Validation of an ELISA method for quantification of the major Timothy grass pollen allergen Phl p 5a (BSP090).

Progress towards standardisation of allergen products has been made in recent years. Nevertheless, no standardised test method to quantify the allergen content of grass pollen allergen products is available at present. One aim of the BSP090 project was to validate a quantitative assay for a major Timothy grass (Phleum pratense) pollen allergen, Phl p 5. Qualification of a candidate ELISA system was performed with regard to range, robustness and cross-reactivity in preliminary studies. The assay specifically detected Phl p 5 with a quantification range from 3.9 ng/mL to 62.5 ng/mL. Suitability to quantify recombinant and natural Phl p 5 was further assessed in a collaborative study including 14 laboratories in Europe and the USA. Precision and accuracy of the assay was satisfactory with 93% of calculated Phl p 5 concentrations and 100% of total recoveries being within the ± 30% acceptance range. Similar results were obtained for spike recoveries, with exclusion of the lowest concentration spike, showing spike recoveries exceeding the acceptance range for six laboratories. Inter-assay (repeatability) and inter-laboratory (reproducibility) variability were satisfactory, in the format used in the present study. Robustness towards different statistical methods for data analysis was demonstrated. In conclusion, the assay can easily be established in routine testing and results of the preliminary testing and collaborative study support the proposal of the assessed Phl p 5-specific ELISA as a European Pharmacopoeia general method.

Allergenicity and cross-reactivity of pine pollen.

BACKGROUND: Pine pollen has long been considered a non-allergenic pollen. The large size of the grain and its low levels of proteins are the main reasons invoked to explain this low allergenicity. The aim of this study was to describe the main allergenic bands of Pinus radiata (PR) and its cross-reactivity with other pine species, other conifers and grass pollen. METHODS: Sixty-five pine-pollen-allergic patients (51% also sensitized to grass pollen) were studied. Skin prick tests (SPT) to a battery of allergens including PR, Pinus pinea, Pinus sylvestris, Pinus nigra and Cupressus sempervirens pollens and specific IgE determination to PR and Pinus strobus were performed. IgE-immunoblotting to a PR extract and other pine pollens was also carried out. UniCAP inhibition and immunoblotting inhibition studies were performed to assess the cross-reactivity between different pollens. RESULTS: The SPTs were positive with all the pine pollen extracts tested in 69% of the patients. Specific IgE was positive to PR or P. strobus in 77% of the patients, and to Lolium perenne in 51%. Nine different allergenic bands were detected. The two main allergens were a 42 kDa band recognized by 85% of the patients and a band of approximately 6-8 kDa recognized by 40%. A high degree of cross-reactivity was observed between different pine pollen species, but not between pines and C. sempervirens pollen. A partial cross-reactivity could be seen between pine and grass pollens only in patients also sensitized to L. perenne. CONCLUSIONS: Pine pollen should be considered as a potential allergenic pollen especially where this pollen is abundant. The detection of a high number of patients that were monosensitized to pine pollen suggests the possibility of treating these patients with specific immunotherapy.

Understanding patient sensitization profiles in complex pollen areas: a molecular epidemiological study.

BACKGROUND: Allergy diagnosis in patients exposed to multiple pollen species is complex and misdiagnosis is often a cause for unsuccessful specific immunotherapy. OBJECTIVE: We studied the sensitization profile of individual allergens (major, minor and pan-allergens) in pollen-sensitized patients in a region with high exposure to olive pollen by investigating the influence of minor allergens on allergic disease and the association between pan- and minor allergen sensitizations. METHODS: A panel of 13 purified allergens, which included the most relevant allergens in the area, as well as minor olive allergens and pan-allergens, were screened using a high-capacity technology (ADVIA-Centaur) in 891 patients. RESULTS: Olive allergy as measured by specific IgE to Ole e 1 was the leading pollinosis in the area. The minor olive allergens Ole e 7 and Ole e 9 were markers of more severe allergic illness. Profilin sensitization was associated mainly with grass allergy, the second most prevalent pollinosis. Salsola kali pollen allergy was the third most common cause of pollinosis in the area. The prevalence of sensitization to the peach allergen Pru p 3, a nonspecific lipid-transfer protein, was notable. CONCLUSION: Epidemiological analysis by component-resolved diagnosis is a new method, which elucidates the interaction between allergen exposure gradient and patient sensitization. High exposure leads to differential sensitization profiles some of which are associated with more severe allergic conditions. Profilin sensitization, related mainly to grass pollinosis, was a marker of more severe grass pollen sensitization.

Degree of olive pollen exposure and sensitization patterns. Clinical implications.

BACKGROUND: Very high levels of exposure to olive pollen in the south of Spain lead to differential allergen sensitization profiles. Therefore, new approaches to allergen standardization, diagnosis, and vaccination are necessary. METHODS: Quantification of minor allergens in extracts, component-resolved patient diagnosis, and IgG4 individual allergen responses were used to evaluate new strategies in the management of olive pollen allergy. RESULTS: Allergen variability observed between different olive cultivars can be used to identify suitable allergen sources that can be combined to yield consistent allergen extracts for diagnosis and immunotherapy. Component-resolved diagnosis can provide a better patient classification. IgG4 levels to major allergens increase significantly, whereas specific IgG4 to minor allergens does not seem to increase, at least during the early phases of immunotherapy. CONCLUSION: Patients exposed to extreme olive pollen levels display a different severity of allergy from those exposed to normal levels, which makes it necessary to follow a different clinical approach. The use of component-resolved diagnosis, better standardized allergen extracts, and new efficacy monitoring techniques will lead to a significant improvement in the management of olive allergy disease.

Prevalence of sensitization to Artemisia allergens Art v 1, Art v 3 and Art v 60 kDa. Cross-reactivity among Art v 3 and other relevant lipid-transfer protein allergens.

BACKGROUND: Artemisia vulgaris is a widespread weed in the Mediterranean area and several allergens have been detected in its pollen. One of them, Art v 3, belongs to the lipid-transfer protein (LTP) family and its prevalence in Artemisia-sensitized patients or its relationship with other LTP allergens is not clear. OBJECTIVE: To assess the pattern of sensitization to an array of mugwort allergens in a Mediterranean population, and to study the cross-reactivity of Art v 3 with Pru p 3 and Par j 1, relevant LTP allergens in the area. METHODS: Skin prick test was performed with whole extracts (A. vulgaris, Parietaria judaica and peach) and pure natural allergens Art v 1, Art v 3, Art v 60 kDa and Par j 1 in 24 mugwort-allergic patients from a Mediterranean area. In vitro assays included measurement of specific IgE and ELISA inhibition among LTP allergens. RESULTS: The three Artemisia allergens elicited a positive skin response in 70-80% of the patients. Seven patients were clearly sensitized to Par j 1 and 11 to Pru p 3. There was no correlation between Par j 1 and Pru p 3 sensitization, but a highly significant correlation was found between peach extract and Art v 3 as regards the skin response. No IgE cross-reactivity was observed between Art v 3/Par j 1 or Pru p 3/Par j 1. In contrast, Art v 3 significantly inhibited the binding to Pru p 3 of IgE from three patients' sera out of six studied, but Pru p 3 was not able to inhibit the IgE binding to Art v 3. CONCLUSION: Art v 3 is a major mugwort allergen and in some patients with IgE to both Art v 3 and Pru p 3, Art v 3 behaves as the primary sensitizing agent.

Cloning and expression of a major allergen from Cupressus arizonica pollen, Cup a 3, a PR-5 protein expressed under polluted environment.

BACKGROUND: This paper describes the cloning and expression of the Cupressus arizonica pollen protein Cup a 3. In addition, we present its modulation under polluted environmental conditions. Species of the Cupressaceae family are important because of their high sensitization prevalence. METHODS: Cup a 3 cloning is based on the sequence of the homologous protein Jun a 3. Cup a 3 was expressed with good yield in the methylotropic yeast Pichia pastoris. RESULTS: Recombinant Cup a 3 (rCup a 3) contains 199 amino acids, 10 potential phosphorylation sites and no glycosylation sites. By immunoblot 63% of cypress allergic patients had specific immunoglobulin E antibodies against rCup a 3 (n = 104). This major allergen is homologous to members of the pathogenesis-related proteins (PR-5 group) and contributes to the overall allergenicity of C. arizonica pollen. Our results show that the increased expression of Cup a 3 is dependent on the pollution in the area where the pollen has been collected, being higher under polluted conditions. CONCLUSIONS: Cup a 3 is a PR-5 protein derived from C. arizonica pollen. The expression of the protein under polluted conditions has a direct incidence on the pollen allergenicity, as has been demonstrated by skin tests and Radioallergosorbent test inhibition.

Is Lolium pollen from an urban environment more allergenic than rural pollen?

BACKGROUND: Allergy to grass pollen is a highly prevalent allergic disease. Hay fever is more predominant in urban than in rural areas, despite the increasingly smaller areas of surrounding grassland. The effect of vehicle exhaust pollutants, mainly diesel particles, and other industrial sources of atmospheric pollution leading to plant damage has been implicated in this phenomenon. OBJECTIVE: This study compared the in vivo and in vitro allergenicity of pooled samples of Lolium perenne grass pollen harvested from 10 different urban areas with that of samples of the same pollen from 10 neighboring rural areas. METHODS: Lolium perenne pollen from different parts of a city and from a nearby rural area was harvested in 1999 and 2000 during the peak pollination period. Protein composition was compared by SDS-PAGE and in vivo and in vitro IgE-binding capacity was compared by skin-prick tests, RAST-inhibition and measurement of the major allergen, Lol p 5. RESULTS: In the two years under study, urban samples contained approximately twice the protein content of the rural samples. Biological activity and Lol p 5 content was higher in urban pollen than in rural pollen and showed differences in the two years under study. CONCLUSIONS: The protein content and allergenicity of Lolium perenne pollen was higher in urban areas than in rural areas. These differences might explain why allergy to grass pollen is more prevalent in urban areas. This finding should be taken into account in diagnosis, preventive measures and specific immunotherapy.

Is Lolium pollen from an urban environment more allergenic than rural pollen?

Background: Allergy to grass pollen is a highly prevalent allergic disease. Hay fever is more predominant in urban than in rural areas, despite the increasingly smaller areas of surrounding grassland. The effect of vehicle exhaust pollutants, mainly diesel particles, and other industrial sources of atmospheric pollution leading to plant damage has been implicated in this phenomenon. Objective: This study compared the in vivo and in vitro allergenicity of pooled samples of Lolium perenne grass pollen harvested from 10 different urban areas with that of samples of the same pollen from 10 neighboring rural areas. Methods: Lolium perenne pollen from different parts of a city and from a nearby rural area was harvested in 1999 and 2000 during the peak pollination period. Protein composition was compared by SDS-PAGE and in vivo and in vitro IgE-binding capacity was compared by skin-prick tests, RAST-inhibition and measurement of the major allergen, Lol p 5. Results: In the two years under study, urban samples contained approximately twice the protein content of the rural samples. Biological activity and Lol p 5 content was higher in urban pollen than in rural pollen and showed differences in the two years under study. Conclusions: The protein content and allergenicity of Lolium perenne pollen was higher in urban areas than in rural areas. These differences might explain why allergy to grass pollen is more prevalent in urban areas. This finding should be taken into account in diagnosis, preventive measures and specific immunotherapy (AU)

Antecedentes: Los pólenes son una causa muy importante de enfermedades alérgicas. La polinosis es mas prevalente en zonas urbanas que en rurales, a pesar de que cada vez hay menos zonas verdes en las ciudades. Se ha valorado el efecto de las partículas diesel y otras fuentes de contaminantes urbanos sobre los pólenes para tratar de explicar este fenómeno. Objetivo: Este estudio compara la alergenicidad de muestras de Lolium perenne recolectado en diferentes zonas de la ciudad de Valladolid con pólenes de la misma especie recogidos en zonas rurales vecinas. Métodos: Se recolectaron pólenes de Lolium perenne de diferentes partes de la ciudad y de zonas rurales circundantes durante su pico de polinización en los años 1999 y 2000. Se mide su reactividad "in vivo" mediante prick tests e "in vitro" por SDS-PAGE, RAST inhibición y medición de su alérgeno principal Lol p 5, y se comparan los resultados obtenidos por todas la técnicas. Resultados: Demostramos una mayor concentración proteica y alergenicidad de los pólenes de Lolium perenne de zonas urbanas comparadas con las rurales cercanas. Conclusión: Sugerimos que esta diferencia en concentración proteica y actividad biológica del polen según el lugar de procedencia debería ser tenida en cuenta para el diagnóstico, medidas de prevención e inmunoterapia específica (AU)

Cross-reactivity between Platanus pollen and vegetables.

BACKGROUND: Several associations have been described between tree and plant pollens and certain foods. The objective of this study is to verify whether there is cross-reactivity between Platanus pollen and vegetable origin foods. METHODS: We selected 56 patients allergic to vegetable foods and subjected them to cutaneous tests with aeroallergens and vegetable foods. A statistical analysis was performed to evaluate the association of Platanus pollen with foods and with other aeroallergens. Later, a specific IgE determination was performed as well as a RAST (radioallergosorbent) inhibition experiment, to verify the existence of cross-reactivity in vitro. RESULTS: In the cutaneous tests we found a positive correlation between Platanus pollen and hazelnut, peanut, banana and celery. The results of the RAST inhibition experiment indicate an important cross-reactivity between the pollen of Platanus acerifolia and hazelnut and banana fruit, and an intermediate cross-reactivity with celery and peanut. CONCLUSION: We have described an association between the pollen of the Platanus tree and some vegetable foods such as hazelnut, banana, peanut and celery. This association could be explained by the in vitro IgE cross-reactivity detected.

Allergy to olive pollen: a study of four family members.

We describe four family members with respiratory and dermatological manifestations of olive pollen allergy. The purpose of this study was 1) to investigate whether these patients' sera react to the same or different olive allergens, and 2) to identify common HLA class II antigens.

Lipid-transfer proteins as potential plant panallergens: cross-reactivity among proteins of Artemisia pollen, Castanea nut and Rosaceae fruits, with different IgE-binding capacities.

BACKGROUND: Lipid-transfer proteins (LTPs), but not Bet v 1 homologues, have been identified as major allergens of apple and peach in the Rosaceae fruit-allergic population in the Mediterranean area. Many of these patients show cosensitization to mugwort pollen. LTPs have an ubiquitous distribution in tissues of many plant species, and have been proposed as a novel type of plant panallergens. OBJECTIVE: We sought to isolate LTPs from Artemisia pollen and from a plant food not belonging to the Rosaceae family, such as chestnut nut, and to compare their amino acid sequences and IgE-binding capacities with those of apple and peach LTPs. METHODS: Allergens (LTPs) were isolated by different chromatographic methods (gel-filtration, ion exchange and/or reverse-phase HPLC), and characterized by N-terminal amino acid sequencing and MALDI analysis. Specific IgE-quantification and immunodetection, as well as immunoblot and ELISA inhibition assays, were carried out using sera from patients allergic to both apple and peach. RESULTS: Purified LTPs from Artemisia pollen and from chestnut seed showed molecular masses about 9 700d, and 43-50% sequence identity with the equivalent allergens of apple and peach in the first 30 N-terminal residues, which comprise about one third of the total amino acid sequence. A similar degree of sequence identity (50%) was found between the Artemisia and chestnut proteins. Both isolated LTPs bound specific IgE of sera from Rosaceae fruits allergic patients. However, substantially lower values of specific IgE-binding and maximum ELISA inhibition percentages were obtained for Artemisia and chestnut LTPs when compared to those from apple and peach. CONCLUSION: LTPs from Artemisia pollen and chestnut crossreact with allergens (LTPs) of Rosaceae fruits, but significant differences in specific IgE-binding capacities were observed among members of the plant LTP family. Thus, further studies are needed to evaluate the clinical significance of the observed cross-reactivities of plant LTPs.

Lipid-transfer proteins are relevant allergens in fruit allergy.

BACKGROUND: Allergy to apple and Prunus fruits is frequently associated with birch pollinosis, with the principal cross-reacting allergens involved being members of the Bet v 1 family. However, a major 13-kd component, nonimmunologically related to Bet v 1, has been implicated as allergen in patients allergic to Prunoideae fruit but not to birch pollen. OBJECTIVE: We sought to isolate and characterize the 13-kd allergen present in apple and peach. METHODS: Sera from patients allergic to both fruits were selected on the basis of clinical symptoms, skin prick tests responses, and specific IgE levels. Allergens were purified by reverse-phase HPLC and characterized by N-terminal amino acid sequencing, MALDI analysis, specific IgE immunodetection, and immunoblot inhibition assays. RESULTS: A 13-kd protein band was recognized in crude apple and peach extracts by 9 of 10 and 10 of 10 sera from patients allergic to fruit, respectively. The isolation and characterization of the corresponding allergens allowed their identification as lipid-transfer proteins, with a molecular mass of 9058 d for the apple protein and 9138 d for the peach protein. Both purified allergens were recognized by sera from patients allergic to fruit and fully inhibited the IgE binding by the 13-kd component present in the 2 crude fruit extracts. CONCLUSION: Lipid-transfer proteins are relevant apple and peach allergens and, considering their ubiquitous distribution in tissues of many plant species, could be a novel type of panallergen of fruits and vegetables.

Group 5 determination in Pooideae grass pollen extracts by monoclonal antibody-based ELISA. Correlation with biologic activity.

A solid-phase, monoclonal antibody-based ELISA was set up to quantitate group 5 allergens in pollen extracts of wild and cultivated Pooideae grasses. The method was able to evaluate group 5 concentration in mass units with a sensitivity in the ng/ml range and a practical working range of 1-100 ng/ml. The group 5 ELISA was compared with rocket immunoelectrophoresis for determination of allergen levels in several Phleum pratense extracts, and a very good quantitative correlation was found (r = 0.98; P < 0.0001). A highly significant correlation (r > 0.8) was also obtained in comparing allergenic potency determined by RAST inhibition to group 5 content in several wild and cultivated grass species. The results proved the usefulness of the method in the standardization of Pooideae pollen extracts employed in diagnosis and treatment.

Quantification in mass units of Bet v 1, the main allergen of Betula verrucosa pollen, by a monoclonal antibody based-ELISA.

BACKGROUND: Fagales pollens are considered among the main agents responsible for allergic diseases in many countries of the northern hemisphere and single major allergens have been shown to be responsible for these responses. OBJECTIVE: To develop a solid phase immunoassay for the quantification of Bet v 1, the main allergen from Betula verrucosa (birch), and to assess its suitability for quantitating the equivalent major allergen in other Fagales species as well. METHODS: The assay is based on the use of two different anti-Bet v 1 monoclonal antibodies which were immobilized on the solid phase and, as a primary standard, affinity purified Bet v 1, the protein content of which was determined by amino acid analysis. RESULTS: The ELISA proved to measure less than 0.2 ng/mL of Bet v 1 with a practical range of 0.4-40 ng/mL and could be suitable to quantify the equivalent major allergen in other Fagales species such as Corylus avellana (hazel), Carpinus betulus (horbeam) and Alnus glutinosa (alder). The method was compared with quantitative electrophoresis and rocket immuno-electrophoresis for the determination of the allergen content in several Betula verrucosa extracts, and a very good quantitative correlation was found. Likewise, the Bet v 1 content exhibited a good correlation (r = 0.87; P < 0.005) with the allergenic potency values obtained by RAST inhibition. CONCLUSIONS: The results indicate that the Bet v 1-assay could be useful for standardization purposes in Fagales pollen extracts intended for clinical use.