Gluten fragment detection with a competitive ELISA.

Area of Science:

• Food Science
• Analytical Chemistry
• Immunology

Background:

• Accurate quantification of prolamin proteins, particularly in hydrolyzed forms, is crucial for food safety and regulatory compliance.
• Existing ELISA methods may not reliably detect partially hydrolyzed prolamins, posing challenges for gluten analysis.
• The R5 antibody is widely used for prolamin detection, but its application in competitive ELISA for hydrolyzed forms requires further validation.

Purpose of the Study:

• To evaluate the performance of a second-generation competitive ELISA utilizing the R5 antibody for prolamin quantification.
• To assess the assay's suitability for detecting partially hydrolyzed prolamins in various food matrices.
• To establish a reliable method for converting signal intensities to gluten concentrations according to Codex Alimentarius standards.

Main Methods:

• Development and validation of a competitive ELISA based on the R5 antibody.
• Utilization of a novel calibrator comprising a peptic-tryptic digest of wheat, rye, and barley prolamins.
• Determination of the limit of detection (LOD) and limit of quantification (LOQ) for the assay.
• Analysis of beer samples and a hydrolyzed wheat product, including spiking experiments with defined concentrations of partially hydrolyzed prolamins.

Main Results:

• The assay demonstrated a limit of detection (LOD) of 1.36 mg prolamin/kg food and a limit of quantification (LOQ) of 5.0 mg prolamin/kg food.
• Significantly higher prolamin concentrations were detected in beer and hydrolyzed wheat samples compared to a sandwich ELISA.
• Recoveries in spiking experiments ranged from 92% to 136%, indicating good accuracy for partially hydrolyzed prolamins.

Conclusions:

• The second-generation competitive ELISA with the R5 antibody is a robust and suitable method for quantifying partially hydrolyzed prolamins in food.
• This assay provides more accurate gluten concentration measurements, especially in processed foods, compared to methods detecting only intact prolamins.
• The newly developed calibrator enables reliable conversion of signal intensities to gluten concentrations, meeting regulatory requirements.