I. Ibarrola

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).

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).

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.

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.

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.

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.

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.

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.

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.