Characterization and trialling of a new measurement method for determining the concentration of allergens in workplace atmospheres

Status:

completed 07/2018

Aims:

Exposure to allergens in workplace atmospheres is a suspected cause of increasing cases of occupational disease. In order to determine the magnitude of this exposure and to evaluate the efficacy of protective measures, standardized measurement methods are required by which allergens in workplace atmospheres can be detected reliably.

Before now, atmospheric allergen samples have primarily been taken by means of the filtration method and a volumetric flow rate of 10 l per minute over a sampling duration of at least four hours (GSP system and teflon filter, diameter 37 mm). The filters are then extracted in the laboratory and the allergens in the filter extract detected by means of specific immunological analysis methods (ELISA). Sampling over several hours is not always possible at workplaces. In addition, extraction of the filters may result in sample loss. One objective of development of the new AS 100 sampling head by Holbach-Umweltanalytik (Wadern) and the BMA laboratory (Bochum) was therefore to produce an allergen sampling system by means of which an adequate air volume for allergen detection (100 l/min) can be obtained within the shortest possible time. In order to avoid losses owing to transport or extraction, the samples are separated as before directly within the reaction vessel for subsequent laboratory analysis (microtiter plates).

The project was to study the physical separation properties and to determine the efficiency of biochemical separation by the new sampling head. The new sampling method is also to be tested in comparison with the filtration methods commonly used to date with regard to its suitability for measuring allergens in workplace atmospheres.

Activities/Methods:

Preliminary tests conducted by the developers of the sampling head for the detection of mould and mite allergens showed the new method to be a promising means by which atmospheric exposure to allergens of biological origin can also be measured at workplaces. The sampling head (AS 100, Holbach-Umweltanalytik) was therefore to be trialled in workplace measurements in comparison with the filtration methods normally used to date.

Exposure to cow hair allergens often leads to severe diseases of the respiratory tract among workers in the relevant businesses. Detection of the Bos d 2 cow hair allergen was well suited to the planned studies, for a number of reasons. Firstly, a commercial test system was already available for analytical detection of this allergen. Secondly, allergen concentrations differing in magnitude were to be anticipated in different areas of businesses involving cattle husbandry (high in animal sheds, lower in the shed office, lowest in living areas). The new measurement method could therefore be tested under real-case conditions in areas exhibiting different concentrations.

In cooperation with the umbrella association of the accident insurance institutions for the agricultural sector (LSV, now the SVLFG, Social insurance for agriculture, forestry and landscaping), comparative measurements for determining the atmospheric concentration of cow hair allergens were performed in four member companies by means of both sampling methods. For the purpose of these studies, the Competence Centre Allergology of the Institute for Prevention and Occupational Medicine (IPA) first optimized the extraction method for the teflon filters and adapted it to the analysis method (detection of cow hair allergens by means of the Bos d 2 ELISA kit (EL-BD 2), Indoor Biotechnologies Inc.). Filter samples were then analysed at the IPA and in the BMA biological and medical analysis laboratory in Bochum by means of the same extraction protocol. Analysis of the microtiter plates (sample holders from the new AS 100 sampling head) was performed solely by the BMA laboratory in Bochum. During the course of the project, the opportunity arose in addition for the AS 100 to be trialled by means of further measurements in the AllergoVet study into the exposure of veterinary students to various animal hair allergens, which was conducted by the German Social Accident Insurance Institution for the public sector in Hesse in conjunction with the IPA in Bochum. For this purpose, atmospheric samples were taken in July/August 2015 and March 2017 at a number of measurement points in treatment rooms and animal sheds at the veterinary faculty of the University of Giessen, and the allergen concentrations of Bos d 2 and further animal hair allergens (Can f 1, Fel d 1) determined. The results of these measurements are reported in the AllergoVet study.

Results:

Bos d 2 was detected by both measurement systems in four measurements in different shed areas in samples taken simultaneously during typical tasks such as feeding and bedding changes. The discrete results of these measurements varied strongly, lying between 57.0 and 1,700 ng Bos d 2 m-3 air for filter samples (n=28) and 0.38 to 429 ng Bos d 2 for samples obtained with the AS 100 (n=30).

The AS 100 generally returned lower allergen concentrations than the filtration method. Several explanations are possible for this result:

Firstly, the concentration that can be measured when separation is performed directly in the reaction vessel is limited compared to the filter method: once all allergen binding sites are occupied, a higher concentration can no longer be measured. For higher concentrations, filtration followed by extraction and the option of dilution of the sample would be more suitable. Conversely, at allergen concentrations below such a saturation concentration, loss-free separation in the reaction vessel may be preferable.

Secondly, where ELISA was used for allergen detection, the "sandwich" variant was deliberately not employed, i.e. the microtiter plates were not coated with a layer of capture antibodies. The absence of this coating had the advantage that the sample holders (microtiter plates) could be transported uncooled, i.e. at room temperature, before and after measurement. The particles containing the allergens were thus extracted from the air into the microtiter plates with the aid of the AS 100 sampling head by the virtual impaction principle, and retained in the microtiter plates solely by adhesion to the vessel walls. This effect of the material from which the sample holders are manufactured is known and typical (F8 Maxisorp Loose, Nunc Immuno modules, Thermo-Fisher Scientific). The monoclonal antibodies for detection of the cow hair allergen are antigen-specific and detect the antigen even when it is bound to the surface of the vessel rather than to the capture antibodies.

The extent to which detection of the allergen could be improved by coating of the sample holders (microtiter plates) could not be determined in the project; for this purpose, further studies are planned under standardized conditions in a bioaerosol test chamber with test dusts containing allergens.

Two experiments were performed in the project to determine the recovery of the cow hair allergen during use of the direct ELISA detection method (i.e. without capture antibodies).

Firstly, the allergen content of a dilute material sample extract from a cowshed (mixture of cowshed dust and silage) was studied in five dilution stages (n=3 in each stage) with both analysis variants: recovery of Bos d 2 in direct ELISA (without capture antibodies) was 51% of that in sandwich ELISA.

Secondly, the allergen concentrations were identified that can be determined optimally by means of the direct analysis method. For this purpose, an extraction test was performed on a material sample (mixture of cowshed dust and silage) and test dust in different mixture ratios, and on a material sample and test dust serving as a positive and negative control. The best recovery rate in the direct ELISA test was obtained at an allergen content of 10% in the sample (material sample and test dust in a ratio of 1:10).

It was also investigated whether particles from the inhalable fraction in the atmosphere are separated by the AS 100 sampling head into a microtiter plate. For this purpose, a parallel test was performed in a test channel at the IFA for sampling with the GSP system and teflon filters (standard method for allergen sampling) and with the AS 100 sampling head. A standard wood dust of suitable particle size (Jeloxyl haho 120 f) was used as the test dust. The dust quantity separated in each test was determined by weighing.

In a total of 16 tests with dust concentrations between 1 and 16 mg/m3 air, the dust mass collected by the AS 100 sampling head was 10 to 70% of that separated on the filters in the GSP system; on average, the AS 100 collected 45% of the dust collected by means of the filtration method. Whereas the GSP system exhibited good collection properties across all dust concentrations, the AS 100 exhibited superior collection efficiency at lower dust concentrations.

Measurements were then performed in businesses. The two sampling systems were employed here simultaneously for sampling at three measurement locations. Two AS 100 sampling heads used in parallel for measurement yielded results that were highly comparable. Transfer of the Bos d 2 cow hair allergen from the area with the highest allergen concentration (cowshed), via the shed office (appreciably lower allergen concentration) to the living area was demonstrated clearly by both measurement systems. The absolute values of the allergen concentrations measured in each area differed, particularly at the highest and lowest concentrations: in the cowshed, a substantially higher allergen concentration was measured with the GSP system (approx. 2,000 ng/m3) than with the AS 100 (approx. 400 ng/m3). By contrast, low but well reproducible allergen concentrations (3.6 to 4.4 ng/m3) were determined in the living area by means of the AS 100, whereas the results for this area yielded by analysis of the filter samples lay in the region of the detection limit.

Conclusion: The "AllMePro" project represents a feasibility study for the suitability of the AS 100 sampling head for measuring allergens in workplace atmospheres.

The Bos d 2 cow hair allergen was collected in measurements in businesses by means of the new sampling head, and its presence demonstrated with the aid of a commercial ELISA test kit, which was used in modified form. The results of the feasibility study indicate that the measurement method is well suited to use at lower atmospheric allergen concentrations, such as at the indoor workplace.

It was not possible to address all sub-aspects conclusively within the project term. Questions as yet unanswered are:

• What dust component (respirable, inhalable), i.e. what particle sizes are collected by means of the AS 100?
• What allergen concentration represents the upper limit of the range for atmospheric measurement with use of the AS 100?
• Can detection of the allergens be improved by the use of capture antibodies?

Follow-on projects are in preparation. These are to validate sampling and analysis methods in a bioaerosol test chamber by means of test dusts containing cow hair allergens and mould allergens.

Last Update: