Investigation of the stability of apple allergen extracts

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.

Section: Discussionmentioning

confidence: 99%

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

Zuidmeer¹,

Leeuwen²,

Budde³

et al. 2006

“…On the one side, in vitro tests may fail to detect sIgE and thereby overlook sensitization towards the allergen. This has been observed in the diagnosis of food allergies, e.g.against apple and carrot [8, 9], and the reasons may be quite trivial ones such as low stability of the allergen or varying concentrations in the raw material used for extraction. Standardization of allergen extracts for prick testing and serum IgE determination is therefore a challenging and serious task [10].…”

Section: The Malaise Of Allergy Diagnosis By In Vitromeasurement Of Igementioning

confidence: 99%

The Role of Protein Glycosylation in Allergy

Altmann¹

2006

342

373

The asparagine-linked carbohydrate moieties of plant and insect glycoproteins are the most abundant environmental immune determinants. They are the structural basis of what is known as cross-reactive carbohydrate determinants (CCDs). Despite some structural variation, the two main motifs are the xylose and the core-3-linked fucose, which form the essential part of two independent epitopes. Plants contain both epitopes, insect glycoproteins only fucose. These epitopes and other fucosylated determinants are also found in helminth parasites where they exert remarkable immunomodulatory effects. About 20% or more of allergic patients generate specific anti-glycan IgE, which is often accompanied by IgG. Even though antibody-binding glycoproteins are widespread in pollens, foods and insect venoms, CCDs do not appear to cause clinical symptoms in most, if not all patients. When IgE binding is solely due to CCDs, a glycoprotein allergen thus can be rated as clinical irrelevant allergen. Low binding affinity between IgE and plant N-glycans now drops out as a plausible explanation for the benign nature of CCDs. This rather may result from blocking antibodies induced by an incidental ‘immune therapy’ (‘glyco-specific immune therapy’) exerted by everyday contact with plant materials, e.g. fruits or vegetables. The need to detect and suppress anti-CCD IgE without interference from peptide epitopes can be best met by artificial glycoprotein allergens. Hydroxyproline-linked arabinose (single β-arabinofuranosyl residues) has been identified as a new IgE-binding carbohydrate epitope in the major mugwort allergen. However, currently the occurrence of this O-glycan determinant appears to be rather restricted.

“…Symptoms are usually restricted to the oral cavity (oral allergy syndrome, OAS) [1], most likely because the responsible Bet v 1-homologous food allergens are very labile and do not survive digestion in the gastrointestinal tract [2, 3]. In recent studies on several hundreds of subjects with Rosaceae-induced OAS, a subgroup with deviating characteristics was identified [4, 5].…”

Section: Introductionmentioning

confidence: 99%

Lipid Transfer Protein: A Pan-Allergen in Plant-Derived Foods That Is Highly Resistant to Pepsin Digestion

Asero

Mistrello

Roncarolo

et al. 2001

110

Background: Lipid transfer proteins (LTPs) are stable and highly conserved proteins of around 10 kD. They have recently been identified as allergens in fruits of the Rosaceae family. Objective: The aim of this study was to investigate whether the highly conserved structure of LTPs justifies a designation as a true pan-allergen, and to study the role of protein stability in allergenicity. Methods: Thirty-eight patients with a positive skin prick test to Rosaceae fruit extracts were characterized by interviews and skin prick tests. To investigate IgE cross-reactivity between Rosaceae and non-Rosaceae LTPs, RAST and RAST inhibition as well as ELISA and ELISA inhibition were performed, using whole food extracts and purified natural and recombinant LTPs. To address the role of protein stability in the allergenicity of LTP, fruit extracts and LTPs were digested with pepsin. Results: IgE antibodies to Rosaceae LTPs cross-reacted with a broad range of non-Rosaceae vegetable foods. Inhibition studies with purified natural and recombinant LTPs confirmed the role of LTP in this cross-reactivity. Many of the patients with this type of cross-reactive IgE antibodies had a clinical food allergy. In contrast to the typical birch Rosaceae cross-reactive patients, the oral allergy syndrome was frequently accompanied by more severe and systemic reactions. IgE reactivity to LTP was shown to be resistant to pepsin treatment of the allergen. Conclusion: LTP is a true pan-allergen with a degree of cross-reactivity comparable to profilin. Due to its extreme resistance to pepsin digestion, LTP is a potentially severe food allergen.