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Gut. May 2000; 46(5): 661–669.
PMCID: PMC1727915

Purification, biochemical, and immunological characterisation of a major food allergen: different immunoglobulin E recognition of the apo- and calcium-bound forms of carp parvalbumin

Abstract

BACKGROUND—Almost 4% of the population suffer from food allergy which is an adverse reaction to food with an underlying immunological mechanism.
AIMS—To characterise one of the most frequent IgE defined food allergens, fish parvalbumin.
METHODS—Tissue and subcellular distribution of carp parvalbumin was analysed by immunogold electron microscopy and cell fractionation. Parvalbumin was purified to homogeneity, analysed by mass spectrometry and circular dichroism (CD) spectroscopy, and its allergenic activity was analysed by IgE binding and basophil histamine release tests.
RESULTS—The isoelectric point (pI) 4.7 form of carp parvalbumin, a three EF-hand calcium-binding protein, was purified to homogeneity. CD analysis revealed a remarkable stability and refolding capacity of calcium-bound parvalbumin. This may explain why parvalbumin, despite cooking and exposure to the gastrointestinal tract, can sensitise patients. Purified parvalbumin reacted with IgE of more than 95% of individuals allergic to fish, induced dose-dependent basophil histamine release and contained, on average, 83% of the IgE epitopes present in other fish species. Calcium depletion reduced the IgE binding capacity of parvalbumin which, according to CD analysis, may be due to conformation-dependent IgE recognition.
CONCLUSIONS—Purified carp parvalbumin represents an important cross reactive food allergen. It can be used for in vitro and in vivo diagnosis of fish-induced food allergy. Our finding that the apo-form of parvalbumin had a greatly reduced IgE binding capacity indicates that this form may be a candidate for safe immunotherapy of fish-related food allergy.


Keywords: food allergy; parvalbumin; circular dichroism; epitopes; antibodies; immunochemistry

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Selected References

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Figures and Tables

Figure 1
Subcellular localisation of parvalbumin in carp muscle and liver by immunogold electron microscopy. Bright field micrograph of ultrathin sections of carp muscle labelled with a monoclonal antibody against parvalbumin (A) and with an isotype matched control ...
Figure 2
Detection of parvalbumin in subcellular carp muscle fractions. (A) Detection of parvalbumin (mαparv.) and histone (αhistone) immunoreactivity in total (E), cytoplasmic (Cy), and nuclear (Nu) western-blotted carp muscle extracts with monoclonal ...
Figure 3
Purification of carp parvalbumin. (A) Purification of carp parvalbumin isoforms by DEAE chromatography monitored by SDS-PAGE and Coomassie blue staining. Parvalbumin-enriched soluble carp muscle protein fraction obtained by ammonsulphate precipitation ...
Figure 4
Characterisation of pI isoforms of carp parvalbumin by IEF. (A) Total carp muscle extract (lane E), soluble fraction after extract boiling (lane A) and after ammonsulphate precipitation (lane B), and a single purified carp parvalbumin isoform present ...
Figure 5
Mass spectroscopic analysis of purified carp parvalbumin pI 4.7. The x axis shows the mass/charge ratio; signal intensity is displayed on the y axis as a percentage related to the most intensive signal obtained in the investigated mass range. ...
Figure 6
Induction of basophil histamine release with purified carp parvalbumin pI 4.7. Basophils from a patient allergic to fish (A) and from a non-atopic individual (B), were incubated with various concentrations of purified carp parvalbumin pI 4.7 and ...
Figure 7
Calcium-dependent IgE recognition of purified carp parvalbumin pI 4.7. Nitrocellulose-blotted carp parvalbumin pI 4.7 was exposed to serum IgE from four patients allergic to fish (Nos 1, 2, 3, and 4) and to a mouse ...
Figure 8
Far-ultraviolet circular dichroism analysis of purified carp parvalbumin pI 4.7. (A) Calcium-bound parvalbumin pI 4.7 at +20°C, +98°C and +20°C after cooling from +98°C. (B) Carp parvalbumin pI 4.7 in ...

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