Towards a Comprehensive Validation of ELISA Kits for Food Allergens: Case 1—Egg

Abstract

There are several enzyme-linked immunosorbent assay (ELISA) kits in the market that have been proven to be useful for the determination of egg in foods. However, inconsistent results that are obtained when different kits are used make the selection of one kit over another very difficult. Two different approaches were used to help understand why results vary among kits. Different kits were used to analyze spiked egg material [NIST reference material (RM) 8445] in wheat flour (raw ingredients) and cookies containing egg as an ingredient baked for different periods of time (processed food). These results were compared with immunoblotting using conjugated antibodies from the commercial kits to determine the antibody specificity and sample extraction efficiency. ELISA results can be difficult to compare because reporting units differ among kits. Results from both ELISA and immunoblotting are in agreement regarding the decreased detection of proteins in baked cookie extracts. Moreover, immunoblotting showed that this reduction is due to reduced protein content in these extracts. However, a properly selected extraction solution may help improve the solubility of certain egg proteins in processed foods. Harmonization of the reporting unit system along with the use of a common reference material is recommended as the path forward in the standardization of detection methods for food allergens. This would assist the end user in making an informed decision regarding the selection of the most appropriate kit for his or her purpose.

Effect of Processing on Recovery and Variability Associated with Immunochemical Analytical Methods for Multiple Allergens in a Single Matrix: Dark Chocolate

“…Although the mean measured recoveries of the ES, MO, and TE kits were 67.9% (ES), 88.6% (MO), and 81.0% (TE) in tempered chocolate, these kits performed better in this matrix compared to other processed matrices. 29 At the lowest incurred level in this study (1.2 ppm), mean measured protein and percent recoveries of the test kits were ES (0.9 ppm, 74.9%), MO (1.16 ppm, 96.8%), NE (4.09 ppm, 340.5%), RB (4.13 ppm, 343.8%), and (1.95 ppm, 162.7%). On the basis of these results, all of the kits were able to detect egg protein in the tempered dark chocolate matrix at or close to the LOQs claimed by the manufacturer, although some kits were more accurate than others.…”

“…Recovery varied among test kits, however, which is also observed when using these kits with other processed foods. 18,29 The NE and RB kits overestimated egg protein in tempered chocolate at all incurred levels with mean recoveries of 285.4 and 256.8%, respectively ( Figure 1B). Although the mean measured recoveries of the ES, MO, and TE kits were 67.9% (ES), 88.6% (MO), and 81.0% (TE) in tempered chocolate, these kits performed better in this matrix compared to other processed matrices.…”

Detection of Allergens in a Multiple Allergen Matrix and Study of the Impact of Thermal Processing

“…Reduced protein solubility following thermal processing due to protein structural changes and the reduced ability of the extraction buffers used to solubilise the heat treated proteins provide some explanations. Diaz-Amigo [31] reported a decrease in detection of egg allergens in baked cookie extracts using ELISA kits due to the reduced quantity of protein extracted with the buffers employed. Other workers have shown changes in secondary structure and sulfhydryl-disulfide interchange reactions on thermal treatment of egg proteins which affected protein antigenicity [6].…”

“…For kit manufacturers it continues to highlight the importance of optimizing the protocol for detecting allergens in both raw and cooked samples. Buffer selection in the preparation of ELISA kits and the conditions employed during testing may help to improve the extraction efficiency of processed food proteins [31].…”

“…Recoveries detected using the Veratox kit decreased from 74.5% for the cookies baked at 350 o F to 35.3% for the cookies baked at 450 o F. For the Biokits, the recoveries were even lower, decreasing from 55% for the cookies baked at 350 o F to 20% for the cookies baked at 450 o F. Both kits performed well on the casein-free samples and no false positives were determined for the casein-free dry mix sample, cookie dough, and baked cookies. In another study [25], the effect of food processing on the detection of milk proteins in incurred peanut butter cookies baked at various times was compared using different ELISA kits (Tepnel, Neogen, r-Biopharm and Morinaga). The author also reported low and variable results in the ability of all tested ELISA kits to detect heat-treated milk proteins in baked cookies.…”