Allergenicity Assessment of Apple Cultivars: Hurdles in Quantifying Labile Fruit Allergens

Abstract

Background: Assessment of allergenicity of foods is important for allergic consumers and regulators. Immunoassays to measure major food allergens are widely applied, often giving variable results. Using the major apple allergen Mal d 1 as a model, we aimed to establish at the molecular level why different immunoassays for assessing allergenicity of apple cultivars produce conflicting outcomes. Methods: Mal d 1 was measured in 53 cultivars from Italy and 35 from The Netherlands, using four different immunoassays. Purified Mal d 1 standards were molecularly characterized by size-exclusion chromatography (SEC) and mass spectrometry (MS). Results: Three immunoassays using an identical standard gave similar results. Minor differences in sample preparation already resulted in significant loss of allergenicity. The fourth assay, using a different Mal d 1 standard, gave 10- to 100-fold lower outcomes. By SEC, this standard was shown to be almost fully aggregated. This aggregation was accompanied by a decrease of the mass of the Mal d 1 molecule by ∼1 kDa as analyzed by MS. The deviating immunoassay was shown to selectively recognize this aggregated form of Mal d 1, whereas the other three assays, including the one based on IgE antibody recognition, preferentially bound non-aggregated allergen. Conclusions: Variable and poorly controllable major allergen modification in both extracts and standards hamper accurate allergenicity assessments of fruits.

Measurement of Lipid Transfer Protein in 88 Apple Cultivars

“…The variation in Mal d 1 levels in this same apple cultivar extracts were reported earlier [13]. The maximum difference between low- and high-allergenic cultivars for Mal d 1 was similar to that now reported for LTP: up to 100-fold.…”

“…Additionally, the even lower allergen concentrations observed in RI compared to the cELISA suggest that IgE epitopes might be relatively labile in extracts compared to the purified standard. For Mal d 1, poor conservation of IgE-binding epitopes in extracts is well established [13, 30]. The results described in this study suggest that this may – to a lesser extent – also be true for LTP.…”

“…Apples of 35 different cultivars from Wageningen (The Netherlands) and 53 cultivars from Ferrara (Italy) were harvested, stored and extracted as described previously [13], essentially according to the method of Bjorksten [18]. Five-millimeter-thick peels were used from 5 apples per cultivar.…”

“…Moreover, knowledge about genotypic differences in Mal d 1 levels in different cultivars can support strategies to breed novel low-allergenic cultivars. A recent study reported Mal d 1 levels in 88 different apple cultivars, but at the same time demonstrated discrepancies between results obtained with different assays for the measurement of the allergen [13]. These discrepancies were largely explained by differences in assay fine specificity, and to some extent by interlaboratory variation.…”

Fireblight (Erwinia amylovora) affects Mal d 1-related allergenicity in apple

“…This general increase in the mRNA level during storage might overrule the initial pathogeninduced upregulation of Mal d 1. Mal d 1 is regarded as a protein highly susceptible to protease degradation and thus reliable quantification of the native protein is difficult to perform (Vieths et al 1995;Sancho et al 2006;Zuidmeer et al 2006). Thus, absolute data have to be treated with caution.…”