Allergenicity and immunochemical characterization of six varieties of Olea europaea

BACKGROUND: The inhalation of Olea europaea pollen is one of the most important causes of allergic respiratory diseases in the Mediterranean basin. The objective of this study was to investigate the antigenic and allergenic composition of six different O. europaea varieties collected in southern Spain. METHODS: The varieties included in the study were: Acebuche (wild olive), Carrasque?o, Nevado, Hojiblanco, Manzanillo and Picual. Extracts of these six varieties were prepared. Twenty-nine olive individuals with an immunoglobulin(Ig)E-mediated allergy to olive pollen were skin tested with these extracts. The antigenic profile of these extracts was evaluated by SDS-PAGE; the allergenic profile was investigated by immunoblotting using the serum of these 29 individuals. The Ole e 1 content was established by ELISA inhibition using purified Ole e 1 and rabbit polyclonal antibodies and by scanning densitometry. RESULTS: The extracts that induced the smallest wheal size were Acebuche and Hojiblanco, being significantly different from the rest of the extracts. The antigenic and allergenic profiles of the extracts also varied. The Ole e 1 content ranged from 0.050 in Hojiblanco to 0.232 in Manzanillo, measured by ELISA inhibition and from 0.153 in Hojiblanco to 0.677 in Nevado, measured by scanning densitometry. CONCLUSIONS: The different varieties of O. europaea pollen studied demonstrated great differences in the in vivo and in vitro potency of the extracts. There were significant differences in the Ole e 1 content, while the protein content remained very similar in these extracts. This study confirms previous observations of a great variability in the antigenic and allergenic composition of O. europaea pollen extracts and establishes significant differences in Ole e 1 content.     

Determination of Ole e 1 by enzyme immunoassay and scanning densitometry. Validation by skin-prick testing

Ole e 1 is an important allergen in Olea europaea pollen extracts. This study describes the development of two new methods that can be used to estimate the Ole e 1 content in olive tree pollen extracts. They are based on (1) an enzyme immunoassay that uses rabbit polyclonal, monospecific antibodies and purified Ole e 1, and (2) scanning densitometry of SDS-PAGE gels. Twelve extracts were evaluated by in vivo and in vitro methods. The in vivo biological potency was estimated by prick skin testing 17 allergic individuals; the in vitro allergenic potency by direct IgE and IgE inhibition assays. The enzyme immunoassay showed an operative range of 0.03-100 microg/ml and demonstrated to be specific for Ole e 1. The Ole e 1 content ranged from 1% to 5% of the total protein in the 12 extracts. The amount of Ole e 1, assessed by gel scanning densitometry significantly correlated with the Ole e 1 content obtained by the immunoassay (r = 0.92; p < 0.001). The Ole e 1 content showed a significant correlation with the total allergenic potency of the extracts, evaluated by direct IgE, specific IgE inhibition and skin-prick testing. These two methods can be used to determine the Ole e 1 content in olive pollen extracts. The content of Ole e 1 can vary from 1% to 5% of the total protein in the extracts.     

Rapid release of Ole e 1 from olive pollen using different solvents

BACKGROUND: Solubility is an important characteristic of allergenic molecules. The aim of this study was to investigate the solubility of Ole e 1, a major allergen of Olea europaea, using different solvents. MATERIAL AND METHODS: Olea europaea pollen was placed in a glass column and extracted using three different solvents: deionized water, phosphate buffer 0.01 M (PBS) and normal saline (NaCl 0.9%). Several fractions were collected after extraction with each solvent and pooled based on individual protein content. Each fraction corresponded to a different elution profile, as determined by linear regression analysis. After 130 min of extraction, the pollen that remained in the column was further extracted overnight. A control olive pollen extract was also prepared with each solvent. The antigenic and allergenic profiles of all the eluted and pooled fractions were analysed by SDS-PAGE and inmmunoblots. Protein and Ole e 1 content and the amount of protein needed to produce 50% inhibition were also calculated. Ten patients were skin prick tested with the fractions obtained with deionized water. RESULTS: Four elution profiles were obtained using deionized water as the extracting solution and three with the two other solvents. The three solvents produced different kinetics of allergen release. Ole e 1 was rapidly released when water was used, obtaining a total of 256 micro g of Ole e 1/ml after only 7 min of extraction (fraction EC1). Using PBS, or NaCl 0.9%, the release of Ole e 1 started after 4 and 9 min of extraction, respectively. The highest amount ofOle e 1 was eluted after 44 and 26 min, with a total concentration of 162 and 203 micro g of Ole e 1/ml, respectively. The presence of Ole e 1 in each phase was verified by SDS-PAGE and immunoblot analyses. CONCLUSIONS: The extracting solution seems to determine the antigenic profile of olive pollen extracts. Ole e 1 is rapidly released from the pollen grain after extraction in deionized water. The solubility seems to be affected by the use of other solvents. These techniques could be used to manipulate the Ole e 1 content in O. europaea extracts.     

Monoclonal antibody-based method for measuring olive pollen major allergen Ole e 1.

BACKGROUND: Olive tree pollen is an important cause of inhalant allergy in Mediterranean countries. The major allergen of this pollen, Ole e 1, has caused reactions in the sera of >80% of olive-sensitive patients. Accurate standardization of allergenic products for diagnosis and immunotherapy is essential to guarantee their quality, and measurement of the major allergen content is becoming an important aspect of standardization procedures. OBJECTIVE: To develop a two-site enzyme-linked immunoadsorbent assay (ELISA) for the quantification of Ole e 1. METHODS: BALB/c mice were immunized with purified natural Ole e 1. After fusion and screening by direct ELISA, one of the monoclonal antibodies (5A3) was selected as the capture antibody in an ELISA for Ole e 1 quantification. Bound allergens were detected by a combination of biotinylated Ole e 1-specific polyclonal rabbit antibody and peroxidase-conjugated streptavidin. This ELISA was subsequently evaluated and compared with other techniques. RESULTS: The developed ELISA was highly reproducible and sensitive, with a detection limit of 0.5 ng/mL and a practical range of 1 to 10 ng/mL. The Ole e 1 content ranged from 3 to 50% of the total protein among the nine Olea europaea pollen extracts studied. The assay also detected Ole e 1-like proteins in pollen from other Oleaceae. Correlation was good between the Ole e 1 content determined by ELISA and scanning densitometry and the immunoglobulin E-binding activity of the extracts. CONCLUSION: The described Ole e 1 ELISA is sensitive, reproducible, specific, and reliable, and therefore, can be helpful for standardization of olive pollen extracts intended for clinical use.     

Identification of a 36‐kDa olive‐pollen allergen by in vitro and in vivo studies

BACKGROUND: Ole e 1 has been considered the major allergen of olive (Olea europaea) pollen. Some other relevant allergens (Ole e 2, 3, 4, and 6) have been recently described. This work aimed to study the IgE-binding frequency of a 36-kDa protein from O. europaea pollen in a large population of olive-allergic patients, its allergenic reactivity in vivo, and its presence in olive pollens of different origin, as well as in other relevant allergenic pollens. METHODS: Identification of IgE-binding components from O. europaea pollen extracts was elucidated by inhibition of SDS-PAGE immunoblotting using recombinant profilin (Ole e 2) and Ole e 1 molecules. The IgE-binding frequency of the 36-kDa protein was estimated by Western blot in a sample of 120 sera from olive-allergic patients. The cutaneous test with the 36-kDa protein was performed by intradermoreaction in allergic patients and control subjects. RESULTS: Exactly 83% of the sera from O. europaea-allergic patients recognized a protein with an apparent molecular weight of 36 kDa, under reducing conditions. It was detected by sera from monosensitized and polysensitized patients, showing a higher IgE frequency than the major allergen Ole e 1 (59%) and the minor profilin (Ole e 2) allergen (27%). Similar reactivity rates (79%) was found by intradermal test. Extracts from olive pollens collected in California presented a much higher amount (around 16-fold on average) of the 36-kDa protein than those from pollens of Spanish origin. The presence of similar allergens was detected only in closely related species (Syringa, Fraxinus, Ligustrum), and not in other common allergenic pollens. CONCLUSIONS: The 36-kDa protein constitutes a major allergen for olive-sensitized patients, but it is not equally represented in O. europaea pollens of different origins.     

Allergenicity and cross-reactivity of Russian olive pollen (Eleagnus angustifolia)

BACKGROUND: The purposes of this study were: to determine the prevalence of sensitization and immunochemical characterization of Eleagnus angustifolia pollen (Russian olive) that belongs to the family Eleagnaceae. METHODS: A total of 134 patients with rhinoconjunctivitis and/or asthma were studied. Its allergenicity, cross-reactivity with olive pollen and the presence of Ole e 1 and Ole e 4-like molecules were evaluated. RESULTS: Eleagnus angustifolia pollen was detected from May to June. Seventy-three of 134 (30.5%) had positive skin test to E. angustifolia, all of them were positive to olive. There was a good correlation between specific immunoglobulin (Ig)E levels to E. angustifolia and Olea europaea (r = 0.77, P = 0.002). Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) immunoblots revealed major IgE-binding bands in the E. angustifolia extract of 43 and 63.7 kDa. The E. angustifolia extract was not able to inhibit olive, whereas O. europaea inhibited E. angustifolia up to 41%. The presence of Ole e 1- and Ole e 4-like allergens in E. angustifolia extract was confirmed by enzyme-linked immunosorbent (ELISA) inhibition assays. Nasal challenge with E. angustifolia was positive in three of six patients with positive skin test to both pollens and negative in five patients with positive skin test only to O. europaea. CONCLUSIONS: This study confirms that E. angustifolia is capable of sensitizing individuals in Madrid. A minimal-to-moderate cross-reactivity with olive pollen was established, suggesting some cross-reactivity but not excluding co-sensitization.     

Detection of airborne allergen (Ole e 1) in relation to Olea europaea pollen in S Spain

BACKGROUND: In recent years, it has been demonstrated that the air carries not only airborne pollen but also plant particles of smaller size that have allergenic activity, and, being within the respirable range, these particles can trigger rapid attacks in the lower respiratory tract. The study of particles according to size (0.7-40 micro m) could provide valuable information on the real allergenic activity in the atmosphere. OBJECTIVE: The purpose of this study was to analyse the dynamics of airborne Olea europaea pollen in contrast to the allergenic activity of Ole e 1 in the atmosphere. METHODS: The analyses were carried out with a Hirst-type volumetric collector and a cascade impactor simultaneously during the MPS of the olive. The indirect ELISA was used to detect the allergenic activity. The sampling was performed in Granada city centre (S Spain), in the Science Faculty building on the University of Granada from 30 April to 26 June 2005. RESULTS AND CONCLUSIONS: This research demonstrates that both the allergenic activity as well as the pollen particles follow in a similar curve, except in periods before or succeeding the main Olea pollen season. The study of the distribution of the allergenic particles according to their sizes reveals that the highest concentrations are between 3.3 and <0.7 micro m, thus indicating that allergenic activity primarily involves paucimicronic particles.     

Monoclonal antibodies against Olea europaea major allergen: allergenic activity of affinity-purified allergen and depleted extract and development of a radioimmunoassay for the quantitation of the allergen.

Several monoclonal antibodies (MAbs) were raised against Olea europaea pollen-extract components. Two of these antibodies, named OL 2 and OL 7, recognize two nonoverlapping, nonrepeating epitopes on the olive-allergen Ole e I, as demonstrated by different techniques. The allergen was purified in a single step by MAb-based affinity chromatography, and the allergen revealed a band at molecular weight 20 kd as well as a minor band at 18 kd on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The contribution of allergen Ole e I to the allergenic activity of O. europaea pollen extracts was determined from the effect of allergen depletion by affinity chromatography on skin reactivity and a histamine-release test. The removal of allergen caused a large reduction in the activity of the preparation in 25 monospecific olive-allergic patients. In agreement, the affinity-purified allergen demonstrated a similar response when it was compared with the whole extract in these assays. The results indicated that Ole e I is by far the most important olive-pollen allergen. A two-site solid-phase radioimmunoassay was developed for the quantitation of the allergen Ole e I in mass units. The assay was based on the MAbs, OL 2 and OL 7, and had a detection limit in the nanogram range. A good correlation was found between allergenic activity, as determined by RAST inhibition, and allergen content in 18 olive-pollen extracts. This result indicates that the assay can be a good alternative to RAST inhibition for the standardization of O. europaea extracts.     

Correlation between Olea europaea and Parietaria judaica pollen counts and quantification of their major allergens Ole e 1 and Par j 1-Par j 2.

BACKGROUND: In patients with pollinosis, allergic symptoms are often correlated with the number of airborne pollen grains, although this correlation is not always close. The direct measurement of the concentration of aeroallergens has only recently been introduced and is an important advance in public health information systems. OBJECTIVE: To compare specific quantification of aeroallergens Ole e 1 and Par j 1-Par j 2 Olea and Urticaceae pollen counts. METHODS: The Hirst method sampler and the Burkard Cyclone sampler were used for pollen count and allergen quantification, respectively. The aerosol was extracted and quantified for Ole e 1 and Par j 1-Par j 2 content using enzyme-linked immunosorbent assay procedures. RESULTS: Day-to-day variations were observed in both the pollen count and the amount of allergens. Pollen counts and aeroallergen quantification were closely correlated with 99% significance (Olea/Ole e 1: R = 0.892, P < .001; Urticaceae/Par j 1-Par j 2: R = 0.734, P < .001). CONCLUSION: The technique for the sampling and quantification of aeroallergens presented in this article, based on enzyme-linked immunosorbent assay and applied to the protein extracts directly obtained from the bioaerosol, represents an important advance in the epidemiologic study of allergic respiratory diseases.     

Immunological activity of recombinant Ole e 1 in patients with Olea europaea pollinosis

BACKGROUND: Recombinant allergens have potential advantages over conventional allergenic extracts. However, these recombinant allergens should be evaluated for their antigenic activity and compared with their natural counterparts before being used for clinical purposes. METHODS: We studied 33 patients with seasonal rhinitis and/or bronchial asthma and a positive skin prick test to Olea europaea pollen extract, 10 atopic patients with no history of pollinosis and a negative skin prick test to O. europaea extract and 10 healthy controls. Skin prick tests and determination by ELISA of specific IgE to natural Ole e 1 (nOle e 1) and recombinant Ole e 1 (rOle e 1) expressed in Pichia pastoris were performed in all patients and controls. Inhibition assays were performed between nOle e 1 and rOle e 1 by ELISA. RESULTS: All patients with O. europaea pollinosis had positive skin test responses to both commercial O. europaea extract and nOle e 1 allergen, and all reacted to rOle e 1 on the skin prick test. The nonatopic and atopic control subjects with negative olive pollen skin test results did not react to rOle e 1 on the skin prick test, even at the highest concentrations, confirming the specificity of this test. We found a weak correlation between the wheal surface area produced by the prick test with nOle e 1 and the wheal surface area produced by rOle e 1 at 10 microgram/ml (r = 0.42, p < 0.05). Comparison of specific IgE against both nOle e 1 and rOle e 1 in the patients did not reveal any significant difference. There was a strong correlation between the amount of specific IgE against nOle e 1 and rOle e 1 (r = 0.99, p < 0.01). The two proteins displayed the same extent of binding inhibition to IgE antibodies in ELISA inhibition experiments. CONCLUSIONS: These results confirm the immunological activity of rOle e 1 expressed in P. pastoris and indicate that Ole e 1 is one of the major allergens in O. europaea pollinosis as evaluated by skin prick test and serological methods. The correlation between rOle e 1 and nOle e 1 in skin test results and serologic data indicates the potential of recombinant allergens for clinical applications and diagnosis of O. europaea pollen allergy.     

Isolation of the main allergen Fra e 1 from ash (Fraxinus excelsior) pollen: comparison of the natural and recombinant forms.

BACKGROUND: Fra e 1 is a major allergen for ash pollen-sensitized individuals in northern and central Europe. It belongs to the Ole e 1-like family and displays high cross-reactivity with taxonomically related members. OBJECTIVES: To isolate and characterize natural Fra e 1 (nFra e 1) from ash pollen and to compare its structural, antigenic, and allergenic properties with those of its recombinant form (rFra e 1). METHODS: The allergen was isolated by means of gel permeation chromatography and reverse-phase high-performance liquid chromatography columns. Molecular characterization was performed by means of Edman degradation, mass spectrometry, circular dichroism, concanavalin A lectin reaction, and anti-horseradish peroxidase polyclonal antibody. Immunologic characterization was performed using immunoblotting and enzyme-linked immunosorbent assay, inhibition experiments, and histamine release assays with serum samples from allergic patients with well-known reactivity to Fra e 1 or Ole e 1 and with polyclonal antiserum and monoclonal antibodies against Ole e 1. The protein used as a reference was rFra e 1, which was produced in the yeast Pichia pastoris. RESULTS: Purified nFra e 1 appeared as 5 variants with different glycosylation degrees. Both nFra e 1 and rFra e 1 were equivalently folded as deduced from the spectroscopic analysis using circular dichroism. Both molecules share the antigenic and allergenic epitopes after the purification process, and the glycan group of nFra e 1 is a potential epitope. Natural Fra e 1 displayed strong cross-reactivity with Ole e 1. CONCLUSIONS: Natural Fra e 1 is a heterogeneously glycosylated protein with high allergenic relevance. It displays structural, antigenic, and allergenic similarity with rFra e 1. Both proteins could be used for clinical purposes.     

Cloning and Expression of the Olea europaea allergen Ole e 5, the pollen Cu/Zn superoxide dismutase

BACKGROUND: Recombinant DNA technology does provide pure, well-defined and reproducible products to be used for clinical purposes, by cloning and expressing the cDNA of allergens present in a specific extract. Ole e 5 is a pollen allergen of Olea europaea with an IgE-binding frequency of about 35%, which has been identified as a superoxide dismutase (SOD). The aim of this study was to clone the cDNA of Ole e 5, to express Ole e 5 in Escherichia coli and to characterize its immunoreactivity. METHODS: cDNA of Ole e 5 was amplified by nested 3'-RACE PCR and cloned in pGEX vector 6P expression vector. After sequencing of some clones and homology analysis, the rOle e 5 was produced in an E. coli strain as a fusion protein with GST and purified. Then, the protein immunoreactivity was evaluated by patients' IgE binding (ELISA, ELISA inhibition, and immunoblotting) and by rabbit anti-rOle e 5 binding (immunoblotting and immunoblotting inhibition). RESULTS: The sequence analysis of Ole e 5 cDNA confirmed that Ole e 5 is a Cu/Zn SOD, with an identity from 90 to 80% with SOD from other species. rOle e 5 was recognized by IgE from 39% of olive pollen-allergic patients tested; moreover, this binding was inhibited by the olive pollen extract. An anti-rOle e 5 antiserum raised in rabbit strongly reacted with a natural component of about 16-kDa molecular weight present in the olive pollen extract; moreover, this binding was inhibited by the recombinant protein. CONCLUSIONS: Ole e 5 is the first Cu/Zn SOD identified as an allergen in a pollen source. Due to the widespread presence of this enzyme, rOle e 5 allergen, cloned and expressed in a complete form in E. coli, could represent a good tool to investigate the allergen cross-reactivity between O. europaea pollen and other allergenic sources, such as plant foods and other pollens.     

Hypersensitivity to black locust (Robinia pseudoacacia) pollen: "allergy mirages".

BACKGROUND: The allergenicity of the ornamental tree Robinia pseudoacacia, or black locust, is unknown. OBJECTIVE: To evaluate the prevalence of sensitization to R. pseudoacacia pollen, its possible allergenic cross-reactivity with other common pollens, and the potential implication of pollen panallergens (profilin, polcalcin, and 1,3-beta-glucanase) as a cause of sensitization to R. pseudoacacia pollen. METHODS: Skin prick testing with R. pseudoacacia pollen was performed in 149 patients with pollinosis. Nasal challenge with R. pseudoacacia pollen was performed in 10 patients. The prevalence of sensitization to the recombinant forms of profilin (rChe a 2), polcalcin (rChe a 3), and the N-terminal of the 1,3-beta-glucanase (rNtD of Ole e 9) was investigated. Immunoblotting, enzyme-linked immunosorbent assay, and competitive inhibition assays were performed with R. pseudoacacia pollen and recombinant pollen allergens. RESULTS: Sixty-four patients (43%) had positive skin prick test reactions to R. pseudoacacia pollen. Nasal challenge results were positive in 5 sensitized patients and negative in 4 controls and 1 sensitized patient. The allergenic profile of R. pseudoacacia pollen comprises at least the panallergen profilin, a calcium-binding protein, and a 1,3-beta-glucanase. The prevalence of sensitization to rChe a 2, rChe a 3, and rNtD of Ole e 9 was 60%, 33%, and 87%, respectively, among patients sensitized to R. pseudoacacia pollen. Binding of IgE to R. pseudoacacia extract was completely inhibited by Robinia, Chenopodium, Olea, Cupressus, and Lolium extracts. CONCLUSIONS: The high prevalence of R. pseudoacacia pollen sensitization in patients with pollinosis is likely to be due to cross-sensitization to panallergens (profilin, polcalcin, and 1,3-beta-glucanase) from other common pollens. This phenomenon may lead to a diagnosis of "allergy mirages."     

Ole e 2 and Ole e 10: new clinical aspects and genetic restrictions in olive pollen allergy

BACKGROUND: The clinical characteristics in olive pollen allergy are dependent on the antigenic load, the allergens profile, and the genetic restrictions. Our objective was to determine specific response pattern in Ole e 2 and Ole e 10 sensitization at those levels. METHODS: We studied 146 patients with seasonal rhinitis and/or asthma and positive prick test to Olea europaea pollen. IgE against Ole e 2 and Ole e 10 were detected by skin prick test and ELISA. HLA-DRB1 and HLA-DQB1 loci were typed by polymerase chain reaction sequence-specific primers method. RESULTS: A total of 102 (69.9%) and 79 (54.0%) patients showed significant IgE antibody response against Ole e 2 and Ole e 10, respectively. There was a significant association between Ole e 2 (OR 2.2, P = 0.04) and Ole e 10 reactivities (OR 2.8, P = 0.007) with asthma. In addition, total and specific IgE antibody levels significantly correlated with asthma (P < 0.05). Patients who reacted to both allergens reached the highest asthma risk factor (OR 4.3, P = 0.002). Phenotypic frequency of DR7 (OR 5.4, Pc = 0.003) and DQ2 (OR 3.6, Pc = 0.02) were increased in positive Ole e 2 patients compared with control subjects. DR2(15) phenotypic frequency was significantly increased (OR 5.6, Pc = 0.02) in positive Ole e 10 patients compared with control subjects. CONCLUSIONS: Our data suggest an association of Ole e 2 and Ole e 10 with bronchial asthma. Also, we found a genetic control of Ole e 2 and Ole e 10 IgE-specific responses that could be relevant to clinical disease in olive pollen allergy.     

Are Ca2+‐binding motifs involved in the immunoglobin E‐binding of allergens? Olive pollen allergens as model of study

BACKGROUND: Several Ca2+-binding proteins, which possess EF-hand sites with a high sequence similarity, have been found to be able to induce Type-I allergy. OBJECTIVE: To study whether the common EF-hand sequential motifs can be involved in the IgE-reactivity of these proteins, thus being responsible of a degree of cross-reactivity among different Ca2+-binding proteins. METHODS: Two olive pollen allergens, Ole e 3 and Ole e 8, have been used in the study. Parvalbumin and calmodulin were included in immunological analyses. Sera from patients allergic to olive pollen, as well as Ole e 3- and Ole e 8-specific rabbit antisera were used in indirect enzyme-linked immunosorbent assay (ELISA), ELISA inhibition assays and immunoblotting. Conformational analyses (circular dichroism spectra and thermal stability) and specific immunodetection assays were performed in the presence and the absence of Ca2+. Chemical breakdown and high-performance liquid chromatography (HPLC) was used to obtain fragments from Ole e 3 containing a single EF-hand motif. RESULTS: Thirty-four (17%) and 16 (8.2%) out of 195 sera from patients allergic to olive pollen contained specific IgE against Ole e 3 and Ole e 8, respectively. The IgE-binding of 12 allergic sera diminished up to 22% for Ole e 3 and to 82% for Ole e 8, when depleted Ca2+. A pool of these sera recognized the two olive allergens and parvalbumin, but at very different extent. Inhibition of the IgE-binding was only achieved between two olive allergens. No structural relationships between Ole e 3 and Ole e 8 were established when specific polyclonal antisera against both proteins were used. CONCLUSION: EF-hand Ca2+-binding sites can not be considered as general allergenic motifs responsible for the cross-reactivity between Ca2+-binding allergens. Different families of Ca2+-binding allergens have specific epitopes that could be involved in the cross-reactivity among members of the same family.     

Purification and fine characterization of a major allergen from Olea europaea pollen extract

Olea europaea (olive) pollen extract was prepared by aqueous extraction and characterized by biochemical and immunochemical methods. Two components, displaying respective mol. wt. of 17000 and 19000, were the most reactive allergens, being the doublet (designated Ole e I) recognized by most sera tested. The 19000 mol. wt. component, purified by conventional biochemical procedure and lectin-affinity chromatography from the Ole e I doublet, was deglycosylated and analyzed by SDS–PAGE and by ELISA inhibition. The results obtained suggest that the 19000 mol. wt. component represents the glycosylated form of the 17000 component.     

The main allergen of Olea europaea (Ole e I) is also present in other species of the oleaceae family

Three major pollen allergens from Fraxinus excelsior, Ligustrurn vulgare and Syringa vulgaris belonging to the Oleaceae family were purified. Monoclonal antibodies previously raised against the main allergen of Olea europaea (Ole e I) were used for their purification by affinity chromatography. The three new purified allergens were able to bind human IgE from serum of olive-allergic patients in a way analogous to Ole e I. Crossed radioimmunoelectrophoresis of the four allergens, using anti-olive extract rabbit serum, showed a unique immunoprecipitation arc with the same characteristics. The four purified proteins had similar molecular weights on SIX-PAGE and the N-terminal sequences for the first 20 amino acids were identical. Furthermore, the concentration of the allergens could be determined using a two-site solid phase assay previously developed for the allergen Ole e I. Our results indicate that the four purified proteins share, to a great extent, antigenic and allergenic epitopes leading to cross-reactivities which could cause common clinical manifestations. We propose for the newly purified allergens the nomenclature of Fra e I, Lig v I and Syr v I.     

Identification and characterization of Che a 1 allergen from Chenopodium album pollen

BACKGROUND: Pollinosis to Chenopodium album has been reported, but no data are available on its allergenic proteins. METHODS: An allergen from C. album pollen has been isolated by means of gel permeation and reverse-phase high-performance liquid chromatography. Molecular characterization was achieved by concanavalin A reaction, mass spectrometry, Edman degradation and cDNA sequence. Antigenic analyses were performed by immunoblotting, ELISA, and ELISA inhibition, using sera from allergic patients, two Ole e 1-specific monoclonal antibodies and an Ole e 1-specific polyclonal antiserum. RESULTS: The isolated allergen, Che a 1, is a glycoprotein of molecular mass 17.088 kD and 143 amino acid residues, whose sequence exhibits 27-45% identity with known members of the Ole e 1-like protein family. 77% of sera from patients allergic to chenopod pollen were reactive to Che a 1. No correlation was found between the IgE reactivities to Che a 1 and Ole e 1, the major allergens from olive pollen, and both allergens display low, although detectable, IgE and IgG cross-reactivities. CONCLUSIONS: Che a 1, a relevant allergen from chenopod pollen, is structurally related to the Ole e 1-like protein family, but exhibits significant differences on its polypeptide sequence that could explain its different antigenic behavior and limited cross-reactivity.     

Cross-reactivity of Olea europaea with other Oleaceae species in allergic rhinitis and bronchial asthma

Cross-reactivity between pollen extracts of four species of Oleaceae was studied: olive (Olea europaea) , ash (Fraxinus excelsior) , privet (Ligustrum vulgare) , and lilac (Syringa vulgaris). Thus, 51 patients and 13 atopic controls were studied, by means of intracutaneous skin tests, histamine-release tests against the four extracts, and specific IgE to O. europaea. The proteic content of the four extracts was assessed by SDS-PAGE and immunoblotting, and similarity of all the extracts studied was observed after electrophoresis and immunodetection. Six common bands were found to be responsible for the cross-reactivity, with apparent molecular weights of 49.6, 40, 36.7, 19.7, 16.7, and 14 kDa, respectively. The cross-reactivity was also corroborated by immunoblotting inhibition and FEIA inhibition. The patients had a similar response to the four allergenic extracts used, although the response to Olea was greatest. When the patients were compared by their geographic origin (northern or southern Spain, according to the distribution of areas of olive pollen influence), there were no significant differences between the two groups in skin reactivity, but a higher histamine release was observed for the four extracts in the southern group, although it was significant only for Fraxinus and Ligustrum. This work corroborated the practicality of the diagnostic methods used and the cross-reactivity between the four species studied, as demonstrated by the different methods used. Therefore, we suggest that only O. europaea extract be used in diagnosis and immunotherapy in Oleaceae pollen allergy.     

Antigenicity of the pollen proteins of various cultivars of Olea europaea

The incidence of positive skin test responses among alopic subjects with suspected respiratory allergy was investigated with commercial and autochthonous pollen extracts of various cultivars of Olea europaea . Pollen was collected from olive trees of well-defined cultivars, extracted, and separated by SDS-PAGE. Immunoblots were used to identify the various IgE-binding proteins of the pollen extracts of the various cultivars. The results revealed six predominant IgE-binding bands, some of which appear in all the cultivars examined. The 18–20-Da band (Ole e 1) appeared in only eight of the cultivars, but not in the nine others. The presence of specific IgE-binding bands in the various pollen extracts and their correlation with the incidence of positive skin tests are discussed.