## 2 Materials and Methods
### Gluten Analysis and Quantification  
The amount of gluten in every food product was evaluated using RIDASCREEN(r) Gliadin (Art. No. R7001), which is an R5 sandwich enzyme immunoassay based on a specific monoclonal antibody to celiac toxic amino acid prolamin sequences to determine gliadin as a measure of gluten in food, and the cocktail solution R-7006 was used for the extraction (R-Biopharm, Darmstadt, Germany).  
The kit is based on the antigen-antibody reaction. The general procedure is as follows: Plate wells are coated with R5 antibodies specific to gliadins. When standards and samples are loaded into the wells, gliadin is bound by these antibodies, forming an antigen-antibody complex. Unbound components are removed in the washing step. Then, the R5 antibody bound to peroxidase is added (conjugate) and the antibody-antigen-antibody (sandwich) complex is formed, after which the washing step is repeated. The enzyme is incubated by adding a substrate and chromogen. The bound conjugate turns the colorless chromogen into a blue color. Coloring is stopped with a stopping reagent.  
Before starting the analysis phase, concentrated solutions such as \"Sample diluent buffer\", \"Wash buffer\", and \"Enzyme conjugate\" in the kit were diluted as indicated in the protocol and used. Samples and standards included in the kit were pipetted at 100 \(\mu\)L into the plate wells. Three washing processes were carried out manually with phosphate buffer solution (PBS) following a 30 min incubation period at room temperature. Each well received a dose of enzyme conjugate before the plate underwent a 30 min incubation period. Then, the washing step was repeated. Following the addition of the substrate and chromogen, each well underwent a 30 min incubation. Following the use of the \"Stop solution,\" the reaction was stopped, and a microplate reader was used to measure theoptical density in the plate wells. At 450 nm, the absorbance was measured, and the data were generated using the RIDA(r) SOFT (R-Biopharm, Darmstadt, Germany) Win software program to create a calibration curve from the standards; the samples were analyzed according to this standard curve to calculate the gluten content. The intricate composition of gluten, which comprises various proteins with distinct solubility levels, necessitates the selection of an appropriate calibrator specific to the target protein to accurately measure its concentration. The calibration graph is established based on this complex structure, and the spectrophotometric method is utilized to validate the generated calibrations.  
Gluten-free samples were extracted using cocktail solution, and the ELISA procedure was performed. Gluten-free samples were run 10 times each, where the average of the concentrations obtained should be less than 3 mg/kg, as specified in the kit procedure. The results of the studies were calculated as LOD (mean + 3 SD). Repeatability in the limit of detection/limit of identification studies (mean + 10 SD) should be less than 5 mg/kg. In the studies, appropriate results were obtained below these values in the acceptance criteria.  
The results were adjusted to fall within the calibration range by diluting samples with absorbance values higher than the highest standard. As the result of the device was read as the gliadin concentration g/mL, the dilution factor was added and converted to mg/kg (ppm). The gluten concentration was calculated by multiplying the outcome by two. The kit's diagnostic limit for gluten is 5 mg/kg (ppm).  
Food samples were classified into 3 groups according to the gluten concentration. The first group comprised samples with gluten levels below 20 ppm, which were considered gluten-free. The second group comprised samples whose gluten level was between 20 and 100 ppm. This group was considered to have a low gluten content. The third group comprised samples with a gluten content of more than 100 ppm, which were considered to have serious gluten contamination.