Antigenic/allergenic analysis of basidiomycete cap, mycelia, and spore extracts

Since airborne basidiospores may be important inducers of respiratory allergy, extracts of spores, caps and mycelia from Pleurotus ostreatus were studied by immunologic techniques. Crossed immunoelectrophoresis of the Pleurotus spore extract showed it to be a complex mixture containing at least 27 precipitating antigens. Crossed-line immunoelectrophoresis comparing Pleurotus spore extract with extracts of Pleurotus cap or mycelia demonstrated both common antigens and antigens unique to the spore extract. Inhibition of Pleurotus spore RAST by extracts of Pleurotus spore, cap, and mycelia suggested that these extracts contained common allergenic components. However, wheal and flare skin reactivity of allergic patients to extracts of P. ostreatus or Cantharellus cibarius demonstrated little correlation between reactivity to cap and spores. These results demonstrate that basidiomycete spore extracts are the best diagnostic reagents to use in clinical studies, although cap and mycelia extracts may provide useful material for further allergen analysis.     

Basidiospore allergens: determination of optimal extraction methods

Five methods were tested by RAST for allergen extraction from seven basidiospore species Armillaria tabescens, Chlorophyllum molybdites, Coprinus quadrifidus, Pleurotus ostreatus, Calvatia cyathiformis, Pisolithus tinctorius , and Scleroderma sp. With each basidiospore type, extracted allergen activity varied according to the method employed. In general, defatting of spores with ethyl ether, followed by homogenization in 0.125 m NH₄HCO₃ buffer, resulted in greatest allergen yield. In order to compare extracts from different isolates, batches of Pleurotus ostreatus spores obtained from different locations and over different time periods were analysed. Spores harvested from the same basidiomycete species in different areas varied in allergen (RAST) and protein (HPLC profile) content. The spores obtained from the same location over a 1–2 year period did not differ significantly. These results indicate that basidiospores can be stored for several years and that spore extracts from different locations can vary. These studies will help in the future to provide better characterized extracts for clinical studies.     

Basidiospore extracts: evidence for common antigenic/allergenic determinants

Spore extracts, prepared from Armillariella tabescens, Pleurotus ostreatus, Coprinus quadrifidus, Amanita muscaria, Ganoderma lucidum, Psilocybe cubensis, Pisolithus tinctorius, Scleroderma sp. and Calvatia cyathiformis, were examined for antigenic/allergenic relationships by Ouchterlony and radioallergosorbent testing (RAST) inhibition, respectively. Ouchterlony, using hyperimmunized rabbit sera, demonstrated a high degree of cross-antigenicity among the extracts tested; however, some unique antigens were also present. RAST inhibition, evaluated by comparing extract concentrations which inhibited the RAST by 50% (IC-50), varied with the allergen tested. P. cubensis was the most potent inhibitor (IC-50 ranged from 0.034 mg/ml for A. tabescens RAST to 0.29 mg/ml for G. lucidum RAST). P. tinctorius was the least potent inhibitor, failing to reach IC-50 at 10 mg/ml for any basidiospore extract. Evaluation of slopes and intercepts of the dose-response lines demonstrated qualitative and quantitative differences among allergens in these extracts. These results indicate the presence of shared allergenic epitopes, and suggest that representative extract panels could be developed for future use in diagnosis and treatment of basidiospore-sensitive individuals.     

Immunoblot analysis of allergens in crude mosquito extracts

Whole body extracts of Aedes albopictus (AAL), Aedes aegypti (AAE) and Culex quequectates (CQU) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. At least 28, 37 and 26 polypeptides, molecular weights (MWs) ranging from 16 to 120 kilodaltons (kD), respectively, were visible following Commassie brilliant blue staining. Sera of two mosquito-sensitive patients were examined for IgE binding to mosquito proteins by immunoblotting. Sera from both patients revealed IgE binding to 52- and 56-kD proteins of AAL. In addition, serum from patient B also bound 48 kD of AAL and did not bind any components of AAE and CQU. Serum from patient A also bound 32 and 58 kD of AAE, and bound only 58 kD of CQU. Sera from 2 control subjects, nonatopic and mosquito-negative atopic, did not bind specific human IgE to any proteins, as demonstrated by negative autoradiographs. Immunoblot analysis showed unique individual IgE-binding patterns and suggested that both genus- and species-specific mosquito allergens exist.     

Identification of spore allergens from the indoor mould Aspergillus versicolor

Background:  Indoor mould growth and dampness are associated with respiratory health effects and allergies and several studies demonstrated that mainly Aspergillus versicolor and Penicillium expansum are responsible for indoor mould exposure. In contrast, commercialized test systems to diagnose allergic reactions to this mould species are not available. In this study, allergenic proteins from spores of the indoor relevant species A. versicolor and P. expansum should get detected and identified.
Methods:  We used two-dimensional (2D)-gel electrophoresis of spore proteins and immunoblotting with sera from patients participating in an epidemiologic study about indoor exposure of moulds and their influence on the development of allergies (ESTERSPEGA). Sera were screened for IgE antibodies specific for proteins from A. versicolor , A. fumigatus and P. expansum in one-dimensional blots and in 2D immunoblots. From the 2D gels, the corresponding spots were picked and identified by mass spectrometry.
Results:  More than 20 allergens from A. versicolor were identified; in particular, seven major allergens were selected, which were detected by more than 90% of the positive sera. The most abundant allergen was glyceraldehyde-3-phosphate dehydrogenase, followed by an unnamed protein, which displays a high homology to sobitol/xylose reductase. The other allergens were identified as catalase A, hypothetical protein AN6918.2, enolase, hypothetical protein AN0297.2 and a protein with homology to a fungal malate dehydrogenase.
Conclusions:  The results indicate an important role of spore proteins from A. versicolor for sensitization against indoor moulds and identification of the major allergens might enable species-specific diagnosis of allergic reactions.     

Sublingual immunotherapy: from biological extracts to recombinant allergens

Sublingual vaccines based on biological extracts from various natural allergen sources are effective in the treatment of respiratory allergies. These vaccines comprise a complex mixture of proteins and glycoproteins that require dedicated standardization procedures to ensure batch-to-batch consistency. Because of the lack of correlation between the potency of an allergen extract and the quantity of major allergen content, standardization is achieved predominantly by determining the global IgE binding capacity of the extract in vitro . New proteomic technologies can be used to further characterize the most abundant proteins present in an extract. Second-generation sublingual vaccines based on recombinant allergens are under development. The aim is to produce molecularly defined vaccines that exhibit superior efficacy, while allowing for simplified immunization schedules. In this approach, recombinant DNA technology is used to express highly purified allergens in their native (i.e. wild-type) conformation. The recombinant allergens are then formulated with ad hoc adjuvants and/or mucoadhesive galenic excipients so that they specifically target oral Langerhans cells and induce allergen-specific regulatory T cells.     

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

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

河虾主要过敏组分的分离、纯化、鉴定及其致敏性分析

目的:鉴定河虾中的过敏原组分,对主要过敏原组分进行纯化并分析其致敏性强弱。方法:用磷酸盐缓冲液(PBS)制备河虾蛋白粗提液,将其与11例虾过敏症患者血清IgE进行Westernblot,鉴定各种分子量的河虾过敏原组分;河虾蛋白粗提液用硫酸铵沉淀、G-50凝胶层析和阴离子交换层析等方法纯化其主要过敏原组分,再用虾过敏患者血清IgE进行Westernblot鉴定;并将纯化的相对分子质量(Mr)为21000、36000、800003种主要过敏原组分与虾过敏症患者血清IgE做间接ELISA,以分析其致敏性强弱。结果:West-ernblot结果显示,河虾蛋白粗提液出现9个阳性条带;其中3种主要过敏原组分21000、36000、80000与患者血清的反应率为36.4%,63.6%,45.5%;将这3种Mr的蛋白组分分别做间接ELISA,结果显示21000、36000、80000过敏原组分与11例虾过敏症患者的混合血清IgE结合的吸光值都显著高于河虾蛋白粗提液。结论:河虾中至少存在9个过敏原组分;21000、36000、8000等3种蛋白组分为河虾的主要过敏原组分,其中以36000过敏原组分致敏率和致敏性最强。进一步研究将探明21000、36000、80000等3种河虾过敏原组分的共同抗原表位,以期为明确食物过敏原检测、临床诊断和虾过敏原疫苗设计提供基础。     

The stability of house dust mite allergens in glycerinated extracts

BACKGROUND: Mite allergen vaccines are important diagnostic and immunotherapeutic reagents. Previous studies on mite allergen stability under different storage conditions have yielded contradictory results. OBJECTIVE: We sought to compare, over a 12-month period, the stability of mite allergens reconstituted in 50% glycerol and stored at different temperatures and to examine the role of protease inhibitors in enhancing allergen stability. METHODS: Lyophilized allergen extracts were reconstituted in 50% glycerol, with and without protease inhibitors, and stored at -70 degrees C, -20 degrees C, 4 degrees C, or 37 degrees C for 12 months. At 6 and 12 months, the extracts were compared with freshly dissolved extracts by competition ELISA with pooled allergic sera, 2-site ELISA with mite-specific mAbs, and immunoblot analyses. RESULTS: The overall potencies of the stored extracts measured by competition ELISA were stable at -20 degrees C and 4 degrees C. As determined by means of the immunoblot and 2-site ELISA, Der f 1 levels decreased at 4 degrees C. Levels of Der f 2, Der p 1, and Der p 2 decreased in at least one of the allergen-specific assays. Storage at 37 degrees C led to overall loss of potency and allergen content, whereas storage at -70 degrees C was associated with a moderate loss of potency that increased with multiple freeze-thaw cycles. Protease inhibitors had no effect on allergen stability. CONCLUSION: Although overall potency of the extracts, as measured by competition ELISA, was preserved at -20 degrees C and 4 degrees C, allergen-specific assays indicated loss of allergens. These findings suggest that the competition ELISA is insensitive to decreases in the concentrations of individual allergens.     

Extraction and analysis of coffee bean allergens

Workers in the coffee industry can develop occupational allergic disease upon exposure to dust associated with coffee manufacturing. Since controversy exists as to the source or chemical nature of these allergens, the mouse model of reaginic antibody production was used to assess the potential sources of allergens in samples obtained from a local coffee manufacturing plant. Mice were immunized with extracts of coffee dust and beans and the resulting reaginic antibody response determined by the passive cutaneous anaphylaxis reaction. Cross-reacting allergens were detected in samples of coffee dust, cleaner can debris and green coffee beans, but not in chaff or roasted coffee beans. None of the allergens detected in coffee samples cross-reacted with extracts of castor beans, although these extracts contained the potent castor bean allergen. Green coffee bean allergens partially purified by gel filtration were heterogeneous with respect to molecular size, although quite similar in their reactivity with reaginic antiserum. These results suggest that the green coffee bean is the major source of allergen in coffee manufacturing plants. This allergen is heterogeneous with respect to size and heat lability, and is immunochemically different from the castor bean allergen.     

Analysis of the mitochondrial and nuclear genomes of two basidiomycetes,Coprinus cinereus and Coprinus stercorarius

Summary The mitochondrial and nuclear genomes of Coprinus stercorarius and C. cinereus were compared to assess their evolutionary relatedness and to characterize at the molecular level changes that have occurred since they diverged from a common ancestor. The mitochondrial genome of C. stercorarius (91.1 kb) is approximately twice as large as that of C. cinereus (43.3 kb). The pattern of restriction enzyme recognition sites shows both genomes to be circular, but reveals no clear homologies; furthermore, the order of structural genes is different in each species. The C. stercorarius mitochondrial genome contains a region homologous to a probe derived from the yeast mitochondrial var1 gene, whereas its nuclear genome does not. By contrast, the C. cinereus nuclear, but not mitochondrial, genome contains a region homologous to the var1 probe. Only a small fraction of either the nuclear or mitochondrial genomes, perhaps corresponding to the coding sequences, is capable of forming duplexes in interspecies solution reassociations, as measured by binding to hydroxylapatite. Those sequences capable of reassociating were found to have approximately 15% divergence for the mitochondrial genomes and 7%–15% divergence for the nuclear genomes, depending on the conditions of reassociation.     

Allergenic extracts to diagnose and treat sensitivity to insect venoms and inhaled allergens

Objective

To review allergenic extracts used to diagnose or treat insect allergies, including how the extracts are manufactured and their measurements of potency or concentration.

花粉变应原的分类和相关的进化分析

目的 分析各种花粉变应原所属的蛋白家族,统计各类蛋白作为变应原出现的次数和在各科植物间的分布情况,结合进化树分析,以了解花粉变应原在自然界分布的一般规律.方法 通过NCBI数据库获取目前已知的所有变应原.用批处理(Batch Entrez)获得全部的氨基酸序列.将每个序列与Pfam数据库比对,以确定各种花粉变应原所属的蛋白家族.对成员众多的Profilin、Ex-pansin家族,应用BLAST搜索变应原的同源序列,获取序列号,Batch Entrez获得全部的氨基酸序列,最后用MEGA4.0软件生成进化树.结果 目前已知的168个花粉变应原,分属于26个蛋白家族.其中,Profilin、pollen_allerg_1和EF hand是3种成员最多的变应原家族,分别有25、20、19种变应原,占总数的38%.10个排名靠前的蛋白家族,变应原数量占总数的79%.在花粉变应原家族中,既有Profilin般分布广泛的,几乎涉及所有的科;也有局限于某科植物的如Ribonuclease、FAD_binding pro-tein、Amb_V、Thaumatin等.通过进化分析可知,各种Profilin变应原高度同源,变应原序列在不同物种间具有高度保守性.禾本科花粉的β-Expansin与无变应原性的Expansin分开进化.结论 通过对花粉变应原的蛋白家族分类,可为变态反应学的基础研究、过敏性疾病的临床诊疗提供参考,并有助于快速发现新的致敏物种和新的变应原.Profilin的高度保守性可能是交叉反应(cross reactivity)发生的主要原因之一.     

Crossed radioimmunoelectrophoretic studies of distinct allergens in two extracts of Cladosporium herbarum

Cladosporium herbarum was supplied from two sources and extracted identically. Antisera against the extracts were produced in rabbits and two reference patterns were established using crossed immunoelectrophoresis (CIE). Both patterns showed more than 60 precipitates but less than 50% of the detectable antigens appeared to be identical in the two extracts. The allergens were identified by means of crossed radioimmunoelectrophoresis (CRIE) using sera from 35 individuals with proven or suspected allergy to C. herbarum. Four important and 10-20 less important allergens were demonstrated. Among the allergens present, there were none reacting with all patient sera. Only 1 out of 3 rabbits immunized with a suspension of broken cells of C. herbarum showed precipitating antibodies to the statistically most important allergen, while 9 rabbits immunized with aqueous extracts of the mold did not. The composition of the two extracts with respect to allergens differed. Allergens present in one extract were not always detectable in the other. The experiments also showed how CIE/CRIE with various combinations of antigens and antisera may be combined with CRIE inhibition, radioallergosorbent test (RAST) and RAST inhibition for comparing complex allergen extracts at the molecular level.     

Proteomic analysis of putative latex allergens

BACKGROUND: Extensive analysis of allergenic proteins is generally time-consuming and labor-intensive. Accordingly, a rapid and easy procedure for allergen identification is required. As sequence information on proteins and genes is accumulated in databases, it is becoming easier to identify a candidate protein using proteomic strategies, i.e. two-dimensional gel electrophoresis, site-specific fragmentation, mass spectrometry and then database search. In this study, we evaluated the usefulness of a proteomic strategy for identifying putative allergens through its application to latex proteins. METHODS: Latex proteins were separated with two-dimensional gel electrophoresis, and putative allergens were visualized by IgE immunoblotting using pooled serum from latex-sensitive patients. The IgE-interactive proteins were cut out from the negatively stained two-dimensional gel and subjected to in-gel digestion by trypsin. Then the resulting peptides were analyzed with mass spectrometry. Based on the mass spectrometric data we obtained, the allergen candidates were assigned by a database search. RESULTS: Five previously reported allergens and five new allergen candidates were identified with the proteomic approach without isolating the individual proteins. Less than 1 mg of crude latex protein was sufficient for the entire protocol. Because plural proteins can be processed in parallel, analysis of about 50 IgE-interactive proteins was accomplished within 1 week. CONCLUSIONS: Analysis of putative allergens with proteomic strategies (allergenomics) is a promising avenue for rapid and exhaustive research. The high resolving power of two-dimensional gel electrophoresis is superior to conventional gel electrophoresis. Moreover, the notable sensitivity and speed of mass spectrometry have pronounced advantages over the N-terminal sequencing that has generally been used for protein identification.