Effects of Extraction Buffer on the Solubility and Immunoreactivity of the Pacific Oyster Allergens

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.

Recombinant Tropomyosin from the Pacific Oyster (Crassostrea gigas) for Better Diagnosis

“…In this study, 21 patients were analysed for their IgE reactivity against the raw and cooked extract of the Pacific oyster. Similarly to the findings of previous studies [6,8], more IgE-binding proteins were observed in the raw compared to the heated protein extract. However, the heated extract demonstrated higher IgE binding intensity than the raw extract for the same IgE-binding proteins.…”

“…While food challenges are regarded as the gold standard of food allergy diagnosis, their application has been associated with risks of severe reaction in the case of shellfish challenge due to high allergen potency and adult patient co-morbidities [26]. Several mollusc IgE-reactive proteins have been identified in previous studies [6,8]. However, they have not been fully characterised, limiting their application as diagnostic tools for mollusc allergy.…”

“…Raw and heated oyster extracts were prepared according to the method described in detail in a previous study [8]. Briefly, five grams of minced fresh Pacific oysters were homogenized in 25 mL of TBSN buffer (25 mM Tris; 3.0 mM KCl; 1 M NaCl, pH 7.4) and stirred at 4 • C overnight.…”

“…However, the knowledge of their allergens and possible allergenic cross-reactivity remains very limited [5][6][7]. In previous studies [6,8], a serum pool from shellfish allergic patients demonstrated immunological reactivity to a 38 kDa protein band. Subsequent analysis using mass spectrometry identified tropomyosin (TM) as the major allergenic protein component.…”

Effect of Processing on Fish Protein Antigenicity and Allergenicity

“…Different extraction strategies should be conducted against different proteins’ properties to ensure their extractability. It is reported that a larger amount of IgE-reactive bands was observed from oyster when it was extracted using high-salt buffers or high-pH buffers [ 91 ]. The addition of 5 mM EDTA in the extraction solution increased salmon parvalbumin extractability significantly [ 23 ].…”