Abstract
Despite recent technological advances, novel allergenic protein discovery is limited by their low abundance, often due to specific physical characteristics restricting their recovery during the extraction process from various allergen sources. In this study, eight different extraction buffers were compared for their ability to recover proteins from Pacific oyster (Crassostrea gigas). The protein composition was investigated using high resolution mass spectrometry. The antibody IgE-reactivity of each extract was determined using a pool of serum from five shellfish-allergic patients. Most of the investigated buffers showed good capacity to extract proteins from the Pacific oyster. In general, a higher concentration of proteins was recovered using high salt buffers or high pH buffers, subsequently revealing more IgE-reactive bands on immunoblotting. In contrast, low pH buffers resulted in a poor protein recovery and reduced IgE-reactivity. Discovery of additional IgE-reactive proteins in high salt buffers or high pH buffers was associated with an increase in allergen abundance in the extracts. In conclusion, increasing the ionic strength and pH of the buffer improves the solubility of allergenic proteins during the extraction process for oyster tissue. This strategy could also be applied for other difficult-to-extract allergen sources, thereby yielding an improved allergen panel for increased diagnostic efficiency.
Highlights
Shellfish allergy is caused by overreaction of the human immune system to harmless shellfish proteins resulting in allergic sensitization and a range of different clinical presentations including urticaria, angioedema, bronchospasm, hypotension, and even life-threatening anaphylaxis or occasionally death [2]
Upon subsequent exposure to the human immune system, allergenic proteins trigger the production of more allergenspecific IgE antibodies, which bind to specific receptors on the surface of mast cells and basophils
Addition of salt up to 1 M to the phosphate-buffered saline (PBS) and tris-buffered saline (TBS) buffers significantly increased the ability of the buffers to retrieve soluble proteins (p < 0.05)
Summary
Shellfish allergy is caused by overreaction of the human immune system to harmless shellfish proteins resulting in allergic sensitization and a range of different clinical presentations including urticaria (hives), angioedema (swelling of throat or other tissues), bronchospasm (trouble breathing), hypotension (low blood pressure and dizziness), and even life-threatening anaphylaxis or occasionally death [2]. Upon subsequent exposure to the human immune system, allergenic proteins trigger the production of more allergenspecific IgE antibodies, which bind to specific receptors on the surface of mast cells and basophils. When these allergenic proteins bind to receptor-bound antibodies, subsequent cross-linking results in activation of these cells leading to mediator release and clinical symptoms [3]. Over 2000 allergenic proteins have been identified, and almost are analysed in detail and have been assigned a unique code by the WHO
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