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J A Asturias

Publications and source records attributed to J A Asturias.

At least 19 recordsLinked to original sources
PubMed2006

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.

Allergens↗
PubMed2006

Purified allergens vs. complete extract in the diagnosis of plane tree pollen allergy.

BACKGROUND: Plane tree pollen allergy is a clinical disorder affecting human population in cities of Europe, North America, South Africa, and Australia. OBJECTIVE: To compare IgE-reactivity of the natural and recombinant forms of two major plane allergens, Pla a 1 and Pla a 2, with the reactivity of Platanus acerifolia pollen extract. METHODS: Forty-seven patients with P. acerifolia allergy, 15 of them monosensitized, and 24 control subjects were included in the study. Natural Pla a 1 and Pla a 2 were purified by standard chromatographic methods and recombinant proteins were expressed in Escherichia coli. Skin prick test and determination of specific IgE were performed with commercial P. acerifolia extract and natural and recombinant purified allergens. RESULTS: Pla a 1 and Pla a 2 were responsible for 79% of the IgE-binding capacity against P. acerifolia pollen extract. A high correlation has been found between the IgE response to nPla a 1 (R = 0.80; P < 0.001) or nPla a 2 (R = 0.79; P < 0.001) vs. P. acerifolia extract as well as between natural and recombinant Pla a 1 (R = 0.89; P < 0.001). Skin testing showed no significant differences between extract and nPla a 2, whereas a higher reactivity was found with nPla a 1. In contrast, rPla a 1 revealed markedly reduced sensitivity in comparison with extract by skin prick test and specific IgE. The sensitivity of the mix Pla a 1+Pla a 2 was 100% and 87.5% for monosensitized and polysensitized patients, respectively, with no false-positive reactions detected. Conclusion Pla a 1 and Pla 2 are sufficient for a reliable diagnosis of P. acerifolia in most patients and induce comparable skin test reactivity as a whole extract.

Adolescent↗
PubMed2006

Biological standardization and maximum tolerated dose estimation of an Alternaria alternata allergenic extract.

BACKGROUND: The manufacture of allergenic extracts from the mold Alternaria alternata is influenced by factors such as strain variability, allergenic origin, culturing conditions and extraction process, which affect the reproducibility of the preparations intended for diagnostic and therapeutic use. OBJECTIVES: To select the most adequate antigenic source of A. alternata extracts and determine its maximum tolerated dose (MTD) to be used in a subsequent immunotherapy efficacy clinical trial. METHODS: Twenty-one patients monosensitized to A. alternata were involved in a biological standardization process of A. alternata extracts. Four different mold strains were cultured and used to produce extracts by three different methods, each incorporating proteins from different origins: culture filtrate, buffer extractable fraction and cellular antigens. The selected extract, characterized as in-house reference (IHR) preparation was used in a MTD finding immunotherapy study. Serum IgE, IgG, IgG1 and IgG4 specific of complete extract and purified natural and recombinant forms of Alt a 1 were determined by different EIA methods. RESULTS: Culture filtrate extract containing the allergens secreted to the spent medium was shown to be the most adequate option for establishing an IHR preparation for A. alternata extract manufacturing. A maximum dose of 1670 UBE, equivalent to 0.1 microg Alt a 1, was determined as MTD for immunotherapy. One year of administration of such a dose at monthly intervals elicited pronounced immunological changes with statistically significant decreases in IgE and increases in IgG4, both estimated with whole extract or purified Alt a 1. CONCLUSION: A high quality natural A. alternata extract has been developed and preliminarily tested to define its MTD for subsequent determination of the optimal dose in an immunotherapy efficacy clinical trial.

Adolescent↗
PubMed2005

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.

Allergens↗
PubMed2005

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.

Adult↗
PubMed2005

L-selectin expression on neutrophils from allergic patients.

BACKGROUND: L-selectin (CD62L) is an adhesion molecule involved in leucocyte attachment to endothelium at sites of inflammation, and it has been demonstrated that L-selectin is rapidly shed after neutrophil activation. Recently, it has been reported that there is increasing evidence of neutrophil participation in asthma and the allergic process. OBJECTIVE: The present study was designed to determine whether an IgE-dependent mechanism can modulate L-selectin expression on the surface of neutrophils. Moreover, we analyse the potential implication of intracellular signal-transduction pathways and whether specific immunotherapy (IT), glucocorticoids and antihistamines might regulate this process. METHODS: Peripheral blood neutrophils from three groups of donors (asthmatic group without IT treatment, IT-treated asthmatic group and healthy group) were used. Cells were challenged in vitro with the specific allergen that produced clinical symptoms in asthmatic patients and also with the allergen to which the patients were not sensitive. Neutrophils from healthy donors were also challenged with allergens. Expression of CD62L on the neutrophil surface was analysed by flow cytometry, and soluble CD62L (sCD62L) in culture supernatant by ELISA. In an attempt to discover which IgE receptor is involved, we also challenged the neutrophils with monoclonal antibody to FcepsilonRI, FcepsilonRII (CD23) and galectin-3 receptors. RESULTS: When neutrophils from allergic patients were challenged with specific allergens that produce clinical allergy symptoms, L-selectin was down-regulated from the surface of those cells, accompanied by a concomitant up-regulation of soluble L-selectin in the supernatant. The challenge with antibodies against FCepsilonRI, FCepsilonRII (CD23) and galectin-3, induces down-modulation of L-selectin on the surface of the neutrophils in all three cases. Calphostin C, wortmannin and manoalide attenuated CD62L down-regulation, suggesting the potential implication of protein kinase C, phosphatidylinositol 3-kinase and phospholipase A(2) in the process. IT and glucocorticoids modulated allergen-dependent CD62L down-regulation, whereas antihistamines (terfenadine, loratadine and cetirizine) or nedocromil sodium did not affect the shedding of L-selectin. CONCLUSIONS: We present evidence that the neutrophil surface expression of CD62L can be modulated by an allergen-dependent mechanism. The modulation of CD62L expression can be induced through the three receptors of IgE. This process can be affected by IT.

Adolescent↗
PubMed2004

Quantification of the major allergen from cypress (Cupressus arizonica) pollen, Cup a 1, by monoclonal antibody-based ELISA.

BACKGROUND: Cypress pollen allergy is an important cause of rhinoconjunctivitis and asthma in Mediterranean countries. Cypress allergenic extracts are difficult to produce since they have low protein and high carbohydrate content, thus accurate standardization of them is essential to guarantee their quality. The aim of this study is to develop a sandwich ELISA for the quantification of Cup a 1, the major allergen of cypress (Cupressus arizonica) pollen extract. METHODS: Monoclonal antibodies directed to purified Cup a 1 were produced. Two of them (9C7 as capture antibody and 3D2 as the tracer) were selected to develop a quantitative sandwich ELISA. This ELISA was subsequently evaluated and compared with other techniques. RESULTS: The described ELISA is very sensitive with a detection limit of 8.7 ng/ml and a practical working range of 62.5-1,000 ng/ml. The assay is also highly reproducible with intra-assay and interassay coefficients of variation of less than 10%. The purified Cup a 1, used as standard, presents pectate lyase enzymatic activity. The assay also detected Cup a 1-like proteins in pollen from other Cupressaceae. A good correlation was obtained between Cup a 1 content of 12 C. arizonica pollen extracts and their IgE-binding activity. CONCLUSIONS: The described Cup a 1 ELISA is sensitive, specific and reproducible and can be used for the quantification of Cup a 1 in C. arizonica and other related pollen extracts. It also provides a reliable indication of the allergenic activity of the whole cypress pollen extract.

Allergens↗
PubMed2004

Biological characterization of glutaraldehyde-modified Parietaria judaica pollen extracts.

BACKGROUND: Allergoids are widely used in specific immunotherapy (SIT) for the treatment of IgE-mediated allergic diseases, but all techniques for standardization of conventional allergic extracts may not be appropriate for standardization of a glutaraldehyde (GA)-modified extract because of the unique characteristics of these extracts. OBJECTIVE: To assess an accurate methodology for standardization of chemically modified extracts. METHODS: GA-modified extracts from Parietaria judaica pollen were purified by diafiltration. Biochemical properties were investigated by determination of amino groups, chromatography, and SDS-PAGE. The IgE-binding activity was determined by skin prick test, enzyme allergosorbent test inhibition, basophil activation, and histamine release tests. Peripheral blood mononuclear cells (PBMCs) from P. judaica pollen-allergic subjects were stimulated with either native or allergoid extracts, and proliferation was measured. RESULTS: Biochemical data indicated a high degree of allergen polymerization resulting in extract components higher than 100 kDa. IgE-binding activity, both in vivo and in vitro, was reduced by more than 99.8%. Both allergen and allergoid induced PBMC proliferation and synthesis of blocking IgG antibodies at similar rates. Moreover, no evidence of introduction of new determinants by chemical modification was found. CONCLUSIONS: The preparation of GA-modified extracts by diafiltration is faster and more reliable than previous chromatographic methods. These modified extracts have drastically reduced their allergenicity while maintaining their immunogenicity, and therefore they can be used in safer and shortened schedules of SIT.

Adolescent↗
PubMed2004

PCR-based cloning and immunological characterization of Parietaria judaica pollen profilin.

Profilin has been described as an allergen present in pollen of trees, grasses and weeds. Since Parietaria judaica profilin has a molecular mass similar to other Parietaria allergens (Par j 1 and Par j 2) in the 14-10 kDa range, it is difficult to assess the prevalence of profilin by immunoblotting or to obtain sufficient amounts of purified native profilin for investigation and diagnosis. The aim of this study was to identify P. judaica profilin by PCR-based cDNA cloning and to elucidate its allergenic characteristics. Two cDNA clones encoding P. judaica pollen profilin were isolated by polymerase chain reaction (PCR) amplification using degenerate primers. Sequencing of both clones (Par j 3.0101 and Par j 3.0102) demonstrated a high amino acid sequence homology. Immunodetection of P. judaica pollen after isoelectrofocusing and incubation with rabbit antiserum against profilin indicated the existence of at least 2 isoforms. Expression in Escherichia coli BL21 (DE3) was carried out using a vector based in the T7 expression system, and the recombinant allergen was isolated by affinity chromatography on poly-(L-proline)-Sepharose. Cross-reactivity has been found between recombinant P. judaica pollen profilin and profilins from other botanical unrelated plants.

Base Sequence↗
PubMed2004

IgE reactivity to profilin in Platanus acerifolia pollen-sensitized subjects with plant-derived food allergy.

BACKGROUND: The presence of profilin-specific IgE antibodies is a cause of cross-reactivity between botanically-unrelated allergen sources. Recently, the association between Platanus acerifolia pollinosis and plant-derived food allergy has been described. The aim of this study was to ascertain whether the P. acerifolia profilin is involved in such cross-reactivity. METHODS: Twenty-three patients suffering from Platanus acerifolia pollinosis and plant-derived food allergy were evaluated in an allergy department. Specific IgE levels to P. acerifolia pollen, P. acerifolia profilin and food extracts were measured. Molecular masses of IgE-binding proteins were calculated by Western blotting and cross-reactivity studies among P. acerifolia profilin and different food extracts were evaluated by Enzyme AllergoSorbent Test (EAST)-inhibition assays. Also, EAST-inhibition assays with the two known P. acerifolia allergens, Pla a 1 and Pla a 2, were performed. RESULTS: Surprisingly, a high IgE-binding prevalence (90%) of P. acerifolia profilin was found. EAST-inhibition showed high inhibition values when Platanus acerifolia pollen extract was used as free phase and plant-derived food extracts as solid phase, whereas the other way round showed low inhibition values. IgE reactivity to profilin was studied using a pool of patient sera, by EAST-inhibition assays with hazelnut, apple peel, peanut, chickpea and peanut extracts as solid phase and no inhibition was obtained when P. acerifolia profilin was used as inhibitor phase. The same results were obtained when purified Pla a 1 and Pla a 2 were also used as inhibitor phase. CONCLUSIONS: The clinical association observed between Platanus acerifolia pollen and plant-derived food could be explained by the in vitro IgE cross-reactivity detected by EAST-inhibition. However, it appears that neither P. acerifolia profilin nor the two major allergens described (Pla a 1 and Pla a 2) can explain such a strong cross-reactivity.

Adult↗
PubMed2003

Par j 1 and Par j 2, the major allergens from Parietaria judaica pollen, have similar immunoglobulin E epitopes.

BACKGROUND: Parietaria judaica is the main cause of allergy in Mediterranean countries. The major allergens from P. judaica pollen, Par j 1 and Par j 2, have amino acid sequence identity of 45% and contain eight cysteine residues involved in disulphide bonds that compact the structure. OBJECTIVE: The aim of the study was to identify IgE-binding epitopes on Par j 1 and Par j 2, the major allergens from P. judaica pollen and correlate them with the three-dimensional structure of the proteins. METHODS: Overlapping peptides representing the complete length of Par j 1 and Par j 2 were synthesized on a cellulose-derivatized membrane. Sera from 17 P. judaica-allergenic patients were used to identify IgE-binding epitopes. Homology modelling of the three dimensional structure of both allergens was generated using the Swiss-Model software on the basis of previously reported crystal structures. RESULTS: Five and eight IgE-binding epitopes were identified on Par j 1 and Par j 2 allergens, respectively. Both proteins belonged to the non-specific lipid transfer proteins (ns-LTP) family and therefore a three-dimensional model of both allergens was constructed on the basis of the maize ns-LTP crystal structure. Par j 1 and Par j 2 allergens have three similar allergenic epitopes with high homology and identical conformation. CONCLUSION: Three similar IgE-epitopes of major allergens from P. judaica have been described. They could be good candidates for designing of IgE haptens as therapeutic agents with reduced anaphylactic side-effects or for developing hypoallergenic variants of these major allergens.

Allergens↗
PubMed2003

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.

Aceraceae↗
PubMed2002

Ultrastructural localization of Ani s 1, a major allergen from the fish parasite Anisakis simplex.

No data about the nature or function of the first major Anisakis simplex allergen, named Ani s 1, is available. The aim of this study was to investigate the ultrastructural localization of this protein, to obtain further information about it. Ani s 1 was detected in secretory granules of the excretory gland and occasionally lining the main excretory canal. Our results suggest that Ani s 1 could be a secretory product and could have an enzymatic function related to mechanisms of infection.

Allergens↗
PubMed2002

Purification and characterization of Pla a 1, a major allergen from Platanus acerifolia pollen.

BACKGROUND: Plane trees, as Platanus acerifolia, are an important source of airborne allergens in cities of the United States and Western Europe. Little is known about the relevant allergens of this pollen. The aim of this study was to identify relevant allergens from P. acerifolia pollen and purify and characterize a major allergen of 18 kDa. METHODS: P. acerifolia pollen extract was fractionated using ion exchange, gel filtration, and reverse-phase chromatography. Analyzes were carried out by EAST, SDS-PAGE, isoelectric focusing, immunoblotting and amino-acid sequencing. RESULTS: An 18-kDa protein from the P. acerifolia pollen extract, which we named Pla a 1, was purified. This nonglycosylated protein had an isoelectric point value higher than 9.3 and was recognized by up to 92% of monosensitized Platanus allergic patients and 83% of polyzensitized patients. Sequencing of its N-terminal yielded an amino acid sequence which showed no homology to the known proteins in the databases. Other relevant allergens detected in monosensitized patients were proteins of 43 and 52 kDa, with immunoglobulin (Ig)E-binding prevalences of 83 and 42%, respectively. Profilin was an important allergen in polyzensitized patients. CONCLUSIONS: The most relevant allergens from the P. acerifolia pollen have been determined. A major allergen, specific of this pollen, and named Pla a 1, has been purified and characterized.

Adolescent↗
PubMed2002

The allergenic relevance of profilin (Ole e 2) from Olea europaea pollen.

Many works have dealt with the study of the allergenic relevance of profilin from allergenic extracts, mainly derived from pollens and vegetable foods. Olive pollen extracts also contain a profilin allergen (Ole e 2). This protein has been characterized in detail, so the amino-acid sequence of three isoforms and the structural model of one of them are already known. The prevalence of Ole e 2 for olive allergenic patients has been evaluated by different in vivo and in vitro methods, and the results compared with those obtained for another pollen profilins.

Allergens↗
PubMed2002

Assessment of profilin as an allergen for latex-sensitized patients.

BACKGROUND: The presence of the actin-binding protein, profilin, has been demonstrated in natural latex extracts; but the clinical significance of this molecule as an allergen for latex-allergic patients is not clear. We studied the allergenic relevance of isolated latex natural and recombinant profilin, by in vivo and in vitro techniques, in two populations of spina bifida children (SB) and adults allergic to latex (AL). METHODS: Profilin is present in small amounts in latex extracts obtained from low ammoniated (LA) natural latex. Its purification by affinity chromatography resulted difficult due to Heb v 1 unspecific binding. Therefore a method was developed to obtain natural profilin from natural latex, combining affinity chromatography (PLP, poly-L-proline Sepharose column) and previous ammonium sulfate fractionation. Alternatively, latex c-serum containing a low amount of Hev b 1 and a relatively higher profilin content could be used. Recombinant latex profilin isoform (rHev b 8) was cloned by PCR amplification. The entire coding region of Hev b 8 was subcloned into the expression vector pKN172 and a non-fusion form of Hev b 8 was expressed in Escherichia coli BL21 (DE3). Purified recombinant protein was obtained after a single passage through PLP-Sepharose column. RESULTS: Natural and recombinant purified Hev b 8 were tested cutaneously by intradermoreaction (ID) in 17 SB and 14 AL patients. They were positive in 15 SB and 14 AL patients. No wheals were produced when tested in nonatopic control patients. Only 42% of sera from latex-allergic patients revealed specific IgE titers of class 1 or higher by enzyme immunoassay and only 39% of them exhibited IgE binding by SDS-PAGE immunoblotting with any natural or recombinant Hev b 8 forms. CONCLUSION: It seems that profilin is a relevant allergen for both groups of patients from a frequency point of view, but with scarce presence in natural latex extracts and raw sources, with a subsequent low IgE induction capacity.

Adult↗
PubMed2001

Quantification in mass units of group 1 grass allergens by a monoclonal antibody-based sandwich ELISA.

BACKGROUND: Grass pollen extracts currently used for allergy diagnosis and immunotherapy are a complex mixture of proteins of which only a few have allergenic activity. Lol p 1 is one of the most important allergens in grass pollen extracts. OBJECTIVES: To develop a two-site enzyme-linked immunosorbent assay for the quantification of Lol p 1 and other group 1 allergens from grass species, and to assess its suitability for quantifying this group of allergens. METHODS: Balb/c mice immunized with recombinant Lol p 1 were used for the production of monoclonal antibodies. Screening of hybridomas was performed by direct ELISA, and selected monoclonal antibodies were immobilized on ELISA plates and incubated with samples containing group 1 allergens. Bound allergens were detected by a combination of biotinylated Lol p 1-specific monoclonal antibody and peroxidase-streptavidin conjugate. RESULTS: The assay is based on three Lol p 1-specific monoclonal antibodies with different epitope specificities. The optimized ELISA measured Lol p 1 concentrations ranging from 125 to 1000 ng/mL and could quantify group 1 allergen from grass species belonging to the Pooidea subfamily. The assay does not depend on anti-sera production or availability of human sera and thus reactives can be produced in unlimited amounts. CONCLUSION: This sensitive and specific Lol p 1 assay will be helpful both for quantifying the group 1 allergen content of Pooideae pollen extracts intended for clinical use and for studying cross-reactivities among pollen extracts.

Allergens↗