Tissue-based skin prick test extracts from Atlantic salmon containing occupationally relevant allergens

Abstract

Introduction: Diagnosis of salmon allergy often includes skin prick testing using commercially available extracts. Multiple studies have shown that these may contain highly variable amounts of important allergens. In this study we aimed to produce skin prick test extracts containing salmon allergens relevant to the occupational exposure situation. The extracts were characterized with respect to the presence of known salmon allergens. The effect of heat-treatment on the antibody-binding of allergens was also assessed.

Method: Allergenic proteins were extracted from muscle, skin, and outer mucus coating from Atlantic salmon (Salmo salar). Electrophoresis was used for protein separation with subsequent silver staining and immunoblotting for the detection of known allergenic proteins with antibody-binding activity to commercially available antibodies. The identity of allergens was confirmed by mass spectrometry.

Results: Our extraction methods were successful in detecting the allergenic proteins aldolase, collagen, enolase, and parvalbumin from muscle, skin, and outer mucus coating from Atlantic salmon. Our work also demonstrates that these allergens have various tolerance towards heating.

Conclusion: By using multiple fish tissues for extraction, we were able to produce skin prick test extracts containing allergens of presumed relevance in diagnosis of work-related sensitization.

Keywords: allergen extracts; occupational allergy; salmon allergy; salmon processing; skin prick testing; thermolabile allergen.

Figure 1

Protein content in tissue extracts from Atlantic salmon, separated by SDS-PAGE in a 4%–12% Bis-Tris gel and stained with Coomassie. R, raw muscle; Co, heated muscle (100°C 30 min); M, mucus; and S, skin. Letters in lowercase corresponds to bands that were analyzed using mass spectrometry, matching the monomeric form of the four allergens collagen (∼110 kDa): a, e, i, and j, enolase (∼50 kDa): b and f, aldolase (∼40 kDa): c and g, and parvalbumin (∼12 kDa): d and h. Extracts used were RM1, M1, CM1, and S1 from Supplementary Table S1.

Figure 2

Western blot for detection of allergenic proteins in a raw muscle tissue extract from Atlantic salmon, using commercial antibodies for (A) collagen, (B) enolase, (C) aldolase, and (D) parvalbumin. Letters in lowercase corresponds to bands that were analyzed using mass spectrometry, matching the monomeric form of the four allergens. Extract used was RM2 from Supplementary Table S1.

Figure 3

Western blot for detection of allergenic proteins in a mucus tissue extract from Atlantic Salmon, using commercial antibodies for (A) collagen, (B) enolase, (C) aldolase, and (D) parvalbumin. Letters in lowercase corresponds to bands that were analyzed using mass spectrometry, matching the monomeric form of the four allergens. Extract used was M2 from Supplementary Table S1.

Figure 4

Western blot for detection of allergenic proteins in a skin tissue extract from Atlantic salmon, using a commercial antibody for collagen. Letters in lowercase corresponds to bands that were analyzed using mass spectrometry. Extract used was S2 from Supplementary Table S1.