Labsystems multiskan ex

The SOD enzyme activity in the liver tissue samples was measured using a SOD determination kit (FLUKA, St. Louis, MO, Cat. No: 19160). The kit uses a highly water-soluble tetrazolium salt, WST-1 (2-(4-lodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt), which produces a water-soluble formazan dye upon being reduced by superoxide anions. Briefly, 20 μL of sample solution, 200 μL of WST-1 working solution and 20 μL of enzyme working solution were added to each well of a 96-well microplate and incubated at 37 °C for 20 min to produce a water-soluble formazan dye. The absorbance at 450 nm was measured using an enzyme-linked immunosorbent assay plate reader (Labsystems Multiskan EX; Thermo Labsystems, Finland, Vartaa, Finland). SOD activity (inhibition rate) was calculated using the formula {[(Ablank 1–Ablank 3)—Asample–Ablank 2)]/(Ablank 1–Ablank 3)} × 100.
The CAT activity of the samples was determined according to the method described previously (Goth 1991 (link)). Briefly, the supernatant was incubated in the substrate solution containing H₂O₂ (65 μmol per mL in 50 mmol/L phosphate buffer, pH 7.0) in a water bath at 37 °C for 60s. The enzymatic reaction was blocked by the addition of ammonium molybdate, and a yellow complex of hydrogen peroxide was obtained. The absorbance of this yellow complex was measured at 405 nm using the spectrophotometer.

The affinities of the horse plasmas (2^nd immunization samples) or IgG antivenoms were measured using ELISA, following the methodology described above, with the inclusion of a potassium thiocyanate (KSCN) elution step [20 (link), 21 (link), 22 (link)]. After the serum incubation step, dilutions of KSCN (0.0 to 5.0 M, in intervals of 1.00 M) in distilled H₂O were added to the wells and incubated for 30 min at room temperature. The remaining bound antibodies were detected with rabbit peroxidase-conjugated anti-horse IgG (whole molecule) (Sigma Aldrich, St. Louis, MO) diluted (1:20,000) in PBS plus 0.1% BSA (100 μL/well). After three washes with the wash buffer, 50 μL/well of substrate buffer was added, and the plates were incubated at room temperature for 15 min. The reaction was terminated with 50 μL/well of 4 N sulfuric acid. The absorbance at 492 nm was recorded using an ELISA plate reader (Labsystems Multiskan Ex, Thermo Fisher Scientific Inc., Waltham, MA). The results are expressed as follows: affinity index (AI) = % bound antibodies at KSCN 5 M.

Cytotoxicity assays were carried out on Jurkat T lymphocytes cells in 96-well microtiter plates with flat-bottomed wells by MTT assay. In total, 25,000 cells resuspended in 200 µL of culture medium were seeded into each well. Plates were incubated for 24 h at 37 °C in a 5% carbon dioxide atmosphere and then the medium was replaced with a fresh medium containing NHs at the final concentration of 225 µg/mL or 112 µg/mL per well. After 24, 48, or 72 h, 20 µL of a 5 mg/mL of MTT solution were added to each well. After a new incubation period (3 h), the dark blue precipitate was isolated by centrifugation and re-dissolved in DMSO. Absorbance measurements were taken at 570 nm with a Labsystems Multiskan EX plate reader (ThermoFisher, Madrid, Spain) and the values were corrected with reference to the measurement at 630 nm. Solvent alone and untreated cells were included as negative controls. Assays were carried out in quadruplicate. Data were expressed as a percentage of the absorbance of the untreated control.