Allergenicity and Stability of 6 New Korean Bony Fish Extracts

Abstract

Purpose: Diagnostic tests for allergen sensitization should reflect real exposure. We made 6 new bony fish extracts, which are consumed popularly in Korea, and evaluated their allergenicity and stability.

Methods: We manufactured fish extracts from codfish, mackerel, common eel, flounder, cutlass, and catfish. Protein and parvalbumin (PV) were evaluated by Bradford assay, 2-site enzyme-linked immunosorbent assay, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and anti-PV immunoblotting. The immunoglobulin E (IgE) reactivities of the extracts were evaluated with ImmunoCAP and IgE immunoblotting using sera from 24 Korean fish allergy patients, 5 asymptomatic sensitizers, and 11 non-atopic subjects. Stability of the extracts stored in 4 different buffers were evaluated for up to a year.

Results: The protein concentrations of commercial SPT fish extracts varied with up to a 7.5-fold difference. SDS-PAGE showed marked differences in the PV concentrations of commercial SPT reagents. Specific IgE measurements for the following investigatory fish extracts-iCodfish, iMackerel, and iEel-were concordant with that of their corresponding Phadia ImmunoCAP measurements. ImmunoCAP results showed marked IgE cross-reactivity among the fish species, and the overall sensitivity of ImmunoCAP with the investigatory fish extracts for identification of culprit fish species was 85.7%. The protein and PV concentrations in the investigatory extracts were highly stable in saline with 0.3% phenol-50% glycerol at 4°C for up to a year.

Conclusions: The commercial SPT fish extracts exhibited considerable variation in terms of allergenicity, which may impact on diagnostic accuracy. Our new fish extracts have sufficient allergenicity and stability and may be adequate to various clinical applications.

Keywords: Allergenicity; bony fish; extracts; fish allergy; fish parvalbumin; food allergy.

Fig. 1. SDS-PAGE (A), anti-parvalbumin IgG immunoblotting (B), and IgE immunoblotting with pooled sera from fish allergy patients (C) of fish extracts. Each lane of the SDS-PAGE gel was loaded with 20 μL of the commercial skin prick test reagents or the new extracts listed in Table 1.

M, marker; A, allergopharma; L, lofarma; H, Hollister-Stier; Inv, investigatory; SDS-PAGE, sodium dodecyl sulphate–polyacrylamide gel electrophoresis; Ig, immunoglobulin.

Fig. 2. ImmunoCAP measurements for 6 new fish extracts. (A) All ImmunoCAP measurements of 24 fish allergy patients and 5 asymptomatic sensitizers. (B) The ImmunoCAP measurements for 14 corresponding culprit allergens to the 11 patients' claim. Hatched box in the figures indicate negative range for ImmunoCAP measurement (< 0.35 kU/L).

sIgE, specific immunoglobulin E.

Fig. 3. sIgE levels against 3 Phadia ImmunoCAP fish species and the 6 new fish extracts in sera from 24 fish allergy patients, 5 asymptomatic sensitizers and 11 negative controls. All of the sIgE measurements were performed using the ImmunoCAP platform.

IgE, immunoglobulin E; sIgE, specific immunoglobulin E.

Fig. 4. Protein stability of the new fish extracts stored at room temperature (A-F) and 4°C (G-L) for up to a year. Extracts were stored in 4 different buffer compositions: normal saline, saline with 50% glycerol, saline with 0.3% phenol, and saline with 0.3% phenol–50% glycerol.

Fig. 5. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis of the new fish extracts stored in saline with 0.3% phenol–50% glycerol at room temperature (A-F) and 4°C (G-L) for up to a year.

M, marker.

Fig. 6. Anti-parvalbumin immunoglobulin G immunoblotting of the new fish extracts stored in saline with 0.3% phenol–50% glycerol at room temperature (A-F) and 4°C (G-L) for up to a year.

M, marker.

Fig. 7. Two-site enzyme-linked immunosorbent assay measurements of parvalbumins in the new fish extracts stored at room temperature and 4°C (A-F) for up to a year.