A Novel Extraction Buffer for Use in Gluten Immunoassays

Introduction: In an effort to address public health concerns regarding gluten, the FDA has recently established regulatory threshold limits for gluten content in foods labeled "gluten-free." Observance of this labeling law requires the use of immuno-based methodologies that can accurately quantify gluten levels in different food matrices. A key factor affecting the performance of these testing methods is the efficiency of the extraction buffer. For gluten, a class of proteins with unique solubility properties, extraction typically relies on the use of ethanol solutions (40-60%) to extract, thereby requiring additional dilution steps and presenting hazardous material concerns.

Purpose: To develop an effective gluten extraction buffer and extraction method for use in immunoassays that does not rely on the use of ethanol solution.

Methods: We developed an extraction buffer consisting of buffered 25% isopropanol and detergent that was directly compatible with downstream applications (including lateral flow assay and ELISA) without the need for dilution. In tandem, an extraction protocol was developed consisting of 1 min heat treatment at 95°C. Using a panel of complex food matrices, our buffer and test methods were directly compared with extraction/test methods from commercial ELISA kits based on Skerrit and R5 monoclonal antibodies.

Results: The MEI/IEH gluten extraction buffer performed comparably to the 40% ethanol (15 min. at 60°C) and 60% ethanol (~120 min. at room temperature) commercial extraction buffers when performed at 95°C for 1 min. In addition, as the MEI/IEH gluten extraction buffer is effectively buffered and formulated with lower solvent content, extracts can be directly applied to ELISA microwells and Lateral Flow Devices, allowing for more sensitive detection capabilities.

Significance: To obviate the need for 40-60% ethanol-based solutions, we have developed a novel extraction buffer based on 25% isopropanol content that yields efficient recovery results using a 1 min. extraction procedure without the need for additional dilution steps. With the increased demand for gluten residue testing following FDA regulations, we anticipate a need for a more rapid and streamlined extraction process.