Versamax elisa plate reader

ELISA was performed as previously described [66 (link)]. In brief, either 20 µg/mL of parasite protein lysate, 20 µg/mL of mouse organ tissue extracts, or 5 µg/mL of BSA were coated for 16 h at 4 °C in 0.05 M carbonate buffer (pH 9.6) in 96-well plates (NUNC MaxiSorb^TM, Roskilde, Denmark). After four washes with 1% (v/v) Tween 20 in PBS (PBST), the plates were blocked for 2 h at RT with 5% (w/v) of skimmed milk in PBST. Subsequently, plates were incubated with scFv 6B6 antibody (25 μg/mL) or plasma from patients with CCD, cutaneous leishmaniasis (CL), or non-infected (NI) individuals (diluted 1:250–1:100) for 2 h at 37 °C at 30 rpm shaking speeds. After four washes with PBST, the wells were incubated with mouse anti-6×His-HRP antibody (1:3000, clone HIS-1, Sigma Aldrich, St. Louis, MO, USA) or mouse anti-human IgG coupled to Peroxidase (HRP) (1:3000, Sigma-Aldrich, USA) for 1 h at 37 °C. All antibodies and plasma were diluted in 1% (w/v) skimmed milk in PBST. After incubation with 3,3′,5,5′-tetramethylbenzidina (TMB, Sigma-Aldrich, USA) plus H₂O₂ as substrate, the reaction was stopped with 4 N H₂SO₄. Optical density (OD) was measured at 450 nm using a VERSAmax^® ELISA plate reader (Molecular Devices Corporation, San Jose, CA, USA).

To study whether pectins can activate or inhibit mTLR2, activation or inhibition assays were performed with pectins using HEK‐Blue cells expressing mTLR2 (Invivogen). HEK‐Blue mTLR2 cells were seeded in 96 well plates at 2.8 × 10⁵ cells mL⁻¹ in 180 µL/well and were incubated overnight in DMEM medium. The next day, the DMEM medium was replaced by DMEM medium containing pectins in the concentrations of 0.5, 1, or 2 mg mL⁻¹. Activation of mTLR2 was studied by treating the cells with the pectins for 24 h. Inhibition of the mTLR2 was studied by pre‐treating the cells with pectins for 1 h followed by addition of 20 µL of the Pam3CSK4 (10 ng mL⁻¹; Invivogen). Culture medium was used as negative control and the TLR2 specific agonist Pam3CSK4 was used as positive control. After 24 h of incubation, media supernatant was mixed with QUANTI‐Blue (Invivogen) in a ratio of 1:10. After 1 h of incubation, NF‐κB activation was quantified at 650 nm using a Versa Max ELISA plate reader (Molecular Devices, Sunnyvale, CA, USA). Incubation steps were performed at 37 °C and 5% CO₂. TLR activation data was represented as fold change compared to culture medium only. TLR inhibition data was represented as fold change compared to Pam3CSK4.

Esterase activity was evaluated with two different substrates: α-naphthyl acetate (for α-Esterase) and β-naphthyl acetate (for β-Esterase), according to the method described by van Asperen (1962) [53 (link)]. Ten μL of protein extract from treated larvae was transferred to a plate (Biofloat™ 96-Well Plate). Then, 190 μL mixture (100 μL of 40 mM sodium phosphate buffer, pH 7.2, 80 μL of distilled water, and 10 μL of 30 mM α -naphthyl or β -naphthyl acetate substrate in acetone, depending on the enzyme being studied, α or β-Esterase) was added.
To construct the standard curve, 10 μL of products in acetone (α- or β-naphythol) was added to each well in the following amounts: 0; 0.001; 0.0025; 0.005; 0.0075; 0.01; 0.015; 0.02 micromoles. Then, 190 μL of the mixture (100 μL of 40 mM sodium phosphate buffer, pH 7.2, 80 μL of distilled water, and 10 μL PBS 0.1% Triton X-100) was added to each well containing the products.
The plate was incubated for 30 min at 30 °C. After incubation, 50 μL of staining reagent (3.4% SDS and 0.3% Fast Blue) was added to each well of the plate and the plate was incubated for 5 min at 30 °C. The absorbance of the samples was measured at 570 nm using a Versamax ELISA plate reader (Molecular Devices, Versamax, Biloxi, MS, USA). The activity was expressed as µU/µg protein.