microtiter plate (Nunc, Roskilde, Denmark). The skin biopsies were incubated overnight at room temperature to allow MF to emerge. The solution was then transferred onto a slide for microscopic examination [14] (link), [36] (link). The biopsies were weighed using a Sartorius electronic balance (Göttingen, Germany) and MF load was calculated per mg skin. Blood smears from all patients were screened for their cellular compositions and malaria infections using standard Giemsa staining protocols. The presence of further helminth infections were assessed using standard diagnostic tests on stool and urine samples [37] (link).

The microtiter plate (Nunc, Roskilde, Denmark) was coated with 2 μg purified protein overnight at 4°C. After 5 washes with PBS containing 0.05% Tween 20 (PBST), the supernatant/lysate of transfected cells was added to each well and incubated at room temperature (RT) for 2 hrs. The wells were then washed 5 times with PBST. Primary antibody was added, and incubation continued at RT for 2 hrs. The wells were washed again, and 100 μL of secondary IgG-horseradish peroxidase was added. After incubation at RT for 1 hr, a commercial peroxidase substrate system was used (Kirkegaard & Perry Laboratories, Inc., MA, Cat. No# 50-76-11), and the optical density at 450 nm was measured with an ELISA reader.
The interaction between AaHig and DENV-2 virions was also measured by ELISA. In the procedure, the plate was coated with 2 μg of purified AaHig protein at 4°C overnight. After 5 washes with PBST, 2 μg of purified inactivated DENV-2 virions (MicroBix, Canada, Cat. No# EL-22-02-001) in PBS was added to each well and incubated for 2 hrs at 4°C. After washing with PBST, a flavivirus E protein 4G2 mAb was added, and further incubated for 2 hrs at 4°C. The analysis followed the procedure is outlined above.