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

Fahlbusch, B; Rudeschko, O.; Müller, Wolf‐Dieter; Schlenvoigt, G.; Vettermann, Stefan; Jäger, Lothar

doi:10.1159/000237128

Cited by 28 publications

(17 citation statements)

References 12 publications

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“…Affinity chromatography also results in a mixture of different isoforms, as was reported for Mal d 1 from apple [25]. Another purification method for the natural Mal d 1 allergen is described by Fahlbusch et al [18], who used RP-HPLC and anion exchange chromatography. These methods do not exclude that isoforms are lost by selective extraction, which may lead to an altered immune reactivity as compared to the isoform mixture in the source tissue.…”

Section: Resultsmentioning

confidence: 90%

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Purification and characterization of natural Bet v 1 from birch pollen and related allergens from carrot and celery

Bollen

García

Cordewener

et al. 2007

Molecular Nutrition Food Res

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Birch pollen allergy is predominantly caused by the major allergen Bet v 1 and can lead to crossreactions with homologous proteins in food. Two major cross-reactive food allergens are Dau c 1 from carrot and Api g 1 from celery, which have never been purified from their natural source. Here, we describe a non-denaturing purification method for obtaining natural Bet v 1, Dau c 1 and Api g 1, comprising of ammonium sulfate precipitation, hydrophobic interaction chromatography and size exclusion chromatography. This method resulted in 98-99% pure isoform mixtures for each allergen. Characterization of these isoform mixtures with Q-TOF MS/MS clearly showed earlier reported isoforms of Bet v 1, Dau c 1 and Api g 1, but also new isoforms. The presence of secondary structure in the three purified allergens was demonstrated via circular dichroism and showed high similarity. The immune reactivity of the natural allergens was compared with recombinant proteins by Western blot and ELISA and showed similar reactivity.

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Section: Resultsmentioning

confidence: 90%

“…acetone used for precipitation [17,] and TFA and ACN, used as components in RP-HPLC protocols [18]. Other methods such as affinity chromatography may be selective for specific epitopes and might thus lead to the loss of isoforms during purification.…”

Section: Introductionmentioning

confidence: 99%

Purification and characterization of natural Bet v 1 from birch pollen and related allergens from carrot and celery

Bollen

García

Cordewener

et al. 2007

Molecular Nutrition Food Res

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“…An allergic reaction to apples that consists mainly of oral itching and swelling is very common in people who are also allergic to birch pollen (Lahti et al, 1980;Dreborg, 1988;Ebner et al, 1991;Vieths et al, 2002). Immunoblotting studies show sharing of allergenic epitopes leading to the cross-reactivity of IgE antibodies (van Ree and Aalberse, 1993;Fahlbusch et al, 1995).…”

Section: Introductionmentioning

confidence: 99%

“…N-terminal sequencing of Mal d 1 showed that the sequence shared 62% (over 25 amino acids; Vieths et al, 1994a) and 67.6% identity (over 37 amino acids; Fahlbusch et al, 1995) to Bet v 1. Furthermore, antibodies to Bet v 1 also cross-reacted with this protein (Ebner et al, 1991Vieths et al, 1994b).…”

Section: Introductionmentioning

confidence: 99%

Characterisation of Mal d 1-related genes in Malus

et al. 2004

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It has been suggested that there are at least 15 Mal d 1-related (PR10) genes in one genotype of apple (Malus x domestica Borkh). We sequenced cDNA libraries of cultivar 'Royal Gala' and identified 12 members of the Mal d 1 family, including the previously reported Mal d 1b and Mal d 1d, an allelic variant of the previously reported Mal d 1a. Eight Mal d 1 gene products were expressed in tree-ripened fruit, in either the cortex or the skin, and most of these were also expressed in leaves in response to challenge with Venturia inaequalis -a fungal disease of apple. Mal d 1 gene products were identified from a large number of different tissues. Degree of ripeness as measured by standard parameters was shown not to predict either the amount of protein able to bind to a specific monoclonal antibody 5H8, previously shown to bind to an allergenic epitope in Mal d 1b and a/d, or the amount of Mal d 1 mRNA present. Mal d 1d and Mal d 1b were the most highly expressed isoforms in 'Royal Gala', particularly in the skin of fruit, and these isoforms were also predominant in other cultivars and species of apple. Genotypes, however, differed in relative predominance of Mal d 1b and Mal d 1d. The predominantly expressed Mal d 1 genes in ripe apple fruit were translated in vivo into proteins and proteins binding to the antibody were found in all cultivars and species examined. New Mal d 1 proteins were identified that bound to the 5H8 antibody. At least two new subfamilies have been identified, and while some structural differences are predicted between groups of isoforms, the P-loop motif is identical in all except two isoforms. A role in intracellular signalling in plants is suggested and in vitro expression of the isoforms should help in assessing their relative roles in disease, allergic responses, senescence and nucleotide-, cytokinin- and brassinosteroid-binding.

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“…The MAbs 1D6, 2B2 and 3F8 were obtained after immunization of BALB/c mice with purified Mal d 1 as described (Fahlbusch et al, 1995). All antibodies belonged to the IgG1 subclass.…”

Section: Monoclonal Antibodiesmentioning

confidence: 99%

Monoclonal Antibodies Raised against the Major Apple Allergen, Mal d 1, are Useful Tools for Epitope Studies

Son

Fahlbusch

Müller

et al. 2001

Food and Agricultural Immunology

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Mal d 1, the major apple allergen shows strong cross-reactivity with IgE specific for the major birch pollen allergen, Bet v 1, and is responsible for birch pollen related food allergy to apple. In contrast to other food and pollen allergens, only a few data on the B-cell epitopes of Mal d 1 are available. To obtain data on the antibody binding epitopes of Mal d 1 and to gain insights to the structures responsible for its B-cell cross-reactivity to Bet v 1, the binding characteristics were studied with 3 monoclonal antibodies (MAbs) raised against Mal d 1 and with patients' IgE directed against Mal d 1 and Bet v 1 by immunoblotting and ELISA. The different binding characteristics of these three MAbs indicated that the MAbs 1D6, 2B2 and 3F8 recognized different epitopes on the major apple allergen. MAb 2B2 cross-reacted with Bet v 1 on immunoblots, whereas 1D6 and 3F8 did not. Since 1D6 and 2B2 (but not 3F8) always competed with IgE from the same apple-allergic patients, 1D6 may be a useful tool for Mal d 1 epitope studies and 2B2 for an epitope that cross-reacts with IgE specific for Bet v 1.

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

Cited by 28 publications

References 12 publications

Purification and characterization of natural Bet v 1 from birch pollen and related allergens from carrot and celery

Purification and characterization of natural Bet v 1 from birch pollen and related allergens from carrot and celery

Characterisation of Mal d 1-related genes in Malus

Monoclonal Antibodies Raised against the Major Apple Allergen, Mal d 1, are Useful Tools for Epitope Studies

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