Lipid Transfer Proteins from Fruit: Cloning, Expression and Quantification

Zuidmeer, Laurian; Leeuwen, W.A. van; Budde, Ilona Kleine; Cornelissen, Jessica; Bulder, Ingrid; Rafalska, Ilona; Besolí, Noèlia Telléz; Akkerdaas, Jaap H.; Asero, Riccardo; Mancebo, Eloina Gonzalez; Ree, Ronald van

doi:10.1159/000086420

Cited by 43 publications

(35 citation statements)

References 76 publications

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“…The monoclonal antibody used as capturing agent in the sELISA has been reported to be cross-reactive to other fruit LTP, i.e. to recognize a highly conserved epitope on the LTP molecule [20]. It is therefore likely that different isoforms [29] of LTP are captured with similar sensitivity and subsequently detected by polyclonal rabbit antibodies.…”

Section: Discussionmentioning

confidence: 99%

“…Purified natural apple LTP [19] was coated onto microtiter plates. Binding of apple LTP-specific rabbit IgG [20] was inhibited by apple extracts. Purified apple LTP was used as a standard.…”

Section: Methodsmentioning

confidence: 99%

“…Sandwich ELISA (sELISA) was performed as described previously [20]. Apple LTP-specific mouse monoclonal antibody 6B5 was used as capturing antibody and rabbit polyclonal antiserum against apple LTP as source of detecting antibodies.…”

Section: Methodsmentioning

confidence: 99%

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Measurement of Lipid Transfer Protein in 88 Apple Cultivars

Sancho

Ree

Leeuwen

et al. 2007

Int Arch Allergy Immunol

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Background: Fruits are a major cause of food allergy in adults. Lipid transfer proteins (LTP) are implicated in severe allergic reactions to fruits, but little is known about LTP content in different cultivars. Objective: Determination of the levels of LTP in a wide range of apple cultivars. Methods: LTP was measured in apples from 53 cultivars grown in Italy and 35 grown in The Netherlands, using three different immunoassays: a competitive ELISA (cELISA), a sandwich ELISA (sELISA) and a RAST inhibition (RI). Selected cultivars were evaluated using the basophil histamine release test (BHR), skin prick test (SPT) and double-blind, placebo-controlled food challenge (DBPCFC). Results: LTP levels measured with the three immunoassays were significantly correlated, as judged by Pearson’s correlation (0.61 < Rp < 0.65; p < 0.0001), but differed with respect to the actual quantities: 3.4–253.2 (sELISA), 2.7–120.2 (cELISA) and 0.4–47.3 µg/g tissue (RI). Between cultivars, LTP titers varied over about a two-log range. Pilot in vitro and in vivo biological testing (BHR, SPT and DBPCFC) with selected cultivars supported the observed differences in LTP levels. Conclusions: Around 100-fold differences in LTP levels exist between apple cultivars. Whether the lowest observed levels of LTP warrant designation as hypo-allergenic requires more extensive confirmation by oral challenges. Determination of cultivar variation in LTP levels provides important information for growers and consumers. Comparison to earlier reported Mal d 1 levels in the same cultivars reveals that a designation as low allergenic does not always coincide for both allergens.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Measurement of Lipid Transfer Protein in 88 Apple Cultivars

Sancho

Ree

Leeuwen

et al. 2007

Int Arch Allergy Immunol

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“…pAbs against grass pollen profilin (Lol p 12) were produced and characterized following the same protocol [28]. pAb and mAbs against natural Mal d 3 were produced as described previously [29]. …”

Section: Methodsmentioning

confidence: 99%

“…A cRIA for measuring Mal d 3 using pAb against Mal d 3 was described previously [29]. Finally, for measuring Mal d 4, 50 µl rabbit anti-Lol p 12 (1:8,000 in PBS-AT) was used in combination with Protein A-Sepharose (0.5 mg) and 125 I-radiolabeled rMal d 4 (20,000 cpm) in a final volume of 900 µl PBS-AT.…”

Section: Methodsmentioning

confidence: 99%

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

Zuidmeer¹,

Leeuwen²,

Budde³

et al. 2006

Int Arch Allergy Immunol

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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.

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Breeding of Hypoallergenic Fruits

Gilissen¹

2011

Breeding for Fruit Quality

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Lipid Transfer Proteins from Fruit: Cloning, Expression and Quantification

Cited by 43 publications

References 76 publications

Measurement of Lipid Transfer Protein in 88 Apple Cultivars

Measurement of Lipid Transfer Protein in 88 Apple Cultivars

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

Breeding of Hypoallergenic Fruits

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