Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Abstract

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 µm in diameter in inhalable particulate matter (PM10, particle size ≤10 µm in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles >0.5 µm.

A New Method for the Assessment of the Oxidative Potential of Both Water-Soluble and Insoluble PM

“…Equivalent PM 10 filters from the second sample line were instead subjected to PM 10 toothbrush detachment and suspension in water, as thoroughly described in Massimi et al [46]. This method has been already applied only by Süring et al [47,48], who used this procedure to quantify allergen-loaded particles in PM 10 by flow cytometry. Briefly, each filter was put in a polystyrene Petri dish of 50 mm diameter and overlaid with 10 mL of deionized water.…”

“…The performance of this method for the recovery of the elements in PM 10 was evaluated by Massimi et al [46] after applying the chemical fractionation procedure described above to the obtained PM 10 water suspensions and to equivalent PM 10 samples not subjected to the toothbrush detachment, revealing an efficiency of approximatively 70% for the recovery of the elements. Moreover, this method is described in Süring et al [47,48].…”

“…To this aim, a non-invasive, simple, and efficient method, recently validated by Massimi et al [46] for the recovery of the elements in PM 10, was used for the detachment and suspension in water of intact PM 10 from field filters by using an electrical toothbrush [47,48]. The retrieved PM 10 water suspension was used for the assessment of the overall OP DCFH , OP AA , and OP DTT of intact PM 10 samples (water-soluble and insoluble fraction) and for the evaluation of the real contribution of insoluble particles to PM redox properties.…”

‘Pollen potency’: the relationship between atmospheric pollen counts and allergen exposure

“…Allergens are located on the surface and within the cytoplasm of individual pollen grains (e.g. Behrendt et al, 1999;Süring et al, 2016) and are present on fine (\ 2 lm) pollen fragments released into the atmosphere (e.g. Buters et al, 2010;Schäppi et al, 1997b;Taylor et al, 2002).…”