W.-D. Müller

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

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.

Detection of crossreactive determinants in grass pollen extracts using monoclonal antibodies against group IV and group V allergens.

Monoclonal antibodies (MoAb) to defined allergens of P. pratense were raised. Five of them were selected for detailed studies by means of immunoblotting after SDS–PAGE and IEF of extracts from P. pratense and L. perenne. Three antibodies (1D11, 3B2, 2D1) recognize structures with mol. wt of 29 and 34 kD and pI of 4.4‐7.7, corresponding to group V allergens. Two other MoAbs (2D8, 3C4) are directed against strong basic structures with a mol. wt of 50 kD and pI of 7.7‐9.9 according to group IV allergens. The specificity of antibodies was supported by direct ELISA with purified group V and IV allergens. The isolated allergens were characterized before by SDS‐PAGE and CIE and that allergenicity was detected with sera of patients with allergic rhinitis. Using our selected MoAbs crossreactive epitopes on group V and IV allergens have been excluded. Our antibodies have been used to detect crossreactivity in 14 grass pollen extracts. The evaluation of the pollen extracts has been performed by enzyme immunoassay (EIA) inhibition. One MoAb (3C4) is able to recognize group IV allergens in all grass species analysed whereas the MoAb 2D8 seems to identify group IV structures in selected grasses only. Binding to conserved structures of group V has been proved for MoAb 1D11. Other group V specific MoAbs (2D1, 3B2) identify similar, however incomplete, spectra. These results have been confirmed also by the dot immunobinding assay.

Analysis of Human T Cell Clones Reactive with Group V Grass Pollen Allergens

Twenty-seven T cell clones (TCC) reactive with group V allergens of Phleum pratense (Phlp V) were established from the peripheral blood of 3 patients allergic to grass pollen. Twenty-four TCC showed the helper cell phenotype CD3+, CD4+, CD8-; the remaining clones were CD3+CD4-CD8+. T cell recognition of Phlp V was exclusively HLA-DR restricted. Many of the Phlp V reactive TCC (19 of 27; 70%) were stimulated additionally by other group V allergens isolated from Lolium perenne, Poa pratensis, and Dactylis glomerata. These data indicate the existence of cross-reacting T cell epitopes among group V allergens of different grasses. The Phlp V triggered cytokine production demonstrated in 13 out of 24 CD4+ TCC a Th2-like pattern (high interleukin 4/gamma-interferon ratios) suggesting group V allergens as important targets of grass pollen specific IgE.

In situ localization of a high molecular weight cross-reactive allergen in pollen andplant-derived food by immunogoldelectron microscopy

BACKGROUND A high molecular weight (60 kd) allergen has been recently identified as a cross-reactive moiety in pollen and plant-derived food. While the cross-reactive allergen has been characterized by immunochemical techniques, little is known concerning its biologic properties. OBJECTIVE In this investigation we studied the in situ localization of the 60 kd cross-reactive allergen in tree, grass, and weed pollen, as well as in plant-derived food (apple and celery). METHODS A monoclonal antibody (3A4) that was raised against the major mugwort pollen allergen, Art v 1, was used to demonstrate the presence of related allergens in nitrocellulose-blotted pollen and plant-food extracts. The tissue localization of the cross-reactive allergen was investigated by immunogold electron microscopy. RESULTS Monoclonal antibody 3A4 recognized IgE epitopes of the 60 kd mugwort allergen and cross-reacted with moieties of comparable molecular weights in birch and timothy grass pollen, as well as in apple and celery extracts. In pollen and plant-derived food the allergen could be localized intracellularly in ribosome-rich areas in the mitochondria and the nucleus. No labeling was observed in the pollen or cell walls or in organelles that are engaged in storage (e.g., starch granules and lipid particles). CONCLUSION Tree, grass, and weed pollen, as well as plant-derived foods, contain a high molecular weight Art v 1-cross-reactive allergen that maps to similar cell compartments.

A two‐site monoclonal antibody ELISA for the quantification of group V allergens in grass extracts

A two‐site solid‐phase enzymimmunoassay was used for the quantification of group V allergen in grass pollen extracts in mass units. The assay is based on the monoclonal antibodies (MoAbs), 1D11 and 3B2 which recognize different epitopes on the standard Phl p V. The MoAb‐ELISA is very sensitive (15 100ng ml Phl p V)and highly specific for group V allergens. Six pollen extracts of different grasses demonstrated parallel binding curves. The group V content ranged between 73 and 673 μg/ml in the extracts. The International Standard of Phleum pratense (IS 82 520) contains 400 μg/nil Phl p V. A good correlation was observed between the group V content and RAST inhibition, with the exception of Poa pratensis.

Group 5 Allergens of Timothy Grass (Phl p 5) Bear Cross-Reacting T Cell Epitopes with Group 1 Allergens of Rye Grass (Lol p 1)

Selected human T cell clones reactive with group 5 allergens of timothy grass (Phl p 5) were cross-stimulated in specific proliferation assays with group 1 allergens of rye grass (Lol p 1). Such interspecies cross-reactivities result obviously from structural motifs presented on defined Phl p 5 fragments as shown with recombinant Phl p 5 products.

Monoclonal antibody immunoassay for quantitative analysis of group V allergens in grass pollen extracts

A two‐site monoclonal antibody (MoAb) ELISA has been developed for the quantification of the Phleun pratense major allergen, Phl p V. The assay is based on two MoAbs which recognize different non‐overlapping epitopes on the Phi p V molecule; one antibody (1D11) was immobilized on the solid phase and the other (3B2) was biotinylated. An affinity‐purified Phi p V preparation (purity of 95%) was used as standard. The assay has a sensitivity of 10 ng/ml of allergen and is suitable for the detection of group V allergen in aqueous grass extracts. The specificity of the assay was investigated with 14 grass pollen and five non‐grass pollen extracts. Different levels of group V allergen were delected in extracts of grasses, but not in non‐grasses. The assay gives a good correspondence with allergenic activity of extracts as determined by ELISA inhibition using serum pool of allergic patients. The results indicate that the two‐site MoAb ELISA could be very useful in the standardization of allergenic extracts from grass pollen.

Application of reversed-phase high-performance liquid chromatography in the purification of major allergens from grass pollen

Group 1 and 5 allergens of different grasses possess similar physicochemical parameters (molecular weight, pI) and therefore separation with conventional chromatographic methods (gel filtration, ion exchange chromatography) is difficult or impossible to achieve. In this paper we describe the isolation of biologically active group 1 and 5 allergens from extracts of Lolium perenne and Phleum pratense by means of reverse phase chromatography on HPLC. The chromatograms showed very different retention times for group 1 (Rt 19.1-20.5 min) and group 5 (Rt 24.3-26.3 min) containing fractions. In addition, this technique is suitable for the separation of group 5 allergens into 5a and 5b subgroups and for the estimation of the amounts of allergen (groups 1 and 5) in the different extracts.

Characterization of Murine Monoclonal Antibodies to Phospholipase A2 and Mellitin from Bee Venom

9 murine monoclonal antibodies (MoAbs), 7 to phospholipase A2 (Pla2; Api m I) and two to mellitin (Api m III) have been produced. Specificities were demonstrated using immunoblotting and enzyme immunoassays (ELISA). Antibodies specific for Pla2 were characterized in detail. Epitope mapping of Pla2-specific MoAbs identified three independent binding regions. 5 selected MoAbs bound to one of these sites compete with specific human IgE. Two of MoAbs characterized are suitable tools to quantify Pla2 in a two-site binding assay.

Variants of allergen Phlp 5b and reduction of anaphylactogenic potential

Abstract One new approach to improved immunotherapy for type 1 allergy might be the use of modified allergens with reduced IgE reactivity but retained T cell reactivity. By site- directed mutagenesis outside the dominant T cell epitopes, we generated deletion mutants of the grass pollen allergen Phl p 5b. Some of these variants revealed significantly reduced IgE reactivity and histamine releasing capacity compared to the wild-type allergen. Furthermore, in vivo skin prick tests showed that the variants had up to 100 fold lower potency to induce cutaneous reactions than the wild-type allergen. On the other hand, T cell clones and T cell lines from grass pollen-allergic patients showed comparable proliferation after stimulation with these variants and wild-type allergen. Thus, variants of the allergen Phl p 5b with reduced anaphylactogenic potential but retained T cell reactivity could be valuable tools for improved allergen-specific immunotherapy.