Rationale for a five-grass pollen vaccine

Grass pollen allergic patients are naturally exposed and sensitized to multiple pollens from various Pooideae species. The question arises as to whether such patients should be desensitized with extracts based on a single pollen or a pollen mixture. Neither conventional diagnosis based on IgE reactivity nor pollen counts enable the identification of which grass species are involved in patient sensitization. Significant cross-immunogenicity is observed between allergens from Pooideae pollens due to their conserved amino acid sequences (e.g. >90% for group 1, 55–80% for group 5, 30–60% for other allergens, including minor allergens). Nevertheless, pollen allergens also contain species-specific T or B cell epitopes, and there is evidence that at least 50% of allergic patients are sensitized to such distinct epitopes. In addition, substantial quantitative differences exist in the allergen (e.g. group 1, 2/3, 4, 5, 6, 10, 13) content of pollens obtained from distinct grass species. In this context, we recommend a vaccine consisting of a combination of pollens from five common and well characterized Pooideae including Anthoxanthum odoratum , Dactylis glomerata, Lolium perenne, Phleum pratense and Poa pratensis for desensitization purposes. This 5-grass mixture is recommended because, (i) it has been validated, in terms of safety and efficacy, by established clinical practice; (ii) it better reflects natural exposure and sensitization conditions at the molecular level than a single pollen; and (iii) it provides a consistent and well-balanced composition of critical allergens, while extending the repertoire of T and B cell epitopes.     

Cross-reactivity between pollen allergens from common Pooideae grasses and cultivated cereals

In Europe, one-third of desensitization treatments for grass pollen allergy are supplemented with a mixture of cereal pollen extracts ( Triticum sativum , Avena sativa, Zea mais and Hordeum vulgare ). We have re-evaluated this practice in light of the recent availability of a desensitization tablet prepared from a mixture of pollen from five common grass species ( Anthoxanthum odoratum , Dactylis glomerata, Lolium perenne, Poa pratensis and Phleum pratense) from the Pooideae subfamily. Common grasses and cultivated cereals are very closely related phylogenetically, and as a consequence, their pollen exhibit a similar molecular composition. The latter is confirmed by ELISA inhibition and immunobloting experiments. Thus, immunotherapy based on a mixture of five grass pollen allows to treat patients sensitized with pollen allergens from cultivated cereals, without a need for additional cereal pollen extracts.     

Molecular variability of group 1 and 5 grass pollen allergens between Pooideae species: implications for immunotherapy

Background Differences between major allergens from distinct grass species remain to be investigated, both in terms of structure and antigenicity. Methods Group 1 and 5 allergens purified from five common Pooideae species were analysed by mass spectrometry (MS). Major histocompatibility complex (MHC) class II‐restricted T cell epitopes were identified using predictive algorithms and human leucocyte antigen (HLA)‐binding assays. CD4⁺ T cell reactivity and IgE binding were assessed based on the induction of CD154 expression in peripheral blood mononuclear cells and using competitive ELISA assays, respectively. Results MS analysis of group 5 pollen allergens reveals considerable intra‐ and inter‐species variability in amino acid sequence, with 30–50 predominant isoforms found for each species. Differences in the amino acid sequence as well as N‐ and O‐glycosylation contribute to the variability of group 1 allergens, yielding 5–10 main isoforms, depending on the species. Out of 14 MHC class II‐restricted T cell epitopes identified within group 1, only one is conserved among the five grass species. Significant differences in binding affinities for HLA‐DR molecules result in variable CD4⁺ T cell recognition of group 1 and 5 allergens purified from the various species. Up to 38% and 85% of patients exhibit seric IgE responses to species‐restricted (or semi‐restricted) epitopes associated with group 1 or 5 allergens, respectively. Conclusion Major pollen allergens from distinct grass species bear both shared and species‐restricted T and B cell immune epitopes. When compared with single extracts, a five grass pollen extract is thus more suitable for specific immunotherapy, as it contains a broader repertoire of the IgE epitopes to which patients are sensitized. Cite this as: H. Chabre, B. Gouyon, A. Huet, V. Baron‐Bodo, E. Nony, M. Hrabina, F. Fenaille, A. Lautrette, M. Bonvalet, B. Maillère, V. Bordas‐Le Floch, L. Van Overtvelt, K. Jain, E. Ezan, T. Batard and P. Moingeon, Clinical & Experimental Allergy, 2010 (40) 505–519.     

Immunotherapy in Hay Fever with Two Major Allergens 19, 25 and Partially Purified Extract of Timothy Grass Pollen

Forty grass pollen hay fever patients were randomly divided into two equal groups and treated from January 1978 with different timothy grass pollen extracts: whole pollen allergens (WPA) (Alutard® SQ), a partially purified extract and purified pollen allergens (PPA) consisting of the major allergens 19, 25. The effect was evaluated by symptom scores in grass pollen season 1, 1978, and titrated thresholds of skin prick test (SPT) and nasal provocation test (NFT) with extracts WPA, PPA, and FGM (five-grass mixture). Extracts were biologically standardized in the HEP (histamine equivalent prick) system (1 HEP = 1,000 biological units (BU)/ml) and used in concentrations of 0.01-0.1-1.0-10 HEP for tests and treatment. The extracts for treatment were attached to Al (OH)₃. Extracts from the same batch were used throughout the investigation. The two patient groups, WPA and PPA, were comparable us regards age, sex, total dosage (range 2,187–73,887 BU) and frequency of side effects. Fewer symptoms (P=0.0001) were observed with WPA than with PPA. Titrated threshold increase showed a treatment efficacy of 70–100% in SPT compared with 15–65% in NPT and tended to show higher protection after treatment with WPA titan PPA hut equal protection against WPA and FGM, in accordance with common allergens in various grass pollen. In SPT with WPA the threshold increase was correlated (P<0.05) to symptom scores. Frequency of major (urticaria, angioedema, asthma) general side effects was positively correlated (P0.05) to pretreatment NPT thresholds below 1 HEP and standard dosage at 1 HEP or more, but tended to a negative correlation with local side effects. All local and general side effects first appeared 1–5h after injection and a more careful dosage pattern than standard is recommendable at maximum concentration, 10 HEP, and also with extracts 1 HEP at pretreatment NPT thresholds below 1 HEP.     

Quantification in mass units of group 1 grass allergens by a monoclonal antibody-based sandwich ELISA

BACKGROUND: Grass pollen extracts currently used for allergy diagnosis and immunotherapy are a complex mixture of proteins of which only a few have allergenic activity. Lol p 1 is one of the most important allergens in grass pollen extracts. OBJECTIVES: To develop a two-site enzyme-linked immunosorbent assay for the quantification of Lol p 1 and other group 1 allergens from grass species, and to assess its suitability for quantifying this group of allergens. METHODS: Balb/c mice immunized with recombinant Lol p 1 were used for the production of monoclonal antibodies. Screening of hybridomas was performed by direct ELISA, and selected monoclonal antibodies were immobilized on ELISA plates and incubated with samples containing group 1 allergens. Bound allergens were detected by a combination of biotinylated Lol p 1-specific monoclonal antibody and peroxidase-streptavidin conjugate. RESULTS: The assay is based on three Lol p 1-specific monoclonal antibodies with different epitope specificities. The optimized ELISA measured Lol p 1 concentrations ranging from 125 to 1000 ng/mL and could quantify group 1 allergen from grass species belonging to the Pooidea subfamily. The assay does not depend on anti-sera production or availability of human sera and thus reactives can be produced in unlimited amounts. CONCLUSION: This sensitive and specific Lol p 1 assay will be helpful both for quantifying the group 1 allergen content of Pooideae pollen extracts intended for clinical use and for studying cross-reactivities among pollen extracts.     

Heterogeneity of commercial timothy grass pollen extracts

Background The diagnosis and specific immunotherapy of allergy is currently performed with allergen extracts prepared from natural allergen sources.
Objective To analyse commercial timothy grass pollen allergen extracts used for in vivo diagnosis regarding their qualitative and quantitative allergen composition and in vivo biological activity.
Methods Antibodies specific for eight timothy grass pollen allergens (Phl p 1, Phl p 2, Phl p 4, Phl p 5, Phl p 6, Phl p 7, Phl p 12, Phl p 13) were used to detect these allergens in timothy grass pollen extracts from four manufacturers by immunoblotting. ELISA assays were developed and used to quantify the three major allergens (Phl p 1, Phl p 2, Phl p 5) in the extracts. The magnitude of skin responses to the four extracts was studied by skin prick testing in 10 grass pollen-allergic patients.
Results The allergen extracts showed broad variations in protein compositions and amounts (24.1–197.7 μg/mL extract). Several allergens could not be detected in certain extracts or appeared degraded. A considerable variability regarding the contents of major allergens was found (Phl p 1: 32–384 ng/mL; Phl p 2: 1128–6530 ng/mL, Phl p 5: 40–793 ng/mL). Heterogeneous skin test results were obtained with the extracts in grass pollen-allergic patients.
Conclusions Timothy grass pollen extracts from different manufacturers exhibit a considerable heterogeneity regarding the presence of individual allergens and hence yield varying in vivo test results. Problems related to the use of natural grass pollen allergen extracts may be circumvented by using defined recombinant grass pollen allergens.     

Phleum pratense alone is sufficient for allergen-specific immunotherapy against allergy to Pooideae grass pollens

Background Specific immunotherapy is the only causal treatment of allergy available today. Traditionally, therapeutic products based on either a single grass species or a mix of such extracts are used for grass pollen immunotherapy. Investigations comparing the immunological response to these allergen preparations are needed to ensure optimal treatment. The objective of this study was to investigate patterns of T and B cell cross-reactivity to Pooideae single-species extracts and to extract mixes.
Methods IgG4 induced by immunotherapy with Phleum pratense extract was investigated for cross-reactivity using nine single-species extracts and four mixes. For the mixes, studies of IgE cross-reactivity were also performed. T cell cross-reactivity was investigated in lines specific to nPhl p 1 or nPhl p 5 allergens, and the amounts of group 1 and 5 allergens in the extracts were quantified by a single radial immunodiffusion.
Results The levels of treatment-induced IgG4 detected by all the extracts displayed a clear correlation to that detected by the P. pratense pollen extract. The IgE studies confirmed the cross-reactivity of P. pratense -specific B cells towards the allergens contained in the mixes, and the T cell studies demonstrated cross-reactivity towards group 1 and 5 major allergens in extracts of six temperate grass species.
Conclusion Extensive T and B cell cross-reactivity was observed towards the allergens of the Pooideae grasses, and the degree of B cell cross-reactivity was independent of the number of species included in the extract mixes. This implies that treatment with pollen extract of just one Pooideae species will affect the allergic responses caused by any of the temperate grasses in this subfamily.     

Allergen-specific immunotherapy with a monophosphoryl lipid A-adjuvanted vaccine: reduced seasonally boosted immunoglobulin E production and inhibition of basophil histamine release by therapy-induced blocking antibodies

BACKGROUND: Allergen-specific immunotherapy represents a causal form of treatment for IgE-mediated allergies. The allergen extract-based analyses of immunotherapy-induced effects yielded highly controversial results regarding a beneficial role of therapy-induced IgG antibodies. OBJECTIVE: We analysed allergen-specific IgE, IgG subclass, and IgM responses in patients treated with a grass pollen allergy vaccine adjuvanted with monophosphoryl lipid A (MPL), a Th1-inducing agent, and in a placebo group using recombinant timothy grass pollen allergen molecules (rPhl p 1, rPhl p 2, rPhl p 5). RESULTS: The strong induction of allergen-specific IgG1 and IgG4 antibodies observed only in the actively treated group was associated with significant clinical improvement. Therapy-induced allergen-specific IgM and IgG2 responses were also noted in several actively treated patients. An inhibition of allergen-dependent basophil histamine release was only obtained with sera containing therapy-induced allergen-specific IgG, but not with sera obtained before therapy or from placebo-treated patients. Moreover, patients with therapy-induced allergen-specific IgG antibodies showed a reduced induction of allergen-specific IgE responses during seasonal grass pollen exposure. CONCLUSION: Successful immunotherapy with the MPL-adjuvanted grass pollen allergy vaccine is associated with the production of allergen-specific IgG antibodies. These blocking antibodies may have protective effects by inhibiting immediate-type reactions and systemic increases of IgE responses caused by seasonal allergen exposure.     

Development of a sandwich-type ELISA for measuring Pla a 1, the major allergen of Platanus acerifolia pollen

BACKGROUND: Platanus acerifolia is an important cause of pollinosis in Western European cities. Pla a 1, a nonglycosylated 18-kDa protein with a prevalence of 80%, is a major allergen in P. acerifolia pollen extracts. Our aim was to develop a Pla a 1-specific ELISA to quantify this protein in allergenic extracts and preparations for clinical use. METHODS: Pla a 1 was purified by cation exchange at pH 7.0, gel filtration, and anion exchange chromatography at pH 10.0. Monoclonal (mAb) and polyclonal antibodies were obtained by immunizing mice and rabbits with nPla a 1. One (5C1) of the 13 mAb obtained was used as capture antibody at 5 mug/ml and biotin-labeled specific polyclonal antiserum at 0.63 microg/ml served for detection. RESULTS: The prevalence of Pla a 1-specific IgE to purified Pla a 1 among 47 P. acerifolia-allergic patients was 79%. The Pla a 1-ELISA developed has a linear range of 3-25 ng/ml, high sensitivity with a detection limit of 0.5 ng/ml and is highly specific as none of the 24 pollen, mite, mold, and plant food extracts tested gave positive results. The assay could quantify Pla a 1-like proteins in other planetree pollen extracts. A good correlation was obtained between Pla a 1 content of 11 P. acerifolia pollen extracts (average content 0.69% of the total protein) and their IgE-binding activity. CONCLUSIONS: The described two-site sandwich ELISA to measure Pla a 1 is useful for standardization of planetree pollen extracts intended for clinical use.     

Characterization of allergens from Trisetum paniceum pollen: an important aeroallergen in Mediterranean continental climatic areas

Background Trisetum paniceum is a grass plant which is characteristic of a Mediterranean continental climate and has been descrihed as one of the major causes of type I allergy in the Madrid region. Objectives To identify and characterize the allergens of Trisetum paniceum pollen. Methods Allergenic extracts were prepared by 24 h incubation of pollens in a buffered solution. Proteins were analysed by a new two-dimensional system in which agarose piates were used for isoelectric focusing. Two-dimensionally resolved proteins were electrically transferred to Immobilon membranes and the allergens immunochemically detected. Proteins from six grass pollens were bound to a membrane and incubated with a pool of serum from grass-pollen-sensitized patients. The bound IgE antibodies were then eluted and used to identify the proteins of Trisetum paniceum pollen that allergenically crossreact with allergens from other pollen grasses. Results Relative to total protein content, Trisetum paniceum pollen had a high proportion of reactive proteins. On the basis of their molecular characteristics, allergens could be classified as group 1,2,4 and 5 components yet included an atypical proportion of basic components. All identified allergens were crossreactive with allergens from the remaining grass pollens studied. Conclusions Trisetum paniceum pollen contains a high proportion of allergens and these include a group of basic proteins which are not detected in other phylogenetically related pollens and could be of atlergological interest.     

Clustering of allergenic pollen on the basis of skin responses of atopic patients by matrix analysis

The responses of 148 atopic patients to some 43 different extracts of allergenic pollen were tested by prick tests. The measure of dissimilarity was introduced and calculated for all pairs of allergens. The investigated allergens were clustered into groups, according to their unbiased greatest similarity, by a matrix-structuring method. Results indicate that subgroups of allergens can be distinguished even within groups of closely related pollen allergens that were believed to be fully cross-reactive. A few cases are demonstrated for various varieties of olives, pecans, date palms, and turf grasses and for some wild chenopods and amaranths. The usefulness of the suggested solution for allergy research and for clinical practice is discussed.     

Concentrations of major grass group 5 allergens in pollen grains and atmospheric particles: implications for hay fever and allergic asthma sufferers sensitized to grass pollen allergens

BACKGROUND: Grass pollen allergens are the most important cause of hay fever and allergic asthma during summer in cool temperate climates. Pollen counts provide a guide to hay fever sufferers. However, grass pollen, because of its size, has a low probability of entering the lower airways to trigger asthma. Yet, grass pollen allergens are known to be associated with atmospheric respirable particles. OBJECTIVE: We aimed (1) to determine the concentration of group 5 major allergens in (a) pollen grains of clinically important grass species and (b) atmospheric particles (respirable and nonrespirable) and (2) to compare the atmospheric allergen load with clinical data to assess different risk factors for asthma and hay fever. METHODS: We have performed a continuous 24 h sampling of atmospheric particles greater and lower than 7.2 microm in diameter during the grass pollen season of 1996 and 1997 (17 October 1996-16 January 1997) by means of a high volume cascade impactor at a height of about 15 m above ground in Melbourne. Using Western analysis, we assessed the reactivity of major timothy grass allergen Phl p 5 specific monoclonal antibody (MoAb) against selected pollen extracts. A MoAb-based ELISA was then employed to quantify Phl p 5 and cross-reactive allergens in pollen extracts and atmospheric particles larger and smaller than 7.2 microm. RESULTS: Phl p 5-specific MoAb detected group 5 allergens in tested grass pollen extracts, indicating that the ELISA employed here determines total group 5 allergen concentrations. On average, 0.05 ng of group 5 allergens were detectable per grass pollen grain. Atmospheric group 5 allergen concentrations in particles > 7.2 microm were significantly correlated with grass pollen counts (rs = 0.842, P < 0. 001). On dry days, 37% of the total group 5 allergen load, whereas upon rainfall, 57% of the total load was detected in respirable particles. After rainfall, the number of starch granule equivalents increased up to 10-fold; starch granule equivalent is defined as a hypothetical potential number of airborne starch granules based on known pollen count data. This indicates that rainfall tended to wash out large particles and contributed to an increase in respirable particles containing group 5 allergens by bursting of pollen grains. Four day running means of group 5 allergens in respirable particles and of asthma attendances (delayed by 2 days) were shown to be significantly correlated (P < 0.001). CONCLUSION: Here we present, for the first time, an estimation of the total group 5 allergen content in respirable and nonrespirable particles in the atmosphere of Melbourne. These results highlight the different environmental risk factors for hay fever and allergic asthma in patients, as on days of rainfall following high grass pollen count, the risk for asthma sufferers is far greater than on days of high pollen count with no associated rainfall. Moreover, rainfall may also contribute to the release of allergens from fungal spores and, along with the release of free allergen molecules from pollen grains, may be able to interact with other particles such as pollutants (i.e. diesel exhaust carbon particles) to trigger allergic asthma.     

Side Effects during Immunotherapy with Purified Grass Pollen Extracts

In a 3-year prospective double blind study, grass pollen allergic patients were allocated to perennial hyposensitization with the timothy major allergens Nos. 19 and 25 (2-component extract) or a 20-component timothy extract. The extracts were biologically standardized and adsorbed to aluminium hydroxide for treatment.
Systemic side effects (SSE) had début after 1 1/2–5 h and lasted without treatment 1/2–10 h. Treatment with the 2-component extract showed preponderance of minor SSE (arthralgia, rhinitis, tiredness, headache, conjunctivitis, nausea, flu-like symptoms), but major SSE (urticaria, angioedetma, asthma) were equally distributed between treatment with the two timothy extracts.
Major SSE comiplicated the treatment before the first grass pollen season in 33% of the patients vs. only in 3% during the subsequent perentiial therapy, and developed (92%) at high single dose of ≥1,000 biological units. The majority (69%) were later able to reach the same or higher dose without relapse. Most (62%) patients with major SSE were predicted by high nasal sensitivity before treatment.
Only 18% of the patients had immediate local skin reactions of ≥2 cm, but delayed local side effects of ≥10 cm were recorded in 70%. Immediate skin reactions did not correlate with delayed skin reactions or with SSE, but delayed local side effects tended towards negative correlation with major SSE. A mean area reduction of 50% of the delayed skin reactions was recorded by repetition of a single dose.
Subcutaneous nodules appeared at single doses of ≥5,000 biological units. Only 5% of the patients contracted nodules during initial preseasonal therapy compared with 38% during subsequent perennial dosage. The nodules contained typical benign granulomas, and the frequency in the two groups was proporitonate to the quantity of aluminium in the two extracts.     

Measurement of IgE antibodies against purified grass pollen allergens (Lol p 1, 2, 3 and 5) during immunotherapy

Background IgE titres tend to rise early after the start of immunotherapy, followed by a decline to pre-immunotherapy levels or lower. Objectives We were interested to ktiow whether the early increase in IgE antibodies includes new specificities of IgE, and whether these responses persist. Methods Sera of 64 patients undergoing grass pollen immunotherapy were tested for IgE against four purified grass pollen allergens: Lol p 1. 2, 3, and 5. At least two serum samples were taken, one before the start of therapy and one between 5 and 18 months after the first immunization (mean: 10 months). Results The mean IgE responses to Lol p 1, 2 and 3 showed a moderate but not significant increase. In contrast, the mean IgE response to Lol p 5 showed a significant decrease of >30%. IgE against total Lolium perenne pollen extract moderately increased (>20%), showing that a RAST for total pollen is not always indicative for the development of IgE against its major allergens. For >40% of the patients it was found that IgE against one or more of the four allergens increased, while IgE against the remaining allergen(s) decreased. Eor 10 sera the ratio of IgE titres against at least two allergens changed by at least a factor of 5. The changes in specific IgE also included conversions from negative (< 0.1 RU) to positive (0.6 to 5.0 RU) for five patients. For two patients, the induction of these ‘new’ IgE antibodies against major allergens was shown to result in a response that was persistent over several years. Conclusion Although active induction of new IgE specificities by immunotherapy was not really proven, the observations in this study indicate that monitoring of IgE against purified (major) allergens is necessary to evaluate changes in specific IgE in a reliable way.     

Melon sensitivity shares allergens with Plantago and grass pollens

Possible associations between allergy to pollen and that to food allergens were studied in 262 patients sensitized to pollen. Forty-four patients (16.7%) showed some allergic symptoms after testing with fruits and vegetables, melon being the food most frequently involved (24 patients), followed by sunflower seed (12 patients). Skin testing was done by the prick method with natural fruit or vegetable, and also with commercial food extracts. We found in our region that the distribution of sensitivity to pollens in the group of patients with allergy to fruits or vegetables does not coincide with the prevalence in pollen-allergic subjects in general, since in the first group -subjects allergic to food - there was a major prevalence of allergy to Plantago ( P < 0.01). In particular, in the group of subjects allergic to melon, the prevalence of sensitivity to grass and especially to Plantago was larger than in pollen-allergic subjects in general ( P < 0.05 and P 0.001, respectively). The use of fresh food produced better results than commercial extracts. A positive skin test to fresh melon closely correlated with positive CAP results. CAP inhibition experiments were carried out, and we found that Dactylis and Plantago extracts inhibited the binding of the melon-positive pool to solid-phase melon. The results suggest the existence of common antigenic epitopes in melon and Plantago pollen, and in melon and grass pollen.     

Immunotherapy with Grass Pollen Major Allergens

Perenial hyposensitization with a partially purified timothy extract resulted in a statistically significantly higher degree of clinical protection than treatment with the two timothy major allergens (Nos. 19 and 25) and protected better from the second—than during the first grass pollen season. The extracts were standardized biologically and adsorbed to aluminium hydroxide for administration. The therapy had a more beneficial influence on sneezing than on rhinorrhoea and blockage of nasal airways, and an excellent effect on grass pollen asthma was obtained with the partially purified timothy extract. Associated birch pollen allergy was not influenced by hyposensitization with grass pollen.     

Occupational asthma induced by garlic dust

Background: Garlic dust has not been a frequently encountered cause of IgE-mediated disease. Objective: We report on 12 patients (all of them garlic workers) with the clinical criteria for occupational asthma. Methods: Skin prick tests and serum-specific IgE determinations were performed with common inhalants, garlic, and other members of the Liliaceae family (onion, leek, and asparagus). Bronchial challenge test with garlic powder was performed in all patients. Garlic and onion extract proteins were separated by sodium dodecylsulfate–polyacrylamide gel electrophoresis. Immunoblot and IgE immunoblot inhibition analyses were performed with patients' sera on extracts of garlic, onion, and pollens of Phleum pratense and Chenopodium album. Results: Garlic sensitization was demonstrated by bronchial challenge test in seven patients (group 1) and ruled out in the remaining five (group 2). Clinical data were similar in both groups. The patients with garlic allergy had a mean age of 27 years, and all of them had pollen allergy; sensitization to other members of the Liliaceae family was also common. Electrophoresis of garlic extract revealed two major protein bands at approximately 12 and 54 kd. During IgE immunoblotting, the pool of sera reacted with garlic proteins mainly at 54 kd. Preincubation with onion, Phleum, and Chenopodium partially abolished the IgE binding to several allergens of garlic. Conclusion: We report on seven patients in whom an occupational garlic allergy was demonstrated. Garlic allergy is relatively rare but seems to affect young subjects with pollen allergy, and sensitization to other members of the Liliaceae family is common. The results of this study confirm the presence of some structurally similar allergens in garlic, onion, and certain pollens. (J Allergy Clin Immunol 1997;100:734-8.)     

Monoclonal antibody-based ELISA to quantify the major allergen of Artemisia vulgaris pollen, Art v 1

BACKGROUND: Pollen of Artemisia vulgaris (mugwort) is a relevant cause of pollinosis in temperate and humid regions. Recently, the major allergen of this pollen, Art v 1, has been characterized. OBJECTIVE: To develop a monoclonal antibody (mAb)-based enzyme-linked immunosorbent assay (ELISA) to quantify Art v 1, and to assess the correlation of Art v 1 content with the biological activity of mugwort pollen extracts. METHODS: Art v 1-specific mAbs were obtained from a BALB/c mouse immunized with high-performance liquid chromatography (HPLC)-purified Art v 1. One of these antibodies (Av 3.7), which recognizes the N-terminal defensin-like domain of Art v 1, was used as the capture antibody in an ELISA method for allergen quantitation. An anti-A. vulgaris rabbit serum was used as the second antibody. Art v 1 was purified by immunoaffinity chromatography and used as the standard in the assay. RESULTS: The purity and identity of the affinity-purified Art v 1 was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), mass spectrometry, amino acid composition, and N-terminal amino acid sequencing. The prevalence of specific IgE against Art v 1, determined by radioallergosorbent test (RAST) in a population of 44 mugwort-allergic patients, was 79%. The Art v 1-ELISA developed displays a detection limit of 0.1 ng/ml, and a practical working range of 0.2-10 ng/ml. The concentration of Art v 1 was measured in 10 A. vulgaris pollen extracts, and a good correlation was observed between the Art v 1 content and the allergenic activity of the extracts. CONCLUSIONS: The results prove the usefulness of the Art v 1-ELISA for the standardization of A. vulgaris pollen extracts intended for clinical use.     

Quantification of the major allergen from cypress (Cupressus arizonica) pollen, Cup a 1, by monoclonal antibody-based ELISA

BACKGROUND: Cypress pollen allergy is an important cause of rhinoconjunctivitis and asthma in Mediterranean countries. Cypress allergenic extracts are difficult to produce since they have low protein and high carbohydrate content, thus accurate standardization of them is essential to guarantee their quality. The aim of this study is to develop a sandwich ELISA for the quantification of Cup a 1, the major allergen of cypress (Cupressus arizonica) pollen extract. METHODS: Monoclonal antibodies directed to purified Cup a 1 were produced. Two of them (9C7 as capture antibody and 3D2 as the tracer) were selected to develop a quantitative sandwich ELISA. This ELISA was subsequently evaluated and compared with other techniques. RESULTS: The described ELISA is very sensitive with a detection limit of 8.7 ng/ml and a practical working range of 62.5-1,000 ng/ml. The assay is also highly reproducible with intra-assay and interassay coefficients of variation of less than 10%. The purified Cup a 1, used as standard, presents pectate lyase enzymatic activity. The assay also detected Cup a 1-like proteins in pollen from other Cupressaceae. A good correlation was obtained between Cup a 1 content of 12 C. arizonica pollen extracts and their IgE-binding activity. CONCLUSIONS: The described Cup a 1 ELISA is sensitive, specific and reproducible and can be used for the quantification of Cup a 1 in C. arizonica and other related pollen extracts. It also provides a reliable indication of the allergenic activity of the whole cypress pollen extract.     

An enzyme-linked immunosorbent assay (ELISA) for the quantitation of sugi pollen and Dermatophagoides mite allergens and its application for standardization of allergen extracts

A simple enzyme-linked immunosorbent assay (ELISA) has been developed for the quantitation of the major allergens of sugi pollen, Cry j I and of Dermatophagoides mites, Der I (Der p I/Der f I) and Der II (Der p II/Der f II) for use in the in vitro standardization of allergen extracts. Polystyrene microplates coated with a IgG fraction of rabbit antiserum were incubated first with allergen extracts and then with biotinylated antiserum IgG. The bound allergen-biotinylated antibody complex was detected with commercially available streptavidin-enzyme conjugate followed by the addition of colorimetric substrate. The assay was very sensitive (-0.2 ng/ml) and reproducible (CV% = 1.9-13.8%). The ELISA was compared with the radioimmunoassay previously described, and the results showed a very good correlation between the assays (r = 0.967-0.990). The allergen content in three sugi pollen and three house dust extracts measured by the ELISA also demonstrated a good agreement with the relative potency of these extracts as determined by the intradermal skin test. These results indicate that the ELISA could be useful in the standardization of allergen extracts.