Isolation and immunochemical characterization of the major allergen of birch pollen (Betula verrucosa)

The classification of some of the extractable birch pollen antigens as allergens was established by crossed radioimmunoelectrophoresis (CRIE). In CRIE the major allergen (antigen 23) exhibited the strongest “radiostaining” and only a few other components of birch pollen extract were visibly radiostained. The major allergen and a preparation containing mainly the minor allergens, antigens 25 and 19, were isolated from a crude aqueous birch pollen extract by a combination of anion-exchange, size-exclusion, and chelate chromatography. Antigen 23 was purified to near homogeneity. The molecular weights and the pIs of antigens 23, 25, and 19 were determined to be 17,000 daltons, pI 5.25 (5.5, 5.0); 25,000 daltons, pI 5.0 (4.9, 5.4); and 29,000 daltons, pI 6.2 (5.4), respectively. The classification of antigen 23 as the major allergen in birch pollen was supported by results of RAST inhibition experiments, RAST screening, and skin prick testing.     

Immunotherapy with partially purified and standardized tree pollen extracts

Fifty-four adult patients with tree pollen-induced rhinitis (28), asthma (1), or rhinitis and asthma (25) were selected for immunotherapy with standardized and partly purified tree pollen extracts using a double blind protocol. The selection was based on clinical history, results of nasal or bronchial challenge, skin prick tests and RAST. Further, based on crossed radio-immunoelectrophoresis, sex, age and severity of symptoms, the patients were allocated in matched pairs and the treatment alternatives were randomly distributed within the pairs. Twenty-three patients treated with extracts composed of any combination of alder, birch and hazel pollen which matched their IgE response in CRIE (Group 1 (ABC)) and 22 patients receiving birch pollen extracts (Group 2 (B)) completed all 3 years of treatment. The in vivo results comprising symptom and medicine consumption scores are given here. Changes in specific skin and nasal reactivity as well as in immunological parameters are presented separately. No significant differences were demonstrated between the treatment groups in the two parameters. Both extracts were effective and reduced in general the symptom scores to one tenth of the starting level. Expressed another way, at the end of the study, the patients tolerated 30 times more pollen until symptoms of the same severity were elicited, compared to before. In the Nordic countries, spring-time asthma and rhino-conjunctivitis caused by pollen from deciduous trees can be effectively treated with an extract of birch pollen alone.     

PCR based cloning and sequencing of isogenes encoding the tree pollen major allergen Car b I from Carpinus betulus, hornbeam.

Cloning of the gene encoding the major allergen, Car b I, from Carpinus betulus (hornbeam) pollen was performed using the Polymerase Chain Reaction (PCR) to specifically amplify the gene of interest using single stranded cDNA as template. Specific primers, deduced from the aminoterminal sequence of the purified protein, were tailored to facilitate direct expression of plasmic clones, and the large fraction of positive clones obtained, revealed the presence of isogenic variation. Three clones were characterized in detail by antibody based assays and nucleotide sequencing. The recombinant allergens were shown by crossed immunoelectrophoresis (CIE) to precipitate with monospecific polyclonal rabbit antibodies raised against purified Bet v I, by crossed radioimmunoelectrophoresis (CRIE) to bind tree pollen allergic patient serum IgE, and by immunoblotting to bind murine monoclonal antibodies, raised against purified Car b I from pollen. Car b I is encoded by a 159-triplets open reading frame. The molecular masses (M(r) = 17272, 17355 and 17217 Da, respectively), the amino acid composition, and the aminoterminal sequence of the predicted polypeptides agree well with data obtained by analysis of the protein purified from pollen. The deduced amino acid sequences show pronounced homology (73, 75 and 74% identities respectively) to Bet v I, the major allergen from Betula verrucosa (white birch) pollen. Soluble recombinant Car b I, without a fusion partner, was produced in Escherichia coli with an immunochemical reactivity closely resembling that of the native pollen allergen. The tree pollen major allergens therefore constitute an ideal system for the study of allergenic epitopes.     

The international collaborative study establishing the first international standard for timothy (Phleum pratense) grass pollen allergenic extract

A selected candidate international reference preparation of timothy grass (Phleum pratense)-pollen extract was studied together with two other freeze-dried timothy pollen allergenic extracts in a multinational study. The collaborators used RAST inhibition, histamine release, quantitative immunoelectrophoresis (crossed immunoelectrophoresis/crossed radioimmunoelectrophoresis and rockets), isoelectric focusing, and other methods. The total allergenic potencies measured in RAST inhibition were evaluated for validity of linearity and parallel-line response. The relative concentrations of some important individual allergenic components were measured. The relative potencies for the total allergenic activity and the timothy components studied in each preparation were expressed relative to the selected candidate. This preparation was established in 1983 by the World Health Organization expert committee on biologic standardization as the international standard for timothy grass-pollen extracts with assigned units of 100,000 IU per ampule.     

Crossed radioimmunoelectrophoretic analysis of cultivated rye (Secale cereale) pollen allergens

Cultivated rye pollen extracts were characterized by means of crossed immunoelectrophoresis (CIE) and crossed radioimmunoelectrophoresis (CRIE). 32 antigens were identified in CIE and among these 16 were radiostained in CRIE analysis of a rye pollen sensitive patient panel. Crossed line immunoelectrophoresis revealed that some of the rye pollen antigens were immunological partially identical with antigens of wheat flour and rye flour.     

Influence of the pH of the extraction medium on the composition of birch (Betula verrucas a) pollen extracts

Extracts from birch ( Betula verrucosa ) pollen were prepared at different pH, with constant pH monitoring and adjustment to preset values in the range 5.5-8.5. The total protein content of these extracts was directly correlated with the pH. Coomassie brilliant blue-stained isoelectric focusing and SDS-PAGE gels and immunoblot analysis demonstrated qualitative differences: some proteins were lost while others appeared when pH was changed. At pH 8.5, formerly unknown birch pollen allergens were detected with pi 9, 9.10, and 9.30 by about 30% of birch pollen-sensitive sera. Birch pollen extracts prepared at a pH close to neutrality, namely, 6.5 and 7.5, showed the greatest protein and different allergen diversity. Thus, extraction pH values are necessary to analyze the whole pattern of allergenic components in an extract.     

Comparative studies on tree pollen allergens. XIV. Characterization of the birch (Betula verrucosa) and hazel (Corylus avellana) pollen extracts by horizontal 2-D SDS-PAGE combined with electrophoretic transfer and IgE immunoautoradiography

The crude aqueous extracts of birch (Betula verrucosa) and hazel (Corylus avellana) pollen were characterized by horizontal isoelectric focusing (IEF) and one-dimensional and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1-D and 2-D SDS/PAGE). Isoelectric focusing reference patterns obtained in pH 3.5 to 9.5 gradient gels contained 31 and 41 bands while the 2-D patterns consisted of 51 and 72 protein spots, respectively. Electrophoretic transfer to nitrocellulose membranes and subsequent 125I-immunoautoradiography demonstrated IgE binding in the region between pH 4.8 and 5.4 for both extracts. The main allergenic reactivities of this pH region were distributed by SDS/PAGE to the molecular weights of 29.5, 17, and 12.5 kilodaltons for birch and 15.5 and 12 kilodaltons for hazel. The high resolving power and the independence of rabbit antibodies were considered the major advantages of the described 2-D methodology as compared with conventional crossed immunoelectrophoretic and radioimmunoelectrophoretic techniques.     

The Use of a Reference Allergenic Extract in the Evaluation of Allergen Products

Two commercially available mugwort (Artemisia vulgaris (Av] pollen extracts Av-ALK and Av-PS were compared with a dialysed reference mugwort pollen extract (Av-REF) by means of crossed immunoelectrophoresis (CIE), crossed radio immunoelectrophoresis (CRIE), RAST-inhibition, RAST-screening and skin prick test. Av-ALK and Av-PS both contained approximately half the number of precipitation arcs detectable in Av-REF, and Av-PS seemed to lack two intermediate allergens and two minor allergens, while Av-ALK seemed to lack one minor allergen. The RAST-inhibition curve obtained for Av-PS did not parallel the curves obtained for Av-ALK and Av-REF, indicating that the allergen composition of Av-PS differs from that of the others. Further, RAST-screening experiments support the CIE observation that Av-PS seemed to lack two of the intermediate allergens. The HEP values determined for Av-ALK and Av-PS diverge considerably from those stated by the manufacturers. The results obtained demonstrate that the outcome of a comparison between a reference allergenic extract and allergenic products depends very much on the method employed.     

Antigens and Allergens in Birch Pollen Extract

More than 70% of the total allergenic activity of a birch pollen (BP) extract was detected within the first 30 min of extraction. Fractionation of the BP extract by gel filtration and analysis of the eluted antigens by a fused rocket immunoelectrophoresis revealed at least three antigens with molecular weights of about 29 000, and 17 000-10 000, corresponding to antigens Nos. 7-8 and No. 2, respectively, in crossed-immunoelectrophoresis (CIE) and in crossed-radioimmuno-electrophoresis (CRIE). Gel isoelectrofocusing of the pooled allergenic fractions revealed two major protein bands with pI's around 5.6 and 5.7, probably corresponding to antigens Nos.7-8 and No. 2, respectively. Antigens Nos. 7-8 were thermoresistant, while antigen No. 2 was thermolabile. The allergenic activity was determined by prick skin testing and by the RAST inhibition method. More than 90% of the allergenic activity in the fractions was located in the protein peak C (mol. wt. 10 000-17 000) containing antigens 7-8. About 30% of the total allergenic activity of the extract (1:10 w/v) was recovered in the peak C fractions, and only less than 0.5% outside these fractions. Higher allergenic activity was obtained for the peak B fractions (mol. wt. 29 000) by skin prick testing than by the RAST. Peak B contained allergens (antigen 2) distinct from those of peak C by the CRIE and by the RAST. The allergenic material in the low molecular weight fractions of peak D (mol. wt. 2000-5000) was allergenically similar to that of peak C in the RAST. Only weak and even negative skin reactions were observed with the peak D fractions in allergic subjects.     

Comparative studies on tree pollen allergens. VIII. Immunological properties of the alder (Alnus incana) pollen extract

The immunological properties of the aqueous crude alder pollen extract (AI crude) and gel filtration fractions AI 3, AI 4 and AI 34 (pool of fractions AI 3 and AI 4) were examined by immuno- and radioimmuno-electrophoretic techniques, RAST titration, RAST inhibition and skin prick tests (SPT). In CIE, the AI crude extract and AI 34 displayed reference precipitate patterns consisting of 27 and 24 visible Coomassie brilliant blue stained lines, respectively. The CRIE allergogram performed by incubation with 18 individual reaginic sera detected three IgE-binding antigens characterized by different IgE-binding properties. Antigen No. 7 (Ag 7) was demonstrated to be the major IgE antibody-binding antigen of alder pollen, while Ag 1 and Ag 11 were classified as intermediate allergens. The allergens of alder pollen were located in fractions AI 3 and AI 4 of the gel filtration chromatogram. Ag 7 was present in both fractions as demonstrated by FRIE with autoradiography (FRIEWA) on the gel filtration fractions and tandem-CRIE of AI 3 and AI 4. The CRIE allergogram, RAST, RAST inhibition and SPT demonstrated fraction AI 34 to be allergenically representative of the AI crude extract both qualitatively and quantitatively. Thus, fraction AI 34 was considered an optimal purified allergen extract of alder pollen, a suitable material for further biochemical characterization and trials on purification of the allergenic reactive antigens.     

Allergenic cross-reactivity of olive pollen

BACKGROUND: Sera of patients allergic to olive (Olea europaea) pollen were used to analyze the IgE cross-reactivity between olive-pollen extract and other pollens obtained from phylogenetically unrelated species. METHODS: We used IgE immunostaining of pollen extracts blotted to nitrocellulose membranes after SDS-PAGE and inhibition analysis of this binding. RESULTS: A high inhibition of the IgE binding on olive-pollen extract was exhibited by birch, mugwort, pine, and cypress pollens, suggesting that these extracts contain proteins which share common epitopes and thus can be recognized by olive-allergic sera. IgE binding to Gramineae pollen extracts was not inhibited by olive-pollen extract, indicating a primary sensitization of the patients to these species. From the inhibition assays, the presence of an allergen of 45 kDa in the olive pollen, which has no homologous counterparts in other allergenic species, has been inferred. CONCLUSIONS: Olive pollen contains allergens which cross-react with pollens from unrelated species, a fact that could simplify the diagnosis and treatment of pollinosis.     

Immunological Investigation of Possible Structural Similarities between Pollen Antigens and Antigens in Apple, Carrot and Celery Tuber

Crossed line immunoelectrophoresis (CLIE) was used to show that a minor allergen present in birch pollen and another present in timothy pollen shared common epitopes with antigens in apple, carrot and celery tuber. Major pollen allergens were not involved. Structural similarities were also noticed in some mugwort pollen antigens and antigens in apple, carrot and celery, but none of these mugwort antigens acted as an allergen. In crossed immunoelectrophoresis (CIE) of samples of apple, carrot or celery extracts towards antibodies against birch pollen, grass pollen or mugwort pollen, four to 14 distinct precipitates were observed and shown to be specific. In crossed radioimmunoelectrophoresis (CRIE) none of the precipitates could be shown to act as an allergen. Using allergen detection on nitrocellulose paper, the apple, carrot and celery extracts were shown to possess IgE-binding capacity.     

Allergens from birch pollen and pollen of the European chestnut share common epitopes

Type I allergy to pollen of the European chestnut (Castanea sativa) represents a major cause of pollinosis in (sub) Mediterranean areas. Using sera from 14 patients with established allergy to pollen of the European chestnut, 13/14 sera (92%) showed IgE-binding to a 22 kD protein, 2/14 (14%) displayed additional binding to a 14 kD protein and 1/14 (7%) bound only to the 14 kD protein of European chestnut pollen extract. Two monoclonal mouse antibodies, BIP 1 and BIP 4, directed against different epitopes of Bet v I (the major birch pollen allergen), and a rabbit antibody to recombinant birch profilin (rBet v II) were used to characterize the proteins of the European chestnut pollen. The recombinant birch pollen allergens, rBet v I and rBet v II (profilin) were employed to show common allergenic structures on proteins from both birch and European chestnut pollen by IgE-inhibition experiments. Despite the fact that the 22 kD protein displayed a higher molecular weight in comparison to the 17 kD major birch pollen allergen, Bet v I, we could demonstrate reactivity of both monoclonal antibodies, BIP 1 and BIP 4, with this protein. A complete inhibiton of IgE-binding to this 22 kD protein was shown by pre-incubating sera with purified recombinant Bet v I. In addition, the 14 kD protein could be identified by IgE-inhibition studies with recombinant Bet v II and by using a rabbit anti-profilin antibody as the profilin from pollen of the European chestnut.     

Fagales pollen sensitization in a birch-free area: a respiratory cohort survey using Fagales pollen extracts and birch recombinant allergens (rBet v 1, rBet v 2, rBet v 4)

BACKGROUND: Birch allergy is one of the most common pollinosis in areas where exposure to high levels of birch pollen is common. Little is known about birch sensitivity in areas without birch pollen exposure and reactivity to birch-related species within the Fagales order. OBJECTIVE: the aim was to evaluate Fagales reactivity within a population not exposed to birch pollen using epidemiological, diagnostic, and laboratory approaches by means of extracts and allergenic molecules. METHODS: A cohort of 5335 respiratory allergic patients was screened by means of skin testing birch, hazel, and oak pollen extracts. Patients were from a birch-free area, but exposed to other Fagales pollen species. A subset of patients was from an intensively cultivated hazel area. A sample of the Fagales allergic population was tested with other Fagales pollen extract (alder, hornbeam, beech, chestnut) and with apple and hazelnut. IgE detection was performed with birch, hazel, oak, apple, and hazelnut extracts, and with Bet v 1, Bet v 2, Bet v 4, and bromelain. IgE immunoblots were performed using birch and hazel extracts. Epidemiological, clinical, and laboratory data were analysed by stratifying the allergic population. RESULTS: Twenty-five percent of the pollen allergic cohort was skin test positive to at least one of the three Fagales species. Combined reactivity to the three species was recorded in 80% of this cohort. Isolated hazel pollen reactivity was recorded in 13.5% of the Fagales allergic patients. Sixty-six percent of these subjects were from the intensively cultivated hazel area. Reactivity to apple and hazelnut was detected by skin test (40%) and IgE reactivity (60%), but only 19% of the positive patients reported symptoms related to at least one of the two foods. Reactivity to Bet v 1 was recorded in 84% of the birch/hazel/oak co-reactivity group, and in 28% of the subjects with the same co-reactivity, but associating a multiple pollen sensitization. IgE to Bet v 2 (50%) and Bet v 4 (23%) panallergens were recorded positive in the latter subset. Bet v 1 prevalence ranged between 48% and 21% among subgroups of patients coming from different areas. Furthermore, an IgE reactivity to hazel-restricted allergenic components was detected among subjects coming from the same area and having a hazel isolated reactivity. CONCLUSION: Fagales allergy can be found in birch-free areas caused by the exposure to other Fagales species. Birch allergens can be useful for mimicking the allergenic extract, but are also the exclusive tools for a fine diagnostic and epidemiological approach to Fagales pollen allergy. Allergenic molecules from the hazel family will increase the panel of available reagents for the molecule-based approach to allergy diagnosis and therapy.     

Isolation and identification of pollen allergens of Artemisia scoparia

The allergenic proteins of Artemisia scoparia pollen were separated and identified with ammonium sulfate precipitation, ion-exchange chromatography, gel filtration, and RAST-inhibition techniques. The important allergenic component Artemisia VI b that constitutes 29% of total protein in the extract was purified to homogeneity. It was found to be an acidic protein with isoelectric point 3.8 and molecular weight of 14,300. It was rich in carbohydrate, but the carbohydrate portion did not appear to be important for allergenicity. In the crossed immunoelectrophoresis reference pattern of the whole pollen extract, 37 precipitin lines could be identified on the anodic side, whereas Artemisia VI b could be observed as a single precipitin line. Immunologically, the whole pollen extract of A. scoparia demonstrated shared antigenic and allergenic determinants with Ageratum conyzoides-pollen extract. The use of fast protein liquid chromatography in partial purification of allergenic components is also discussed.     

Composition and stability of allergenic extracts made from gamma-irradiated rye grass (Lolium perenne) pollen

BACKGROUND: Phenol is commonly added to allergenic extracts as a bacteriostatic agent, but it is poisonous and also detrimental to proteins, which accelerates extract degradation. Sterilization by gamma-irradiation of the source material could be an alternative to the use of phenol. OBJECTIVE: To analyse the potential effects of gamma-irradiation of pollen on the composition, potency, and stability of the resulting extract, and compare them with those of phenol. METHODS: Ryegrass (Lolium perenne) pollen was sterilized by gamma-irradiation at a dose of 25 kGy. Extracts prepared from the irradiated pollen were then compared by electrophoresis techniques and RAST inhibition to extracts, without or with 0.5% phenol, from nonirradiated pollen. In addition, proteolytic activity was compared in extracts from irradiated and nonirradiated pollen. To evaluate the stability of extracts on storage, they were analysed after forced degradation for up to 7 days at 37 degrees C. RESULTS: When fresh extracts were analysed, there were no noticeable differences between the three types, as judged by immunoblotting and RAST inhibition experiments. However, on storage, extracts from irradiated pollen appeared to be superior to extracts from nonirradiated pollen, as some proteins were more stable in the former. This could be related to the lower proteolytic activity we have also observed in extracts from irradiated pollen. In contrast, extracts containing phenol degraded much faster, as proven by all our methods of investigation. CONCLUSION: Gamma-irradiation of pollen did not influence the IgE-binding capacity of the resulting extracts, but did yield extracts with somewhat improved stability, probably by reducing the proteolytic activity. It may be concluded that gamma-irradiation of the source material represents a good alternative to the use of phenol for the preparation of allergenic extracts.     

椰子花粉过敏原的分离、鉴定与纯化

目的 对椰子花粉的变应原组分进行初步的分离、鉴定及纯化.方法 提取椰子花粉粗提液,用十二烷基硫酸钠.聚丙烯酰胺凝胶电泳(sDS-PAGE)分离椰子花粉的蛋白质组分并测定其相对分子质量,采用免疫印迹法鉴定其变应原成分,并通过离子交换层析对椰子花粉变应原进行初步分离纯化,免疫印迹进行检测.结果 SDS-PAGE显示椰子花粉粗提液有10条蛋白带,其中相对分子质量(肘,)为60 000、50 000、35 000、28 000、19 000、16 000和14 000的蛋白可与椰子花粉过敏性病人血清IgE结合,且M,50 000、16 000和14 000为主要变应原;离子交换层析结果显示主要过敏原成分主要分布在V峰中.结论 对椰子花粉变应原进行了初步的分离、鉴定和纯化,为临床椰子花粉变态反应疾病的诊断和治疗奠定了基础.     

Selection of patients for biological standardization as exemplified by standardization of mugwort, goosefoot and English plantain pollen allergen extracts/preparations

The biological activity of a partly purified, biochemically/immunochemically characterized mugwort pollen allergen preparation and crude pollen extracts of mugwort, goosefoot and English plantain was determined by means of skin prick test (SPT). The patient inclusion criteria with mugwort were a well-defined positive clinical history and a positive SPT. Symptoms related to goosefoot/English plantain pollens are difficult to define, as these weeds flower during the grass pollen season. Thus patients tested with these allergens did not fulfill the most important inclusion criterion for so-called biological standardization. To elicit a wheal of the same size as that produced by histamine 1 mg/ml required 100 to 10,000 times more material from these weeds, than from mugwort and other pollen allergen extracts investigated earlier. One thousand Biological Units/ml (BU/ml) corresponded to 8.3 micrograms dry weight (dw/ml) of the crude and 1.8 micrograms dw/ml of the purified mugwort pollen allergen preparation. Only 7/22 goosefoot-and English plantain-tested patients were positive at conjunctival or nasal challenge. All three weeds showed a similar composition with 5-10 allergens by CIE/CRIE analysis and 10-13 by immunoblotting analysis. One dominating allergen (approx. 15,000 d), could be identified for each weed species by protein gel blot after separation by SDS g-PAGE. There was no other explanation for the difference in biological activity than the criteria of selection. If there is no obvious clinical history, which is the main patient inclusion criterion in biological standardization, then additional criteria should be used.     

A study of allergens in celery with cross-sensitivity to mugwort and birch pollens

Sixty-one sera with positive RAST to mugwort pollen ( Artemisiae vulgaris ) were submitted to RASTs for birch pollen ( Betula verrucosa) and celery ( Apium graveolens ). In 36 cases RAST results were positive for celery. In addition, 23 sera presented specific IgE to birch pollen. The binding of specific IgE to individual allergens in celery, mugwort pollen and birch pollen was studied by the immunoblotting technique. This involved electrophoretic separation of allergenic extracts, electrotransfer of proteins onto nitrocellulose sheets and sensitive immunoenzymatic detection. Eighteen sera had specific IgE binding to two celery components of molecular weight around 15 kD. All these sera also detected a 15 kD allergen in mugwort and two allergens in birch of 14 kD and 16 kD molecular weight. The sera that did not detect the 15 kD bands in celery failed to react with both the 15 kD mugwort component and the 14 and 16 kD birch components. Specific cross-inhibitions of the detection of these allergens on immunoblots were obtained by pre-incubation of the sera with crude extract of the three species. These results strongly suggest that such allergens display some structural identity and that they could be at the origin of some cases of crossed hypersensitivity to celery, mugwort pollen and birch pollen.     

The effect of cysteinyl leukotrienes on growth of eosinophil progenitors from peripheral blood and bone marrow of atopic subjects

BACKGROUND: Therapeutic allergen extracts are frequently stored as mixtures to provide preparations used for specific immunotherapy. Substantial protease activity, found in certain mold extracts, has been suspected to cause a loss of allergenic activity as a result of self-degradation, as well as by means of degradation of allergens from pollen and other allergenic sources. OBJECTIVE: This study was performed to address possible deterioration of individual pollen allergens during storage of extract mixtures, with a mold extract as the source of proteolytic activity. METHODS: Aqueous birch and timothy pollen extracts were stored for 60 days at 6 degrees C with and without addition of an extract derived from the mold Fusarium culmorum. The stability of the pollen allergens Bet v 1, Bet v 6, Phl p 1, and Phl p 5, as well as 2 to-date-undefined F culmorum allergens was examined by using immunoblotting analysis with sera from allergic patients and allergen-specific mAbs. Furthermore, the residual allergenic activity of the pollen extracts was monitored by using the rat basophilic leukemia cell-mediator release assay. Proteolytic activity of extracts was determined by using a commercial protease assay and gelatinase zymography. RESULTS: Pollen extracts were very stable, corresponding to the low proteolytic activity of these extracts. In contrast, high proteolytic activity was found for the F culmorum extract, resulting in self-degradation of mold proteins and deactivation of allergens. Similarly, the mixtures showed a strong decrease of allergenic potency in the mediator release assay. Bet v 1 and Phl p 1 were relatively stable, whereas Bet v 6 and Phl p 5 were almost entirely degraded within 1 day. CONCLUSIONS: Proteases of the mold F culmorum clearly affected the overall allergenic activity of pollen extracts within a short time period. Apart from general objections against the use of mixtures of non-cross-reacting allergens, mixing of pollen extracts with extracts derived from molds for immunotherapy is not recommended unless they are applied directly after preparation of the mixture.