Component-resolved in vitro diagnosis in carrot allergy: does the use of recombinant carrot allergens improve the reliability of the diagnostic procedure?

BACKGROUND: In Europe, pollen-related food allergy is the most frequent form of food allergy in adults. Reliability of current diagnostic procedures, however, is poor and therapeutic options are not available. OBJECTIVES: In the present study, we created a panel of recombinant allergens from carrot and evaluated its potential in component-resolved in vitro diagnosis of carrot allergy. METHODS: Recombinant (r) Dau c 1.0104, Dau c 1.0201 and Dau c 4 were cloned by a polymerase chain reaction strategy, expressed in Escherichia coli and purified. Carrot lipid transfer protein (LTP) was expressed in the yeast Pichia pastoris. Sera from 40 carrot-allergic patients were investigated. Twenty-one birch pollen-allergic subjects with negative open provocation to carrot and 20 non-allergic subjects were included as controls. IgE binding to recombinant allergens as well as to cross-reactive carbohydrate determinants (CCD) was measured by ELISA. Cross-reactivity between Dau c 1 isoforms and Bet v 1 was assayed by ELISA inhibition. Biological activity of the recombinant carrot allergens was assessed by histamine release assay and peripheral blood mononuclear cells stimulation. RESULTS: Ninety-eight percent of the carrot-allergic patients were positive to at least one recombinant allergen; 98% reacted to rDau c 1.0104, 65% to rDau c 1.0201, 38% to rDau c 4 and 20% had IgE against CCD. Specificity using the recombinant allergens was high when compared with non-allergic controls, but low compared with birch-sensitized subjects without carrot allergy. Sensitization to Dau c 1.0201, however, proved to be highly specific for clinically relevant sensitization. Inhibition assays indicated the absence of LTP in carrot root extract, and epitope diversity between Dau c 1.0104, Dau c 1.0201 and Bet v 1. CONCLUSIONS: Our panel of recombinant allergens from carrot can provide a standardized tool for in vitro diagnosis of carrot allergy, and for epitope studies