Allergens in fine particles may cause symptoms inallergic asthmatics. In order to assess the exposureof susceptible persons, a method to measure theallergen load in fine and coarse particles wasdeveloped. Aerosols are collected with a high-volume air samplerby multistage impaction. They are separated into fivesize classes, ranging from >10 μm to 10 μm). In smallersized fractions, the allergen load is often close tothe detection limit. When clearly detectable amountsof allergen are present, in the fine size fraction theallergen load shows only a weak correlation to thepollen counts and the allergen concentrations in thecoarse particle fractio

Koller, Theo

Monn, Christian

Riediker, Michael

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Aerobiologia 16: 251–254, 2000.© 2000 Kluwer Academic Publishers. Printed in the Netherlands. 251Determination of birch pollen allergens in different aerosol sizesMichael Riediker, Theo Koller & Christian MonnInstitute for Hygiene and Applied Physiology, Environmental Hygiene Section, Swiss Federal Institute of Techno-logy, Zürich, Switzerland(author for correspondence: r-mail: riediker@iha.bepr.ethz.ch; fax: +41 1 632 1173)Received 15 October 1999; accepted in final form 15 February 2000Key words: allergens, birch, determination, ELISA, fine particles, pollenAbstractAllergens in fine particles may cause symptoms in allergic asthmatics. In order to assess the exposure of susceptiblepersons, a method to measure the allergen load in fine and coarse particles was developed.Aerosols are collected with a high-volume air sampler by multistage impaction. They are separated into five sizeclasses, ranging from>10 m to <1 m and sampled on glass fibre filters. After sampling, filters are crushed intoa fine powder using a hydraulic press. Allergens are then eluted on a shaker into Tween-20-containing phosphatebuffered saline. After microfiltration, the eluate is ready for analysis with ELISA-techniques (Enzyme LinkedImmuno Sorbens Assay).Two different methods are used for the analysis of allergens: One is a sandwich-ELISA using monoclonal IgG-antibodies, the other is a competitive ELISA based on polyclonal IgE-antibodies obtained from patients allergicto birch pollen. Using the monoclonal antibodies information on the amount of one particular allergen (the majorallergen Bet v1) is obtained. On the other hand the competitive ELISA using the polyclonal IgE is much moresensitive and indicates the total birch pollen allergens. Data obtained during spring 1998 show good correlationof pollen counts and allergen content in the coarse particle fraction containing intact pollen (>10 m). In smallersized fractions, the allergen load is often close to the detection limit. When clearly detectable amounts of allergenare present, in the fine size fraction the allergen load shows only a weak correlation to the pollen counts and theallergen concentrations in the coarse particle fraction.IntroductionExposure to pollen allergen loaded fine particles (<about 10 m) is related to allergic airway responsein pollen-sensitised asthmatics (Rosenberg, Rosenthalet al., 1983). Mainly particles smaller than 10 mcan penetrate into the lower parts of the airways(Schlesinger, 1985).In order to assess the exposure of susceptiblepersons, a method was developed to measure theallergen load in fine and coarse particles containingvery little or a large number of intact pollen, respec-tively.Materials and methodsAerosols were collected with a cascade impactor withrectangular slots mounted on a high-volume sampler(565 l/min, DIGITEL DH77, Digitel AG, Switzer-land). The sampler was positioned on a rooftop incentral Zurich. The cut-points of the sampling stagesas indicated by the supplier were 10.2 m (stage 1),4.2 m (stage 2), 2.1 m (stage 3) and 1.4 m (stage4) (Marple and Liu, 1974; Willeke, 1975). A backupfilter after stage 4 collected all remaining particles.Stage 2 was coated with silicone grease and used as atrap to catch bounced and blown-off pollen from stage1 to prevent contamination of lower stages with intactpollen. On all other stages, particles were collected onglass fibre filters.252Figure 1. Competitive ELISA: A dilution series of affinity purified Bet v1 was run together with a dilution series of birch extract (in equivalentsof Bet v1). For comparison, the standard curve of the sandwich-ELISA is shown.Every day during the pollen season the samplerwas started at 7:00 AM and run for 24 hours.After sampling filters were folded twice and storedin polypropylene bags (MIGROS, Switzerland) at−40 C until extraction. For extraction a modifiedpulverisation technique according to Jensen et al.,1989 was used. Filters were first crushed into a finepowder using a hydraulic press (3 times 5 tons). Aller-gens were then eluted over night on a shaker at 30 Cinto phosphate buffered saline (pH = 7.4) containingTween-20 (0.5%) and sodium azide (0.01%). Aftermicrofiltration (0.45 m, celluloseacetate, Schleicher& Schuell, Germany), extracts were stored at −40 Cuntil analysis with ELISA-techniques.Two different methods were used for the analysisof allergen concentrations: One is a sandwich-ELISAusing monoclonal IgG-antibodies (Schäppi, Monn etal., 1996), the other is a newly developed versionof the competitive ELISA (Crowther, 1995) basedon polyclonal IgE-antibodies obtained from patientsallergic to birch pollen and a standardised birch pollenextract. For the sandwich-ELISA concentrations of themajor allergen Bet v1, and for the competitive ELISAequivalents of Bet v1 of total protein are obtained.Statistical analysis was performed using Systat 8.0.To account for the skewed distribution of pollen andallergen data, Spearman rank-order correlation coeffi-cients were calculated. This measure is a nonpara-metric version of the Pearson correlation coefficient,based on the ranks of the data and is independent ofthe distribution of the underlying data.ResultsIn a competitive ELISA, a dilution series of affinitypurified Bet v1 was run together with a dilution seriesof standardised birch extract (Figure 1). Concentra-tions of the extract are indicated as equivalents ofBet v1. Figure 1 indicates, that most of the compe-titive signal can be attributed to Bet v1. Comparisonwith the sandwich-ELISA shows, that the competi-tive ELISA is about ten times more sensitive than thesandwich-ELISA.Figure 2 shows the results for the birch pollenseason 1998 in the coarse particle fraction (>10.2 mPollen counts were obtained from MeteoSwiss (SMA)Zurich (Burkard trap). For the sandwich ELISA,concentrations of the major allergen Bet v1, and forthe competitive ELISA total protein from the birchextract are shown. Both allergen tests and the pollencounts are correlated during the pollen season (r> 0.8,p < 0.0001). In smaller sized particles, the allergenload is often below or close to the detection limit,especially for the sandwich-ELISA). Figure 3 showsthe time course of the fine particles for the compe-253Figure 2. Time-course plot of pollen at SMA Zurich and allergen concentrations in the first stage of the sampler (>10.2 m).Figure 3. Time-course plot of pollen at SMA Zurich and allergen concentrations in the lower stages of the sampler for the competitive ELISA.254titive ELISA. On days with detectable amounts ofallergen load in fine particles (competitive ELISA) theload is only weakly correlated with the pollen counts(4.2m to 2.2m: r = 0.57, p < 0.05) and the allergenconcentrations in the coarse particle fraction.Discussion and conclusionsUsing the monoclonal antibodies in the sandwich-ELISA, information on the amount of one particularallergen (the major allergen Bet v1) is obtained. Onthe other hand the competitive ELISA using the poly-clonal IgE is much more sensitive but not specific forone particular epitope.Data obtained during spring 1998 show that onmost days birch pollen counts parallels the birchallergen load in the particle fraction containing intactpollen (>10.2 m). The correlation, however, ismuch poorer for particles of smaller size than intactpollen (<10.2 m). Allergen concentrations in thesestages are often close to detection limit. It thereforeremains unclear whether this weaker correlation is dueto limitations of the ELISA or due to differences inthe distribution of allergens on particles smaller thanpollen.AcknowledgementsThis study is part of a co-operative project with theAllergy Unit of the University Hospital Zurich (Prof.B. Wüthrich). We would like to acknowledge Aller-gopharma Joachim Ganzer KG, Reinbek, Germanyfor providing allergens and the antibodies for thesandwich-ELISA.ReferencesCrowther J.R.: 1995, ELISA: Theory and Practice. Human PressInc., Totowa, NJ.Jensen J., Poulsen L.K. et al.: 1989, Immunochemical estimationsof allergenic activities from outdoor aero-allergens, collected bya high-volume air sampler. Allergy 44(1), 52–59.Marple V.A. and Liu B.Y.H.: 1974, Characteristics of laminar jetimpactors. Environmental Science and Technology 8(7), 648–654.Rosenberg G.L., Rosenthal R.R. et al.: 1983, Inhalation challengewith ragweed pollen in ragweed-sensitive asthmatics. Journal ofAllergy and Clinical Immunology 71(3), 302–310.Schäppi G., Monn C. et al.: 1996, Direct determination of aller-gens in ambient aerosols: Methodological aspects. InternationalArchives of Allergy and Immunology 110, 364–370.Schlesinger R.B.: 1985, Comparative deposition of inhaled aero-sols in experimental animals and humans: A review. Journal ofToxicology and Environmental Health 15(2), 197–214.Willeke K.: 1975, Performance of the slotted impactor. AmericanIndustrial Hygiene Association Journal (Sept.), 683-691.

Determination of birch pollen allergens in different aerosol sizes

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Determination of birch pollen allergens in different aerosol sizes

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