Gold nanoparticle-based lateral flow immunoassay for the rapid and on-site detection of wheat allergen in milk

Wheat gliadin is one of the most important causes of gluten sensitivity. This study aimed to develop a sandwich enzyme-linked immunosorbent assay (ELISA) and gold nanoparticle-based lateral flow (LFIA) strips to detect wheat allergen in food. Through cell screening, ten monoclonal antibodies (mAbs) against gliadin and after optimization were obtained, a pair of mAbs suitable for its detection (capture antibody: mAb 7; detection antibody: HRP-labeled mAb 6) were identified. Based on two of these antibodies, a sandwich ELISA with limit of detection (LOD) of 60 ng/mL in negative milk, and more importantly, negligible cross-reactivity to other allergens was developed. The average recoveries for gliadin in negative milk were 99.16%–100.07% using the sandwich ELISA. We also developed LFIA strips for the rapid detection of gliadin with visual limit of detection (vLOD) of 25 ng/mL and calculated LOD value of 6.56 ng/mL in negative milk. The test results of positive samples obtained from the LFIA strips were highly consistent with those of the sandwich ELISA. Thus, the developed LFIA strip is an effective and reliable tool for the rapid and on-site detection of wheat allergen in milk.