Immunoblotting analysis of wheat allergens: control of side reactions through wheat polypeptides naturally present in dried cow milk

Immunoblotting analysis of wheat allergens can be a two-step procedure: an incubation with a human serum and one with an anti-IgE-horseradish peroxidase (HRP) conjugate, visualised by chemiluminescence. Different procedures produced side reactions that appeared as non-specific additional bands which hampered the identification of specific IgE-reacting proteins. We then showed that the rabbit anti-human IgE-HRP conjugate most likely contains anti-wheat antibodies that bind with the proteins. We investigated another procedure to accurately identify wheat specific IgE-reacting proteins. The use of polyvinylpyrrolidone-40 throughout the procedure and 3% cow milk for the conjugate incubation provided signals clear-cut enough to detect proteins up to the allelic level. Tested against patients, the procedure identified IgE-reacting proteins. It also provided new data in terms of gliadins for patients suffering from hypersensitivity to hydrolysed wheat proteins. Lastly, we investigated why dried cow milk blocks. Using rabbit serum containing anti-wheat antibodies, we detected wheat cross-reacting polypeptides in it