S. Kespohl

A New Method to Bind Allergens for the Measurement of Specific IgE Antibodies

Background: Detection of allergen-specific IgE antibodies in patients’ sera plays a key role for the diagnosis of IgE-mediated allergy. If no validated test system is available, diagnostic tools must be developed, usually by coupling or binding the allergens to a solid phase. Streptavidin ImmunoCAP™ is a new solid phase for binding of allergens which can be used in the Pharmacia CAP® system. Objective: It was the aim of this study to assess the diagnostic validity of Streptavidin ImmunoCAP. Methods: Biotinylation and allergen concentration for binding to Streptavidin ImmunoCAP were optimized and IgE obtained with natural rubber latex, obeche wood, wheat and rye flour Streptavidin ImmunoCAP were compared with the results of ImmunoCAP™ and Enzyme Allergo-Sorbent Test (EAST) using sera from patients complaining of workplace-related respiratory symptoms. Results: While the relation of biotin-label and protein was critical (best results were obtained with a 5- fold molar excess), labelled protein for coupling to streptavidin ImmunoCAP was applicable in a wide concentration range. On average, IgE values with streptavidin ImmunoCAP were as high as with ImmunoCAP but considerably higher than values obtained by EAST. Conclusion: Streptavidin ImmunoCAP is a valuable tool for sensitive and specific measurement of IgE binding to new allergens superior to cellulose disk-based methods.

Monitoring of occupational and environmental aeroallergens - EAACI Position Paper Concerted action of the EAACI IG Occupational Allergy and Aerobiology & Air Pollution

Exposure to high molecular weight sensitizers of biological origin is an important risk factor for the development of asthma and rhinitis. Most of the causal allergens have been defined based on their reactivity with IgE antibodies, and in many cases, the molecular structure and function of the allergens have been established. Significant information on allergen levels that cause sensitization and allergic symptoms for several major environmental and occupational allergens has been reported. Monitoring of high molecular weight allergens and allergen carrier particles is an important part of the management of allergic respiratory diseases and requires standardized allergen assessment methods for occupational and environmental (indoor and outdoor) allergen exposure. The aim of this EAACI task force was to review the essential points for monitoring environmental and occupational allergen exposure including sampling strategies and methods, processing of dust samples, allergen analysis, and quantification. The paper includes a summary of different methods for sampling and allergen quantification, as well as their pros and cons for various exposure settings. Recommendations are being made for different exposure scenarios.

Impact of cross‐reactive carbohydrate determinants on wood dust sensitization

Background Occupational wood dust exposure can induce allergy and may be one cause of respiratory health problems among woodworkers.

Natural rubber latex and chestnut allergy: cross-reactivity or co-sensitization?

Background:  Chestnut and natural rubber latex (NRL) allergy are often associated in the latex‐fruit syndrome.

Biochemical and immunological analysis of mould skin prick test solution: current status of standardization

Sensitization prevalence to moulds reached from less than 10% in the general population to more than 25% in atopic and/or asthmatic subjects. To diagnose IgE‐mediated mould sensitization, skin prick test (SPT) and specific IgE (sIgE) measurement are recommended. However, concordance of SPT and sIgE results is often less than 50% and standardization of the extracts is required to achieve reliable test results.

Identification of an obeche (Triplochiton scleroxylon) wood allergen as a class I chitinase

Background:  Wood dust is known to cause allergic occupational asthma and obeche (Triplochiton scleroxylon) is a prominent exponent in this field. However, the knowledge about wood allergens is still limited. The aim of this study was to identify and characterize obeche wood allergens.

Sensitization due to Gum Arabic (Acacia senegal): The Cause of Occupational Allergic Asthma or Crossreaction to Carbohydrates?

Background: A pharmaceutical industry worker was exposed to dust of gum arabic in the tablet coating plant and complained of work-related shortness of breath, chest tightness, runny nose, itching and redness of the eyes. This case was investigated for allergy to gum arabic and compared with a control group. The aim of the study was to identify the IgE-binding components responsible for the work-related symptoms. Methods: Skin prick tests (SPTs)and specific IgE (sIgE) measurements with environmental and occupational allergens, spirometry and a specific bronchial challenge with gum arabic were performed. One hundred and nineteen control subjects underwent SPT with gum arabic and 43 controls were tested for sIgE. Crossreactivity between gum arabic and horse radish peroxidase was investigated by IgE CAP inhibition. A combined procedure of immunoblotting and periodate treatment was applied to identify the epitope nature of gum arabic. Results: Allergy to gum arabic was shown by SPT, presence of sIgE and a positive bronchial challenge with gum arabic. Sensitization to gum arabic was demonstrated by SPT or sIgE in 7 and 5 controls, respectively. The results of inhibition with horse radish peroxidase, immunoblotting and periodate treatment suggest that gum arabic sIgE of the patient and 1 SPT-positive control subject were directed to the polypeptide chains of gum arabic. In contrast, gum arabic sIgE of the other controls reacted to carbohydrate components. Conclusions: Sensitization to gum arabic carbohydrate structures occurs casually in atopic patients with pollen sensitization without obvious exposure to gum arabic. This study suggests that allergy to gum arabic is mediated preferentially by IgE antibodies directed to polypeptide chains of gum arabic.

Development and Application of Mold Antigen-Specific Enzyme-Linked Immunosorbent Assays (Elisa) to Quantify Airborne Antigen Exposure

The aim of our study was to develop specific enzyme-linked immunosorbent assays (ELISAs) and apply these to assess mold antigen exposure in composting plants. Sandwich ELISAs based on polyclonal antibodies to Aspergillus fumigatus (Af), Penicillium chrysogenum (Pc), and Cladosporium herbarum (Ch) antigens were developed and validated. Reactivity to 18 different mold species was tested. To optimize extraction procedure, inhalable dust samples taken by a parallel sampler were extracted with or without homogenization. In 31 composting plants stationary pumps were installed at 4 sites to collect 124 inhalable dust samples. The newly developed ELISAs were used in addition to an anti β-1,3-glucan ELISA to quantify mold antigens. The Cladosporium ELISA showed less than 0.04% reactivity to extracts from other fungal genera, while the Af ELISA demonstrated a reactivity of up to 3.6% and the Pc ELISA reacted up to 11% to other mold species. Extraction of parallel sampled filters gave higher antigen amounts with homogenization. The increase was highest for Pc-antigens, followed by Af-antigens, and lowest for Ch-antigens. Mean lower detection limits of homogenized inhalable dust samples were 5 ng/m3 (Af), 0.6 ng/m3 (Pc), 0.2 ng/m3 (Ch), and 0.6 ng/m3 (β-1,3-glucan). The ELISAs were able to detect antigens in 43% (Af), 37% (Pc), 94% (Ch), or 100% (β-1,3-glucan) of the 124 airborne dust samples. Inhalable dust, β-1,3-glucan, and Af-, Pc-, and Ch-antigen concentrations were significantly correlated. The newly developed mold antigen ELISAs are thus able to measure airborne exposure levels in composting plants and differentiate between distinct fungi genera.

Occupational IgE-mediated softwood allergy: characterization of the causative allergen.

Allergic reactions to wood dust allergens are rare, and only few in vitro diagnostic tools and information about relevant allergens are available. To differentiate between protein-based allergy and probably clinically silent glycogenic sensitization, it is helpful to characterize the relevant protein allergens and specify IgE binding. The current case report deals with the occupational softwood allergy of a carpenter exposed to different wood dusts. Skin tests and IgE tests against wood were performed with specifically tailored ImmunoCAPs and cross-reactive carbohydrate determinants. Potential allergens were identified by IgE blots and tandem mass spectrometry. The clinical relevance was verified by challenge tests. Specific IgE to softwood (spruce, pine and larch wood), beech wood, natural rubber latex (NRL) and horseradish peroxidase (HRP) were detected. Allergens in spruce wood, the dominant allergen source, were identified as peroxidases. Softwood were the strongest inhibitors. HRP reduced IgE binding to softwood to <50%, indicating predominantly proteinogenic epitopes, whereas IgE binding to NRL and beech wood was reduced to >50% by HRP, indicating predominantly glycogenic IgE epitopes. Skin and challenge tests underlined that softwoods were the source of sensitization. For the polysensitized patient, a clinically relevant softwood allergy was diagnosed, not only by challenge tests but also with specifically tailored in vitro tools.

How to diagnose mould allergy? Comparison of skin prick tests with specific IgE results.

Diagnosis of mould allergy is complicated due to the heterogeneity of the test material and the decrease in the number of commercial mould skin test solutions that are currently available.