Spectrophotometer epoch microplate reader

The antioxidant reducing capacity of the syrups was determined spectrophotometrically on the basis of the Folin-Ciocalteu assay (reagent: mixture of phosphowolframic and posphomolybdenic acid) and expressed as gallic acid equivalents. For this purpose, a methanolic:water solution (75:25, v/v) of freshly prepared gallic acid was prepared, with a concentration ranging between 2 and 500 mg/L. About 1 g of the sample was diluted in 10 mL of deionized water (Milli-Q purification system) and for the measurements, 15 μL of sample and 165 μL of Folin-Ciocalteu assay (1:9, v/v in water) were placed on the microplate. Deionized water was used as a blank. After 3 min, 140 μL of sodium carbonate (9%, w/w) was added and the microplate was placed in a dark place for 60 min. Using a Spectrophotometer Epoch Microplate reader (BioTek Instruments, USA), the absorbance was measured at 765 nm. The method was validated with a determined uncertainty of 10%.

Antioxidant activity was determined spectrophotometrically on the basis of the measurement of DPPH radical scavenging activity as described by Bhave et al. (48 (link)) with slight modifications. 1 g of sample was diluted with 10 mL deionized water (Milli-Q purification system; Millipore). To determine antioxidant activity, a methanolic solution containing DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals (52 mg/100 mL) was used. To calculate the concentration of antioxidants, expressed as equivalents of ascorbic acid, an aqueous solution of freshly prepared L-ascorbic acid, with a concentration ranging between 1 and 20 mg/L was prepared. Deionized water was used as a blank. The reduction of DPPH free radicals to DPPH₂ (diphenylpicrinehydrazine) indicated by a change in color from violet to yellow was measured after 60 min at 517 nm using a Spectrophotometer Epoch Microplate reader (BioTek Instruments, USA). The method was validated with a determined uncertainty of 10%.

Nunc 96–Well non–surface treated Microplates (Thermo Fisher Scientific, MA, USA) were coated with 100 μl (12 ng/well) of the recombinant NiV–G in 0.06 M carbonate/bicarbonate buffer (pH 9.6) overnight at 4°C. After washing 5 times with PBS–T, plates were blocked with 5% commercial porcine serum (Life Technologies, New Zealand) in Casein blocking buffer (Sigma–Aldrich, SL, USA). A commercial normal pig serum was used as a negative control, and one serum collected at 28 dpi from NiV–B inoculated pig (#7) was used as a positive control in the cELISA. Negative and positive serum controls are included in each ELISA plate to calculate percent inhibition (PI) results for test samples. Thus after blocking, test serum samples, positive, and negative serum controls (50 μl/well, 1:10 in PBS–T) were added in duplicate, then an equal volume of the hybridoma culture supernatant (F20NiV−65, 1:500 in Casein blocking buffer) were added at the same time. Following 1 h incubation and washing, the HRP–conjugated anti–mouse IgG (1:500, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) was added. Then 3,3′,5,5′–Tetramethylbenzidine (TMB, Pierce Biotechnology Inc. IL, USA) was added. The reaction was stopped using 2N Sulfuric acid and the OD was determined at 450 nm using a BioTek Epoch Microplate Spectrophotometer reader. Results of PI were calculated based on the following formula: