The TNF-α assay was performed using the DeQuantoTM Human TNF-α ELISA kit, (# QT 4001) according to the manufacturer’s instructions. The ELISA technique used in the study was the sandwich method. After thawing the tube for each sample, two 0.5 mL aliquots were made that were stored at −30 °C for the ELISA assay. All reagents were maintained at room temperature with microplate preheated for 15 min (96-well microplates). To prepare a 750 mL solution, a 30 mL concentrated wash buffer was prepared with 720 mL distilled water. The prepared solution was centrifuged at 10,000× g for a minute followed by preparation of working solution at 500 pg/mL using 1 mL standard and sample diluents for reference and left for 10 min. Dilutions were made in 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 0 pg/mL sequence. Biotinylated diluents (Elabscience, Houston, TX, USA, E-EL-H0109) were used to prepare the concentrated solution of 1x from a 100x diluted solution. Similarly, a 100x concentrated HRP conjugate working solution was diluted to 1x concentration with its diluents.

Well plates were incubated at 37 °C for 90 min. After filtering the liquid, a biotinylated detection antibody at 100 µL was added and incubated for 1 h at 37 °C. Subsequently, the solution aspired was washed thrice and added to the 100 µL HRP conjugate (Elabscience, Houston, TX, USA), followed by incubation at 37 °C for 30 min. An amount of 90 µL of substrate reagent (Elabscience, Houston, TX, USA) was added and incubated for 15 min at 37 °C. The plate’s outcome was read at 450 nm and calculated following the addition of stop solution (50 µL of 0.16 M sulfuric acid).

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