Immunologic characterization of purified recombinant timothy grass pollen (Phleum pratense) allergens (Phl p 1, Phl p 2, Phl p 5)1

Vrtala, Susanne; Susani, Markus; Sperr, Wolfgang R.; Valent, Peter; Laffer, Sylvia; Dolecek, Christiane; Kraft, Dietrich; Valenta, Rudolf

doi:10.1016/s0091-6749(96)80156-4

Cited by 101 publications

(96 citation statements)

References 16 publications

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“…The prevalence of IgE and IgG subclass reactivity to recombinant carp parvalbumin or, for control purposes, to rPhl p 5, an immunologically unrelated timothy grass pollen allergen (37), was determined in sera from fish-allergic patients, grass pollen-allergic patients, and nonatopic individuals by ELISA. ELISA plates (Nunc Maxisorb, Roskilde, Denmark) were coated with the recombinant proteins (5 g/ml in 0.1 M sodium bicarbonate (pH 9.6)) and blocked with 1% human serum albumin in TBST.…”

Section: Elisa For Quantification Of Ige and Igg Subclass Reactivitiementioning

confidence: 99%

Recombinant Carp Parvalbumin, the Major Cross-Reactive Fish Allergen: A Tool for Diagnosis and Therapy of Fish Allergy

Swoboda¹,

Bugajska-Schretter²,

Verdino

et al. 2002

The Journal of Immunology

218

168

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IgE-mediated reactions to fish allergens represent one of the most frequent causes of food allergy. We have constructed an expression cDNA library from carp (Cyprinus carpio) muscle in phage λgt11 and used serum IgE from a fish allergic patient to isolate 33 cDNA clones that coded for two parvalbumin isoforms (Cyp c 1.01 and Cyp c 1.02) with comparable IgE binding capacities. Both isoforms represented calcium-binding proteins that belonged to the β-lineage of parvalbumins. The Cyp c 1.01 cDNA was overexpressed in Escherichia coli, and rCyp c 1.01 was purified to homogeneity. Circular dichroism analysis and mass spectroscopy showed that rCyp c 1.01 represented a folded protein with mainly α-helical secondary structure and a molecular mass of 11,416 Da, respectively. rCyp c 1.01 reacted with IgE from all fish-allergic patients tested (n = 60), induced specific and dose-dependent basophil histamine release, and contained most of the IgE epitopes (70%) present in natural allergen extracts from cod, tuna, and salmon. Therefore, it may be used to identify patients suffering from IgE-mediated fish allergy. The therapeutic potential of rCyp c 1.01 is indicated by our findings that rabbit Abs raised against rCyp c 1.01 inhibited the binding of IgE (n = 25) in fish-allergic patients to rCyp c 1.01 between 35 and 97% (84% mean inhibition) and that depletion of calcium strongly reduced IgE recognition of rCyp c 1.01. The latter results suggest that it will be possible to develop strategies for immunotherapy for fish allergy that are based on calcium-free hypoallergenic rCyp c 1.01 derivatives.

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Section: Elisa For Quantification Of Ige and Igg Subclass Reactivitiementioning

confidence: 99%

Recombinant Carp Parvalbumin, the Major Cross-Reactive Fish Allergen: A Tool for Diagnosis and Therapy of Fish Allergy

Swoboda¹,

Bugajska-Schretter²,

Verdino

et al. 2002

The Journal of Immunology

218

168

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“…Comparable amounts of extracted proteins (ϳ100 g/cm) were separated by a preparative 14% SDS-PAGE under reducing conditions and blotted onto nitrocellulose (35)(36)(37). Recombinant Phl p 5A and rPhl p 6 were expressed in E. coli BL21 (DE3) and purified as described (25,28). Recombinant Phl p 5B and a recombinant fragment comprising the 136 C-terminal amino acids of Phl p 5B were expressed in E. coli using plasmid pMalc and purified as described (38).…”

Section: Preparation Of Pollen Extracts Recombinant Allergens and Amentioning

confidence: 99%

“…Plasmids pLNOH2 and pLNOK allowed transient coexpression of the Fab-derived variable regions as IgG1 heavy chain and light chain, respectively, after cotransfection into COS-7 cells (40). Supernatants of transfected COS-7 cells were checked for the presence of human IgG1 Abs with specificity for the recombinant Phl p 5A fragment by ELISA as described (28).…”

Section: Expression Of a Recombinant Human Ige Fab And A Complete Fabmentioning

confidence: 99%

“…Phl p 5A represents a major grass pollen allergen that is recognized by Ͼ80% of grass pollen-allergic patients. In sensitized patients it accounts for up to 60% of grass pollen-specific IgE, it induces strong IgE responses in experimental animal systems, and exhibits a surprising potency to activate allergic effector cells and to elicit immediate type skin and nasal reactions (27)(28)(29)(30); V. Niederberger and R. Valenta, unpublished data). By allergen gene fragmentation we identified a Phl p 5A domain that contains the binding site for the human monoclonal IgE Fab.…”

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confidence: 99%

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A Human Monoclonal IgE Antibody Defines a Highly Allergenic Fragment of the Major Timothy Grass Pollen Allergen, Phl p 5: Molecular, Immunological, and Structural Characterization of the Epitope-Containing Domain

Flicker

Vrtala

Schmitz

et al. 2000

The Journal of Immunology

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Almost 90% of grass pollen-allergic patients are sensitized against group 5 grass pollen allergens. We isolated a monoclonal human IgE Fab out of a combinatorial library prepared from lymphocytes of a grass pollen-allergic patient and studied its interaction with group 5 allergens. The IgE Fab cross-reacted with group 5A isoallergens from several grass and corn species. By allergen gene fragmentation we mapped the binding site of the IgE Fab to a 11.2-kDa N-terminal fragment of the major timothy grass pollen allergen Phl p 5A. The IgE Fab-defined Phl p 5A fragment was expressed in Escherichia coli and purified to homogeneity. Circular dichroism analysis revealed that the rPhl p 5A domain, as well as complete rPhl p 5A, assumed a folded conformation consisting predominantly of an α helical secondary structure, and exhibited a remarkable refolding capacity. It reacted with serum IgE from 76% of grass pollen-allergic patients and revealed an extremely high allergenic activity in basophil histamine release as well as skin test experiments. Thus, the rPhl p 5A domain represents an important allergen domain containing several IgE epitopes in a configuration optimal for efficient effector cell activation. We suggest the rPhl p 5A fragment and the corresponding IgE Fab as paradigmatic tools to explore the structural requirements for highly efficient effector cell activation and, perhaps later, for the development of generally applicable allergen-specific therapy strategies.

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“…It is evident from electrophoretic separation that the preparations being obtainable commercially are purified and therefore contain a lower quantity of proteins than raw pollen extract. However, proteins of certain molecular weight (84 and 55,6 kDa) occur in all observed pollens and they present the main allergens apparently 20,21,22 , i.e. allergens with which at least 50 per cent of allergic patients specific react.…”

Section: Discussionmentioning

confidence: 99%

Influence of Bacterial Immunomodulators on the Induction of Specific Igg With Patients During a Peroral Hyposensitization Therapy

Hradilová¹

2001

BIOMED PAP

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Peroral administration of allergen during hyposensitization therapy with allergic patients is in comparison with an administration by injection of a wilder inductor of specific IgG antibodies. The peroral administration of bacterial immunomodulators increases IgG formation significantly. The described experiment examines the influence of bacterial immunomodulator Olimunostim on the level of specific antibodies during peroral hyposensitization therapy with allergic patients.

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Immunologic characterization of purified recombinant timothy grass pollen (Phleum pratense) allergens (Phl p 1, Phl p 2, Phl p 5)1

Cited by 101 publications

References 16 publications

Recombinant Carp Parvalbumin, the Major Cross-Reactive Fish Allergen: A Tool for Diagnosis and Therapy of Fish Allergy

Recombinant Carp Parvalbumin, the Major Cross-Reactive Fish Allergen: A Tool for Diagnosis and Therapy of Fish Allergy

A Human Monoclonal IgE Antibody Defines a Highly Allergenic Fragment of the Major Timothy Grass Pollen Allergen, Phl p 5: Molecular, Immunological, and Structural Characterization of the Epitope-Containing Domain

Influence of Bacterial Immunomodulators on the Induction of Specific Igg With Patients During a Peroral Hyposensitization Therapy

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