Recombinant Carp Parvalbumin, the Major Cross-Reactive Fish Allergen: A Tool for Diagnosis and Therapy of Fish Allergy

Abstract

IgE-mediated reactions to fish allergens represent one of the most frequent causes of food allergy. We have constructed an expression cDNA library from carp (Cyprinus carpio) muscle in phage λgt11 and used serum IgE from a fish allergic patient to isolate 33 cDNA clones that coded for two parvalbumin isoforms (Cyp c 1.01 and Cyp c 1.02) with comparable IgE binding capacities. Both isoforms represented calcium-binding proteins that belonged to the β-lineage of parvalbumins. The Cyp c 1.01 cDNA was overexpressed in Escherichia coli, and rCyp c 1.01 was purified to homogeneity. Circular dichroism analysis and mass spectroscopy showed that rCyp c 1.01 represented a folded protein with mainly α-helical secondary structure and a molecular mass of 11,416 Da, respectively. rCyp c 1.01 reacted with IgE from all fish-allergic patients tested (n = 60), induced specific and dose-dependent basophil histamine release, and contained most of the IgE epitopes (70%) present in natural allergen extracts from cod, tuna, and salmon. Therefore, it may be used to identify patients suffering from IgE-mediated fish allergy. The therapeutic potential of rCyp c 1.01 is indicated by our findings that rabbit Abs raised against rCyp c 1.01 inhibited the binding of IgE (n = 25) in fish-allergic patients to rCyp c 1.01 between 35 and 97% (84% mean inhibition) and that depletion of calcium strongly reduced IgE recognition of rCyp c 1.01. The latter results suggest that it will be possible to develop strategies for immunotherapy for fish allergy that are based on calcium-free hypoallergenic rCyp c 1.01 derivatives.

Cloning, Expression, and Mapping of Allergenic Determinants of αS1-Casein, a Major Cow’s Milk Allergen

“…B, Sera from an additional nine IgE-positive patients (nos. 6,7,11,15,27,31,44,46,66) were preadsorbed with all available peptides (Cas1-Cas6 plus Cas1.2-Cas5.6). IgE levels to ELISA plate-bound r␣S1-casein are displayed as OD values (y-axis).…”

Expression of a Biologically Active GFP-αS1-Casein Fusion Protein in Lactococcus lactis

“…Of these four proteins, α S1 -casein thought to be the most potent for inducing a specific IgE response [1]. α S1 -casein is considered as a true food allergen, also classified as class I food allergen, which is comparable to the major fish allergen parvalbumin [21], the major peanut allergens [13], and the major shrimp allergen [15], and it can induce severe and life-threatening anaphylactic reactions. α S1 -casein, which is a single-chain linear phosphoprotein of 199 amino acid residues, has only a small amount of secondary structure and lacks disulfide bonds, which results in a reduction of tertiary interactions [8].…”

Identification of Der p 23, a Peritrophin-Like Protein, As a New Major Dermatophagoides Pteronyssinus Allergen Associated with the Peritrophic Matrix of Mite Fecal Pellets

“…A rabbit antiserum was raised against rDer p 23 using Freund's adjuvant (Charles River Laboratories, Kisslegg, Germany), and the ability of the Der p 23-specific rabbit Abs to inhibit binding of D. pteronyssinus allergic patients' IgE to the allergen was examined by ELISA competition assays (29). ELISA plate-bound Der p 23 (5 μg/ml) was preincubated with 1:100 in PBS, 0.05% (v/v) Tween 20 (PBST), and 0.5% (w/v) BSA diluted rabbit antiDer p 23 Abs, or rabbit preimmune serum and incubated at 4°C overnight.…”

“…Purified Der p 23 was analyzed by MALDI mass spectrometry (piChem, Research and Development, Graz, Austria) as described previously (29).…”