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EFFECTS OF THERMAL TREATMENTS ON ELISA DETECTION OF MILK PROTEINS
Title
EFFECTS OF THERMAL TREATMENTS ON ELISA DETECTION OF MILK PROTEINS
Creator
Lu, Yingshuang
Date
2011-07-27, 2011-07
Description
Cow’s milk allergy is one of the most prevalent food allergies in the United States. Commercial ELISA test kits, based on antigen-antibody...
Show moreCow’s milk allergy is one of the most prevalent food allergies in the United States. Commercial ELISA test kits, based on antigen-antibody reactions, are increasingly used by food manufacturers to detect the presence of allergen residues. Milk is a common ingredient used in a variety of foods that are subjected to different degrees of cooking. How thermal processing may affect the quantitative analysis of milk allergens by ELISA test kits needs to be determined. This study first examined the performance of four total protein assays (Pierce 660nm, Modified Lowry, Coomassie, BCA) for quantification of protein residues in thermally processed milk. BCA and Lowry assays were affected by lactose and its Maillard reaction byproducts resulting in the overestimation of protein concentrations in the samples. Pierce 660 nm assay was not affected and therefore was picked as the method for total protein analysis when evaluating commercial ELISA kits. Performances of four ELISA test kits (Veratox for Total Milk Allergen, BioKits BLG Assay Kits, ELISA SYSTEMS Casein and Beta-Lactoglobulin Residue assays) in comparison with the Pierce 660 nm assay for detection of thermally treated milk samples was also evaluated. NIST non-fat milk powder standard reference material 1549 and Backpacker’s Pantry Powdered Whole Milk were either heated in water at 60◦C, 63◦C, boiled, or autoclaved for 10 or 30 min, dry-heated in a mini-oven at 100 - 232◦C for 10 min, or fried in corn oil at 150◦C or 180◦C for 3 min in a deep fryer. Milk proteins in the oil samples were analyzed either directly or after extraction with phosphate-buffered saline with 0.05% Tween (PBST) followed by partitioning in hexane to remove residual oil. The results show that all four ELISA test kits were able to accurately quantify the amount of milk proteins in uncooked oils. Inclusion of the PBST/hexane extraction step prior to test kit analyses did not improve the detection. Elevated heat resulted in a lower level of proteins extracted. While the amount of protein residues determined by Veratox and ELISA SYSTEMS Casein kits were similar to those obtained by the Pierce assay, the BioKits BLG kits registered the highest amount of proteins in samples. On the other hand, ELISA SYSTEMS Beta-Lactoglobulin assay registered the lowest level of proteins in these samples. These results suggest that heat treatment could affect the solubility and possibly the antigenic properties of proteins. Depending on the test kits used, these changes could result in over- or underestimation of protein residues in thermally treated foods.
M.S. in Food Safety and Technology, July 2011
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