Validation of a Competitive Elisa Method on Supplemental Enzyme Matrices

Abstract

Introduction: Supplemental enzymes are becoming increasingly used in the food industry. Consequently, they also need to be analyzed for gluten due to labeling reasons for food manufacturers to provide food allergen detection. Gluten is analyzed by using a Sandwich ELISA using the R5 antibody. However, sandwich ELISAs are not suitable for the analysis of fragmented gluten since detection is based on the size of the fragments. As a result, competitive R5 ELISAs have been implemented for use. When a competitive ELISA is used to analyze enzymes without gluten, the results for gluten contamination are very high and its cause is unknown. It has been suggested that the enzymes destroy antibodies of the test format and therefore false positive results are obtained. This study aimed to investigate if the competitive ELISA can be used for the gluten analysis in supplemental enzymes by the adaption of the extraction method. Methods: Enzyme solutions were spiked with known concentrations of gluten then tested for gluten content using sandwich ELISA kits and competitive ELISA kits per manufacturer’s instructions. Additional enzyme samples were inactivated by raising the extraction temperature to 100°C to inactivate the enzymes and also tested using both sandwich and competitive ELISA kits. Results: Enzymes were spiked with gluten and analyzed with the two different ELISAs showed false negative results with the sandwich ELISA and false positive results with the competitive ELISA. Preincubation experiments showed that the enzymes destroyed the antibody used in the competitive ELISA. On the other hand, extracts extracted at 100°C did not show that effect. Conclusion: In conclusion, competitive ELISA kits may be used to test fermentation products such as enzymes when the adapted extraction method is used. Spiking experiments clearly showed a good recovery of gluten in the competitive ELISA with the modified extraction, showing that the boiling step does not affect existing gluten content in the samples. This method can be used for supplemental enzymes for the analysis of gluten content in such products.