Development and Validation of a Lateral Flow Test Kit for Detection of Hydrolyzed Gluten Residues in Food

Introduction: Gluten intolerance disorders such as celiac disease require complete avoidance of gluten in the diet.  However, strict elimination of gluten is problematic due to contamination that can occur during food processing. Accordingly, numerous countries have established a regulatory threshold limit for gluten content in foods labeled "gluten-free.” Observance of the limit requires the use of antibody-based immunoassays that can detect gluten from a variety of foods. This objective is somewhat problematic for foods containing hydrolyzed proteins.

Purpose: To develop a rapid test kit capable of reporting hydrolyzed gluten residues from swabs down to 2.0 µg/swab and 20 µg/g (or µg/ml or ppm) for foods in under 20 min.

Methods: Prolamins were prepared by Osborn and quantified by Kjeldahl method. To generate monoclonal antibodies, BALB/c mice were repeatedly immunized with rye prolamins, splenocytes were fused with a mouse myeloma cell line, and ensuing colonies were screened against triticeae-derived prolamins. An IgG-secreting hybridoma, 25A5, was identified and raised in ascites. The mAb was purified by FPLC on a protein G column, characterized, and then used to develop a lateral flow device (LFD) for the detection of gluten. Concordance was assessed using a commercial ELISA kit. 

Results: The MEI/IEH competitive gluten lateral flow test method demonstrated a sensitivity of 2.0 µg/swab and 20.0 µg/mL for foods. Specificity analysis revealed no cross-reactivity with common commodities except for teff. The assay was equally sensitive as the commercial R5-based ELISA kit in its ability to report gluten residues from hydrolyzed foods, and required less time (< 20 min) to perform.

Significance: The development of a highly sensitive and rapid test method capable of detecting trace amounts of hydrolyzed gluten residues in under 20 min should aid food manufacturers’ and regulatory entities in monitoring for gluten residues, particularly in testing of fermented foods.