Background: In order to enable reproducible and comparable exposure measurements of fungal alpha-amylase (alpha-amylase) in different laboratories and countries, the entire procedure from sampling of airborne dust to measuring extracted samples (including standards and the used enzyme) immunoassays must be standardized. The aim of this study was to establish optimal elution and assay conditions.
Methods: A parallel sampler was used for simultaneous collection of 10 samples of inhalable dust in bakeries and mills in Germany, England, the Netherlands and Spain. Three enzyme-immunoassays (EIAs) for detection of fungal alpha-amylase based on monoclonal antibodies or polyclonal antibodies were used for the measurement of the parallel-sampled filters (n=432) extracted using several methods. The results were analysed by regression analysis of variance. Additional filters (n=54) were extracted and analysed using two EIAs to investigate the storage stability of the extracts.
Results: Although alpha-amylase concentrations correlated well (r> or =0.88), differences were found between the EIAs in the sensitivity and nominal values (up to a mean factor 5.8). The best elution medium for airborne filters (phosphate-buffered saline 'PBS' with 0.05% Tween-20) led to 1.2 to two times higher alpha-amylase allergen yields than extraction in PBS only, while higher Tween-20 concentrations decreased the extracted alpha-amylase yield. During storage of frozen dust/filter extracts for 3-4 months at -20 degrees C, a loss of approximately 40% of measurable alpha-amylase was observed, which could be partially prevented by addition of 0.1% casein to the medium directly after extraction.
Conclusion: Although the effects of only a few of many possible causes of variation were investigated, for these factors a clear choice could be made with regard to optimal elution conditions and the use of validated EIAs with calibrated standards, thus making significant progress towards a completely standardized procedure for airborne alpha-amylase measurements.