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PubMed1977

Allergenic structure of allergen M from cod. II. Allergenicity of the limited tryptic hydrolysis peptides of fragment TM 2.

The peptides P1B3 (amino acid residues No. 88-113) and LT3N1 (residues No. 88-108), obtained from the limited tryptic hydrolysis of fragment TM 2 from codfish allergen M inhibited the interaction between allergen M and reaginic IgE antibodies both in vitro (87 and 62% RAST inhibition, respectively) and in vivo (total PK test inhibition). The peptide A7B1 (residues No. 97-113) overlapping the former at the COOH-terminus, was inactive in PK test inhibition experiments and produced only a minor inhibition of RAST (15%). The findings suggest that the nonapeptide residue No. 88-96 is partially indispensible for the allergenic activity of fragment TM 2, and is likely to incorporate allergenic determinant(s).

Allergens↗
PubMed1996

Domestic allergens in public places. II: Dog (Can f1) and cockroach (Bla g 2) allergens in dust and mite, cat, dog and cockroach allergens in the air in public buildings.

BACKGROUND: Sensitization and exposure to indoor allergens are the major risk factors for asthma. It is possible that significant exposure to domestic allergens occurs outside the home. OBJECTIVES: To investigate the levels of Can f 1 and Bla g 2 in the dust from carpeted floors and upholstered seats in public buildings and public transport and the airborne concentrations of Der p 1, Fel d 1, Can f 1 and Bla g 2 in schools and offices. METHODS: Can f 1 and Bla g 2 were measured in the dust collected by vacuuming a 1 m2 area of carpet, as well as upholstered seats in five schools, six hotels, four cinemas, six pubs, three buses and two trains. Dust was also collected from the bedroom carpet, living room carpet, mattress and sofa in 20 homes with and 20 homes without a dog in the same area. Personal airborne sampling (2 L/min) was conducted for 8 h in offices (n = 16) and classrooms (n = 9). In addition, airborne samples in schools were collected using a high volume pump (60 L/min) for 1 h in three classrooms immediately after the children vacated the school. Can f 1, Bla g 2, Der p 1 and Fel d 1 were assayed using a two-site monoclonal antibody-based ELISA. RESULTS: Can f 1 was detected in all dust samples from public places, ranging from 0.2 to 52.5 micrograms/g. Significantly higher levels were found in upholstered seats (geometric mean--GM 9.4 micrograms/g) than in carpets (GM 1.5 micrograms/g; P < 0.001), and levels of Can f 1 > 10 micrograms/g were found in 40% of upholstered seats in public places. Can f 1 was significantly higher in upholstered seats in public places than in sofas in homes without a dog (GM 1.8 micrograms/g; P < 0.001). Detectable levels of Bla g 2 were found in all of the schools (GM 2.4 U/g, range 0.8-4.4 U/g). Bla g 2 concentration greater than 2U/g (provisional threshold level representing risk of sensitization) was measured in 65% of the classrooms sampled. Der p 1 and Bla g 2 were below the detection limit in all airborne samples. However, airborne Fel d 1 and Can f 1 were detected in schools and offices, albeit in low concentrations. CONCLUSIONS: Upholstered seats from public places constitute a reservoir for the accumulation of dog allergen, and a source of exposure to Can f 1 inside public buildings or on public transport. Exposure to cockroach allergens in schools may be important for cockroach sensitized asthmatic children.

Air Pollution, Indoor↗
PubMed2003

Prevention of allergen-specific IgE production and suppression of an established Th2-type response by immunization with DNA encoding hypoallergenic allergen derivatives of Bet v 1, the major birch-pollen allergen.

In atopic patients, programming towards a preferential Th2 immunity leads to IgE antibody production and cellular Th2 immunity against otherwise harmless antigens. We report the development of prophylactic and therapeutic DNA vaccines for the major birch-pollen allergen, Bet v 1. We constructed three DNA vaccines, coding for the complete cDNA, coding for two hypoallergenic fragments or coding for a hypoallergenic Bet v 1 mutant. The protective effect was studied in mice pretreated by intradermal DNA injections, then sensitized with Bet v 1 protein. Mice pretreated with any of the three Bet v 1-specific DNA vaccines were protected against allergic sensitization to Bet v 1. Protection was characterized by a lack of Bet v 1-specific IgE production, a lack of basophil activation and an enhanced IFN-gamma expression. DNA vaccines with wild-type Bet v 1 induced strong Bet v 1-specific antibody responses whereas DNA vaccines with hypoallergenic Bet v 1 derivatives induced no (fragments) or only transient (mutant) Bet v 1-specific antibody responses. A therapeutic approach with the fragment-DNA vaccine reduced IgE production and stimulated a sustained Th1 cytokine milieu. Our results demonstrate that DNA vaccines with hypoallergenic forms of the allergen specifically protect against sensitization and suppress established Th2-type responses. This concept may be applied for the development of safe and specific DNA vaccines for the prophylaxis and therapy of allergic diseases.

Allergens↗
PubMed2005

Identification of human T cell epitopes in Japanese cypress pollen allergen, Cha o 1, elucidates the intrinsic mechanism of cross-allergenicity between Cha o 1 and Cry j 1, the major allergen of Japanese cedar pollen, at the T cell level.

BACKGROUND: Pollens from species of Cupressaceae family are one of the most important causes of respiratory allergies worldwide. In Japan, many patients with pollinosis have specific IgE to both pollens of Japanese cypress (Chamaecyparis obtusa) and Japanese cedar (Cryptomeria japonica). The sequences between Cha o 1 and Cry j 1, the major allergens of Japanese cypress and Japanese cedar pollens, respectively, are 80% identical. OBJECTIVE: This study was undertaken to identify T cell epitopes in Cha o 1, and to elucidate the mechanism of cross-allergenicity between Cha o 1 and Cry j 1, at the T cell level. METHODS: T cell epitopes in Cha o 1 were identified by the reactivity of T cell lines, generated from 19 patients, to stimulation with overlapping peptides. The subsets of T cell clones specific to rCha o 1 were determined according to their ability to produce IL-4 and IFN-gamma. Peptide specificities of two T cell clones were determined by stimulation with the peptides from Cha o 1 and Cry j 1. RESULTS: Four dominant and six subdominant T cell epitopes were identified in Cha o 1. While four T cell epitopes, p11-30, p211-230, p251-270 and p331-350, were common to Cha o 1 and Cry j 1, 4 T cell epitopes, p61-80, p71-90, p311-330 and p321-340, were considered to be unique to Cha o 1. The subsets of T cell clones were predominantly of T helper2-type. One T cell clone recognized p16-30, which is common to Cha o 1 and Cry j 1, but another recognized p321-330, which is unique to Cha o 1. CONCLUSION: Presence of both T cells reactive to T cell epitopes common to Cha o 1 and Cry j 1 and T cells specific to T cell epitopes unique to Cha o 1 in patients with pollinosis contributes to prolonged symptoms after the cedar pollen season in March and the following cypress pollen season in April.

Adult↗
PubMed1989

[A clinical study of two cases with immediate hypersensitivity to apple-pulp and an investigation of cross-allergenicity between apple-pulp allergen and some other pollen allergens].

In Japan there are no case reports of immediate hypersensitivity to apple-pulp. Here, we report on two patients: a 23-year-old woman and a 14-year-old girl with 3-year and 7-year histories, respectively, of anaphylactic reactions to apple-pulp. In the first patient, eating raw apples immediately elicited itching and tingling of the lips and mouth with severe edema of the lips and tongue, irritation of the throat and slight colic in the upper abdomen. In the second, nausea and vomiting occurred after ingestion of apples. In skin prick tests, they showed positive reactions to apple-pulp extract prepared by the method of Björkstén et al. and to fresh apple juice. In addition, IgE antibody to the apple-pulp extract was detected in the sera of both patients. They also possessed IgE antibodies to some other species of both pollens and fruits/vegetables in their sera. In the present study, cross-allergenicity between apple-pulp and five pollen species, which consisted of birch, Japanese cedar, orchard grass, mugwort and ragweed pollen, was investigated by RAST inhibition. The apple-pulp extract effectively inhibited RASTs to all the pollens except Japanese cedar pollen. These results suggested that the four pollens might have shared allergen with the apple-pulp extract.

Adolescent↗
PubMed1982

Comparative studies on tree pollen allergens. II. Isolation of alder (Alnus incana) pollen allergens: purification and some characteristics of the major allergen pI 4.78.

The aqueous extract of alder (Alnus incana) pollen was found to consist of a protein mixture amounting to 3% of the dry pollen weight. Separation by gel permeation chromatography on an AcA-54 column resulted in 6 fractions designated chronologically to their relative elution volumes (AI 1 to AI 6). The immunological reactivity of these fractions was demonstrated in both IgE and IgG test systems. Being both immunochemically and quantitatively dominant fraction AI 4 was selected for further purification by a semipreparative isoelectric focusing column. A well-resolved and apparently homogeneous component with a pI value of 4.78 was eluted from the IEF column. The homogeneity of this fraction was demonstrated by one sharp band in thin layer IEF, one single band in HVE, one distinct sharp peak in HPLC and a symmetrical precipitate in CIE. The molecular weight was estimated to 22,500 daltons. Fraction pI 4.78 was shown to be a major allergen of alder pollen, it elicited a 1.17 histamine wheal reaction in skin prick test at a concentration of 1 mg . ml-1 and showed a significantly high RAST inhibition in all of the reaginic sera used. Fraction pI 4.78 was found to be an adequate model allergen of alder pollen well suited for further immunochemical characterization.

Allergens↗
PubMed2002

[Use of conjugated allergen-polymer vaccines (pollen allergens of new generation) for allergen-specific immunotherapy].

AIM: To assess effectiveness of new pollen allergotropins for allergen-specific immunotherapy (ASI). MATERIAL AND METHODS: The allergotropins were produced basing on conjugation of pollen allergoids (timothy, birch, mugwort pollen) and polyelectrolite (polyoxidonium) with immunomodulating properties. Pollen allergotropins (timpol, berpol) were designed to treat pollenosis caused by sensitization to timothy and birch pollen. Pollen allergotropins were studied in ASI given to 140 patients with pollenosis and 10 patients with bronchial asthma. RESULTS: In the group of pollenosis patients with hypersensitivity to birch pollen the response to berpol was excellent in 55% patients (the disease symptoms disappeared, antihistamine drugs intake was stopped), good--in 33%, satisfactory--in 12%. Timpol produced a marked response in all the patients treated for hypersensitivity to timothy pollen. CONCLUSION: Pollen allergotropins have a high therapeutic effect under low risk of provoking reactions of anaphylactic type in patients on ASI.

Adult↗
PubMed2002

Comparison of skin prick test results between crude allergen extracts from foods and commercial allergen extracts in atopic dermatitis by double-blind placebo-controlled food challenge for milk, egg, and soybean.

Skin Prick Test (SPT's) are performed to identify the causes of allergy. However, low diagnostic accuracy is a limitation to SPT, for which many possible causes have been suggested. The protein composition and allergenicity of crude allergen extracts from foods and commercial allergen extracts for SPT were analyzed. Clinical significances of SPT using crude allergen extracts from foods were compared with those using commercial allergen extracts. A total of 292 atopic dermatitis patients were involved in this study. Crude allergen extracts were prepared from milk, egg white, egg yolk, and soybean. The protein composition of food allergen extracts and commercial allergen extracts of milk, whole egg, white, egg yolk, and soybean were compared by SDS-PAGE. The allergenicity was tested by the immunoblotting method using immune sera. SPTs were performed using crude and commercial allergen. Double-blind placebo- controlled food challenge (DBPCFC) was performed to verify the SPT results and to compare the clinical significance of crude and commercial allergen extracts. Protein composition differed markedly between crude and commercial allergen extracts. By immunoblotting, crude and commercial allergen extracts showed different allergenicity. The SPT results using crude and commercial allergen extracts showed significant differences. The prevalence of milk, egg and soybean allergy was over 35% in atopic dermatitis. The accuracy of SPT using crude allergen extracts from foods was significantly higher than that using commercial allergen extracts. In the case of soybeans, the result of SPT using commercial allergen extract was clinically insignificant for the prediction of soybean allergy. The source of allergen extract was very important for the appropriate SPT in food allergy. The accuracy of SPT might be improved using the appropriate allergen source for food allergy.

Adolescent↗
PubMed1999

Pollen-related food allergy: cloning and immunological analysis of isoforms and mutants of Mal d 1, the major apple allergen, and Bet v 1, the major birch pollen allergen.

BACKGROUND: Mal d 1, the major apple allergen, cross-reacts with IgE specific for the major birch pollen allergen, Bet v 1, and is responsible for birch pollen related food allergy to apple. Isoforms of Bet v 1 showing minor sequence variations display different binding capacity for specific IgE antibodies from allergic patients. Moreover, strain-dependent variation of allergenicity has been reported for apples. OBJECTIVE: To investigate the occurrence of strain-dependent isoforms of Mal d 1 which may differ in their allergenic potential, to obtain data on structures essential for binding of Mal d 1 to the antibody, and to gain insights into the structures responsible for its IgE cross-reactivity to Bet v 1. METHODS: The cDNA of Mal d 1 from various apple strains was amplified by a PCR strategy based on conserved regions of known Mal d 1-sequences, and sequenced. Two major isoforms of Mal d 1 were expressed as recombinant proteins and purified, as were different variants of the major birch pollen allergen, Bet v 1. Together with already existing recombinant birch pollen and apple allergens, these were subjected to allergenicity testing by IgE-immunoblotting, enzyme allergo sorbent test and dose related mediator release. "Hot-spots" for IgE-reactivity were identified by site-directed mutagenesis. RESULTS: Twelve Mal d 1-clones were sequenced from 7 apple varieties and compared to 3 known Mal d 1 sequences. The clones were clustered into two groups, each showing a high degree of sequence identity to one of the known sequences and specific differences to the third sequence. No strain-specific sequences were identified. In contrast, apple strains with reported differences in allergenicity showed different expression levels of the major allergen. Immunologic testing of recombinant allergens revealed high IgE binding capacity of 2 major isoforms, named GD26 and GS29, with a slightly higher IgE binding capacity of GD26. Moreover, the allergenicity was similar to another r Mal d 1 reported in the literature, representing the isoform divergent from our clones. Mutational analysis of our Mal d 1 allergens identified serine in position 111 as essential for IgE binding. Allergenicity was almost depleted by changing this residue into a proline. Moreover, the corresponding serine residue, present in position 112 of Bet v 1, was in a similar manner crucial for the allergenicity of the birch pollen allergen. CONCLUSION: We conclude that divergent allergenicity of apple strains mainly depends on different expression levels of the major allergen. Introduction of a proline residue in position 111 of Mal d 1 and in position 112 of Bet v 1 led to a drastic reduction of allergenicity of both the pollen and the food allergen, obviously also removing the cross-reactive epitope. Mutants with reduced IgE-reactivity but maintained T-cell reactivity may represent new candidates for a safer specific immunotherapy with reduced side-effects.

Allergens↗
PubMed2000

Allergen immunotherapy for asthma.

BACKGROUND: Allergen specific immunotherapy has long been a controversial treatment for asthma. Although beneficial effects upon clinically relevant outcomes have been demonstrated in randomised controlled trials, there remains a risk of severe and sometimes fatal anaphylaxis. The recommendations of professional bodies have ranged from cautious acceptance to outright dismissal. With increasing interest in new allergen preparations and new methods of delivery, it was time to conduct a systematic review of allergen specific immunotherapy for asthma. OBJECTIVES: Allergen specific immunotherapy involves injecting an extract of the allergen under the skin. It is also known as hyposensitisation or desensitisation, and carries a risk of potentially fatal anaphylaxis. The objective of this review was to assess the effects of allergen specific immunotherapy for asthma. SEARCH STRATEGY: We searched the Cochrane Airways Group trials register up to 1997, Dissertation Abstracts, WorldCat and ArticleFirst. SELECTION CRITERIA: Randomised trials using various forms of allergen specific immunotherapy to treat asthma. DATA COLLECTION AND ANALYSIS: Eligibility of studies for inclusion was decided by three reviewers independently. Quality assessment of studies was performed by two reviewers independently. MAIN RESULTS: Fifty-four trials were included. There were 25 trials of immunotherapy for house mite allergy; 13 pollen allergy trials; eight animal dander allergy trials; two Cladosporium mould allergy and six trials looking at multiple allergens. Concealment of allocation was assessed as clearly adequate in only 11 of these trials. Significant heterogeneity was present in a number of comparisons. Overall, there was a significant reduction in asthma symptoms and medication following immunotherapy. There was also a significant improvement in asthma symptom scores (standardised mean difference -0.52, 95% confidence interval -0.70 to -0.35). People receiving immunotherapy were less likely to report a worsening of asthma symptoms than those randomised to placebo (odds ratio 0.27, 95% confidence interval 0.21 to 0.35). People randomised to immunotherapy were less likely to require medication than those randomised to placebo (odds ratio 0.28, 95% confidence interval 0.19 to 0.42). Allergen immunotherapy reduced allergen specific bronchial hyper-reactivity, with some reduction in non-specific bronchial hyper-reactivity as well. There was no consistent effect on lung function. REVIEWER'S CONCLUSIONS: Immunotherapy may reduce asthma symptoms and use of asthma medications, but the size of the benefit compared to other therapies is not known. The possibility of adverse effects (such as anaphylaxis) must be considered.

Allergens↗
PubMed2002

The allergen profile of ash (Fraxinus excelsior) pollen: cross-reactivity with allergens from various plant species.

BACKGROUND: Ash, a wind-pollinated tree belonging to the family Oleaceae, is distributed world-wide and has been suggested as a potent allergen source in spring time. OBJECTIVE: The aim of this study was to determine the profile of allergen components in ash pollen in order to refine diagnosis and therapy for patients with sensitivity to ash pollen METHODS: The IgE reactivity profile of 40 ash pollen-allergic patients was determined by immunoblotting. Antibodies raised to purified pollen allergens from tree and grass pollens were used to identify cross-reactive structures in ash pollen extract. IgE immunoblot inhibition studies were performed with recombinant and natural pollen allergens to characterize ash pollen allergens and to determine the degree of cross-reactivity between pollen allergens from ash, olive, birch, grasses and weeds. RESULTS: The allergen profile of ash pollen comprises Fra e 1, a major allergen related to the major olive allergen, Ole e 1, and to group 11 grass pollen allergens, the panallergen profilin, a two EF-hand calcium-binding protein, a pectinesterase-like molecule and an allergen sharing epitopes with group 4 grass pollen allergens. Thus, the relevant allergens of ash are primarily allergens that share epitopes with pollen allergens from other tree, grass and weed species. CONCLUSIONS: Allergic symptoms to ash pollen can be the consequence of sensitization to cross-reactive allergens from other sources. The fact that ash pollen-allergic patients can be discriminated on the basis of their specific IgE reactivity profile to highly or moderately cross-reactive allergens has implications for the selection of appropriate forms of treatment.

Adolescent↗
PubMed2006

Comparative genomics of fungal allergens and epitopes shows widespread distribution of closely related allergen and epitope orthologues.

BACKGROUND: Allergy is a common debilitating and occasionally life threatening condition. The fungal kingdom contains a number of species that produce a wide range of well defined protein allergens although the vast majority of fungal species have unknown allergenic potential. The recent genome sequencing of a variety of fungi provides the opportunity to assess the occurrence of allergen orthologues across the fungal kingdom. Here we use comparative genomics to survey the occurrence of allergen orthologues in fungi. RESULTS: A database of 82 allergen sequences was compiled and used to search 22 fungal genomes. Additionally we were able to model allergen structure for representative members of several highly homologous allergen orthologue classes. We found that some allergen orthologue classes that had predicted structural congruence to allergens and allergen epitopes were ubiquitous in all fungi. Other allergen orthologues classes were less well conserved and may not possess conserved allergen epitope orthologues in all fungi. A final group of allergen orthologues, including the major allergens Asp f 1 and Alt a 1, appear to be present in only a limited number of species. CONCLUSION: These results imply that most fungi may possess proteins that have potential to be allergens or to cross react with allergens. This, together with the observation that important allergens such as Asp f 1 are limited to genera or species, has significant implications for understating fungal sensitization, and interpreting diagnosis and management of fungal allergy.

Allergens↗
PubMed2002

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.

Allergens↗
PubMed2004

Hev b 5 and Hev b 13 as allergen markers to estimate the allergenic potency of latex gloves.

BACKGROUND: Sensitization to natural rubber latex has been linked to proteins from medical latex gloves. Various assays to estimate the amount of residual allergenic proteins extractable from latex gloves to assess their potential exposure hazard have inherent weaknesses. OBJECTIVE: This investigation was aimed at developing 2-site immunoenzymetric assays and identifying appropriate protein markers to assess the allergenic potential of latex gloves. METHODS: The presence of 6 latex allergens--Hev b 1, 2, 3, 5, 6, and 13--was measured in a cross-section of commercial latex medical gloves by using monoclonal and polyclonal antibody-based 2-site immunoenzymetric assays. The overall allergenic potential of these gloves was assessed by IgE-inhibition assay. Stepwise multiple regression analyses were performed to identify marker allergens that best explained the variation in latex glove allergenicity. RESULTS: All 6 latex allergens were detected in at least some of the glove samples. Hev b 5 and Hev b 13 were identified as the marker allergens that combined best to explain the variation in the glove allergenicity. The significant multiple correlation (R=0.855) between these 2 markers and glove allergenic potency forms the basis of an assay to gauge latex glove allergenicity. CONCLUSION: The overall allergenic potential of latex gloves can be estimated by using Hev b 5 and Hev b 13 as indicator allergens. The correlation between glove allergenicity and the level of these allergens was maintained for low-protein gloves (<200 microg/g). This estimation of glove allergenicity was superior to that obtained by using total protein readings.

Allergens↗
PubMed1997

Performance characteristics of a new automated enzyme immunoassay for the measurement of allergen-specific IgE. Summary of the probability outcomes comparing results of allergen skin testing to results obtained with the HYTEC system and CAP system.

BACKGROUND: Measurement of allergen-specific IgE may assist in the diagnosis of allergy in selected patients. The development of new assays for determination of allergen-specific IgE should be optimized with respect to analytical sensitivity, precision, automation, and reporting of test results in mass units. OBJECTIVE: The objectives of this study were to evaluate the analytical performance of the new automated HYTEC method for the measurement of allergen-specific IgE and to compare the performance of this new assay to that of the Pharmacia CAP system. METHODS: To demonstrate parallelism, allergen-specific IgE dilution curves were generated by diluting four patient sera containing cat (19.8 kU/L), D. pteronyssinus (5.4 kU/L), birch pollen (2.6 kU/L), and timothy grass (1.0 kU/L) with equine serum. The precision study utilized sera from three different atopic donors with low levels (0.62 +/- 0.05 kU/L), moderate levels (1.45 +/- 0.18 kU/L), and high levels (13.59 +/- 0.89 kU/L) of allergen-specific IgE to three common inhalant allergens: D. pteronyssinus, timothy grass, and birch. Sera for outcome probability determinations were obtained from 54 patients who were evaluated for the presence of inhalant allergy by skin prick test and physical examination. Only sera from patients with at least one positive skin prick test that clinically correlate with the physician's evaluation of case history and physical examination were selected for study material. In this study, sensitivity and specificity are conditional probabilities describing performances of the CAP system and the HYTEC system to skin prick testing. The lowest threshold, 0.35 kU/L, recommended for detection of allergen-specific IgE was used for both systems. RESULTS: The HYTEC system accurately detected a reduction in allergen-specific IgE antibody with different serum concentrations of allergen-specific IgE ranging from 0.10 kU/L to 20 kU/L. The median interdilutional coefficient of variation of 12.5% was obtained with assay samples containing 19.8 to 0.1 kU/L allergen-specific IgE antibody. During a 20-day trial period, three standard allergen-specific IgE controls, 0.62, 1.45 and 13.39 kU/L, respectively, demonstrated a mean coefficient of variation less than 18%. In 20% of the patients, duplicate assay determinations of allergen-specific IgE measurements resulted in a one-class discrepancy in the lowest assay range, 0.35 to 0.70 kU/L, only. The correlation coefficient between the HYTEC and CAP allergen-specific IgE assays was 0.77, rank correlation coefficients, being allergen-dependent and ranging from 0.62 to 0.91. Allergen-specific IgE assay sensitivity of the HYTEC system ranged from 0.78 to 1.00, whereas the assay sensitivity for the CAP system ranged from 0.50 to 1.00. Assay specificity ranged from 0.66 to 0.93 for the HYTEC system and from 0.70 to 0.87 for the CAP system. Eighty-nine percent of HYTEC-positive patients had a positive skin prick test (3% standard error) and 87% of CAP-positive patients had a positive skin prick test (4% standard error). Ninety-two percent of HYTEC-negative patients had a negative skin prick test (4% standard error), whereas 76% of CAP-negative patients had a negative skin prick test (5% standard error). CONCLUSION: The HYTEC system fulfills the current analytical requirements necessary to measure allergen-specific IgE antibody quantitatively and qualitatively, and compares favorably in performance with the CAP system.

Allergens↗
PubMed1999

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.

Air Pollutants↗
PubMed1995

Isolation and characterization of the 18-kDa major apple allergen and comparison with the major birch pollen allergen (Bet v I).

The major allergen from birch pollen, Bet v I, and the cross-reacting 18-kDa major allergen from Golden Delicious and Granny Smith applies were isolated by micropreparative SDS-PAGE followed by electroelution. In the case of apples, highly active, low-temperature extracts were used. The purity of the allergens was checked by analytic SDS-PAGE and immunoblotting with allergic patients' sera, as well as by N-terminal amino acid microsequencing, and the allergens were found to be very pure. The strong immunologic activity of the isolates was determined by the enzyme allergosorbent test (EAST) and EAST inhibition assays; this activity was, in the case of Bet v I, similar to that of a preparation obtained by monoclonal antibody affinity chromatography. The allergenic potency of Bet v I and of the cross-reactive apple allergen was determined by EAST inhibition and dose-related histamine release. With both assay systems, the allergenic reactivity of Bet v I was considerably higher than that of the major apple allergen. Furthermore, skin prick tests with the purified allergens and with whole allergenic extracts were performed on a group of 33 patients suffering from birch-pollen and apple hypersensitivity, and on a control group of 10 patients. The frequency of positive prick test results in the allergic patient group ranged from 73% for the major allergen from Golden Delicious apples to 97% with Bet v I and whole birch pollen extract, respectively. In contrast to our low-temperature extracts, commercial prick test solutions of four different manufacturers were found to be unreliable for the diagnosis of apple allergy. The skin test results again indicated the strong immunologic activity of the allergen isolates and the predominance of the major allergens in context with birch-pollen and apple hypersensitivity. Taken together, the results support the view that the 18-kDa major allergen represents most of the allergenicity of the the apple fruit, and that all allergenic epitopes of the apple proteins are present on Bet v I.

Allergens↗
PubMed1991

Group V allergens in grass pollens. II. Investigation of group V allergens in pollens from 10 grasses.

In an earlier study an allergen from Phleum pratense (timothy) pollen, Phl p V, has been isolated and physicochemically characterized. In this study Phl p V and immunochemically similar components from other grass pollens (group V allergens) have been investigated using immunoelectrophoretic techniques. To study the allergenic importance of the group V allergens, the allergenic compositions of 10 grass pollen extracts were investigated in crossed radioimmunoelectrophoresis (CRIE) using 20 sera from grass pollen-allergic donors. Group V allergens were identified using monospecific rabbit antibodies raised against Phl p V, anti-Phl p V, which react with other group V allergens usually producing dense precipitates in immunoelectrophoresis. In this way group V allergens were identified in eight extracts, and when present the precipitate corresponding to the group V allergen was the dominant IgE binding precipitate. All identified group V allergens bound IgE in at least 17 of the 20 investigated sera. Monospecific rabbit antibodies raised against the group I allergen of Lolium perenne (rye grass), anti-Lol p I, do not precipitate group V allergens, indicating that there are no immunochemical similarities between group I and group V allergens. In SDS-PAGE anti-Phl p V identifies IgE-binding components with molecular weights between 26 and 33 kD. In contrast, anti-Lol p I binds to components of slightly higher molecular weight. Apparently, the group V components are allergens that are physicochemically and immunochemically distinct from group I allergens.

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