Monoclonal antibody-based ELISA to quantify the major allergen of Cynodon dactylon (Bermuda grass) pollen, Cyn d 1.

BACKGROUND: Pollen of Bermuda grass (Cynodon dactylon) is an important cause of pollinosis in many areas of the world. Most patients show sensitivity to the major allergen Cyn d 1, a glycoprotein composed of a number of isoforms with a molecular mass of 31-32 kDa. The aim of this work was to develop a monoclonal antibody (mAb)-based ELISA to quantify Cyn d 1, and to assess the correlation of the allergen content with the biological activity of C. dactylon pollen extracts. METHODS: After fusion of myeloma cells with spleen cells from a BALB/c mouse immunized with C. dactylon pollen extract, Cyn d 1-specific mAbs secreting hybridomas were selected, and the antibodies characterized. One of them (4.4.1) was used as the capture antibody in an ELISA method for Cyn d 1 quantitation. An anti-Cyn d 1 rabbit serum was used as the second antibody. Cyn d 1 was purified by immunoaffinity chromatography with mAb 4.4.1, characterized, and used as the standard in the assay. RESULTS: The identity, purity and isoallergen composition of affinity-purified Cyn d 1 was confirmed by N-terminal amino acid sequencing, SDS-PAGE, Western blot and 2D electrophoresis. The Cyn d 1 ELISA is highly specific and sensitive, with a detection limit of 0.24 ng/ml and a linear range of 1.1-9.2 ng/ml. An excellent correlation was found when the content of Cyn d 1, measured in 16 different extracts, was compared with the allergenic activity of the same extracts determined by RAST inhibition. CONCLUSIONS: The results prove the usefulness of the Cyn d 1 ELISA for the standardization of C. dactylon-allergen products on the basis of major allergen content.

Monoclonal antibody-based ELISA to quantify the major allergen of Artemisia vulgaris pollen, Art v 1.

BACKGROUND: Pollen of Artemisia vulgaris (mugwort) is a relevant cause of pollinosis in temperate and humid regions. Recently, the major allergen of this pollen, Art v 1, has been characterized. OBJECTIVE: To develop a monoclonal antibody (mAb)-based enzyme-linked immunosorbent assay (ELISA) to quantify Art v 1, and to assess the correlation of Art v 1 content with the biological activity of mugwort pollen extracts. METHODS: Art v 1-specific mAbs were obtained from a BALB/c mouse immunized with high-performance liquid chromatography (HPLC)-purified Art v 1. One of these antibodies (Av 3.7), which recognizes the N-terminal defensin-like domain of Art v 1, was used as the capture antibody in an ELISA method for allergen quantitation. An anti-A. vulgaris rabbit serum was used as the second antibody. Art v 1 was purified by immunoaffinity chromatography and used as the standard in the assay. RESULTS: The purity and identity of the affinity-purified Art v 1 was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), mass spectrometry, amino acid composition, and N-terminal amino acid sequencing. The prevalence of specific IgE against Art v 1, determined by radioallergosorbent test (RAST) in a population of 44 mugwort-allergic patients, was 79%. The Art v 1-ELISA developed displays a detection limit of 0.1 ng/ml, and a practical working range of 0.2-10 ng/ml. The concentration of Art v 1 was measured in 10 A. vulgaris pollen extracts, and a good correlation was observed between the Art v 1 content and the allergenic activity of the extracts. CONCLUSIONS: The results prove the usefulness of the Art v 1-ELISA for the standardization of A. vulgaris pollen extracts intended for clinical use.

Ensayo de cuantificación del alergeno mayoritario de alternaria alternata, alt a 1 / Quantification assay for the major allergen in alternaria alternata, alt a 1

Antecedentes y objetivos: Los extractos alergénicos de hongos poseen una gran variabilidad como consecuencia de la existencia de diferentes subtipos de cada especie, así como del resultado de las diferentes condiciones de cultivo y de los distintos procedimientos de extracción. El resultado de esta intrínseca complejidad es la dificultad que conlleva la estandarización y caracterización de estos extractos para su uso en el diagnóstico y tratamiento de enfermedades alérgicas. Con el objetivo de contribuir a esta importante tarea, hemos desarrollado un ELISA en fase sólida para la cuantificación del alergeno mayoritario de Alternaria alternata, Alt a 1. Material y métodos: Se inmunizaron ratones BALB/c con un extracto de A. alternata y se procedió a fusionar las células del bazo del ratón que tenía mejor título frente a A. alternata con células del mieloma SP2/0-Ag 14. Los hibridomas resultantes fueron seleccionados por su capacidad de secretar anticuerpos específicos frente a Alt a 1 mediante un radioimmunoanálisis de captura de antígeno utilizando el alergeno marcado radiactivamente. Una selección posterior mediante un análisis de inhibición competitiva entre los anticuerpos monoclonales (AcMo) nos permitió identificar dos AcMo (Aa 1.1.1 y Aa 4.4.1) que reconocían epítopos diferentes en la molécula de Alt a1. Estos dos AcMo fueron utilizados en el desarrollo del ELISA para la cuantificación de Alt a 1, adsorbiendo uno de ellos a la fase sólida (Aa 4.4.1) y usando el segundo anticuerpo (Aa 1.1.1) como anticuerpo trazador conjugado con biotina. El AcMo Aa 1.1.1 fue purificado y adsorbido a un soporte sólido para purificar Alt a1 a homogeneidad mediante cromatografía de afinidad y usar este material purificado como estándar en la construcción de la curva de valoración, previo análisis de su pureza y de su actividad biológica. Resultados: El ensayo es específico y sensible; posee un límite de detección de 0,5 ng/mL y un rango de cuantificación entre 1 y 25 ng/mL. Se halló una excelente correlación (r=0,9767; p<0,0001) cuando se valoró el contenido de Alt a 1 en 13 extractos de A. alternata por este método y se comparó con la actividad alergénica de los mismos determinada por RAST de inhibición. Conclusiones: El ELISA que hemos desarrollado es especialmente útil para monitorizar la calidad y estabilidad de los extractos de A. alternata de una manera simple y fiable, y como herramienta potencial para valorar Alt a1 en muestras ambientales y en múltiples sistemas biológicos (AU)

Monoclonal antibodies against the major allergen of Plantago lanceolata pollen, Pla l 1: affinity chromatography purification of the allergen and development of an ELISA method for Pla l 1 measurement.

BACKGROUND: Plantago lanceolata (English plantain) pollen is a relevant cause of pollinosis in temperate regions. The major allergen of this pollen, Pla l 1, is recognized by the specific IgE from more than 80% of plantain-sensitive patients. It displays significant sequence homology with the major olive-pollen allergen Ole e 1. The objective was to develop a monoclonal antibody-based ELISA to quantify Pla l 1, and to assess the correlation of Pla l 1 content with the biologic activity of plantain pollen extracts. We also aimed to establish the specificity of the monoclonal antibodies against the potentially cross-reactive allergen Ole e 1, and to investigate the presence of Pla l 1-like proteins in psyllium and melon that have been reported to cross-react with P. lanceolata pollen. METHODS: After fusion of myeloma cells with spleen cells from a BALB/c mouse, two Pla l 1-specific monoclonal antibodies secreting hybridomas were selected, and the antibodies characterized. One of them (2A10) was used as the capture antibody in an ELISA for Pla l 1 quantitation. An anti-P. lanceolata rabbit serum was used as the second antibody. Pla l 1 was purified by immunoaffinity chromatography and used as the standard in the assay. RESULTS: The ELISA developed was highly reproducible and sensitive, with a detection limit of 0.1 ng/ml, and a practical working range of 0.4-12 ng/ml. The specificity was demonstrated against a large battery of allergens, including Ole e 1. The concentration of Pla l 1 was measured in 19 extracts of P. lanceolata pollen, and a good correlation was observed between the Pla l 1 content and the allergenic activity of the extracts. Pla l 1 was not detected in psyllium or melon extracts. CONCLUSION: The results prove the usefulness of the Pla l 1-ELISA for the standardization of extracts of P. lanceolata pollen intended for clinical use.

Monoclonal antibody-based method to quantify Gly m 1. Its application to assess environmental exposure to soybean dust.

BACKGROUND: The demonstration that some asthma epidemics have been caused by allergens of soybean-hull dust prompted us to develop a two-site ELISA, suitable for the quantification of the major allergen (Gly m 1), to be used for the prevention of new episodes. METHODS: BALB/c mice were injected with Gly m 1 purified from soybean hulls. After fusion and screening, 10 monoclonal antibodies (mAbs) were obtained that were shown to be specific for Gly m 1 Two of them (6G1 as the capture antibody; 1G10 as the tracer) were selected to develop a quantitative two-site ELISA for the indoor and outdoor determination of Gly m 1. RESULTS: The two-site ELISA developed is very sensitive, with a detection limit of less than 0.2 ng/ml and a practical working range of 0.4-10 ng/ml. The assay is also highly reproducible with an intra-assay coefficient of variation of 3.5% and an interassay coefficient of variation of 12.5%. The method was applied to measure the concentration of Gly m 1 in air-sampler filters and in house-dust samples. Our results illustrate that there is a good correlation between the content of Gly m 1 in a number of samples and the allergenic activity as measured by ELISA inhibition. CONCLUSIONS: A specific and sensitive method is presented that can be used for the quantification of Gly m 1. The application of this method may allow the establishment of risk limits for soybean dust, and thus may contribute to the control of environmental contamination and to the prevention of new asthma epidemics.

Isolation by mAb based affinity chromatography of two Par j I isoallergens. Comparison of their physicochemical, immunochemical and allergenic properties.

We report the identification and separation of two isoallergen components of Par j I, the major allergen from Parietaria judaica pollen. First, electrophoretic conditions for consistently separating both isoforms in an SDS-PAGE system were established, and mol. wt values of 13,000 (isoallergen IA) and 10,500 (isoallergen IB) were estimated. Immunoblot, after SDS-PAGE experiments, with individual P. judaica-sensitive human sera revealed a slightly different IgE-binding pattern for each isoallergen. Four anti-Par j I mAbs were obtained from BALB/c mice immunized with a purified Par j I preparation comprising IA and IB isoallergens. Three mAbs were directed to an epitope shared by both isoallergens, and the fourth one recognized specifically one epitope on Par j IB. Dot-blot experiments with the deglycosylated allergen showed that the mAbs did not recognize the carbohydrate prosthetic group of the molecules. Affinity chromatography using the mAbs allowed the separation of the isoallergens that retained their IgE-binding ability after the purification process. Amino acid composition analyses and partial N-terminal sequencing demonstrated an extensive homology and also the existence of some structural differences between Par j I isoallergens, which is in agreement with the high, but not complete, cross-reactivity observed in competition ELISA experiments. Finally, skin prick tests performed on 28 P. judaica-sensitive patients showed that all of them recognized both isoforms and that allergenic epitopes present in Par j IA and IB are responsible for most of the allergenic activity of the whole extract.

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.

Studies on the biochemical structure of the major cat allergen Felis domesticus I.

The major cat allergen, Fel d I, was purified to homogeneity from cat dander extract by sequential mAb affinity chromatography and HPLC size exclusion. The purity and allergenic activity of the preparation was demonstrated by different techniques such as HPLC, RAST inhibition, skin prick tests and CIE/CRIE. Fel d I showed a mol. wt of about 35,000 by HPLC gel filtration and of 18,000 by SDS-PAGE, confirming that it is a non-covalently linked dimer. However, SDS-PAGE analysis under reducing conditions as well as labelling experiments with 14C-iodoacetamide of 2-ME-reduced Fel d I showed that each mol. wt 18,000 monomer is comprised of two covalently S-S bound polypeptides with apparent mol. wt. of 4000 (alpha-chain) and 14,000 (beta-chain). Reduction and alkylation of Fel d I obliterated most of its allergenic activity, as determined by RAST inhibition and immunoblotting, suggesting that most of the IgE-binding sites are conformational. On the other hand, treatment of Fel d I by N-glycanase under reducing and non-reducing conditions indicated the presence of N-linked oligosaccharides in the beta-chain. Carbohydrate analysis data of the whole Fel d I molecule showed the presence of a relatively high carbohydrate content (approximately 20%). RAST inhibition experiments of native and deglycosilated allergen suggest that most IgE epitopes are located in the protein moiety of the molecule. However, the deglycosilated allergen showed a 2-4 fold reduction in its inhibition capacity of RAST as compared to the native allergen, suggesting that carbohydrates could have some role in keeping the active conformation of those epitopes. The N-terminal amino acid sequence of the beta-chain (20 residues) and most of the alpha-chain (40 residues) were determined. Both chain sequences showed no homology with other known protein sequences.

Monoclonal antibody based radioimmunoassay for the quantitation of the main cat allergen (Fel d I or Cat-1).

A two-site solid-phase radioimmunoassay has been developed for the quantitation of the main cat allergen, Fel d I or cat allergen 1. The assay is based on two different monoclonal antibodies which recognize different epitopes on the Fel d I molecule; one antibody (C5/24) was immobilized on the solid phase and the other (C5/8) was labeled with 125I, being the allergen molecule sandwiched between them. The assay is specific for the Fel d I molecule and sensitive enough to detect as little as 0.25 ng/ml of allergen. The Fel d I RIA was compared with a radial immunodiffusion technique for the determination of allergen levels in several cat extracts and a good quantitative correlation was found. The same good correlation was found when the results obtained with the Fel d I RIA were compared with the determination of the total allergenic activity by RAST inhibition. The results indicate that the MAb RIA could be very useful in the standardization of allergenic extracts from feline origin.

Monoclonal antibodies against Fel d I and other clinically relevant cat allergens.

Several mouse monoclonal antibodies (MAbs) were obtained which specifically recognized allergen molecules from cat dander extract. Two of them (C5.8 and C5.24) were specific for the main cat allergen, Fel d I. One (C5.25) recognized an important allergen with approximate molecular weight of 30000 Da and a pI between 3.9 and 4.3. A third group of MAbs comprised several hybridomas which were specific either for cat albumin or cat immunoglobulin. The immunochemical characterization and the clinical significance of the cat dander components recognized by these MAbs is presented and discussed.

Characterization of the main IgE-binding components of cat dander.

Cat dander extract (CDE) was fractionated by preparative isoelectric focusing (pIEF). All the fractions showed allergenic activity, but the most acidic ones (pH range 3.3-4.3) had the highest specific activity. These fractions also had the highest cat allergen 1 content and a partial antigenic identity as determined by fused rocket immunoelectrophoresis. Immunoblotting of the pIEF fractions and the crude CDE after analytical IEF showed the presence of native proteins with IgE-binding ability all along the pH range 3.3-6.2. However, when the IgE-immunoprecipitated components of the most acidic pIEF fractions were focused in the presence of urea, a neat band was found at pH 3.9. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting of CDE proved the presence of, at least, 9 polypeptides with affinity to IgE, those with molecular weights of 18,000 and 22,000 daltons being the most active ones. These polypeptides presumably belong to cat allergen 1 since they were found in the most acidic pIEF fractions as well as in the crossed immunoelectrophoresis arc corresponding to this allergen. Furthermore, we found a group of active proteins with molecular weights of 10,000, 22,000, 25,000, 50,000 and 65,000 daltons (cat albumin) and with isoelectric points between 3.3 and 6.2.

[Evaluation of the total biological activity and allergenic composition of allergenic extracts]. / Evaluación de la actividad biológica total y composición alergénica de extractos alergénicos.

In the present study, a complete procedure is presented in order to standardize allergenic extracts, the meaning of which is the measurement of the total allergenic activity and the determination of the allergenic composition. The measurement of the biological activity comprises 2 steps: Preparation of Reference Extracts and determination of their "in vivo" activity. Evaluation of the total allergenic activity of extracts for clinical use. Reference extracts were prepared from the main allergens and their "in vivo" biological activity was determined by a quantitative skin prick test in a sample of at least 30 allergic patients. By definition, the protein concentration of Reference Extract that produces, in the allergic population, a geometric mean wheal of 75 mm.2 has an activity of 100 biological units (BUs). The determination of the biological activity of a problem extract is made by RAST inhibition. The sample is compared with the corresponding Reference Extract by this technique and, from this comparison, it is possible to quantify the activity of the problem extract in biologic units (BUs) with clinical significance. Likewise, different techniques have been used to determine the allergenic composition of extracts. These techniques comprise 2 steps: Separation of the components of the extract. Identification of the components that bind specific human IgE. The separation of the components of the extract has been carried out by isoelectric focusing (IEF) and electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE). In order to identify the allergenic components, an immunoblotting technique has been employed. The separated components in the IEF gel or SDS-PAGE gel are transferred to a nitrocellulose sheet and later on, this membrane is overlaid with a serum pool from allergic patients and a mouse monoclonal anti-human IgE, labelled with 125I. Finally, the autoradiography of the nitrocellulose membrane is obtained. In this way it is possible to compare the allergenic composition of an extract with the corresponding Reference Extract and so to employ for clinical use only those extracts with the right allergenic composition.

Monoclonal antibodies to distinct epitopes on human IgA and their use to IgA determination.

Several monoclonal antibodies (MAbs) against human IgA have been obtained which specifically bind to human myeloma and polyclonal IgA. Three of these MAbs have been purified and studied further. They recognize both IgA subclasses and define three distinct epitopes on the IgA molecule. These MAbs were used to develop a solid-phase radioimmunoassay (RIA) in which one MAb was immobilized and the other two were labeled with 125I. The assay has a sensitivity in the nanogram range. A good correlation was found (r = 0.97, P less than 0.001) when the solid-phase RIA was compared with a commercially available immunodiffusion technique for the determination of IgA levels in serum samples.

A competitive solid-phase radioimmunoassay for quantitation of the major allergen of Parietaria pollen.

A competitive solid-phase radioimmunoassay has been developed for quantitation of the major allergen of Parietaria judaica pollen. The assay is based on: (1) the ability of AC/1.1 monoclonal antibody to bind specifically to the P. judaica major allergen, and (2) the ability of crude pollen extracts or purified allergen to inhibit the binding of 125I-labelled allergen to solid-phase-bound AC/1.1 monoclonal antibody. The assay is sensitive enough to detect as little as 10 ng of allergen. A good correlation is found when the results obtained are compared with those produced by RAST inhibition (r = 0.95; P less than 0.001). Thus, this method can also be used for the estimation of the allergenic activity of P. judaica pollen extracts. The assay is easily completed in 2 h, allowing simultaneous analysis of a number of extracts.

Cross-reactivity among Chenopodiaceae and Amaranthaceae.

Pollen extracts from Atriplex latifolia, Beta vulgaris, Salsola kali and Amaranthus retroflexus were compared with an extract from Chenopodium album by both in vivo and in vitro methods. Skin prick tests on 20 C. album-sensitive patients were positive with all extracts. RAST inhibition together with two-dimensional immunoelectrophoresis and two-dimensional radioimmunoelectrophoresis indicate that common allergenic determinants are present. Electrophoretic transfer for detection of IgE binding molecules from sodium dodecyl sulfate-polyacrylamide gel electrophoresis or isoelectricfocusing gels suggests that the common allergenic determinants are present in molecules with various molecular weights or isoelectric points.