The Russian Thistle (Salsola kali) pollen major allergen, Sal k 1, can be quantified in allergenic extracts and airborne pollen.

BACKGROUND: Russian thistle (Salsola kali) pollen is an important cause of pollinosis in areas where rainfall is not abundant. Our aim was to develop an ELISA for quantification of the major allergen of S. kali extracts, Sal k 1, and to assess the correlation of this allergen content with the allergenic activity of extracts. METHODS: Sal k 1 was purified by ion exchange and gel permeation chromatography and identified by mass spectrometry. Monoclonal antibody 4C11 was used for capture at 5 microg/ml and biotin-labeled specific antiserum at 0.25 microg/ml served for detection. The allergenic activity of the pollen extracts was measured by enzyme allergosorbent test inhibition. RESULTS: Sal k 1 reacted to 85% of sera from 40 S. kali-allergic patients and was able to inhibit 92% of the IgE-binding capacity of patients' serum pool to the whole extract. The ELISA had a lineal range between 1.25 and 20 ng/ml of purified Sal k 1. The intra- and interassay coefficients of variation were lower than 5 and 10%, respectively. The assay was very sensitive since it had a detection limit of 0.08 ng/ml. No reactivity was found outside the Amaranthaceae family where only Kochia and Salicornia sp. gave significant reactivity. A good correlation (Spearman's rho = 0.92) was obtained between Sal k 1 content of different S. kali extracts and their IgE-binding activity. CONCLUSIONS: The results proved the usefulness of the two-site sandwich ELISA for aeroallergen control and for the standardization of S. kali pollen extracts intended for clinical use.

Engineering of major house dust mite allergens Der p 1 and Der p 2 for allergen-specific immunotherapy.

BACKGROUND: Specifically designed recombinant allergens with reduced IgE reactivity are promising candidates for a more defined, effective, and safer specific immunotherapy (SIT). OBJECTIVE: We sought to obtain hypoallergenic hybrid molecules which could potentially be applied to house dust mite (HDM) allergy treatment. METHODS: Two hybrid molecules (QM1 and QM2) derived from the two major Dermatophagoides pteronyssinus allergens, Der p 1 and Der p 2, were engineered by PCR, produced in Escherichia coli, and purified. The overall IgE-binding capacity of the hybrids was compared with their single components by Western blot, specific IgE, skin prick test (SPT), and IgE-inhibition assays. T cell proliferation assay were performed to confirm their retention of T cell reactivity. Immune responses to the hybrid molecules were studied in BALB/c mice. RESULTS: The IgE reactivity of both hybrid proteins was strongly reduced as evaluated by in vitro methods. Furthermore, in vivo SPTs performed on 106 HDM-allergic patients showed that the hybrid proteins had a significantly lower potency to induce cutaneous reactions than the individual components. Hybrid molecules induced higher T cell proliferation responses than those produced by an equimolecular mixture of Der p 1 and Der p 2. Immunization of mice with the hybrid proteins induced Der p 1- and Der p 2-specific IgG, which inhibited the binding of allergic patients' IgE to these natural allergens. CONCLUSION: QM1 and QM2 hybrids exhibited less IgE-binding activity but preserved immunogenicity and fulfilled the basic requirements for hypoallergenic molecules suitable for a future SIT of HDM allergy.

An antibody-based ELISA for quantification of Ani s 1, a major allergen from Anisakis simplex.

Anisakis simplex is a nematode parasite that can infect humans who have eaten raw or undercooked seafood. Larvae invading the gastrointestinal mucosa excrete/secrete proteins that are implicated in the pathogenesis of anisakiasis and can induce IgE-mediated symptoms. Since Ani s 1 is a potent secreted allergen with important clinical relevance, its measurement could assess the quality of allergenic products used in diagnosis/immunotherapy of Anisakis allergy and track the presence of A. simplex parasites in fish foodstuffs. An antibody-based ELISA for quantification of Ani s 1 has been developed based on monoclonal antibody 4F2 as capture antibody and biotin-labelled polyclonal antibodies against Ani s 1 as detection reagent. The dose-response standard curves, obtained with natural and recombinant antigens, ranged from 4 to 2000 ng/ml and were identical and parallel to that of the A. simplex extract. The linear portion of the dose-response curve with nAni s 1 was between 15 and 250 ng/ml with inter-assay and intra-assays coefficients of variation less than 20% and 10%, respectively. The assay was specific since there was no cross-reaction with other extracts (except Ascaris extracts) and was highly sensitive (detection limit of 1.8 ng/ml), being able to detect Ani s 1 in fish extracts from codfish and monkfish.

Expression of a recombinant protein immunochemically equivalent to the major Anisakis simplex allergen Ani s 1.

BACKGROUND: Anisakis simplex is a nematode which can parasitize humans, producing anisakiasis and can induce immunoglobulin-(Ig)-E-mediated allergic symptoms. Parasite recombinant proteins, such as the major allergen Ani s 1, may be useful tools to avoid misdiagnosis of A simplex allergy due to cross-reactivity when whole parasite extracts are used. OBJECTIVE: To obtain Ani s 1 allergen as a recombinant protein with IgE-binding properties similar to its natural counterpart. METHODS: Ani s 1-encoding cDNA was amplified by polymerase chain reaction and cloned. The allergen was expressed in Escherichia coli as a nonfusion protein. Natural and recombinant Ani s 1 were investigated by means of Western blotting, enzyme allergosorbent test, enzyme-linked immunosorbent assay (ELISA), and ELISA inhibition using sera from 53 patients with A simplex allergy. RESULTS: Residues of the amino acid sequence of the encoded protein were 99.4% identical to the reported one. Purified rAni s 1 was obtained with a yield of 2 mg/L of culture while the yield of the natural counterpart was only 50 micro/g of larvae. rAni s 1 reactivity was not significantly different from that of the natural allergen; the correlation was excellent (p = 0.92, P < .001). ELISA-inhibition experiments showed that the dose-response inhibition curve obtained with rAni s 1 overlapped with that of nAni s 1. In an enzyme allergosorbent analysis, 86.8% of the A simplex-allergic patient sera reacted to rAni s 1. CONCLUSION: Recombinant Ani s 1 is immunochemically equivalent to its natural counterpart and therefore might be useful for the in vitro diagnosis of anisakiasis and A simplex-mediated allergy.

Cloning, expression and characterization of mugwort pollen allergen Art v 2, a pathogenesis-related protein from family group 1.

Mugwort (Artemisia vulgaris) belongs to the Compositae family, and is one of the main causes of allergy in late summer and autumn. The aim of the study was to characterize the allergen Art v 2 from mugwort pollen. Skin prick tests, performed in 19 patients allergic to mugwort and 10 control patients, showed an Art v 2 sensitization prevalence of 58%, whereas none false-positives were detected among control patients. Art v 2 was purified by standard chromatography and binding to Concanavalin A column and had an apparent molecular mass of 33 and 20 kDa, calculated by gel permeation and SDS-PAGE under denaturing conditions, respectively, showing that the allergen is composed of two identical subunits. Art v 2-encoding cDNA was amplified by PCR using degenerate primers based on reported partial amino acid sequences. Cloned cDNA encoding Art v 2 contains 140 bp that codify for a polypeptide of 15.8 kDa, with a predicted pI value of 5.2, and one potential N-glycosylation site. Protein homology search demonstrated that Art v 2 share 55-42% identical residues with pathogenesis-related protein PR-1 of tomato, potato, rape, wheat and rice. Homology was also found to Ves v 5 (41% identical residues). Bacterial-expressed recombinant Art v 2 was recognized only by 21% of mugwort-allergic patients. In conclusion, Art v 2 from mugwort is the first weed pollen allergen that belongs to the pathogenesis-related protein PR-1 and its recombinant form could help molecular diagnosis of mugwort associated allergy.

Validation of ELISA methods for quantification of the major birch allergen Bet v 1 (BSP090).

To date, the potency of allergen products in Europe is expressed in manufacturer-specific units relative to a product-specific in-house reference. Consequently, cross-product comparability of allergen products from different manufacturers with respect to strength and efficacy is impossible. The Biological Standardisation Programme (BSP) project BSP090 addresses this issue via the establishment of reference standards in conjunction with ELISA methods for the quantification of major allergens in allergen products. Since the initiation of BSP090, the recombinant major allergen Bet v 1 has been adopted by the European Pharmacopoeia Commission as a Chemical Reference Substance (CRS). In parallel, two sandwich ELISA systems for quantification of Bet v 1 were found suitable in preliminary phases of BSP090 to be validated in a large collaborative study. In this study, the candidate ELISA systems were compared with respect to accuracy, precision and variability. Thirteen participating laboratories tested model samples containing the CRS as well as spiked and unspiked birch pollen extracts. Both in pre-testing and in the collaborative study, the 2 candidate ELISA systems confirmed their suitability to quantify recombinant and native Bet v 1. As no clear-cut decision for one of the ELISA systems could be made based on the results of the collaborative study, a post-study testing was performed. Bet v 1 content of 30 birch pollen allergen products was determined in parallel in both ELISA systems. Consequently, 1 candidate ELISA system was selected to be proposed as the future European Pharmacopoeia standard method for Bet v 1 quantification.

Sequence polymorphism and structural analysis of timothy grass pollen profilin allergen (Phl p 11).

Three cDNA clones encoding timothy grass pollen profilin (Phl p 11) were newly isolated. Comparison of the sequences of four cDNA clones, including a previously isolated clone, showed a low level of polymorphism. Isoelectrofocusing of highly purified timothy grass profilin indicated the existence of at least five isoforms. One recombinant profilin showed similar immunological properties to natural timothy grass profilin. Tertiary structure of Phleum pratense profilin was obtained by homology-based molecular modeling.

Sequencing and high level expression in Escherichia coli of the tropomyosin allergen (Der p 10) from Dermatophagoides pteronyssinus.

The cDNA encoding an allergen from the dust mite Dermatophagoides pteronyssinus has been cloned and sequenced. The allergen (Der p 10) is a tropomyosin that shared more than 65% identical residues with other invertebrate tropomyosins. The final recovery of recombinant Der p 10 from the culture media after a single purification step was as much as 26 mg/l. The recombinant allergen is reactive to shrimp antitropomyosin IgG antibodies and has a 5.6% frequency of IgE reactivity in sera from mite-allergic patients.

Cloning and immunological characterization of the allergen Hel a 2 (profilin) from sunflower pollen.

Sunflower (Helianthus annuus) sensitization is not always related with occupational allergy. We have isolated the allergen profilin (Hel a 2) from this Compositae plant, cloned and sequenced five cDNAs encoding for full-length or partial Hel a 2. Natural sunflower profilin reacted with specific IgE in the 121 sera tested, at a frequency of 30.5%. Expression of the cDNA encoding Hel a 2 in Escherichia coli and a simple purification procedure by poly-L-proline chromatography allowed immunological characterization of the recombinant allergen. Binding of monoclonal antibodies against sunflower profilin revealed that some epitopes responsible for antigen-specific IgG production were not present in the recombinant allergen. High cross-reactivity has been found between recombinant Hel a 2 and profilins from other Compositae plants and also from botanically distant plants.

Quantification of profilins by a monoclonal antibody-based sandwich ELISA.

Profilins are plant allergens responsible for cross-reactivities in pollen and fruit-allergic patients. A two-site enzyme-linked immunosorbent assay has been developed for the quantification of profilins and its suitability for quantifying profilin in different plant extracts has been evaluated. The assay is based on two profilin-specific monoclonal antibodies (mAbs) with different epitope specificities. These antibodies were immobilized on ELISA plates and incubated with samples containing profilin. Bound profilin was detected by a combination of biotinylated profilin-specific antiserum and peroxidase-streptavidin conjugate. The optimized ELISA measured profilin concentrations ranging from 4 to 250 ng/ml and could quantify profilins from plant species of a variety of different botanical families. No reactivity to mites, molds, or crustaceans was detected, suggesting that the immunoassay is plant-specific. The results indicate that this sensitive profilin-assay will be helpful both for quantifying the profilin content of allergenic extracts intended for clinical use and for studying cross-reactivities between pollen extracts.

Reactivity of Candida albicans germ tubes with salivary secretory IgA.

Salivary secretory IgA (sIgA) has been shown to react with a group of heat shock mannoproteins preferentially expressed on yeast cells grown at 37 degrees C. Since at this temperature C. albicans can induce germ tubes, we explored the role of germ tube induction on human salivary sIgA reactivity in both germinative and agerminative C. albicans strains, in an attempt to investigate whether the germ tube expressed the heat shock mannoproteins reactive with sIgA. The reactivity with sIgA of the agerminative strain, grown at 25 and 37 degrees C for different times, was measured spectrofluorometrically and was fairly constant with time. Yeast cells grown at 37 degrees C tended to be more reactive than those grown at 25 degrees C. In contrast, when compared with the yeast cells of the germinative strain grown at 25 degrees C, there was a statistically significant decrease in reactivity with sIgA during germ tube formation. Serum IgA and IgG did not show statistically significant changes in reactivity with C. albicans during germination, suggesting differences in reactivity with C. albicans cell wall antigens between mucosal and systemic humoral responses. Cell wall mannoproteins of molecular masses > 60 kDa were characterized by Western blotting as responsible for the decrease in sIgA reactivity observed in the germ tube, and the fall in sIgA reactivity was related to the release of cell wall mannoproteins into the culture medium. The release of these mannoproteins may be a mechanism whereby C. albicans avoids the action of sIgA, and it may play an important role in the post-parasite relationship in oral candidiasis.

Vulvovaginal candidiasis refractory to treatment with fluconazole.

We present the case of an infertile patient, whose first attempt at IVF had to be postponed for 18 months due to a vulvovaginal yeast infection refractory to treatment. The main causative organism was a Candida glabrata strain resistant to all the imidazolic agents tested. The organism and the host's humoral status were studied in depth, looking for possible causes of the refractoriness to treatment.

Tolerance and immunological changes of chemically modified allergen vaccine of Parietaria judaica in accelerated schedules.

The physicochemical modification of allergen vaccines provides a chance for administering higher doses in a shorter period of time. We sought to assess the safety and immunological changes of using a biologically standardized and modified Parietaria judaica pollen extract in accelerated schedules. Two accelerated schedules were tested in 45 P. judaica-allergic patients: 20 patients reached the maximum dose after two visits using two different concentrations and 25 patients reached the maximum dose after only one visit with two injections of the maximum concentration vial. The tolerance was assessed by recording all side effects related with immunotherapy. Specific antibody levels against native extract and rPar j 2 allergen were evaluated at the beginning and the end of the study. Allergenic potency determined by enzyme allergosorbent test (EAST) inhibition and skin prick test showed that modified P. judaica pollen had a 99.9% less allergenicity than native extract. After 650 doses administered, two clinically irrelevant local reactions (diameter<0 x 5 cm) and no systemic reactions were registered. Significant increases in allergen-specific IgG4 and IgG against P. judaica extract and rPar j 2 and significant decrease of specific IgE against Par j 2 were observed. The modified extract of P. judaica is safe to treat sensitive patients, even at accelerated regimens, and induces significant immunological changes.

Diagnosis of Parietaria judaica pollen allergy using natural and recombinant Par j 1 and Par j 2 allergens.

BACKGROUND: Parietaria judaica pollen is one of the main causes of allergic diseases in the Mediterranean area and contains two major allergens, called Par j 1 and Par j 2. OBJECTIVE: To evaluate the diagnostic potential of natural and recombinant forms of Par j 1 and Par j 2 in comparison with standardized P. judaica pollen extract. METHODS: Thirty patients allergic to P. judaica pollen and 15 control patients were investigated. Skin prick tests and determination of specific IgE levels were performed with commercial P. judaica extract, natural Par j 1 and Par j 2, and recombinant forms of both allergens expressed in P. pastoris. RESULTS: The whole group of patients with allergy to P. judaica had a positive skin test reaction to purified nPar j 1-Par j 2 and rPar j 2 at 5 microg/mL, and no false-positive reactions were detected. Natural and recombinant Par j 1 and Par j 2 showed no significantly different responses in skin tests compared with P. judaica extract. A high correlation was found between the serum-specific IgE levels to P. judaica extract vs. natural (R=0.996; P<0.001) and recombinant allergens (R=0.887 and 0.982 for rPar j 1 and rPar j 2, respectively; P<0.001). rPar j 2 displayed a 100% sensitivity and specificity among P. judaica-allergic patients. CONCLUSIONS: In vivo and in vitro diagnosis of P. judaica pollen allergy could be simplified using rPar j 2. This protein showed comparable IgE response and skin prick reactivity with those produced by P. judaica pollen extract.

A sensitive monoclonal antibody-based enzyme-linked immunosorbent assay to quantify Parietaria judaica major allergens, Par j 1 and Par j 2.

BACKGROUND: Parietaria pollen is one of the most important causes of pollinosis in Mediterranean countries. Parietaria judaica pollen extract presents two major allergens, Par j 1 and Par j 2, that belong to the lipid transfer protein family. OBJECTIVE: To develop an ELISA for quantification of both major allergens of P. judaica pollen extracts, and to assert correlation of these allergens content with the allergenic activity of extracts. METHODS: Natural Par j 1-Par j 2 allergens were purified by gel filtration, ion exchange, and affinity chromatography and identified by mass spectrometry. Rabbit antisera were obtained using this protein preparation as antigen and used for immunoaffinity purification of nPar j 1-Par j 2. BALB/c mice were immunized with the immunopurified nPar j 1-Par j 2 and after fusion and screening by direct ELISA, 5D4 monoclonal antibody was selected as capture antibody to develop a quantitative two-site ELISA. Bound proteins were detected by a biotinylated Par j 1-Par j 2-specific polyclonal antibody. RESULTS: The optimized ELISA was developed from 25 to 8000 pg/mL of purified Par j 1-Par j 2, and a linear portion of 200-1000 pg/mL. The intraassay and interassay coefficients of variation were lower than 7% and 14% respectively. The assay was very sensitive and specific as it had a detection limit of 25 pg/mL and did not detect reactivity with the same family plants, as Urtica. Par j 1-Par j 2 allergens content was measured in 14 P. judaica and two P. officinalis pollen extracts showing a significant correlation with their allergenic activity measured by enzyme allergosorbent test inhibition. CONCLUSIONS: The results proved the usefulness of the two-sandwich ELISA for the standardization of Parietaria pollen extracts intended for clinical use, because of its good correlation with allergenic potency.

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.

Pho d 2, a major allergen from date palm pollen, is a profilin: cloning, sequencing, and immunoglobulin E cross-reactivity with other profilins.

BACKGROUND: Up to now, some date palm pollen (DPP) allergens have been described but very few data are available about their molecular nature. The aim of this study was to identify and characterize Pho d 2, a major allergen from this pollen. METHODS: Sera from 25 patients allergic to DPP were analysed by immunoblotting. Purification of DPP profilin was performed by poly-l-proline affinity chromatography. Profilin-encoding cDNA from DPP was cloned by using a RT-PCR strategy and recombinant allergen was expressed as a non-fusion protein in Escherichia coli. Natural and recombinant Pho d 2 were investigated by means of enzyme allergosorbent test to compare the immunologic properties of both allergens and to analyse cross-reactivity with other profilins. RESULTS: A 14.4 kDa protein was identified as a major allergen in DPP extract. Purification, cloning, heterologous expression, and inhibition experiments identified it as profilin (Pho d 2). Pho d 2 comprises 131 amino acids and has high sequence identity with other allergenic food and pollen profilins. The prevalence of specific IgE antibody reactivity to natural Pho d 2 by ELISA was 56% and 64% by skin prick test (SPT). Pho d 2 is an important allergen as it is responsible for more than 70% of the IgE reactivity to the pollen extract. IgE directed against Pho d 2 showed a strong cross-reactivity with other profilins such as those from olive tree and grass pollens. CONCLUSION: Pho d 2, a 14.4 kDa protein identified as profilin, is a major and relevant allergen in DPP, as confirmed by SPT and thereby may elicit clinical symptoms in sensitized patients.

Development of a sandwich-type ELISA for measuring Pla a 1, the major allergen of Platanus acerifolia pollen.

BACKGROUND: Platanus acerifolia is an important cause of pollinosis in Western European cities. Pla a 1, a nonglycosylated 18-kDa protein with a prevalence of 80%, is a major allergen in P. acerifolia pollen extracts. Our aim was to develop a Pla a 1-specific ELISA to quantify this protein in allergenic extracts and preparations for clinical use. METHODS: Pla a 1 was purified by cation exchange at pH 7.0, gel filtration, and anion exchange chromatography at pH 10.0. Monoclonal (mAb) and polyclonal antibodies were obtained by immunizing mice and rabbits with nPla a 1. One (5C1) of the 13 mAb obtained was used as capture antibody at 5 mug/ml and biotin-labeled specific polyclonal antiserum at 0.63 microg/ml served for detection. RESULTS: The prevalence of Pla a 1-specific IgE to purified Pla a 1 among 47 P. acerifolia-allergic patients was 79%. The Pla a 1-ELISA developed has a linear range of 3-25 ng/ml, high sensitivity with a detection limit of 0.5 ng/ml and is highly specific as none of the 24 pollen, mite, mold, and plant food extracts tested gave positive results. The assay could quantify Pla a 1-like proteins in other planetree pollen extracts. A good correlation was obtained between Pla a 1 content of 11 P. acerifolia pollen extracts (average content 0.69% of the total protein) and their IgE-binding activity. CONCLUSIONS: The described two-site sandwich ELISA to measure Pla a 1 is useful for standardization of planetree pollen extracts intended for clinical use.

The major Platanus acerifolia pollen allergen Pla a 1 has sequence homology to invertase inhibitors.

BACKGROUND: Sycamores or plane trees are an important source of airborne allergens in many cities of the United States and Western Europe. Pla a 1 has been described as a major allergen from Platanus acerifolia (London plane tree). OBJECTIVE: To clone and characterize the cDNA for Pla a 1 and to express the recombinant protein. METHODS: Pla a 1 was isolated by cationic exchange, gel filtration, and reverse-phase chromato-graphies. Pla a 1 cDNA was cloned by reverse transcription followed by polymerase chain reaction, using amino acid sequences from tryptic peptides of the allergen. The Pla a 1 encoding sequence has been subcloned into the pKN172 expression vector and expressed in Escherichia coli as a non-fusion protein. Purified recombinant protein has been tested for its IgE-binding capacity in immunoblot, immunoblot inhibition, and ELISA. RESULTS: Pla a 1 reacted with serum IgE from 35 of the 42 (83.3%) Platanus-allergic patients studied and represented 60% of the total IgE-binding capacity of the P. acerifolia pollen extract. The allergen displayed 43% sequence identity to a grape invertase inhibitor and showed a predicted secondary structure characteristic of all-alpha proteins. Serological analysis revealed that both natural and recombinant forms of Pla a 1 displayed similar IgE-binding capacity. CONCLUSIONS: Pla a 1 belongs to a new class of allergens related to proteinaceous invertase inhibitors. Recombinant Pla a 1 binds IgE in vitro like its natural counterpart and, therefore, it can be useful for specific diagnosis and structural studies.