Rational Design, Structure-Activity Relationship, and Immunogenicity of Hypoallergenic Pru p 3 Variants.

SCOPE: Allergies to lipid transfer proteins involve severe adverse reactions; thus, effective and sustainable therapies are desired. Previous attempts disrupting disulfide bonds failed to maintain immunogenicity; thus, the aim is to design novel hypoallergenic Pru p 3 variants and evaluate the applicability for treatment of peach allergy. METHODS AND RESULTS: Pru p 3 proline variant (PV) designed using in silico mutagenesis, cysteine variant (CV), and wild-type Pru p 3 (WT) are purified from Escherichia coli. Variants display homogenous and stable protein conformations with an altered secondary structure in circular dichroism. PV shows enhanced long-term storage capacities compared to CV similar to the highly stable WT. Using sera of 33 peach allergic patients, IgE-binding activity is reduced by 97% (PV) and 71% (CV) compared to WT. Both molecules show strong hypoallergenicity in Pru p 3 ImmunoCAP cross-inhibition and histamine release assays. Immunogenicity of PV is demonstrated with a phosphate-based adjuvant formulation in a mouse model. CONCLUSIONS: An in silico approach is used to generate a PV without targeting disulfide bonds, T cell epitopes, or previously reported IgE epitopes of Pru p 3. PV is strongly hypoallergenic while structurally stable and immunogenic, thus representing a promising candidate for peach allergen immunotherapy.

Phase I study of subcutaneous allergen immunotherapy with Dermatophagoides pteronyssinus in patients with allergic rhinoconjunctivitis with or without asthma.

AIM: A double-blind placebo-controlled study was conducted according to EMA guidelines, to evaluate safety, tolerability and short-term treatment effects of three updosing regimens of Dermatophagoides pteronyssinus subcutaneous allergen immunotherapy. PATIENTS & METHODS: Forty-eight patients were randomized to groups: A (six weekly doses), B (eight weekly doses) or C (eight doses, two clustered doses over 3 weeks).  RESULTS: The most frequent adverse events were local reactions. No serious adverse events were found. Severe systemic reactions were reported more frequently in Group C. Decreased cutaneous responses and increased specific IgGs were shown in all active groups, even within the short-term. CONCLUSION:  Dermatophagoides pteronyssinus subcutaneous allergen immunotherapy in depot presentation exhibited good safety and tolerability. Group A seemed to show the best profile for further clinical development.

Molecular aerobiology - Plantago allergen Pla l 1 in the atmosphere.

INTRODUCTION: Exposure to airborne pollen from certain plants can cause allergic disease, but allergens can also be found in non-pollen-bearing fractions of ambient air. This may explain why the allergic response in susceptible patients does not always coincide with the presence and magnitude of airborne pollen counts. Plantago pollen is an important cause of pollinosis in northern Mediterranean countries, but it is difficult to determine its incidence in allergies because Plantago pollen appears in the atmosphere at the same time as grass pollen. OBJECTIVE: The study aimed to investigate the relationship between the atmospheric concentration of Pla l 1 aeroallergen and Plantago pollen, and its incidence in a population group. MATERIALS AND METHOD: Pollen was sampled using a Hirst-type volumetric trap (Burkard) and Burkard Cyclone sampler (Burkard) for Pla l 1 allergen. Allergen was determined with a Pla l 1-specific ELISA. Serum-specific IgE levels to several plant allergens were measured with the EAST system. RESULTS: The aerobiological dynamics of Plantago pollen grains and Pla l 1 did not follow the same trend, whereas the sum of Plantago with some other pollen types showed a more similar behaviour. Of the 118 subjects tested, sera from 52 contained IgE to Plantago pollen, but only 5 were monosensitized. CONCLUSIONS: The presence of Pla l 1 in the atmosphere depends not only on Plantago pollen but also on the pollen of other species from the Oleaceae family. Knowledge of the behaviour of allergen Pla l 1 in the atmosphere can help understand better asthma exacerbations associated with aeroallergens.

Expression of Alt a 1 allergen from Alternaria alternata in the yeast Yarrowia lipolytica.

Allergies affect almost 25% of the population in industrialized countries. Alternaria alternata is known to be a significant source of aeroallergens and sensitization to this mold is a risk factor for the development of wheezing, asthma, and atopic dermatitis. Diagnosis and treatment of allergies requires the production of large amounts of pure and well defined protein. Yarrowia lipolytica, a non-pathogenic ascomycete able to secrete high levels of enzymes that can grow in inexpensive substrates, has been considered a useful host for heterologous gene expression. In the present work, we have developed two vectors for expressing Alt a 1, the most relevant A. alternata allergen, in Y. lipolytica. One vector is autosomal and one is integrative. With both systems, rAlt a 1 was secreted into the culture medium. The immunological characteristics of the purified recombinant allergen were determined by IgE-blot using sera from 42 A. alternata-allergic patients. We have carried out ELISA-inhibition experiments using sera from four patients to compare the IgE-binding capacity of natural and recombinant allergens. Our results show that Y. lipolytica is able to produce a recombinant Alt a 1 which is immunochemically equivalent to the natural counterpart and could be used for immunotherapy and diagnostics.

Anisakis simplex recombinant allergens increase diagnosis specificity preserving high sensitivity.

BACKGROUND: So far, the frequency of Anisakis simplex-specific IgE antibodies has been determined by skin prick tests (SPTs) and the ImmunoCAP system. These commercial methods have good sensitivity, but their specificity is poor because they use complete parasite extracts. Our aim was to determine the frequency of sensitization to A. simplex using recombinant Ani s 1, Ani s 3, Ani s 5, Ani s 9 and Ani s 10 and to evaluate these allergens for diagnosis, comparing their performance with the commercial methods. PATIENTS AND METHODS: We conducted a descriptive, cross-sectional validation study performed in an allergy outpatient hospital clinic. Patients without fish-related allergy (tolerant patients, n = 99), and A. simplex-allergic patients (n = 35) were studied by SPTs, ImmunoCAP assays and detection of specific IgE to A. simplex recombinant allergens by dot blotting. RESULTS: SPTs and ImmunoCAP assays were positive in 18 and 17% of tolerant patients, respectively. All A. simplex-allergic patients had positive SPTs and ImmunoCAP assays. Specific IgE against at least one of the A. simplex recombinant allergens tested was detected in 15% of sera from tolerant patients and in 100% of sera from A. simplex-allergic patients. Detection of at least one A. simplex recombinant allergen by dot blotting and ImmunoCAP assay using complete extract showed a diagnostic sensitivity of 100% with both methods. However, the specificity of dot blotting with A. simplex recombinant allergens was higher compared with ImmunoCAP (84.85 vs. 82.83%). CONCLUSIONS: There are 15% of tolerant patients with specific IgE against important A. simplex allergens. The recombinant allergens studied here increase the specificity of A. simplex diagnosis while keeping the highest sensitivity. A. simplex recombinant allergens should be included with A. simplex allergy diagnostic tests to improve their specificity.

Detection of airborne Par j 1 and Par j 2 allergens in relation to Urticaceae pollen counts in different bioclimatic areas.

BACKGROUND: In aerobiological studies, the Parietaria pollen type usually includes all Parietaria and Urtica species found in the area. Given that Urtica is a nonallergenic plant, the pollen counts report incomplete information on the presence of allergens in the atmosphere. Discordance between the pollen concentrations of Urticaceae and allergic symptoms has been observed in patients with pollinosis. OBJECTIVE: To compare the Urticaceae pollen counts with the Par j 1 and Par j 2 aeroallergen concentrations from 2 different Spanish geographic areas to determine the allergenic load in the atmosphere. METHODS: Hirst-type volumetric traps and Burkard Cyclone samplers were used for pollen counts and aeroallergen capture, respectively. The quantification of Par j 1 and Par j 2 allergens was performed using specific 2-site antibody enzyme-linked immunosorbent assay. Transmission electron microscopy and immunocytochemical techniques were applied to localize these allergens in the orbicules. RESULTS: Differences between areas and years were obtained in both pollen and aeroallergen concentrations. Despite the lower pollen counts recorded in Cartagena, higher aeroallergen concentrations were registered compared with Ourense. A lower correlation was achieved between Urticaceae pollen concentrations and aeroallergen levels, with a maximum positive significant correlation (adjusted R2 = 0.466, P < .001). Intense labeling of Par j 1 and Par j 2 proteins was observed in the orbicules, the tapetal membrane, and the tapetal tissue remnants. CONCLUSION: This method may be valuable for epidemiologic research to establish correlations between concentrations of Parietaria aeroallergens and clinical symptoms. Therefore, the measurement of aeroallergens should be incorporated into the aerobiological studies with clinical applications.

A new lipid transfer protein homolog identified as an IgE-binding antigen from Japanese cedar pollen.

Japanese cedar (Cryptomeria japonica) pollen is a major cause of seasonal rhinitis and conjunctivitis in Japan, and an understanding of its full allergen repertoire is prerequisite for the development of future molecular diagnostics and immunotherapeutic strategies. Here we report the identification of a new C. japonica pollen IgE-binding antigen (CJP-8) homologous to lipid transfer proteins (LTPs), a class of plant cross-reactive allergens found in foods, latex, and pollen grains. The cjp-8 cDNA encodes a 165-amino acid polypeptide possessing the conserved eight cysteines characteristic of plant LTP family members. Escherichia coli-expressed recombinant CJP-8 (r-CJP-8) reacted with IgE antibody from Japanese cedar pollinosis patients at a 37.5% frequency (6/16).

Par j 1 and Par j 2, the two major allergens in Parietaria judaica, bind preferentially to monoacylated negative lipids.

Par j 1 and Par j 2 proteins are the two major allergens in Parietaria judaica pollen, one of the main causes of allergic diseases in the Mediterranean area. Each of them contains eight cysteine residues organized in a pattern identical to that found in plant nonspecific lipid transfer proteins. The 139- and 102-residue recombinant allergens, corresponding respectively to Par j 1 and Par j 2, refold properly to fully functional forms, whose immunological properties resemble those of the molecules purified from the natural source. Molecular modeling shows that, despite the lack of extensive primary structure homology with nonspecific lipid transfer proteins, both allergens contain a hydrophobic cavity suited to accommodate a lipid ligand. In the present study, we present novel evidence for the formation of complexes of these natural and recombinant proteins from Parietaria pollen with lipidic molecules. The dissociation constant of oleyl-lyso-phosphatidylcholine is 9.1 +/- 1.2 microm for recombinant Par j 1, whereas pyrenedodecanoic acid shows a much higher affinity, with a dissociation constant of approximately 1 microm for both recombinant proteins, as well as for the natural mixture. Lipid binding does not alter the secondary structure content of the protein but is very efficient in protecting disulfide bonds from reduction by dithiothreitol. We show that Par j 1 and Par j 2 not only bind lipids from micellar dispersions, but also are able to extract and transfer negative phospholipids from bilayers.

Double-blind, placebo-controlled Alternaria alternata immunotherapy: in vivo and in vitro parameters.

Few studies have been published on the efficacy and safety of immunotherapy with fungal extracts, possibly because of difficulties arising from antigenic variability among different strains of fungus. The aim of the study was to analyze changes in the in vivo and in vitro parameters in response to immunotherapy with an Alternaria alternata extract. We studied 28 patients with rhinitis, bronchial asthma, or both caused by Alternaria. The patients were randomized to the active immunotherapy or placebo group, and a conventional schedule of immunotherapy was used. We recorded changes for a year in skin reactivity (skin prick test), conjunctival reactivity (conjunctival provocation test), and in vitro parameters (serum-specific IgE, IgG, IgG1 and IgG4 for A. alternata complete extract and for natural and recombinant Alt a 1). Twenty-three patients completed the study and all attained the maintenance dose. There were no changes in skin reactivity in the active treatment group, and reactivity increased at the end of the study period in the placebo group. Conjunctival sensitivity decreased only in the active treatment group when the maintenance dose was reached. Allergen-specific IgE decreased, and IgG, IgG1 and IgG4 increased in all periods of study in the active treatment group, with no changes in the placebo group. Allergen-specific immunotherapy with the A. alternata extract tested here led to a decrease in conjunctival reactivity and induced a significant immunologic response.

Neutrophils as a novel source of eosinophil cationic protein in IgE-mediated processes.

The production of eosinophil cationic protein (ECP) in IgE-mediated diseases has been associated mainly with eosinophils, although no IgE-dependent ECP release has been observed in these cells. Because there is increasing evidence of neutrophil participation in allergic processes, we have examined whether human neutrophils from allergic patients were able to produce ECP by an IgE-dependent mechanism. After challenge with specific Ags to which the patients were sensitized, ECP release was detected in the culture medium. Furthermore, intracellular protein was detected by flow cytometry, immunofluorescence staining, and Western blotting. Expression at both mRNA and de novo protein synthesis were detected, respectively, by RT-PCR and radiolabeling with (35)S. Ag effect was mimicked by cell treatment with anti-IgE Abs or Abs against FcepsilonRI and galectin-3 (FcepsilonRI>galectin-3), but not against FcepsilonRII. These observations represent a novel view of neutrophils as possible source of ECP in IgE-dependent diseases.

Genetically engineered hybrid proteins from Parietaria judaica pollen for allergen-specific immunotherapy.

BACKGROUND: Despite the use of conventional allergen-specific immunotherapy in clinical practice, more defined, efficient, and safer allergy vaccines are required. OBJECTIVE: The aim of the study was to obtain hypoallergenic molecules by deleting B-cell epitopes, which could potentially be applied to Parietaria judaica pollen allergy treatment. METHODS: Three hybrid molecules (Q1, Q2, and Q3) derived from fragments of the 2 major P judaica pollen allergens, Par j 1 and Par j 2, were engineered by means of PCR. Hybrid structures were compared with their natural components by means of circular dichroism, and their biologic activities were compared by using T-cell proliferation assays. Their IgE-binding activity was determined with Western blotting, skin prick tests, and enzyme allergosorbent and ELISA inhibition tests. RESULTS: The hybrid proteins, especially Q2 and Q3, revealed significantly reduced IgE reactivity compared with the natural allergens, as well as with the whole P judaica extract. Furthermore, in vivo skin prick tests showed that the hybrid proteins had a significantly lower potency to induce cutaneous reactions than the whole P judaica extract. Two (Q1 and Q2) of the 3 hybrid proteins induced a comparable T-cell proliferation response as that produced by the whole extract and natural allergens. CONCLUSION: Considering its reduced anaphylactogenic potential, together with its conserved T-cell reactivity, the engineered Q2 protein could be used in safe and shortened schedules of allergen-specific immunotherapy against P judaica pollen allergy. CLINICAL IMPLICATIONS: Recombinant hybrid Q2 is able to induce T-cell proliferation, thus evidencing a potential therapeutic effect. Its reduced IgE-binding capacity envisages an excellent safety profile.

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.

Induction of cyclooxygenase-2 expression by allergens in lymphocytes from allergic patients.

Cyclooxygenase (COX) is a key enzyme in prostaglandin (PG) synthesis. Up-regulation of COX-2 expression is responsible for increased PG release during inflammatory conditions and is thought to be also involved in allergic states. In this study, we demonstrate that in human T, B and natural killer lymphocytes from allergic patients, COX-2 expression became induced upon cell challenge with specific allergens and that this process is presumably IgE dependent and occurs after CD23 receptor ligation. This induction took place at both mRNA and protein levels and was accompanied by PGD2 release. IgE-dependent lymphocyte treatment elicited, in parallel, an activation of the MAPK p38 and extracellular signal-regulated kinase 1/2, an enhancement of calcineurin (CaN) activity, and an increase of the DNA-binding activity of the nuclear factor of activated T cells and of NF-kappaB, with a concomitant decrease in the levels of the cytosolic inhibitor of kappaB, IkappaB. In addition, specific chemical inhibitors of MAPK, such as PD098059 and SB203580, as well as MG-132, an inhibitor of proteasomal activity, abolished allergen-induced COX-2 up-regulation, suggesting that this process is mediated by MAPK and NF-kappaB. However, induction of COX-2 expression was not hampered by the CaN inhibitor cyclosporin A. We also examined the effect of a selective COX-2 inhibitor, NS-398, on cytokine production by human lymphocytes. Treatment with NS-398 severely diminished the IgE-dependently induced production of IL-8 and TNF-alpha. These results underscore the relevant role of lymphocyte COX-2 in allergy and suggest that COX-2 inhibitors may contribute to the improvement of allergic inflammation through the reduction of inflammatory mediator production by human lymphocytes.

Diagnosis of Alternaria alternata sensitization with natural and recombinant Alt a 1 allergens.

BACKGROUND: Diagnosis of Alternaria alternata sensitization is hampered by the variability and complexity of fungal extracts, and thus simplification of the diagnostic procedures with purified allergens should be pursued. OBJECTIVE: We sought to compare A alternata extract and purified natural Alt a 1 (nAlt a 1) and recombinant Alt a 1 (rAlt a 1) allergens for their diagnostic value. METHODS: Forty-two patients allergic to A alternata , 10 atopic patients with negative skin prick test responses to A alternata extract, and 10 healthy subjects were investigated. Skin prick tests and determination of specific IgE levels were performed with nAlt a 1 and 2 different types of rAlt a 1: rbAlt a 1, expressed in Escherichia coli , and ryAlt a 1, expressed in the yeast Yarrowia lipolytica . RESULTS: Prevalence for Alt a 1, Alt a 2, and Alt a 11 by IgE dot-blot testing was 98%, 0%, and 15%, respectively, and therefore Alt a 1 was used as a marker for A alternata sensitization. Immunoblotting and inhibition analysis showed no IgE-binding differences between nAlt a 1 and rAlt a 1. The whole group of patients with allergy to A alternata had positive skin test reactions to purified allergens at 100 microg/mL, whereas no false-positive reactions were detected. Natural or ryAlt a 1 elicited a similar response in skin tests compared with A alternata extract, although a reduced reactivity was observed with rbAlt a 1. Specific IgE levels to nAlt a 1 or rAlt a 1 showed significant correlation and similar sensitivity and specificity. CONCLUSIONS: Alt a 1, either in its natural or recombinant form, is sufficient for a reliable diagnosis of A alternata sensitization and induces skin prick reactivity comparable with that produced by A alternata extract.

Production profile of the major allergen Alt a 1 in Alternaria alternata cultures.

BACKGROUND: The fungus Alternaria is strongly associated with asthma, but the importance of fungal allergen products is frequently underestimated. The profile of allergen release from fungal material is poorly understood. OBJECTIVE: To investigate expression of the major allergen of Alternaria alternata, Alt a 1, during its growth in culture conditions for allergen extract production. METHODS: Allergen expression was examined by Alt a 1-specific 2-site monoclonal antibody enzyme-linked immunosorbent assay, immunoblotting, and potency assays. The release of Alt a 1 was studied by transmission electron microscopy in conjunction with immunogold staining by using antibodies with specificity for Alt a 1. RESULTS: A maximum amount of Alt a 1 was obtained after 4.5 weeks of growing, and it was found predominantly in the spent culture medium. In the same way, total IgE binding activity showed 15-fold more activity in the spent culture medium than in the buffer-extractable antigen fraction. Immunogold electron microscopy provided evidence that Alt a 1 is released from spores and mycelia. CONCLUSIONS: Alternaria alternata allergenic proteins were constantly released into the culture medium, where they accumulated. Alt a 1 was a good marker for checking optimal culture conditions for A alternata extract production intended for clinical use.

Human neutrophils synthesize IL-8 in an IgE-mediated activation.

It has been demonstrated that neutrophils are responsible for the release of large amounts of the inflammatory chemokine interleukin-8 (IL-8), associated with inflammation. To further define the mechanisms implicated, we have analyzed the response of human neutrophils from allergic patients to specific antigens or challenge with anti-immunoglobulin (Ig)E antibodies. Neutrophils showed a dose- and time-dependent production of IL-8. The release of the cytokine was parallel to expression of IL-8 mRNA analyzed by the polymerase chain reaction. This expression was transient-it occurred after 3 h of anti-IgE treatment and was maintained for 18 h. Trifluoperazine, EGTA, reduced nicotinamide adenine dinucleotide phosphate-oxidase inhibitors, and reactive oxygen species (ROS) scavengers inhibited IL-8 production, indicating a critical dependence of calcium and oxidative stress. Moreover, an inhibitory effect of cyclosporin A, an immunosuppressor that inhibits calcineurin activity, on IL-8 release and IL-8 mRNA expression was observed. This is the first evidence of the involvement of ROS and calcium/calcineurin in IgE-dependent IL-8 production. These findings open new perspectives into the functional role of neutrophils in IgE-associated diseases.

Identification of a polygalacturonase as a major allergen (Pla a 2) from Platanus acerifolia pollen.

BACKGROUND: Planetree pollen allergy is a clinical disorder affecting human populations in cities of the United States and Western Europe, but little is known about its relevant allergens. OBJECTIVE: We sought to purify, characterize, and clone the 43-kd allergen from Platanus acerifolia. METHODS: P acerifolia pollen extract was fractionated by using ion-exchange and gel-permeation chromatography. Analyses were carried out by using ELISA, SDS-PAGE, isoelectrofocusing, and immunoblotting. Partial amino acid sequence was obtained by means of Edman sequencing of cyanogen bromide-digested peptides. Specific cDNA was cloned by using reverse transcription, followed by PCR, with amino acid sequences from peptides of the allergen. RESULTS: The allergen isolated from P acerifolia pollen, Pla a 2, is a glycoprotein with an observed molecular mass of 43 kd and an isoelectric point value of 9.3. It is involved in the allergic responses of 84% of patients with planetree-induced pollinosis and represented 52% of the total IgE-binding capacity of the P acerifolia extract. Pla a 2 displays polygalacturonase (PG) activity, being the first PG with functional enzyme activity from an angiosperm plant pollen described as an allergen. The cDNA allergen sequence codified for a 372-residue protein with 56% and 42% sequence identity to PGs from pollen and fruits, respectively. Western blot analysis showed that Pla a 2 is present in pollen and stems and has IgG cross-reactivity with a PG from tomato and pectate lyases from Cupressaceae pollen. CONCLUSION: Pla a 2, a major allergen of P acerifolia pollen with PG activity has been purified, characterized, and cloned.

A sensitive two-site enzyme-linked immunosorbent assay for measurement of the major Alternaria alternata allergen Alt a 1.

BACKGROUND: Alt a 1 is the major allergen in Alternaria alternata, one of the most important fungi associated with allergic diseases. Mold allergenic extracts show considerable heterogeneity, and thus accurate standardization of these extracts is essential to guarantee their quality. OBJECTIVE: To develop an Alt a 1-specific assay and to evaluate the correlation of Alt a 1 content with the IgE-binding activity of A. alternata extracts. METHODS: Recombinant Alt a 1 was produced as nonfusion protein from a polymerase chain reaction-cloned complementary DNA Alt a 1 sequence. Natural Alt a 1 was purified from spent culture medium. Monoclonal and polyclonal antibodies directed to Alt a 1 were produced and used to construct a specific Alt a 1 enzyme-linked immunosorbent assay (ELISA). RESULTS: The ELISA developed was highly reproducible and sensitive, with a detection limit lower than 0.5 ng/mL and a practical working range of 0.5 to 50 ng/mL. The assay was able to detect an Alt a 1-like protein in Stemphylium extracts. Identical parallel dose-response curves were observed when natural Alt a 1 and recombinant Alt a 1 were used as standard. A good correlation was obtained between Alt a 1 content of 13 A. alternata extracts and their IgE-binding activity. Alt a 1 was responsible for 70% of the IgE-binding activity of the whole extract. CONCLUSIONS: This sensitive and specific Alt a 1 assay allows the quantification of this major mold allergen and represents a useful tool for the standardization of A. alternata extracts in mass units. It also provides a reliable indication of the allergenic activity of the whole extract.

Molecular and structural analysis of the panallergen profilin B cell epitopes defined by monoclonal antibodies.

Interactions of five mouse mAb (10A4, 5F2, 9A7, 9G4 and 3H8) and sunflower profilin were characterized using synthetic overlapping peptides. All the continuous B cell epitopes analyzed in this work were 6-10 amino acids in length, and clustered at the N- and C-terminal alpha-helices and a two-stranded segment composed of residues 40-50. Mutational analysis of the epitopes revealed that single amino acid changes within these peptides had dramatic effects on IgG-binding characteristics. A three-dimensional molecular model of sunflower profilin was generated by homology modeling based on the crystal structure of Arabidopsis thaliana profilin. All but one of the murine B cell epitopes defined in this work were located on the surface of the profilin molecule in the alpha-helices (10A4 and 3H8) or in the turns (5F2 and 9G4). In contrast, 9A7 epitope was located in the profilin core and partially buried by the C-terminal. Two mAb (5F2 and 10A4) inhibited the binding of anti-profilin human IgE up to 52%. In contrast, mAb 3H8 seemed to enhance the binding of anti-profilin IgE of sera from allergic patients.

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.