Helmut Rumpold

Common epitopes of birch pollen and apples--studies by western and northern blot.

Eighty-three sera from patients with birch-pollen allergy were investigated for IgE antibodies against apple allergens by means of immunoblotting. In immunoblots, 81 patients (97.6%) exhibited IgE directed against the major allergen of birch, Bet v I (17 kd), and these patients also demonstrated IgE binding to apple allergens in the molecular weight range 17 to 18 kd. Inhibition studies by preincubation of sera with birch-pollen extract led to complete blocking of IgE binding to this 17 to 18 kd protein, whereas preincubation with apple extract could not diminish IgE binding to Bet V I. Furthermore, a 17 kd protein in apple extract could be detected by immunoblotting with a Bet v I-specific monoclonal antibody. Northern blotting with a Bet v I cDNA clone as a probe revealed cross-hybridization of birch and apple allergen coding nucleic acids under conditions of high stringency, suggesting significant homology of the nucleic acid level. Our results support the concept that antigens in birch pollen and apples share allergenic epitopes leading to IgE cross-reactivities that may cause clinical manifestations when a special threshold level of specific IgE antibodies is reached.

Recombinant birch pollen allergens (rBet v 1 and rBet v 2) contain most of the IgE epitopes present in birch, alder, hornbeam, hazel, and oak pollen: A quantitative IgE inhibition study with sera from different populations ☆ ☆☆ ★ ★★

BACKGROUNDnPollen from trees of the order Fagales are important allergen sources in most parts of the world. Clinical, immunochemical, and molecular biology studies indicate that they contain cross-reactive allergens. The major birch pollen allergen, Bet v 1, and birch profilin, Bet v 2, a highly cross-reactive allergen, have been cloned and expressed in Escherichia coli.nnnOBJECTIVEnThe purpose of this study was to demonstrate the presence of allergens in Fagales pollens that share IgE epitopes with recombinant Bet v 1 and Bet v 2 and to determine the percentage of birch, alder, hornbeam, hazel, and oak pollen-specific IgE that can be preabsorbed with rBet v 1 and rBet v 2 from 102 sera of different populations of subjects allergic to Fagales tree pollen.nnnMETHODSnThe presence of rBet v 1- and rBet v 2-homologous allergens in tree pollen extracts was investigated by IgE immunoblot inhibition experiments, and the percentage of tree (birch, alder, hornbeam, hazel, and oak) pollen-specific IgE that was bound by a mixture of rBet v 1 and rBet v 2 was determined by RAST-based quantitative IgE inhibition experiments. The clinical significance of IgE antibody cross-reactivity was studied by skin prick testing with rBet v 1, rBet v 2, and Fagales pollen extracts.nnnRESULTSnNatural birch, alder, hornbeam, hazel, and oak pollen contain allergens that share IgE epitopes with rBet v 1 and rBet v 2. A combination of rBet v 1 and rBet v 2 accounted for 82% of tree pollen-specific IgE on average. Most of the tree pollen-specific IgE was directed against rBet v 1.nnnCONCLUSIONnrBet v 1 and rBet v 2 contain most of the Fagales pollen-specific IgE epitopes and may therefore substitute natural tree pollen extracts not only for diagnosis but also for patient-tailored immunotherapy of tree pollen allergy.

Parvalbumin, a cross-reactive fish allergen, contains IgE-binding epitopes sensitive to periodate treatment and Ca2+ depletion☆☆☆★★★

BACKGROUNDnType I allergy to fish is a severe health problem in countries in which a large percentage of the population derive income from fishing.nnnOBJECTIVEnThe aim of the study was to characterize cross-reactive IgE-binding components in six different fish species (cod, tuna, salmon, perch, carp, and eel). The effect of reducing extraction conditions, periodate treatment, and depletion of Ca2+ on binding of IgE to the allergens was investigated.nnnMETHODSnExtracts were prepared under nonreducing and reducing conditions. IgE-binding components were characterized by IgE immunoblotting, and cross-reactive epitopes were studied by IgE-immunoblot inhibition experiments. To reveal calcium-sensitive or carbohydrate-containing epitopes, nitrocellulose-blotted extracts were exposed to ethylene glycol bis(beta-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA) and periodate.nnnRESULTSnSera from all patients allergic to fish (n = 30) displayed IgE reactivity to parvalbumin, a 12 kd protein present in fish extracts from six different species. Reducing extraction conditions had no effect on IgE binding to parvalbumins, whereas periodate treatment and depletion of protein-bound calcium led to a substantial reduction of IgE binding. Parvalbumins from six different species contained cross-reactive IgE epitopes.nnnCONCLUSIONnParvalbumin represents a cross-reactive fish allergen. It contains IgE epitopes that are sensitive to periodate treatment and Ca2+-depletion.

IgE antibodies to recombinant pollen allergens (Phl p 1, Phl p 2, Phl p 5, and Bet v 2) account for a high percentage of grass pollen-specific IgE.

BACKGROUNDnPollen from different grass species are some of the most potent elicitors of Type I allergy worldwide. The characterization of antigenic structures and IgE epitopes common to different grass species is relevant to define reagents for diagnosis and specific therapy of grass pollen allergy.nnnOBJECTIVEnThe purpose of this study was to estimate the percentage of IgE directed to common, cross-reactive, or both types of epitopes shared by recombinant pollen allergens (Phl p 1, Phl p 2, Phl p 5, and Bet v 2) and natural pollen extracts from nine different monocots (Anthoxanthum odoratum, Avena sativa, Cynodon dactylon, Lolium perenne, Phragmites australis, Poa pratensis, Secale cereale, Triticum sativum, Zea mays) by using sera from different populations.nnnMETHODSnNatural pollen extracts from nine different monocot species were characterized regarding their allergen contents by using specific antibodies and by IgE immunoblot inhibition with recombinant allergens. The percentage of grass pollen-specific IgE that was preabsorbed with a combination of recombinant timothy grass pollen allergens (Phl p 1, Phl p 2, and Phl p 5) and recombinant birch profilin (Bet v 2) was determined by ELISA in sera from 193 European, American, and Asian subjects.nnnRESULTSnIgE to recombinant pollen allergens accounted for a mean 59% of grass pollen-specific IgE. A lower inhibition of IgE binding to certain natural extracts (C. dactylon and Z. mays) could be attributed to the absence of immunologically detectable group 5 and group 2 allergens in these species.nnnCONCLUSIONnWe define four recombinant pollen allergens that account for a substantial proportion of grass pollen-specific IgE. The recombinant pollen allergens characterized may represent candidates not only for diagnosis but also for patient-tailored immunotherapy of grass pollen allergy.

Recombinant allergens for immunoblot diagnosis of tree-pollen allergy

Diagnosis of type I allergy essentially depends on the availability of defined allergens, which can be provided by recombinant deoxyribonucleic acid (DNA) technology. We have previously isolated the c(complementary)DNAs encoding the major birch-pollen allergen, Bet v I, and another allergen with a molecular weight of 14 kd that was identified as birch profilin and designated Bet v II. These cDNAs were isolated from a lambda gt11 expression library by screening with the serum IgE from allergic patients. To obtain expression in Escherichia coli of recombinant allergens without additional fused polypeptides, both cDNAs were inserted into the plasmid pKK223-3. E. coli cells expressing Bet v I and birch profilin (Bet v II) were used for the preparation of recombinant proteins. These proteins were tested for their IgE-binding properties on immunoblots with sera from 100 different birch pollen-allergic patients. All patients sera, which reacted with the natural allergens, Bet v I and Bet v II, demonstrated an identical IgE-binding pattern to recombinant birch-pollen allergens. Recombinant allergens may therefore be useful for the setup of diagnostic tests that allow the discrimination of different IgE-binding patterns as well as for patient-tailored immunotherapy.

Natural latex, grass pollen, and weed pollen share IgE epitopes

BACKGROUNDnBecause of the frequent use of natural latex products, IgE-mediated reactions to latex proteins represent an important health threat in industrialized countries. Although several latex allergens have been characterized and IgE cross-reactivities with allergens present in plant-derived food have been described, limited information is available regarding the presence of common IgE-binding components in latex and plant pollen.nnnMETHODSnBy using serum IgE from 56 individuals with latex allergy, the IgE-binding components in ammoniated latex milk and latex glove extracts were characterized by immunoblotting. The presence of cross-reactive IgE-binding components in the different latex extracts, extracts from mugwort, ragweed, timothy grass pollen, and recombinant birch pollen allergens (Bet v 1 and Bet v 2 [birch profilin]) was studied by immunoblot inhibitions and quantitative competition experiments. The involvement of carbohydrates in the constitution of cross-reactive IgE epitopes was studied by periodate treatment of extracts.nnnRESULTSnAlthough sera from certain individuals with latex allergy showed IgE reactivity with protein bands of different molecular weights in Western-blotted latex milk and glove extracts, both extracts contained common IgE epitopes. Although preincubation with recombinant Bet v 1 and Bet v 2 did not significantly inhibit IgE binding to latex proteins, weed and, in particular, timothy grass pollen extract strongly inhibited IgE binding to latex allergens. The cross-reactive IgE epitopes were sensitive to periodate treatment.nnnCONCLUSIONSnMugwort, ragweed, and timothy grass pollen share IgE epitopes with glycoprotein latex allergens. The presence of common epitopes might in part explain clinical symptoms in patients allergic to pollen on contact with latex.

Calcium-dependent immunoglobulin E recognition of the apo- and calcium-bound form of a cross-reactive two EF-hand timothy grass pollen allergen, Phl p 7

Type I allergy, an immunodisorder that affects almost 20% of the population worldwide, is based on the immunoglobulin E (IgE) recognition of per se innocuous antigens (allergens). Pollen from wind‐pollinated plants belong to the most potent allergen sources. We report the isolation of a cDNA coding for a 8.6 kDa two EF‐hand calcium binding allergen, Phl p 7, from a timothy grass (Phleum pratense) pollen expression cDNA library, using serum IgE from a grass pollen allergic patient. Sequence analysis identified Phl p 7 as a member of a recently discovered subfamily of pollen‐specific calcium binding proteins. Recombinant Phl p 7 was expressed in Escherichia coli and purified to homogeneity as determined by mass spectroscopy. Approximately 10% of pollen allergic patients displayed IgE reactivity to rPhl p 7 and Phl p 7‐homologous allergens present in pollens of monocotyledonic and dicotyledonic plants. Circular dichroism analysis of the calcium‐bound and apo‐rPhl p 7 indicated that differences in IgE recognition may be due to calcium‐induced changes in the protein conformation. The fact that patients mount IgE antibodies against different protein conformations is interpreted as a footprint of a preferential sensitization against either form. The biological activity of rPhl p 7 was demonstrated by its ability to induce basophil histamine release and immediate type skin reactions in sensitized individuals. In conclusion, IgE binding to Phl p 7 represents an example for the conformation‐dependent IgE recognition of an allergen. Recombinant Phl p 7 may be used for diagnosis and perhaps treatment of a group of patients who suffer from allergy to pollens of many unrelated plant species.—Niederberger, V., Hayek, B., Vrtala, S., Laffer, S., Twardosz, A., Vangelista, L., Sperr, W. R., Valent, P., Rumpold, H., Kraft, D., Ehrenberger, K., Valenta, R., Spitzauer, S. Calcium‐dependent immunoglobulin E recognition of the apo‐ and calcium‐bound form of a cross‐reactive two EF‐hand timothy grass pollen allergen, Phl p 7. FASEB J. 13, 843–856 (1999)

Molecular characterization of dog albumin as a cross-reactive allergen

Indoor allergens comprise a group of allergenic proteins that are commonly derived from house dust mite and cat and dog dander. In addition to the two major dog allergens (molecular weights: 19 and 23 kd), dog albumin represents an important allergen for up to 35% of patients who are allergic to dogs. In IgE immunoblot inhibition studies and histamine release tests it has been demonstrated that patients who react to dog albumin exhibit IgE reactivity with purified albumins from cat, mouse, chicken, and rat. The proportion of dog-specific IgE directed against dog albumin was determined for patients allergic to dog albumin, and it ranges from 70% to 90%. By IgE immunoscreening of a lambda gt11 expression library from a dog salivary gland, we identified a number of reactive complementary DNA clones. All patients with IgE reactivity against natural dog albumin displayed IgE reactivity to the beta-galactosidase fusion protein encoded by clone 54c, which was therefore assumed to contain major IgE epitopes of dog albumin. The deduced amino acid sequence of clone 54c was compared with the Swiss-Prot library, and significant sequence homologies were found with albumins from different species (human: 82.6%, pig: 81.8%, cattle: 77.3%, sheep: 78.8%, mouse: 75.8%, and rat: 76.2%). Several other IgE-positive clones hybridized with oligonucleotides that were prepared according to this sequence. Partial complementary DNA coding for dog albumin fragments may be considered a useful tool for further characterization of major IgE epitopes of dog albumin.

IgE cross-reactivities against albumins in patients allergic to animals

BACKGROUNDnType I allergic symptoms and severe asthma in particular are frequently caused by animal hair/dander proteins, among which albumins are possible cross-sensitizing allergenic components.nnnMETHODSnThe significance and degree of IgE-cross-reactivities against various albumins were studied in a representative number (n = 200) of patients allergic to animals with hair/dander extracts, purified albumins from different animals, and a recombinant dog albumin fragment expressed in lysogenic Escherichia coli Y1089 and purified as a beta-galactosidase fusion protein.nnnRESULTSnDespite a high degree of sequence homology among different albumins, a remarkable variability of IgE cross-reactivities was observed, indicating that some patients were sensitized preferentially against certain albumins. Most of the patients allergic to albumins, however, reacted to dog, cat, and horse albumin, which also bound a high percentage of albumin-specific IgE.nnnCONCLUSIONnThe purified recombinant dog albumin fragment, representing 265 amino acids of the mature protein, bound IgE from all 15 patients allergic to albumin tested suggesting its potential usefulness for diagnosis and perhaps therapy.

Monoclonal Antibodies against Birch Pollen Allergens: Characterization by Immunoblotting and Use for Single-Step Affinity Purification of the Major Allergen Bet v I

Two monoclonal antibodies against birch pollen proteins were produced by immunizing BALB/c mice with birch pollen extract. In immunoblotting experiments, antibody BIP 1 reacted with a 17-kilodalton (kD) protein considered to represent the major birch pollen allergen Bet v I. A second monoclonal antibody, BIP 3, reacted with 3 different birch pollen proteins of molecular weights 32, 36 and 68 kD of which the 36- and 68-kD proteins corresponded to minor allergens of birch pollen. Two-dimensional electrophoresis/immunoblotting experiments revealed that BIP 1 reacted with all Bet v I isoallergens, also identified by human IgE antibodies. Using BIP 1 coupled to Sepharose 4B as reverse immunosorbent, Bet v I was obtained in a single-step procedure and characterized as single band by SDS-PAGE.