Optimization of apple allergen preparation for in vivo and in vitro diagnostics

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

“…The supernatant was then filtered through a 0.45-µm filter and dialyzed with phosphate-buffered saline (PBS) overnight at 4°C, concentrated using Amicon Ultra PL-10 (Millipore Corporation, Bedford, Mass., USA) and stored at –70°C. Indian jujube extract was prepared by a modification of the low-temperature procedure [8]. The fresh fruit (100 g) was mechanically homogenized in 200 ml of precooled acetone, filtered, washed twice with acetone-ether (1:1, vol/vol) and lyophilized.…”

Section: Methodsmentioning

confidence: 99%

Allergenic Components of Indian Jujube (Zizyphus mauritiana) Show IgE Cross-Reactivity with Latex Allergen

Lee¹,

Chen

Lan

et al. 2004

Background: ‘Latex-fruit syndrome’ has been well documented. A prevalence of latex allergy among medical workers of 6.8–8.6% had been reported in Taiwan. However, there has been no study to determine the importance and type of fruit hypersensitivity in latex-allergic patients in Taiwan. This study aimed to identify the allergenic components of Indian jujube (Zizyphus mauritiana) and characterize the cross-reactivity of specific IgE antibodies to latex allergen. Methods: Crude extracts were prepared from Indian jujube and from ammoniated natural rubber latex, and six medical workers and one patient with a history of fruit allergy underwent skin testing with routine allergens, latex, Indian jujube and other fruits. Sera from two Indian jujube skin test-positive latex-allergic subjects were used for allergen-specific IgE, immunoblotting, immunoblot inhibition and enzyme-linked immunosorbent assay (ELISA) inhibition studies. Results: Both patients had positive skin test responses and specific IgE assays to Indian jujube and latex extracts. Immunoblotting revealed that IgE from both subjects bound to a 42-kD latex protein and a 42-kD Indian jujube protein. In addition, IgE from one subject bound to a 30-kD Indian jujube protein. Preincubation of atopic sera with Indian jujube or latex extract demonstrated absent and/or marked inhibition of IgE binding. Moreover, anti-Indian jujube protein antibody-based ELISA was able to detect latex extracts. Conclusions: Our results add to findings regarding the ‘latex-fruit syndrome’ described in the literature, and further study of the cross-reacting allergens identified in Indian jujube may help to elucidate the mechanisms underlying this syndrome.

“…Since apple allergens are highly labile, extracts with high allergenic activity first had to be prepared as described by Rudeschko et al [11], These extracts are excellent for both in vitro and in vivo diagnostic studies. Using such concen trated apple extracts, the isolation of the major allergen in an active form was successful using anion exchange chro matography followed by RP-HPLC ( fig.…”

Section: Discussionmentioning

confidence: 99%

“…Serum was collected from 32 patients with a positive history of skin tests and a RAST class to birch > 4 and to apple > 2 [11]. The serum pool had a RAST class to apple o f 3.4, indicating high specific IgE levels for apple allergens.…”

Section: Human Serummentioning

confidence: 99%

“…Purified Bet v 1, kindly provided by Dr. B. Weber (Allergopharma), was used for cross-reac tivity studies [9], Apple extracts were prepared from mature, fresh Golden Delicious apples by a low-temperature method [10] modified in our laboratory [11]. Briefly, 100-g apples were homogenized with a mixing rod and Ultraturrax and treated with 200 ml diacetone alcohol (4-hydroxy-4-methyl-2-pentanon, Aldrich) for 18 h at -20°C.…”

Section: Source Materials Anti Extract Preparationmentioning

confidence: 99%

See 1 more Smart Citation

Purification and Characterization of the Major Allergen from Apple and Its Allergenic Cross-Reactivity with Bet v 1

Fahlbusch¹,

Rudeschko²,

Müller³

et al. 1995

Self Cite

The major allergen from apple extract was concentrated by anion exchange chromatography and further purified by reverse-phase HPLC. A distinct peak with a high degree of homogeneity was obtained. The isolated protein has a MW of 18 kD and specific IgE-binding capacity (immunoblotting, IgE-binding inhibition). N-terminal amino acid analyses of the allergen allowed 37 cleavages and showed 67.6% identity to Bet v 1, the major allergen of birch pollen. Enzyme immunoassay inhibition studies with serum of birch/apple-allergic patients showed that besides cross-reacting structures to Bet v 1, apple-specific IgE antibodies could exist. Monoclonal antibodies (mAbs) were raised against the 18-kD allergen from apple and characterized by means of immunoblotting and ELISA. Only three of the eight studied mAbs reacted with Bet v 1.