M. Carmen Arilla

Cloning and expression of the panallergen profilin and the major allergen (Ole e 1) from olive tree pollen

BACKGROUND Olive tree (Olea europaea) pollen allergy is one of the main causes of allergy in Mediterranean countries and some areas of North America. OBJECTIVE To clone olive allergens and to characterize immunologically the purified recombinant allergens. METHODS Full-length complementary deoxyribonucleic acid (cDNA) strands encoding olive allergens (Ole e 1) were cloned by polymerase chain reaction amplification and sequenced. Recombinant proteins were produced in Escherichia coli by the use of two different expression systems. Immunoreactivity of the recombinant proteins was tested by ELISA and Western blot with serum from patients with allergy to olive. RESULTS Significant sequence polymorphism was found in both allergens. The panallergen profilin was expressed as a nonfusion protein and was purified to homogeneity after a single step of affinity chromatography with a poly-L-proline Sepharose column. One cDNA encoding an Ole e 1 isoform was expressed as a fusion protein consisting of the glutathione S-transferase of Schistosoma japonicum and Ole e 1. The fusion protein was purified to homogeneity by gel filtration chromatography and affinity chromatography with a glutathione-Sepharose column, and digested with thrombin. Both recombinant allergens shared B cell epitopes with the corresponding natural allergens. CONCLUSION IgE-reactive Ole e 1 and olive profilin expressed in bacteria were purified after simple chromatographic procedures and may be useful for diagnostic purposes.

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.

Characterization of recombinant Mercurialis annua major allergen Mer a 1 (profilin)

BACKGROUND Two major allergens (Mer a 1A and Mer a 1B), tentatively identified as profilin, have been described in the euphorbiacea, Mercurialis annua. OBJECTIVES We sought to clone and characterize these major allergens from M. annua pollen and to obtain the immunologically active and soluble recombinant allergen, which could then be used for diagnostic procedures and therapy. METHODS Isolation of cDNA clones was performed by polymerase chain reaction amplification with degenerate primers. Expression in Escherichia coli BL21 (DE3) was carried out with a vector based in the T7 expression system, and the recombinant allergen was isolated by affinity chromatography on poly-(L-proline)-Sepharose. Electrophoretic (sodium dodecylsulfate-polyacrylamide gel electrophoresis, isoelectric focusing, and 2-dimensional polyacrylamide gel electrophoresis) and immunochemical methods (Western blot and ELISA) were used for the characterization of the recombinant allergen. RESULTS Two cDNA inserts coding for M. annua pollen profilin (Mer a 1) were cloned and sequenced. Full-length Mer a 1 cDNA was expressed in E. coli as nonfusion protein. The final yield of recombinant Mer a 1 from the culture media after a single purification step on poly-(L-proline)-Sepharose was as much as 5 mg per liter. The reactivity of recombinant Mer a 1 with IgE antibodies present in sera from patients allergic to M. annua, Olea europaea, and Ricinus communis pollens was comparable to that of the natural counterparts, but latex profilin had no cross-reactivity with M. annua profilin. Recombinant Mer a 1 was shown to share B-epitopes with sunflower profilin. CONCLUSION This approach is suitable for the production of defined and purified recombinant allergens, which could allow more detailed immunologic characterization of these proteins and the development of much more accurate diagnostic measures and specific anti-allergic treatments.

Cloning, Isolation, and IgE-Binding Properties of Helix aspersa (Brown Garden Snail) Tropomyosin

Background: Gastropod consumption is quite frequent in the Mediterranean countries and cross-reactivities with crustaceans have been described, but the mechanism of this allergenic cross-reactivity has not been studied in detail. This study aimed to produce recombinant Helix aspersa (brown garden snail) tropomyosin and investigate its implication for cross-reactivity among invertebrates. Methods: A tropomyosin-specific cDNA encoding H. aspersa tropomyosin was synthetized, and recombinant allergen was overexpressed in Escherichia coli as nonfusion protein. IgE-binding reactivity was studied by immunoblotting and immunoblot inhibition experiments with sera from snail-allergic patients. Results: Cloned brown garden snail tropomyosin shares high homology with other edible mollusk tropomyosins (84–69% identity) as well as with those from arthropods (65–62%), and less homology with vertebrate ones (56% identity). Tropomyosin reacted with 18% of the sera from patients with snail allergy. Inhibition experiments, using natural and recombinant tropomyosins, showed different degrees of cross-reactivity between invertebrate tropomyosins. Sera from snail-allergic subjects recognized tropomyosins in both mollusks and crustacean extracts. Conclusions: Tropomyosin represents a minor allergen in snail extracts, but it is clearly involved in invertebrate cross-reactivity.

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.

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.

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.

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.