Monoclonal IgE antibodies against birch pollen allergens: novel tools for biological characterization and standardization of allergens

BACKGROUND: IgE antibodies are key players in immediate hypersensitivity reactions. Allergen characterization and standardization is usually based on the sera of allergic patients, whereas monoclonal IgE antibodies specific for clinically relevant allergens are very rare. OBJECTIVE: The aim of this study was to establish IgE mAbs specific for birch pollen allergens, because these are important inhalant allergens. METHODS: IgE-producing hybridomas were identified by using the highly sensitive rat basophilic leukemia cell mediator release assay with enhanced allergen stimulation by additional cross-linking with birch pollen-specific IgG antibodies. The obtained IgE mAbs were characterized by immunologic methods and by cDNA sequencing. RESULTS: Seven IgE mAbs specific for the birch pollen allergens Bet v 1 or Bet v 6 were obtained and were all biologically active in mast cell-based assays. Mediator release experiments with mAb combinations indicated that 2 different epitope regions were recognized on Bet v 1, whereas the 2 Bet v 6-specific mAbs bound to the same epitope region. After sensitization of rat basophilic leukemia cells with IgE mAbs, different amounts of Bet v 1 or Bet v 6 were detected in commercial diagnostic allergen reagents, whereas sensitization with polyclonal IgE resulted in similar allergenic potency of all products. CONCLUSIONS: IgE mAbs represent promising novel tools for allergen characterization and component-resolved standardization of allergen extracts.     

Grass pollen allergens

To date, eleven groups of grass pollen allergens eliciting a specific IgE response in atopic individuals have been identified. Groups 1 and 5 allergens are the most critical (major) pollen allergens leading to the sensitization of 90% and 65–85% allergic patients, respectively. Other allergens frequently involved in the IgE response include groups 2/3, 4, 6, 7, 10–13 allergens. Allergens found in various Pooideae exhibit high homology in terms of their amino acid sequence composition, which translates into significant cross-reactivity in terms of antibody (IgE and IgG) as well as T cell responses. Nevertheless, for a given allergen group, there is evidence of both interspecies (i.e. differences in amino acid sequences) and intraspecies (multigenes, post-translational modification, mRNA splicing or editing) molecular variability.     

Key pollen allergens in North America.

OBJECTIVE: To discuss major pollen aeroallergens in North America that are essential for effective immunotherapy and to propose a list of pollen aeroallergens that could be prioritized for allergen standardization. DATA SOURCES: PubMed was used to search the existing medical literature. No date restrictions were used. Keywords included allergy, aeroallergen, taxonomy, cross-reactivity, pollen, and specific genus and species names. RESULTS: Tree species possess relatively unique allergens, and representative members should be chosen at the genus or family level. In the Composite family, there is significant cross-reactivity between ragweed species within the Ambrosia genus. Selection of one species should be sufficient for skin testing and immunotherapy. Extensive allergenic cross-reactivity exists among grasses. Selection of timothy grass alone or in combination with a single northern grass species provides adequate coverage in the northeastern regions of North America. CONCLUSIONS: One of the goals within the field of allergy should be to identify high-priority targets for future development of standardized commercial extracts. The standardization of increasing numbers of allergen extracts potentially benefits the discipline of allergy by facilitating transfer of care among physician practices, improving uniformity of patient care, and providing a template on which geographically specific extract choices can be built.     

Structural characterziation of pollen allergens

The molecular characterization of allergens has accelerated significantly since the wide-spread implementation of modern analytical methods. The combination of gene cloning and heterologous protein expression has generated an extensive array of allergens that is available for comparative analysis, as well as clinical applications. Several internet-accessible allergen databases integrate the accumulated information from biomedical research and clinical practice. Innovations in classical biophysical methods, such as mass spectrometry, X-ray crystallography, and nuclear magnetic resonance spectroscopy, have rendered complex biological macromolecules amenable to detailed structured analysis. The modern scientific era has realized the synthesis of bioinformatics, molecular biology, biochemistry, biophysics, and immunology, and given us the means needed to decipher the remaining mysteries of allergies. This article addresses how the synergism of modern scientific techniques has hastened our understanding of allergies, how these techniques are applied in the identification and characterization of allergens, and how these methods assist the radional development of clinical tools for allergy diagnosis and treatment.     

Characterization of high-molecular-mass allergens in oilseed rape pollen.

BACKGROUND: Oilseed rape pollen allergies have been previously described as the result of cross-sensitization with various pollens. Recently, several proteins have been identified as oilseed rape allergens. The aim of the present work was the characterization of oilseed rape pollen allergens by two-dimensional (2-D) gel analysis and amino acid microsequencing. METHODS: Water extractable proteins from oilseed rape pollen were separated by isoelectrofocusing and then transferred onto a nitrocellulose sheet. Twenty-one human sera from pollen- or mustard-allergic individuals were screened for their reactivity to oilseed rape proteins. Eleven sera possessed IgE which recognized oilseed rape pollen proteins and one serum was selected for further 2-D characterization and amino acid microsequencing of the allergens. RESULTS: The results showed that three molecules from oilseed rape pollen were identified as oilseed rape allergens which have not yet been described. These three proteins were molecules of 70 kD with a pI >8, 40 kD with a pI around 10 and 80 kD with a pI around 5. These proteins displayed identities with the berberine bridge protein, a receptor-like protein kinase and the cobalamin-independent methionine synthetase from Arabidopsis thaliana, respectively. The genes encoding the putative Arabidopsis molecules are located on chromosome 1 (berberine bridge protein) and chromosomes 3 and 4 (receptor-like protein kinases). CONCLUSION: These results show that certain high-molecular-mass proteins from oilseed rape pollen are allergens.     

Identification and partial purification of pollen allergens from Artemisia princeps

The pollen of Artemisia has been considered as the main late summer-autumn allergen source in this country. To identify its allergenic components, Artemisia princeps pollen extracts were separated by 10% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose membrane, where IgE binding components were detected by the reaction with sera of twenty Artemisia-allergic patients and 125I-anti-human IgE, sixteen components in the molecular range of 10,000 and 85,000 daltons were detected. Twelve bands bound to IgE from 50% of the sera tested, and two bands (37,000, 23,000 daltons) showed the highest (85%) frequency of IgE-binding in twenty sera tested. When the gel of SDS-PAGE with Artemisia pollen extracts was sliced into 11 allergenic groups (AG) and the protein of each AG was obtained by the gel elution method, the wormwool-RAST inhibition test showed that the AG 10 demonstrated to be the most potent, and the AG 7 was the next. Six AGs showed significant responses (more than 100% of wheal size to histamine, 1 mg/ml) on the skin prick test in more than 50% of the patients tested. It is suggested that electrophoretic transfer analysis with SDS-PAGE may be a valuable method for Artemisia allergen identification, and the possibility of partial purification of allergens by employing gel elution is discussed.     

The spectrum of olive pollen allergens

Olive pollen is one of the most important causes of seasonal respiratory allergy in Mediterranean countries, where this tree is intensely cultivated. Among the high number of protein allergens detected in this pollen, 8 - Ole e 1 to Ole e 8 - have been isolated and characterized. Ole e 1 is the most frequent sensitizing agent, affecting more than 70% of the patients suffering of olive pollinosis, although others, such as Ole e 4 and Ole e 7, have also been shown to be major allergens. In this context, the prevalence of many olive pollen allergens seems to be dependent on the geographical area where the sensitized patients live. Some of the olive allergens have been revealed as members of known protein families: profilin (Ole e 2), Ca(2+)-binding proteins (Ole e 3 and Ole e 8), superoxide dismutase (Ole e 5) and lipid transfer protein (Ole e 7). No biological function has been demonstrated for Ole e 1, whereas Ole e 4 and Ole e 6 are new proteins without homology to known sequences from databases. cDNAs encoding for Ole e 1, Ole e 3 and Ole e 8 have been overproduced in heterologous systems. The recombinant products were correctly folded and exhibited the functional activities of the natural allergens. In addition to the Oleaceae family, other species, such as Gramineae or Betulaceae, contain pollen allergens structurally or immunologically related to those of the olive tree. This fact allows to detect and evaluate antigenic cross-reactivities involving olive allergens. The aim of this research is the development of new diagnostic tools for olive pollinosis and new approaches to improve the classical immunotherapy.     

Identification of Parietaria judaica pollen allergens

Parietaria judaica pollen allergens, fractionated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes, were identified using 52 sera collected in Australia and Sicily from P. judaica pollen-allergic patients. IgE-binding pollen components transferred to nitrocellulose were detected by reaction with 125I-anti-human IgE and autoradiography. Nine pollen components, ranging in molecular weight (MW) from approximately 10,000 to 80,000 daltons, bound IgE antibodies but the two fastest migrating components sometimes each separated into two very closely migrating bands. The faster of the two components exhibiting doublet formation (MW approximately 10,000 daltons) showed by far the highest frequency of IgE binding, being recognised by 50 of the 52 sera examined. Although patients' IgE reaction patterns to P. judaica allergens were heterogeneous, the degree of heterogeneity was much less than that observed with house dust mite and other pollen extracts studied by electrophoretic transfer analysis. Results with gradient gel-nitrocellulose transfer experiments which showed no IgE-binding components with MWs less than 70,000 daltons, and comparisons of our electroblotting results with crossed radioimmunoelectrophoresis results of others, suggested that the doublet proteins with MWs of approximately 10,000 probably bind to higher MW proteins in P. judaica pollen extracts.     

Comparative studies on tree pollen allergens. XI. Trials on the regulation of IgE response in mice using modified birch pollen allergens

The major allergen of birch pollen, BV45, was isolated and conjugated to 2-o-methoxy polyethylene glycol-4,6-dichloro-5-triazine (mPEG). The molecular-weight variant of 6,000 daltons of the activated mPEG was used in an antigen-specific regulation of IgE response. In these studies 200 genetically high IgE responders (CBA/Ca) female mice were intradermally immunized by the purified BV45, with the use of various adjuvants. Four different sets of experiments were made. In the first experiment 50 mice were immunized on days 1 and 15 with BV45. This was followed by another booster dose of the native BV45 or the modified mPEG-BV45 in Freund's incomplete adjuvants (FIA) on day 72. In the 2nd experiment, a similar procedure was performed except that booster doses were injected on day 135. Subsequently on day 136, BV45/FIA was intradermally injected. In the 3rd and 4th sets of experiments, 2 groups of 50 mice were daily immunized by intraperitoneal injections of 10 successive doses of BV45. On day 33 a dose of BV45 or mPEG-BV45 respectively, was intraperitoneally injected in each mouse. This injection was followed by other 10 successive doses of BV45. In experiment 3 no adjuvants were used, and in experiment 4 aluminium hydroxide gel was used as adjuvant. The mice immune response was assessed by analyses of the serum IgG and IgE levels. In all experiments higher IgG concentrations were shown for the immunized mice as compared to the non-treated control animals. Administration of booster doses of BV45 or mPEG-BV45 induced increases in the IgG levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cross-reactivity between pollen allergens from common Pooideae grasses and rye

In Germany, specific pollen extracts are currently used for immunotherapy of rye ( Secale cereale ) allergy. Like most common grasses, S. cereale belongs to the Pooideae subfamily. Using sera from grass pollen-allergic patients, immunoblotting and ELISA inhibition studies demonstrate that a five-grass allergenic extract from Anthoxanthum odoratum , Dactylis glomerata, Lolium perenne, Poa pratensis and Phleum pratense inhibits over 90% of IgE binding to rye allergens. This result confirms the high degree of homology between allergens from S. cereale and common grass species. We conclude that a five-grass pollen mixture is appropriate for desensitization of patients who are allergic to rye pollen without the need for additional rye pollen extracts.     

Cross-reactivity of pollen allergens: impact on allergen immunotherapy.

OBJECTIVE: To provide guidelines for the rational formulation of allergen immunotherapy extracts based on knowledge of pollen allergen and epitope cross-reactivity. DATA SOURCES: A PubMed search was performed for articles published from 1966 to 2007 using the keywords pollen, allergen, and cross-reactivity. Older literature was found through cross-referencing of older articles and older reviews on pollen cross-reactivity. Study Selection: Articles that dealt with crude pollen extracts and characterized allergens that addressed cross-reactivity were selected for inclusion in this review. RESULTS: In addition to unique allergens, several families of botanic proteins have similarities that allow them to act as pan-allergens. Although frequently these are minor allergens, in some circumstances they may also be major allergens. Recent studies have investigated nonspecific lipid transfer proteins, calcium-binding proteins, pathogenesis-related protein families, and profilins. Calcium-binding proteins and nonspecific lipid transfer proteins are responsible for pollen-fruit interactions and pollen cross-reactivity. Clarification of pollen allergen enzymatic activity helps explain the ubiquitous nature of these proteins. CONCLUSION: Characterization of specific pollen allergens and their protein families has provided insight into cross-reactivity. Clarification of these relationships allows for consolidation or substitution in formulation of inhalant extracts.     

Fractionation and analysis of allergenicity of allergens from Prosopis juliflora pollen

Prosopis juliflora pollen allergen extract was prepared, and its crude allergen extract was fractionated by Sephadex G-100 gel filtration. Six different fractions were obtained which was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Protein and carbohydrate content of each fraction were estimated. Fraction E (MW 20,000) showed a 25% carbohydrate concentration. The amino acid analysis indicated that this fraction was rich in glutamic acid and alanine. Antigenicity or allergenicity of fractionated allergens were checked by gel diffusion test, rocket immunoelectrophoresis, skin prick test, and radioallergosorbent test. All these test indicate that fraction E consisted mainly of allergenic molecules (MW 20,000) of P. juliflora pollen.     

Rapidly released allergens from short ragweed pollen: II. Identification and partial purification

We have identified several basic allergens in 16-min extracts of short ragweed (Ambrosia elatior) pollen and obtained a partially purified fraction (G50 II) using a combination of ion-exchange (CM-Sephadex C25) and gel-filtration (Biogel P30 and Sephadex G50) procedures. G50 II was allergenically and antigenically distinct from known allergens antigen E, Ra3, and Ra5. It gave a highly symmetrical peak on Sephadex G50 (fine), corresponding to a molecular weight of 11,500 daltons, and a single band on SDS polyacrylamide gel electrophoresis under both reducing and nonreducing conditions. However, two cathodic antigens were detectable by both agarose electrophoresis and crossed immunoelectrophoresis. Allergenic activity of G50 II, assessed by puncture testing in ragweed-sensitive subjects, showed a characteristic pattern of response independent of patient response to AgE, Ra3, and Ra5. The major antigenic and allergenic component of G50 II is designated Ra6.