Patients Allergic to Fish Tolerate Ray Based on the Low Allergenicity of Its Parvalbumin

Abstract

Background: Clinical reactions to bony fish species are common in patients with allergy to fish and are caused by parvalbumins of the β-lineage. Cartilaginous fish such as rays and sharks contain mainly α-parvalbumins and their allergenicity is not well understood.

Objective: To investigate the allergenicity of cartilaginous fish and their α-parvalbumins in individuals allergic to bony fish.

Methods: Sensitization to cod, salmon, and ray among patients allergic to cod, salmon, or both (n = 18) was explored by prick-to-prick testing. Clinical reactivity to ray was assessed in 11 patients by food challenges or clinical workup. IgE-binding to β-parvalbumins (cod, carp, salmon, barramundi, tilapia) and α-parvalbumins (ray, shark) was determined by IgE-ELISA. Basophil activation tests and skin prick tests were performed with β-parvalbumins from cod, carp, and salmon and α-parvalbumins from ray and shark.

Results: Tolerance of ray was observed in 10 of 11 patients. Prick-to-prick test reactions to ray were markedly lower than to bony fish (median wheal diameter 2 mm with ray vs 11 mm with cod and salmon). IgE to α-parvalbumins was lower (median, 0.1 kU/L for ray and shark) than to β-parvalbumins (median, ≥1.65 kU/L). Furthermore, α-parvalbumins demonstrated a significantly reduced basophil activation capacity compared with β-parvalbumins (eg, ray vs cod, P < .001; n = 18). Skin prick test further demonstrated lower reactivity to α-parvalbumins compared with β-parvalbumins.

Conclusions: Most patients allergic to bony fish tolerated ray, a cartilaginous fish, because of low allergenicity of its α-parvalbumin. A careful clinical workup and in vitro IgE-testing for cartilaginous fish will improve patient management and may introduce an alternative to bony fish into patients' diet.

Keywords: Basophil activation; Cod; Fish allergy; Food challenge; IgE; Parvalbumin; Ray; Skin prick test.

Figure E1

Diagnostic flowchart for assessing tolerance of ray. Numbers in parentheses represent the numbers of subjects for each step.

Figure E2

Gating strategy in BAT. Basophilic cells were selected from whole blood based on CCR3^high/SSC^low. Activation of basophils was determined by expression of an activation marker CD63. Expression of CD63 for unstimulated control, 2 stimulation controls (anti-FCεRI mAb and fMLP), and stimulation with 100 ng/mL cod parvalbumin is demonstrated for P1 as an example. fMLP, Formyl-methionyl-leucyl-phenylalanine.

Figure E3

Characterization of parvalbumins from bony and cartilaginous fish species. A, Coomassie-stained SDS-PAGE gel of purified parvalbumins. B, Western blot using commercial antiparvalbumin antibodies. C, Circular dichroism (CD) spectra of purified parvalbumins.

Figure E4

BAT. Percentages of CD63⁺ basophils (y-axes) at different concentrations of β-parvalbumins and α-parvalbumins are represented for individual subjects allergic to fish (P1-P18).

Figure E5

Response to positive (anti-FcεRI and fMLP) and negative (stimulation buffer) controls in BAT for subjects with fish allergy. Ctrl, Control; fMLP, formyl-methionyl-leucyl-phenylalanine; Neg, negative; pos, positive.

Figure E6

Detection of fish allergens in ray extract. A, Coomassie-stained SDS-PAGE of extract of raw ray filet and ray filet cooked for 5, 10, or 20 minutes. B-D, Immunoblots with ray extract using antiparvalbumin antibody (Fig E6, B), antienolase antibody (Fig E6, C), and antialdolase antibody (Fig E6, D). E, IgE immunoblots using extract of raw ray filet. P, E, and A stand for controls parvalbumin, enolase and aldolase, respectively. P4 and P6 were additionally tested on extract of cooked ray (4* and 6*). Ab, Antibody.

Figure 1

IgE levels to α-parvalbumins are significantly lower than IgE levels to β-parvalbumins. A, IgE titers (kUA/L) to α-parvalbumins and β-parvalbumins among 18 patients allergic to fish were determined by ELISA. B, Patient-specific IgE recognition patterns to α-parvalbumins and β-parvalbumins. The signal intensity of parvalbumin-specific IgE is represented in a grading log10 scale for concentrations of measured parvalbumin-specific IgE (kUA/L).

Figure 2

IgE antibody cross-reactivity of cod α-parvalbumin to salmon (β) and ray (α) parvalbumin determined by inhibition ELISA in patients allergic to bony fish (n = 10). Microtiter plates were coated with cod β-parvalbumin and sera preincubated with cod β-parvalbumin (A), salmon β-parvalbumin (B), or ray α-parvalbumin (C) at increasing concentrations.

Figure 3

Basophil response to α-parvalbumins was lower than to β-parvalbumins. Data indicate basophil activation (measured as percentage of CD63⁺ basophils) in response to stimulation with different doses of fish parvalbumins in patients allergic to fish (n = 18).

Figure 4

α-Parvalbumins show lower capacity than β-parvalbumins to induce skin reactions in patients allergic to fish. SPTs were performed with different doses of natural purified fish parvalbumins. Parvalbumins were used in concentrations ranging from 0.1 μg/mL to 50 μg/mL. Average wheal diameter of greater than or equal to 3 mm compared with negative control was rated as a positive response. (X) indicates that SPT was not performed.