Spectramax reg id3

The content of free SH group in the control SPI and SPI-polyphenol conjugates were examined according to our previous study (Pi, Sun, Guo, Chen, Cheng, & Guo, 2022 ). In brief, 40 mg DTNB was dissolved in 10 mL Tris-Gly solution (pH 8.0, 0.086 M Tris, 0.09 M glycine, 4 mM EDTA) for Ellman’s reagent. Each freeze-dried sample was diluted with distilled water to 0.25 mg/mL. Next, 0.4 mL of sample solution was mixed with 0.6 mL of phosphate buffer solution (0.1 M, pH 8.0), and then mixed with 10 µL Ellman’s reagent, followed by incubation at 37 °C for 20 min. After taking 200 µL of the resulting sample in the microtiter plate, and the absorbance was measured at 412 nm using the microplate reader (SpectraMax reg iD3, Beckman Coulter, US). The content of free SH group was computed using the following equation: $\mathit{The}contentoffreeSHgroup\left(\mu mol/gofprotein\right)=73.5\times O{D}_{412}\times D/C$
OD₄₁₂ represented the absorbance at 412 nm; D and C were the dilution factor and the content (mg/mL) of samples, respectively.

The IgE binding capacity of the control SPI, SPI–EGCG, and SPI-PC conjugates were further evaluated by ELISA (Lv, Qu, Yang, Liu, & Wu, 2021 ) with slight modification. Firstly, each sample was diluted to 1 μg/ml using carbonate buffer solution (pH 9.6), and was incubated in a 96-wells microtiter plate for 100 μL/well. After incubation at 4 ℃ overnight and washing by PBS (0.01 M, pH7.0) with 0.05 % Tween-20 (PBST) three times, 5 % skim milk powder in PBS was used to block the plates. After washing the plates, the pooled patient serum was added in the well, followed by incubation at 37 ℃ for 1 h. Next, the well was incubated with goat anti-human IgE HRP conjugates at 37 ℃ for 1 h after washing again. Finally, TMB was added to each 100 μL/well after washing again and then incubated at 37 ℃ for 13 min, followed by terminating the reaction with 2 M sulfuric acid. The absorbance of each well was measured at 450 nm using the microplate reader (SpectraMax reg iD3, Beckman Coulter, US). The allergenic capacity (%) was computed using the following equation: $\mathit{The}allergeniccapacity(\%)=\frac{O{D}_{450}(SPI-polyphenolconjugates)}{O{D}_{450}(thecontrolsoybeanproteins)}\times 100\%$