AntigenSampling – validation of the GSP and AS100 sampling system for the detection of antigens in work places

Status:

completed 03/2022

Aims:

A repeated exposure to bioaerosols can lead to the development of various health problems for employees. Allergy induced asthma, allergenic skin diseases and allergic alveolitis are some of the examples, that count as occupational diseases. The dose-response relationship between bioaerosol concentrations and disease development is still not resolved. One approach to gain more information, is the detection of airborne mold antigens and allergen.

The goal of this study was to test and compare two sampling systems, suitable for collection of mold containing aerosols and subsequent analysis via ELISA. Additionally, both systems were evaluated regarding their practicability in the application in occupational settings and the respective protocols for sample processing were tested. As detection and measurement system, a commercial ELISA test kit (AveX), specific for the clade Aspergillus versicolor, was used. To produce controlled concentrations of mold containing aerosol, a bioaerosol chamber and two different aerosol generators were applied. Previous to the comparison experiments, the specificity of the ELISA and influences of growth conditions on the chosen fungal species, were investigated.

Activities/Methods:

As detection and measurement system, a commercial ELISA test kit (AveX), specific for the clade Aspergillus versicolor, was used. To produce controlled concentrations of mold containing aerosol, a bioaerosol chamber and two different aerosol generators were applied. Previous to the comparison experiments, the specificity of the ELISA and influences of growth conditions on the chosen fungal species, were investigated.

This preliminary work showed that none of the ten, at the beginning available isolates, which were identified as Aspergillus versicolor, belonged to the respective species. Rather, the molecular identification showed, that they are closely related and belonged to the clade Aspergillus versicolor and the sublades versicolor and sydowii. The tested growth conditions, showed different effects on the isolates. Additionally, isolates from the same species showed different antigen contents per spore. It was decided to continue the work with the one isolate, with which the ELISA kit was originally developed. This strain was purchased from the ATCC strain collection. Instead of comparing the sampling methods with two isolates of Aspergillus versicolor, the original strain (Aspergillus amoenus) and an Aspergillus fumigatus isolate were used.

Results:

In the project, two sampling systems for the collection of airborne mold particles were investigated and compared to each other. The Gesamtstaubprobenahme (GSP) system, which samples airborne particles onto a Teflon filter and the MBASS30 / AS100 system, which collects the spores in the cavities of a microtiter strip. The experiments with Aspergillus fumigatus were performed in three concentrations, with five repeats each and a commercial Asp f 1 allergen ELISA for detection. For none of the concentrations and sampling systems, concentrations above the detection limit could be measured. The production of higher concentrations of airborne spores was not possible in this case, as the species belongs to the risk group 2 organisms and production of spore dust was therefor not possible. The tests with Aspergillus amoenus were performed in six concentrations and five repeats each. Additionally, for each concentrations three microtiter strips for measurements of antigens after rinsing the cavities and collecting the liquid (indirect measurement), were produced.

The comparison of the sampling systems showed, that the direct measurement in microtiter stips gives results at lower concentrations than for the indirect measurement or the sampling on Teflon filters. Although the results of the indirect measurement were always lower than the results for the direct measurement, both correlate very well. The comparison of the direct antigen detection in microtiter strips and the one on Teflon filters show, that both systems are suited for different ranges of concentrations. With filtration, an about 10 fold higher concentration was measured. But the sampling in microtiter strips is more sensitive than the filter collection. These observations are most probably a result of different absorption areas of the sampling systems and probably sample loss effects. In the measurement campaigns in two Austrian and two German occupational settings, these observations could be confirmed. The test of storage and transport influences on the sample containers was planned, but could only be accomplished for the AS100 microtiter strips. The results give no indication, that there is a negative effect of transport and storage for 21 days.

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