*2.8. Double Antibody Sandwich Enzyme-Linked Immunosorbent Assay (ELISA)*

The allergenicity of WPI was evaluated by determining the content of Jug r 1, the major allergen of walnut, using double antibody sandwich ELISA Kits. WPI dispersions oxidized with 0, 5, 10, 15, and 30 mmol/L AAPH were prepared (3 mg/mL) and centrifuged at 5000× *g*, 4 ◦C for 30 min, and the supernatants were saved as sample solutions. A 50 μL portion of standard and mentioned sample solution were loaded onto the microplate wells, respectively, followed by being closed and incubated at 37 ◦C for 30 min, then the liquid was discarded and the microplates were washed 5 times by wash solution and patted dry. Next, enzyme-labeled reagent (50 μL) was added to each well except the blank well, after incubation and washing, and the chromogenic agents A (50 μL) and B (50 μL) were added to each well, mixed well and incubated in the dark at 37 ◦C for 10 min. Finally, the reaction was terminated by adding a stop solution (50 μL) to each well, and the absorbance at 450 nm was read (TECAN, F50, Männedorf, Switzerland). A standard curve was drawn with the standard concentration as abscissa and OD as the ordinate. This experiment was repeated with three different batches of samples, and the Jug r 1 content was calculated by the standard curve according to the corresponding OD.

#### *2.9. Statistical Analysis*

All experiments were repeated three times, and the results were expressed as average ± standard deviation (SD). The significance was determined by one-way ANOVA, and the graphs were drawn using origin 2021 software (Origin Lab, Northampton, MA, USA).

#### **3. Results and Discussion**
