2.5. Enzyme-linked immunosorbent assay (ELISA) inhibition assay
This protocol is extracted from research article:
Effect of ultrasonic pre-treatment on Ara h 1 in peanut sprouts
Ultrason Sonochem, May 25, 2021; DOI: 10.1016/j.ultsonch.2021.105607

Concentrations of Ara h 1 in peanut sprouts and extracts were measured according to the protocol of the sandwich ELISA kit based on the intensity of a blue to yellow color change measured at 450 nm with a microplate reader. To measure the Ara h 1 concentration of the sample, the kit included a set of calibration standards used to produce a standard curve of OD versus Ara h 1 concentration. The concentration of Ara h 1 in the samples was then determined by comparing the sample ODs to the standard curve.

Ground peanut/peanut sprout samples were mixed with a 20 × volume of sample buffer, extracted at 60  °C for 10 min, and centrifuged at 2500 g and 4  °C for 10 min (SHZ-88, Jintan District Baita Jinchang Experimental Instrument Factory, China). A sample of the supernatant (10 μl) was combined with 40 μl of sample diluent in a well, 100 μl of HRP-conjugate reagent was added, and the mixture was incubated for 60 min at 37 °C. Each well was then aspirated and washed, and the process was repeated four times for a total of five washes. After inverting the microplate and blotting it on clean paper towels, 50 μl each of chromogen solution A and chromogen solution B were added to each well, gently mixed, and incubated for 15 min at 37 °C in the dark. Stop solution (50 μl) was then added to each well, followed by 100 μl of HRP-conjugate reagent, and the mixture was incubated for 60 min at 37 °C. The OD at 450 nm was then measured on a microplate reader within 15 min.

The defatted peanut powder in each sample was calculated using standard curves developed with purified Ara h 1 using the ELISA procedure described above. The final results were calculated as PPM Ara h 1 and were expressed as the mean ± standard deviation of three replications.

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