Incorporating Real World Experiences in an Undergraduate Biochemistry Laboratory CourseDetection of Gluten in Food and Beverages Using an Enzyme-Linked Immunosorbent Assay

Abstract

Celiac disease (CD), an autoimmune disease affecting 1% of the global population, is caused by the consumption of gluten. Gluten is a storage protein that is found in wheat, rye, and barley. The addition of gluten to food products imparts unique viscous and elastic characteristics in foods such as bread. The only recommended treatment for celiac disease is a gluten-free diet. The consumption of gluten-free labeled food products reduces the risk of an autoimmune response and other symptoms associated with celiac disease, including anemia, dermatitis herpetiformia, osteoporosis, muscle weakness, ataxia, and coagulopathy. The Codex Alimentarius and Food and Drug Administration (FDA) state that gluten-free foods must limit gluten content to 20 ppm. Using an enzyme-linked immunosorbent assay (ELISA), quantitative studies can be performed to detect the concentration of gluten present in gluten-free food products. This work uses a direct ELISA to quantify the amount of antigen (gluten) present by measuring absorbance in gluten-containing and gluten-free food products. A color gradient was observed based on the increasing concentration of gluten present in each gluten standard. Multiple substrates (o-phenylenediamine, 3,3',5'-tetramethylbenzidene, aminosalicylic acid, and 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic-acid)) were used to determine the most efficient method for incorporating this technology in an undergraduate biochemistry laboratory experiment where time is restricted and resources may be limited. This experiment was optimized to complete this assay within a 2.5 h lab period, thereby demonstrating a real world application using traditional biochemistry techniques and enhancing the students' laboratory skill set and critical thinking skills.

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General peroxidase reaction.

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Absorbance versus protein concentration (mg/mL) of gluten standards.

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Absorbance versus gluten concentration after reducing incubation times.

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Image of a 96-well plate with 6 different substrates: (A) OPD; (B) ASA; (C)­TMB, BioRad; (D) TMB, BioLegend; (E) ABTS-Liquid; and (F) ABTS-Solid.

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Comparison of five different substrates at different time intervals.