Replacement of murine sera by allergen-specific monoclonal IgE antibodies: a new approach for the characterisation of allergen extracts.

Allergen characterisation that is based on patients' sera or monoclonal allergen-specific IgG antibodies has several disadvantages. Current methods such as immunoblotting or allergen-specific EUSA are non-functional assays and cannot be used to evaluate the biological allergenic activity of allergen products. We have established an in vitro assay based on polyclonal murine IgE and allergen-dependent mediator release of rat basophilic leukaemia (RBL) cells as an alternative to passive cutaneous anaphylaxis (PCA), an animal model of IgE-mediated allergic reactions. The RBL assay is a functional in vitro test which enables the measurement of biological potency of allergen extracts to be made. Up to now, allergen-specific IgE-containing murine sera were used for sensitisation of RBL cells. Sensitisation with allergen-specific IgE monoclonal antibodies (IgE mAbs) would reduce the number of animals necessary for the production of allergen-specific IgE. In addition, IgE mAbs are better defined and will offer more exact determination of allergens. Since allergen-specific IgE mAbs were not available, the aim of this study was to produce such antibodies. As a new strategy to select IgE-producing hybridomas the RBL mediator release assay was used: the cells were incubated with hybridoma supernatant and stimulated with allergen and crosslinking allergen-specific polyclonal IgG antibodies. By this technology IgE mAbs specific for the birch pollen allergens Bet v 1 and Bet v 6 were produced. In conclusion, this novel strategy enables the production of panels of allergen-specific IgE mAbs by immunisation of a limited number of mice to be made. These IgE mAbs in combination with the RBL mediator release assay may serve as new tools for the evaluation of diagnostic and therapeutic allergen extracts.

Phenylcoumaran benzylic ether and isoflavonoid reductases are a new class of cross-reactive allergens in birch pollen, fruits and vegetables.

We investigated the biochemical function of the birch pollen allergen Bet v 6 and its role in the IgE-cross-reactivity between birch pollen and plant foods, and characterized Pyr c 5, a Bet v 6-related food allergen, from pear; the proteins were expressed as His-Tag fusion proteins in Eschershia coli and purified by Ni-chelate affinity chromatography under native conditions. Nonfusion proteins were obtained by factor Xa protease treatment. The highest degree of amino-acid sequence identity of Pyr c 5 and Bet v 6 was found with a plant protein related to a defense mechanism, which we have named phenylcoumaran benzylic ether reductase (PCBER) based on its ability to catalyze the NADPH-dependent reduction of 8-5' linked lignans such as dehydrodiconiferyl alcohol to give isodihydrodehydrodiconiferyl alcohol. Enzymatic assays with recombinant Pyr c 5 and Bet v 6 showed PCBER catalytic activity for both recombinant allergens. Both Pyr c 5 and Bet v 6 allergens had similar IgE binding characteristics in immunoblotting and enzyme allergosorbent tests (EAST), and bound IgE from 10 sera of birch-pollen-allergic patients including six pear-allergic subjects. EAST inhibition experiments with Pyr c 5 as the solid phase antigen suggested that homologous allergens may be present in many vegetable foods such as apple, peach, orange, lychee fruit, strawberry, persimmon, zucchini (courgette), and carrot. In extracts of pear, apple, orange, and persimmon, the presence of proteins of approximately 30-35 kDa containing Bet v 6 cross-reactive epitopes was demonstrated with two Bet v 6-specific monoclonal antibodies. Recombinant Pyr c 5 triggered a strong, dose-dependent mediator release from basophils of a pear-allergic subject, suggesting that Pyr c 5 has the potential to elicit type I allergic reactions.

Pyr c 1, the major allergen from pear (Pyrus communis), is a new member of the Bet v 1 allergen family.

Pear is known as an allergenic food involved in the 'oral allergy syndrome' which affects a high percentage of patients allergic to birch pollen. The aim of this study was to clone the major allergen of this fruit, to express it as bacterial recombinant protein and to study its allergenic properties in relation to homologous proteins and natural allergen extracts. The coding region of the cDNA was obtained by a PCR strategy, cloned, and the allergen was expressed as His-Tag fusion protein. The fusion peptide was removed by treatment with cyanogen bromide. Purified non-fusion protein was subjected to allergenicity testing by the enzyme allergosorbent test (EAST), Western blotting, competitive inhibition assays, and basophil histamine release. The deduced protein sequence shared a high degree of identity with other major allergens from fruits, nuts, vegetables, and pollen, and with a family of PR-10 pathogenesis related proteins. The recombinant (r) protein was recognised by specific IgE from sera of all pear-allergic patients (n = 16) investigated in this study. Hence, the allergen was classified as a major allergen and named Pyr c 1. The IgE binding characteristics of rPyr c 1 appeared to be similar to the natural pear protein, as was demonstrated by EAST-inhibition and Western blot-inhibition experiments. Moreover, the biological activity of rPyr c 1 was equal to that of pear extract, as indicated by basophil histamine release in two patients allergic to pears. The related major allergens Bet v 1 from birch pollen and Mal d 1 from apple inhibited to a high degree the binding of IgE to Pyr c 1, whereas Api g 1 from celery, also belonging to this family, had little inhibitory effects, indicating epitope differences between Bet v 1-related food allergens. Unlimited amounts of pure rPyr c 1 are now available for studies on the structure and epitopes of pollen-related food allergens. Moreover, the allergen may serve as stable and standardised diagnostic material.

Allergy caused by ingestion of zucchini (Cucurbita pepo): characterization of allergens and cross-reactivity to pollen and other foods.

BACKGROUND: Allergy to zucchini (Cucurbita pepo), a member of the Cucurbitaceae family, has not previously been reported. We examined 4 patients complaining of allergic symptoms, such as oral allergy syndrome, nausea, diarrhea, or pruritus, after the intake of zucchini. OBJECTIVE: After the confirmation of food allergy, we wanted to characterize zucchini allergens and examine possible cross-reactions to pollen and food. METHODS: The patients underwent skin prick and prick-to-prick-testing with different allergens, including zucchini, latex, and birch, ragweed, and grass pollen. Moreover a double-blind, placebo-controlled, food challenge was performed to confirm food allergy. Total and specific serum IgE levels were determined by using CAP-FEIA and the enzyme allergosorbent test method (EAST), respectively. Proteins from zucchini reacting with patient IgE were detected by means of immunoblotting. To characterize cross-reacting IgE antibodies, immunoblot- and EAST-inhibition assays were carried out. RESULTS: All patients in this study had positive reactions to zucchini both in prick-to-prick tests and double-blind, placebo-controlled, food challenges. Specific serum IgE levels to zucchini were found in all cases. In blot- and EAST-inhibition assays IgE from two patients revealed binding to zucchini profilin at about 15 kd. Furthermore, in two cases, including one of the profilin-positive patients, IgE directed against cross-reacting carbohydrate determinants was detected. For one patient, no cross-reacting IgE could be found, but IgE from this patient reacted strongly with a zucchini protein at 17 kd. CONCLUSIONS: We report the first 4 cases of food allergy to zucchini. Zucchini allergens can cause systemic reactions and are at least partially heat stable. We suggest that allergy to zucchini can occur as a result of primary sensitization to zucchini, as well as to cross-reactions to the panallergen profilin and cross-reacting carbohydrate determinants.

Molecular cloning and characterization of a birch pollen minor allergen, Bet v 5, belonging to a family of isoflavone reductase-related proteins.

BACKGROUND: Birch pollen is a major cause of pollinosis and is responsible for cross-reactive oral allergies to fruits, nuts, and vegetables. The major allergen, Bet v 1, has been extensively characterized, and 3 minor allergens, Bet v 2, Bet v 3, and Bet v 4, have been cloned and sequenced. Recently, another birch pollen protein with an apparent mass of 35 kd was described as a new IgE-binding protein in birch pollen with cross-reacting homologues in plant foods. OBJECTIVE: The aim of this study was to determine the primary structure of the 35-kd birch pollen allergen and to investigate its immunologic properties. METHODS: On the basis of a known complementary DNA fragment, a PCR strategy was applied to obtain the full-length nucleotide sequence of the coding region. The protein was expressed as His-Tag fusion protein in Escherichia coli and purified by Ni-chelate affinity chromatography. Nonfusion protein was obtained by cyanogen bromide treatment of the fusion protein. IgE-binding characteristics and potential allergenicity were investigated by immunoblot, immunoblot inhibition analysis, rat basophil leukemia-cell mediator release assay, and basophil histamine release and compared with those of natural (n) Bet v 5, recombinant (r)Bet v 1, and rBet v 2. RESULTS: Recombinant Bet v 5 has a mass of 33 kd, an isoelectric point of 9.0, and sequence identity of 60% to 80% to isoflavone reductase homologue proteins from various plants. On immunoblots the recombinant Bet v 5 bound IgE from 9 (32%) of 28 sera from patients allergic to birch pollen with a CAP class of at least 3; Bet v 1 was detected by 89% of these patients. IgE immunoblot and inhibition experiments showed that nBet v 5 and rBet v 5 shared identical epitopes. A rabbit antiserum raised against pea isoflavone reductase and patients' IgE reacted with Bet v 5 and proteins of similar size in several vegetable foods, including exotic fruits. A similar reaction pattern was obtained with 2 Bet v 5-specific mAbs. Furthermore, Bet v 5 triggered a dose-dependent mediator release from rat basophil leukemia 2H3 cells passively sensitized with murine anti-birch pollen IgE and from basophils of a Bet v 5-reactive subject with birch pollen allergy. In contrast, no mediator release could be induced from basophils of a subject who was monosensitized to Bet v 1. CONCLUSIONS: This 33-kd protein, designated as Bet v 5, is a new minor allergen in birch pollen and may be responsible for pollen-related oral allergy to specific foods in a minority of patients with birch pollen allergy. Amino acid sequence comparison and immunoreactivity to anti-isoflavone reductase serum indicate that Bet v 5 is related to isoflavone reductase, a protein family that is involved in plant defense reactions.

Cross-reactivity and epitope analysis of Pru a 1, the major cherry allergen.

A high percentage of birch pollen allergic patients experiences food hypersensivity after ingestion of fresh fruits and vegetables. The cross-reactivity of the major allergens of sweet cherry (Pru a 1), apple (Mal d 1), pear (Pyr c 1), celery tuber (Api g 1) and carrot (Dau c 1) is due to structural similarities which are reflected by high amino acid sequence identities with Bet v 1a, the major birch pollen allergen. Apart from a strong cross-reactivity to Bet v 1a, IgE inhibition experiments with Mal d 1, Pru a 1 and Api g 1 demonstrated the presence of common and different epitopes among the tested food allergens. Secondary structure prediction of all investigated allergens indicated the presence of almost identical structural elements. In particular, the 'P-loop' region is a common domain of the pollen related food allergens and of pathogenesis related proteins. To identify the IgE binding epitopes, five overlapping recombinant Pru a 1 fragments representing the entire amino acid sequence with lengths of approximately 60-120 residues were investigated. Weak IgE binding capacity was measured exclusively with Pru a IF4 (1-120) by immunoblotting, whereas none of the fragments showed allergenicity in the rat basophil leukaemia cell mediator release assay. Site-directed mutagenesis experiments with Pru a 1 revealed that amino acid S112 is critical for IgE binding of almost all patients sera tested. This reduced IgE binding was also observed with a single point mutant of Bet v 1a (S112P) and thus indicated serine 112 as an essential residue for preserving the structure of a cross-reactive IgE epitope. Moreover, two Pru a 1 mutants with an altered 'P-loop' region, showed a lowered IgE binding capacity for IgE from a subgroup of allergic patients. The investigation of essential features for preserving cross-reactive IgE-epitopes provides the structural basis for understanding the clinically observed cross-allergenicity between pollen and fruits. Moreover, non-anaphylactic allergen fragments or variants derived from the IgE-inducing pollen allergens may serve as useful tools for a new strategy of specific immunotherapy.