Recombinant antibody-based sandwich ELISA for the detection of sesame 11S globulin allergens in commercial processed foods: a fully in vitro approach

: Sesame is a major food allergen subject to mandatory labelling in multiple jurisdictions, yet reliable analytical methods to support quality control and verify allergen labelling remain essential. Many commercial immunoassay kits rely on animal-derived polyclonal antibodies, compromising reproducibility and batch-to-batch consistency. Here, we report the development and analytical characterisation of a direct sandwich ELISA for sesame 11S globulin allergen detection using recombinant rabbit monoclonal antibodies generated through an in vitro phage display workflow, without animal immunization. Two high-affinity antibody clones were selected from a naïve scFv library against an 11S-enriched sesame fraction and reformatted as IgGs. The assay demonstrated high specificity and no detectable cross-reactivity against different food ingredients, including seeds and tree nuts containing homologous 11S globulins. The assay achieved a limit of detection (LOD) of 0.087 μg/mL and a limit of quantification (LOQ) of 0.293 μg/mL for sesame protein in solution, and an LOD of 5.47 mg/kg in a thermally processed biscuit model, confirming detection capability after baking. Mean recoveries in representative food matrices ranged from 54.1% to 80.6%, with intra- and inter-day precision meeting accepted criteria for quantitative ELISA. Assay performance was compared to a commercial polyclonal-based ELISA and sesame-specific real-time PCR in the analysis of commercial products with diverse labelling claims. Results were largely concordant, while the developed assay offered the advantage of using a fully defined, reproducible antibody source. These findings support the potential of recombinant antibody-based immunoassays as robust, scalable tools for sesame allergen monitoring and verification of allergen labelling claims in food control settings.