Detection of Mustard, Egg, Milk, and Gluten in Salad Dressing Using Enzyme‐Linked Immunosorbent Assays (ELISAs)

Abstract

Enzyme-linked immunosorbent assay (ELISA) is a commonly used method for the detection of trace amounts of potentially allergenic protein residues in foods. However, food matrices and processing conditions can affect the detection of protein residues. The effects of acidity on the detectability of several allergenic proteins commonly found in salad dressing using ELISAs was investigated. First, recovery experiments were performed on salad dressing formulated with 0 to 1000 ppm mustard flour (mustard). The mean percent recovery for mustard from the salad dressing was only 7.7%+/- 1.6%. When the pH of the salad dressing was adjusted to pH 7 prior to spiking with mustard, recovery improved to 94.1%+/- 7.6%. However, if the pH was adjusted to pH 7 after spiking and extraction, the recovery was only 11.1%+/- 1.7%. When vinegar was spiked with mustard flour at pH 3, 3.5, and 4, detectability of mustard was lowest at pH 3. Basic extraction of mustard proteins from salad dressing did not improve the mustard detection. Acidic salad dressing matrices reduced the detectability of mustard by the mustard ELISA probably because of acid precipitation of mustard proteins that renders them insoluble and nonextractable. Commercial salad dressings containing 100 ppm (mg/kg) of egg, milk, or gluten were analyzed every 2 to 4 d for 90 d using 3 commercially available ELISAs. A decrease in the detection of the egg, milk, and gluten in the salad dressing upon storage was observed. Our study highlighted the importance of evaluating the utility of various ELISAs for specific food matrices and the recovery as a function of product storage.

Shrimp tropomyosin retains antibody reactivity after exposure to acidic condition

“…Reduction in the concentration of soluble protein especially from samples marinated in vinegar at pH 1.0–3.5 on the other hand may be due to the denaturation of myofibrillar proteins, which results in their poor solubility in the extraction buffer . In a study involving the recovery of mustard protein from salad dressing (an acidic food matrix) spiked with 0–1000 ppm mustard flour, mustard protein recovery was maximum (94%) in salad dressing adjusted to pH 7 before spiking and minimum (7.7–11%) in salad dressing spiked at its natural acidic pH (3.2) . The authors attributed this low recovery to the poor solubility and extractability of the mustard protein in an acidic food matrix.…”

“…Similarly, when the single effect of vinegar pH (3.0, 3.5 and 4.0) on the ELISA detection of mustard protein was investigated, the lowest concentration of mustard protein was detected in samples exposed to the lowest vinegar pH . This result was due to the reduced solubility of the protein in the extraction buffer caused by acid denaturation …”

“…In a study to determine the effect of thermal processing on the immunochemical detection of egg, milk and peanut allergens in a baked product matrix, it was found that poor recovery of the soluble allergenic proteins by extraction buffers led to underestimation of the allergen concentration by most ELISA kits . Similarly, when the single effect of vinegar pH (3.0, 3.5 and 4.0) on the ELISA detection of mustard protein was investigated, the lowest concentration of mustard protein was detected in samples exposed to the lowest vinegar pH . This result was due to the reduced solubility of the protein in the extraction buffer caused by acid denaturation …”

“…This has the tendency to reduce the pH of the extraction solution, its buffering capacity and its ability to solubilize myofibrillar proteins. Effective solubilization of allergenic protein is essential for the accurate immunochemical detection of allergenic proteins . Therefore reduced soluble protein recovery may be the main challenge in the immunochemical analysis of acid‐marinated foods or high acid foods.…”

Food Production and Processing Considerations of Allergenic Food Ingredients: A Review

“…Although testing methods for food allergens with excellent sensitivity and selectivity have been developed and commercialized, they are still subject to inaccuracies due to matrix and processing effects and stability issues [27, 28]. Conformational epitopes can be modified by processing or residence time within the food matrix [29], although linear epitopes will generally withstand denaturant conditions in the food.…”