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Quantification of the Aspergillus versicolor allergen in house dust
Journal of Immunological Methods 372 (2011) 89–94
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Journal of Immunological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j i m
Research paper
Quantification of the Aspergillus versicolor allergen in house dust Chunhua Shi, Donald Belisle, J. David Miller ⁎ Ottawa-Carleton Institute of Chemistry, Carleton University, Ottawa, Canada ON K1S 5B6
a r t i c l e
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Article history: Received 21 April 2011 Received in revised form 28 June 2011 Accepted 29 June 2011 Available online 13 July 2011 Keywords: Aspergillus versicolor ELISA Allergen Polyclonal antibody House dust
a b s t r a c t Aspergillus versicolor, a fungus commonly found on damp building materials, produces the allergen, Asp v 13. Here we report a sensitive Asp v 13 capture ELISA for A. versicolor spores and spore- and mycelial fragments in house dust samples. The method is based on a double polyclonal capture ELSIA. The detection limits for Asp v 13 antigen and A. versicolor spores without dust were 2.44 pg and 12 ng (ca. 110 spores). Detection limits for Asp v 13 and A. versicolor spores in sieved house dust samples were 1.0 ng and 7.8 μg per gram dry weight house dust, respectively. This detection limit is lower than for other house dust allergen immunoassays including for Stachybotrys chartarum, Aspergillus fumigatus, but much lower than that from Alternaria alternata. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Between 10 and 30% of homes in North America have moisture problems leading to mold growth and other dampness contaminants such as house dust mites. This leads to exposures to fungal particles not found in outdoor air (Foto et al., 2005). These particles contain allergens, triple helical glucan and cooccurring toxins (Miller et al., 2010; Rand et al., 2010). Such exposures are associated with exacerbation of asthma as well as increased upper respiratory disease (NAS, 2000, 2004; Health Canada, 2004; World Health Organization, 2009). More recent panels have found that the remediation of moisture and mold was justified in terms of improved public health (Jacobs et al., 2010; Krieger et al., 2010; World Health Organization, 2009). Exposure assessment to fungal allergens has been rendered more difficult by recurrent identification of numerous speciesand genus-specific, but minor allergens (Crameri, 2011; SoeriaAtmadja et al., 2010). To avoid this problem, we have focused on major proteins present on the surfaces of spores, spore- and hyphal fragments, present in numerous strains of the target fungi collected over a wide geographic area and screened with a large collection of human sera (e.g. Wilson et al., 2009; Xu et al.,
2007). Using this approach (Liang et al., 2011), an allergen from A. versicolor was identified. The protein was approved by the I.U. I.S. allergen nomenclature sub-committee as Asp v 13. This fungus is characteristically found growing on particular damp building materials worldwide. It produces a number of toxins as well as triple helical glucan that affect lung biology (Liang et al., 2011; Miller et al., 2008; Rand et al., 2010). A number of researchers have reported that proteins from A. versicolor yield IgM monoclonal antibodies (mAb; Liang et al., 2011). Assays for allergens based entirely on IgM antibodies have generally not been successful (Abebe et al., 2006). However, our process includes the development of an enhanced polyclonal antibody (pAb) to the target fungus (e.g. Xu et al., 2008; Liang et al., 2011; Wilson et al., 2009). As an alternative approach to developing a mAb based assay, this paper reports a method based on a novel method using the enhanced pAb developed during the discovery process (Liang et al., 2011). This produced a capture assay reported suitable for measuring the allergen in settled dust. 2. Materials and methods 2.1. Polyclonal antibody preparation
⁎ Corresponding author. Tel.: + 1 613 520 2600x1053; fax: + 1 613 520 2749. E-mail address: [email protected] (J.D. Miller). 0022-1759/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2011.06.034
A goat anti-Asp v 13 polyclonal antibody (pAb) was produced according to the procedure described by Xu et al.
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(2007). Briefly, a goat was injected with 5 mg of A. versicolor spores. After 23 days, the goat was boosted using 1 mg purified Asp v 13 from A. versicolor filtrate, to increase the quantity of the resulting target polyclonal antibody (Liang et al., 2011). Goat boosting was completed at Cedarlane Laboratories, Ltd. (Hornby, Ontario; meets the requirements of the Canadian Council on Animal Care). The final antigen-specific pAb (hereafter PAb-Asp v 13) was purified and decontaminated as follows (Fig. 1, panel A). Step 1:20 ml boosted goat serum was adjusted to pH 7.4, and then loaded to 5 ml Protein G affinity column (GE Healthcare) for affinity purification. After a 100 ml buffer A (50 mM PBS buffer containing 200 mM NaCl) wash, antibody was eluted by buffer B (20 ml 20 mM pH = 2.5 sodium acetate). The eluted solution was immediately neutralized by buffer C (400 mM pH = 8.0 PBS). The antibody solution was then concentrated using a 15 ml 30 kDa molecular weight cutoff filter (Millipore) and the buffer exchanged with buffer A for future usage. Step 2: purified Asp v 13 was prepared as previously reported (Liang et al, 2011). Two milligrams biotinylated Asp v 13 was then applied to a 1 ml streptavidin affinity column (GE Healthcare). The column was washed
with 20 ml Buffer A, loaded with pAb from Step 1 and incubated for 5–10 min at 4 °C, and then washed by 2 × 20 ml buffer A. The antigen-specific pAb was then eluted with buffer B and immediately neutralized by buffer C to pH around 7.0. Step 3: 5 mg antibody from step 1 was biotinylated and applied to 1 ml streptavidin affinity column. After the column was washed with 20 ml Buffer A, pAb from step 2 was loaded to the antigen affinity column and incubated for 5–10 min. Step 4: The flow-through from Step 3 was then loaded to an empty 1 ml streptavidin column to get rid of any leached biotinylated Asp v 13/antibody from the former streptavidin treatment. The resultant antibody from the above 4 step purification process was concentrated, buffer exchanged with buffer A and 50% glycerol added for storage for ELISA and immune blot assays. 2.2. Biotinylation of antibody and antigen Purified 1 ml Asp v 13 (2 mg/ml), pAb purified from protein G sepharose (Step 1) or 0.5 mg pAb-Asp v 13 were reacted with 80 mol excess NHS-LC-LC-Biotin (Pierce) in DMSO according to the manufacture's instruction. The
Fig. 1. pAb-Asp v 13 purification and characterization. Panel A, Cartoon representation of pAb-Asp v 13 purification. Please see the Materials and methods for detailed procedure. Panel B, Western blot characterization of pAb-Asp v 13 using A. versicolor spore extract. Lane M: protein standard with molecular weights marked left; lane 1: CBB staining of spore protein; lane 2: western blot with crude pAb after step 1; lane 3: western blot with pAb-Asp v 13. Panel C and D, Asp v 13 capture ELISA for antigen (C) and A. versicolor spores (D) using pAb-Asp v 13 as coating antibody and biotinylated pAb-Asp v 13 as primary antibody.
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reaction was stopped by adding 0.2 ml 1 M pH = 7.0 Tris buffer and the buffer exchanged using a 5 ml G-25 column (GE Healthcare) to remove unbound biotin molecules. Biotinylated antigen and pAb were then loaded to streptavidin columns to prepare the antigen-biotin and pAb-biotin binding streptavidin columns for antigen-specific pAb purification and antigen-specific pAb decontamination above respectively, while biotinylated pAb-Asp v 13 was used as the primary antibody in the antigen capture ELISA assay. 2.3. SDS-PAGE and Western blot A. versicolor spores were produced as follows. Rice (50 g; Uncle Ben's™) was placed in 500 ml wide-mouth Erlenmeyer flasks, 30 ml of water was added and the mixture was autoclaved for 30 min. Cultures were inoculated with A veresicolor DAOM 235361 and incubated at 25 °C in the dark for 3 weeks, and dried at 30 °C. The provenance of other strains is provided elsewhere (Liang et al., 2011; Xu et al., 2008). Spores were harvested by physical removal from the rice grains and stored in vials at 4 °C. This method is known to produce allergens and toxins reflective of those present on building material (e.g. Wilson et al., 2009). For each test, spores (20 mg) were placed in a vial with 3/8 methacrylate beads (ATS Scientific Inc.), which was milled for 30 min with a Spex-Certiprep mixer mill (Model 5100, Metuchen, NJ). The spore fragments were then dissolved in 0.1 ml of PBS buffer with 0.1% Tween-20 (PBST) and sonicated for 2 h at 4 °C. The resulting solution was centrifuged at 10,000 ×g. SDS-PAGE and Western blot were performed following the methods of Xu et al. (2007). Briefly, 10 μg proteins were separated by 12% SDS-PAGE with SeeBlue® Plus2 Pre-Stained Standard (Invitrogen). The gel was visualized with GelCode Blue Stain Reagent (Pierce) according to manufacturers' instructions or transferred to a Hybond-PVDF membrane (Amersham Biosciences) with a Hoefer miniVE electrotransfer unit (Amersham Biosciences). The transfer was carried out at a constant current of 350 mA for 60 min. 1/20000 or 1/5000 dilutions of pAb-Asp v 13 (1 mg/ml) or 1/2000 dilution of pAb (5 mg/ml) from step 1 was used as primary antibodies followed by corresponding anti-goat-IgG conjugated with alkaline phosphatase (Sigma). Detection was achieved by incubating membranes with BCIP/NBT (Sigma) for 5 to 10 min.
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pH 9.6 carbonate bicarbonate buffer (Sigma) to each well of microplates (NUNC Maxisorp, Nalgene). The plate was incubated overnight at 4 °C, then washed three times with 200 μl PBS buffer with 0.1% Tween-20 (PBST) and blocked with 200 μl PBST solution with 1% BSA for 4 h at room temperature. After washing twice with 200 μl PBST, 100 μl 1:8000 biotinylated pAb-Asp v 13 (0.5 mg/ml) in 1% BSA/ PBST was added and the plate was incubated at room temperature for 2 h. After washing twice with 200 μl PBST, 100 μl streptavidin conjugated with horseradish peroxidase (Sigma) in 1% BSA/PBST (1 mg/ml, 1:16000) was then applied to each well and incubated at room temperature for 30 min. After 4× final wash with 200 μl PBST, 100 μl TMB substrate (Sigma) was added per well and the plate was incubated for 10 min at room temperature for the blue color development. The enzyme reaction was stopped by adding 50 μl 0.5 M H2SO4. The optical density at 450 nm was read using a Molecular Devices Spectra Max 340PC reader (Sunnyvale, CA). Antigen Asp v 13 capture ELISA was performed by coating 20 ng capture antibody pAb-Asp v 13 in 50 mM pH 9.6 carbonate bicarbonate buffer each well in the microplate, which was incubated overnight at 4 °C. After the plate was blocked by 1% BSA, 100 μl different concentrations of Asp v 13, spore extract or dust sample extracts in 1% BSA/PBST were added for antigen capture at room temperature for 2 h. The samples were then analyzed using the indirect ELISA outlined above. 2.6. Analysis of A. versicolor spores in house dust Fine dust was used from samples collected as part of a study where floors were repeatedly vacuumed with a HEPA filter equipped vacuum. The dust was sieved to b300 μm (stainless steel test sieve model 50, Fisher Scientific) and weighed using a Sartorius balance (A120‐S 4; accurate to N0.1 mg (Salares et al, 2009). The dust was spiked with different amounts of A. versicolor spores only or with both A. versicolor and A. ochraceus were milled as above. The spore fragments and spiked dust samples were dissolved in PBST containing 5 μl of fungal protease inhibitor (Sigma) and sonicated for 2 h at 4 °C. The solution was then centrifuged at 10,000 ×g. The supernatants were then loaded to ELISA microplates for indirect and capture ELISA detection.
2.4. Protein concentration assay 3. Results Bradford method was applied to determine the protein concentrations. Briefly, reaction mixtures were prepared in a 96 well microtiter plate (Fisher Scientific) consisting of 150 μl of Quick Start Bradford Dye Reagent (Bio-Rad) mixed with 5 μl of detected samples or 5 μl PBS buffer as the negative control. After the addition of ddH2O to final volume of 300 μl, the plate was incubated at room temperature for 10 min. Absorbance readings were taken at 595 nm using a microplate spectrophotometer (Molecular Devices SpectraMax 340PC). 2.5. Indirect and Asp v 13 capture ELISA Indirect ELISA was carried out by coating 100 μl aliquots of different concentrations of antigen/spore extracts in 50 mM
3.1. Purification, decontamination and characterization of PAb-Asp v 13 Approximately 40 mg pAb was purified from 20 ml Asp v 13 boosted goat sera by protein G sepharose column (Fig. 1A, step 1). Purity of the pAb in this step was N99% by SDS PAGE with CBB staining (data not shown). As would be expected, most of the pAb antibodies were not specific to Asp v 13 antigen. Further purification by allergen affinity chromatography was done using a biotinylated allergen-/streptavidin column (Fig. 1A, step 2). Yield from this step was ~ 5% Asp v 13-specific pAb. After removal of self-interacting pAb using the biotinylated antibody-bound streptavidin column and elimination of any residual biotinylated Asp v 13 or pAb from
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step 3 and step 2, there was a modest loss (henceforth pAbAsp v 13). The final, purified pAb was characterized by Western blot and capture ELISA. The response to the pAb by Western blot using the pAb is shown in Fig. 1. This was purified from protein G sepharose (1:2000 dilutions, Fig. 1B, lane 2) and PAb-Asp v 13 (1:20,000 dilution, Fig. 1B, lane 3). Lane 2 had multiple low density bands without a dominant band, while lane 3 had only a single dominant band around 80 kDa, the dimer of Asp v 13 (Shi and Miller, 2011). The pAb-Asp v 13 was used as the capture antibody and the biotinylated pAb-Asp v 13 as the detection antibody in capture ELISA. Titration curves were established with serial dilutions of the allergen and A. versicolor spores with the optimized capture antibody (20 ng/well, primary at 1:8000 dilution and secondary antibody concentrations at 1:16000 dilution; Fig. 1C and D). The limit of detection (LOD) of the capture ELISA was 24 pg/ml allergen (p = 0.045; Fisher's LSD) with a linear range of 0.05–0.4 ng/ml (R 2 = 0.990). For spores, the LOD was 120 ng/ml (p = 0.0381) with a linear range 0.06–0.48 μg/ml (R 2 = 0.997). The detection limit for the spores in the capture ELISA was ca. 110 spores per well. 3.2. Cross-reactivity Using indirect ELISA, the cross-reactivity of pAb-Asp v 13 to 22 selected fungi spores was examined. Compared to the response from A. versicolor spores (which was set at 100%), the spores of the closely-related A. sydowii gave the highest
response around 71%. A. ochraceus and A. penicillioides and P. chrysogenum and P. decumbens gave intermediate crossreactivity; the remaining species did not cross react (Fig. 2A). When pAb-Asp v 13 was used as the capture antibody for capture ELISA, the intermediate cross-reactive spores such as A. ochraceus, A. penicillioides, P. chrysogenum and P. decumbens had modest cross-reactivity b of the A. versicolor/A. sydowi response. Cross reactivity to the other species was not observed (Fig. 2B). 3.3. Analysis of house dust samples spiked with A. versicolor spores Recovery of A. versicolor spores in dust was reduced to 7.8 μg/g dust and 9.8 μg/g dust when extracted with 50 and 10 mg/ml dust, respectively (Fig. 3A). In similar experiments, the recoveries of allergen were 84.0% ± 5.0 and 85.5% ± 5.5 respectively. The linear ranges for 50 mg/ml and 10 mg/ml dust were between 2 and 15.6 μg/g dust (R 2 = 0.99), and between 9.8 and 78 μg/g dust (R 2 = 0.99), respectively. When A. versicolor and A. ochraceus spores were both present, the response of the capture ELISA was not significantly reduced until a very large proportion of A. ochraceus spores were present. When 250 μg A. ochraceus spores per gram dust sample with half as much A. versicolor spores, the detection limit dropped by 50% (Fig. 4B). As the proportion of A. versicolor spores was increased below 125 μg, (i.e. 50%) there was no effect on the detection of A. versicolor (Fig. 4C).
Fig. 2. The cross-reactivity of pAb-Asp v 13 to 23 selected spores. Panel A, 100 μl selected spore extracts per well was coated directly in plate at 0.156 μg/ml for indirect ELISA; panel B, Asp v 13 capture ELISA response to selected spores and 3.906 ug/ml selected spore extracts were applied. 1:8000 biotinylated pAb-Asp v 13 was used as the primary and 1:16000 Strep-HRP used as the secondary.
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Fig. 3. Analysis of A. versicolor spore-spiked dust by Asp v 13 capture ELISA and demonstration of cross-reactivity. Panel A, A. versicolor spore-spiked dust sample. Diamond: spores only; square: spore-spiked 50 mg/ml dust and triangle: spore-spiked 10 mg/ml dust. Long arrow indicates quantification limit of 0.39 μg/ml with 50 mg/ml dust (p = 0.026 with Fisher's LSD model), while the short arrow indicates quantification limit of 0.098 μg/ml with 10 mg/ml dust (p = 0.000 with Fisher's LSD model). Panel B, the affect of A. versicolor spore detection by A. ochraceus spores. 25 (circle), 5(triangle), 1 (square) or 0 (diamond) μg/ml A. ochraceus spores were applied in the analysis.
4. Discussion Our previous studies using a multistep PCR protocol (Shi and Miller, 2011; Shi et al., 2011) had shown that the dominant allergen from A. versicolor is Asp v 13 (provisionally Avs41; Liang et al., 2011). The allergen is a 403 amino acid protein with multiple antigenic and allergenic epitopes on its surface (Shi and Miller, 2011). A BLAST search of the sequence of Asp v 13 indicated that it is a subtilisin-like alkaline serine protease similar to Asp f1 1 and other alkaline serine proteins from Aspergillus spp. and Penicillium species (Shi and Miller, 2011; Chou et al., 1999). After a goat was immunized with spores of A. versicolor, followed by two sequential boosts with the purified Asp v 13 protein, the response of the polyclonal antibody against Asp v 13 was enhanced. This is due to an increase of particular antibodies resulting from the spore immunization related to the target protein and those not boosted declined (Liang et al., 2011). This response was observed for other fungi (e.g. Wilson et al., 2009; Xu et al., 2008). The polyclonal antibody purified directly from a protein G column was not specific. However using pAb and biotinylated after one purification
Fig. 4. Combinative effect of both cross-reactive spores (A. ochraceus as the representative) and dust to the detection of A. versicolor spores in dust samples. Panel A, A. versicolor spore's assay in dust sample without A. ochraceus spores. Arrow showed the detection limit of 7.8125 μg spores per gram dust (p b 0.05). Panel B, A. versicolor spore assay in dust sample with 250 μg A. ochraceus spores per gram dust. Arrow shows the detection limit of 15.625 μg spores per gram dust (p b 0.05). The variant data and the notched plot box with 95% confidence were calculated by SYSTAT. Panel C, statistical analysis of the effect of 250 μg A. ochraceus spores to different A. versicolor spore's assay. P-values in each concentration with/without A. ochraceus calculated from SYSTAT were shown in the top of the related A. versicolor spore's concentration respectively.
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(step 1, Fig. 1A) as the capture and primary antibody respectively gave high background in the Asp v 13 capture ELISA. This resulted from the strong interaction between capture antibody and primary antibody that obscured the capture ELISA signal (data not shown). Although using pAb after antigen affinity purification (Fig. 1A, step 2) as the capture and primary antibody significantly decreased the noise, the sensitivity of the assay remained inadequate (data not shown) possibly from the leaching of biotinylated antigen from the streptavidin column and trace goat auto-antibodies. The specificity of the pAb to Asp v 13 was significantly improved by the antigen-affinity purification and decontamination steps (Fig. 1A & B). The Asp v 13 limit of quantification was ~ 1 ng Asp v 13 antigen per gram sieved dust (representing ~ 7.8 μg or ~ 7 × 10 4 spores per gram dust). This is more sensitive than our assay for S. chartarum (Xu et al., 2008), to A. fumigatus Asp f1 (Dillon, et al., 2007; Ryan et al., 2001), and is a much more sensitive a published assay for than A. alternata allergen Alt a1 (Salo et al., 2005). The panel that produced “Clearing the air” (NAS, 2000) the desirable method for assessing exposure to fungi must be based on measurement of allergens (antigens). Unlike serious cross-reactivity problem between allied species of Alt a1 polyclonal antibodies (Schmechel et al., 2008; Peters et al., 2008), pAb-Asp v 13 prepared by our method is specific with negligible cross-reactivity to the 21 tested fungi species tested. This included the closely related species such as A. niger, A. fumigatus, A. ochraceus, A. flavus and A. penicillioides. Only A. sydowii spores gave similar signal to A. versicolor. These are closely related taxa (Peterson, 2008) that often co-occur (Miller et al., 2008). As with the closely related S. chartarum and S. chlorohalonata (Xu et al., 2008), the Asp v 13 capture ELISA would detect both A. sydowii and A. versicolor if one or both were present. In summary, we report a new pAb protocol in this case for the allergen Asp v 13. This was achieved by using the enhanced pAb in a capture ELISA detection of A. versicolor spores in dust. The method is suitable for the measurement of the dominant A. versicolor allergen Asp v 13 in house dust. Acknowledgments This work was funded by an NSERC IRC to JDM and a MITACs award to Ms. Natacha Provost and JDM. We are grateful for the contributions of Cedarlane Laboratories, Ltd. who made the antibodies. References Abebe, M., Kumar, V., Rajan, S., Thaker, A., Sevinc, S., Vijay, H.M., 2006. Detection of recombinant Alt a1 in a two-site, IgM based, sandwich ELISA opens up possibilities of developing alternative assays for the allergen. J. Immunol. Methods 30 (312), 111. Chou, H., Lin, W.L., Tam, M.F., Wang, S.R., Han, S.H., Shen, H.D., 1999. Alkaline serine proteinase is a major allergen of Aspergillus flavus, a prevalent airborne Aspergillus species in the Taipei area. Int. Arch. Allergy Immunol. 119, 282.
Crameri, R., 2011. The problem of cross-reactivity in the diagnosis of fungal allergy. Clin. Exp. Allergy 41, 302. Dillon, H.K., Boling, D.K., Miller, J.D., 2007. Comparison of detection methods for Aspergillus fumigatus in environmental air samples in an occupational environment. J. Occup. Environ. Hyg. 4, 509. Foto, M., Vrijmoed, L.L., Miller, J.D., Ruest, K., Lawton, M., Dales, R.E., 2005. Comparison of airborne ergosterol, glucan and Air-O-Cell data in relation to physical assessments of mold damage and some other parameters. Indoor Air 15, 257. Health Canada, 2004. Fungal Contamination in Public Buildings: Health Effects and Investigation Methods. Health Canada, Ottawa, Canada. Jacobs, D.E., Brown, M.J., Baeder, A., Sucosky, M.S., Margolis, S., Hershovitz, J., Kolb, L., Morley, R.L., 2010. A systematic review of housing interventions and health: introduction, methods, and summary findings. J. Public Health Manag. Pract. 16, S5. Krieger, J., Jacobs, D.E., Ashley, P.J., Baeder, A., Chew, G.L., Dearborn, D., Hynes, H.P., Miller, J.D., Morley, R., Rabito, F., Zeldin, D.C., 2010. J. Public Health Manag Pract. 16, S11. Liang, Y., Zhao, W., Xu, J., Miller, J.D., 2011. Characterization of against two related exoantigens from Aspergillus versicolor isolated using sera from atopic patients. Int. Biodeterior. Biodegrad. 65, 217. Miller, J.D., et al., 2008. Mold ecology: recovery of fungi from certain moldy building materials. In: Prezant, B., Weekes, D., Miller, J.D. (Eds.), Recognition, Evaluation and Control of Indoor Mold. American Industrial Hygiene Association Press, Fairfax, VA, p. 43. Miller, J.D., Sun, M., Gilyan, A., Roy, J., Rand, T.G., 2010. Inflammationassociated gene transcription and expression in mouse lungs induced by low molecular weight compounds from fungi from the built environment. Chem. Biol. Interact. 183, 113. NAS, 2000. Clearing the Air. Institute of Medicine, National Academies Press, Washington, DC. NAS, 2004. Damp Indoor Spaces and Health. Institute of Medicine, National Academies Press, Washington, DC. Peters, J.L., Muilenberg, M.L., Rogers, C.A., Burge, H.A., Spengler, J.D., 2008. Alternaria measures in inner-city, low-income housing by immunoassay and culture-based analysis. Ann. Allergy Asthma Immunol. 100, 364. Peterson, S.W., 2008. Phylogenetic analysis of Aspergillus species using DNA sequences from four loci. Mycologia 100, 205. Rand, T.G., Sun, M., Gilyan, A., Downey, J., Miller, J.D., 2010. Dectin-1 and inflammation-associated gene transcription and expression in mouse lungs by a toxic (1,3)-beta-D: glucan. Arch. Toxicol. 84, 205. Ryan, T.J., Whitehead, L.W., Connor, T.H., Burau, K.D., 2001. Survey of the Asp f 1 allergen in office environments. Appl. Occup. Environ. Hyg. 16, 679. Salares, V.R., Hinde, C.A., Miller, J.D., 2009. Analysis of settled dust in homes and fungal glucan in air particulate collected during HEPA vacuuming. Indoor Built Environ. 18, 485. Salo, P.M., Yin, M., Arbes Jr., S.J., Cohn, R.D., Sever, M., Muilenberg, M., Burge, H.A., London, S.J., Zeldin, D.C., 2005. Dustborne Alternaria alternata antigens in US homes: results from the National Survey of Lead and Allergens in Housing. J. Allergy Clin. Immunol. 116, 623. Schmechel, D., Green, B.J., Blachere, F.M., Janotka, E., Beezhold, D.H., 2008. Analytical bias of cross-reactive polyclonal antibodies for environmental immunoassays of Alternaria alternata. J. Allergy Clin. Immunol. 121, 763. Shi, C., Miller, J.D., 2011. Characterization of human antigenic protein Asp v 13 from Aspergillus versicolor. Mol. Immunol. doi:10.1016/j.molimm.2011.05.010. Shi, C., Smith, M.L., Miller, J.D., 2011. Characterization of human antigenic proteins SchS21 and SchS34 from Stachybotrys chartarum. Int. Arch. Allergy Immunol. 155, 74. Soeria-Atmadja, D., Onell, A., Borgå, A., 2010. IgE sensitization to fungi mirrors fungal phylogenetic systematics. J. Allergy Clin Immunol. 125, 1379. Wilson, A.M., Luo, W., Miller, J.D., 2009. Using human sera to identify a 52-kDa exoantigen of Penicillium chrysogenum and implications of polyphasic taxonomy of anamorphic ascomycetes in the study of antigenic proteins. Mycopathologia 168, 213. World Health Organization, 2009. WHO Guidelines for Indoor Air QualityDampness and Mould. Xu, J., Jensen, J.T., Liang, Y., Belisle, D., Miller, J.D., 2007. The biology and immogenicity of a 34 kDa antigen of Stachybotrys chartarum sensu lato. Int. Biodeterior. Biodegrad. 60, 308. Xu, J., Liang, Y., Belisle, D., Miller, J.D., 2008. Characterization of monoclonal antibodies to an antigenic protein from Stachybotrys chartarum and its measurement in house dust. J. Immunol. Methods 332, 121.
Contents lists available at ScienceDirect
Journal of Immunological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j i m
Research paper
Quantification of the Aspergillus versicolor allergen in house dust Chunhua Shi, Donald Belisle, J. David Miller ⁎ Ottawa-Carleton Institute of Chemistry, Carleton University, Ottawa, Canada ON K1S 5B6
a r t i c l e
i n f o
Article history: Received 21 April 2011 Received in revised form 28 June 2011 Accepted 29 June 2011 Available online 13 July 2011 Keywords: Aspergillus versicolor ELISA Allergen Polyclonal antibody House dust
a b s t r a c t Aspergillus versicolor, a fungus commonly found on damp building materials, produces the allergen, Asp v 13. Here we report a sensitive Asp v 13 capture ELISA for A. versicolor spores and spore- and mycelial fragments in house dust samples. The method is based on a double polyclonal capture ELSIA. The detection limits for Asp v 13 antigen and A. versicolor spores without dust were 2.44 pg and 12 ng (ca. 110 spores). Detection limits for Asp v 13 and A. versicolor spores in sieved house dust samples were 1.0 ng and 7.8 μg per gram dry weight house dust, respectively. This detection limit is lower than for other house dust allergen immunoassays including for Stachybotrys chartarum, Aspergillus fumigatus, but much lower than that from Alternaria alternata. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Between 10 and 30% of homes in North America have moisture problems leading to mold growth and other dampness contaminants such as house dust mites. This leads to exposures to fungal particles not found in outdoor air (Foto et al., 2005). These particles contain allergens, triple helical glucan and cooccurring toxins (Miller et al., 2010; Rand et al., 2010). Such exposures are associated with exacerbation of asthma as well as increased upper respiratory disease (NAS, 2000, 2004; Health Canada, 2004; World Health Organization, 2009). More recent panels have found that the remediation of moisture and mold was justified in terms of improved public health (Jacobs et al., 2010; Krieger et al., 2010; World Health Organization, 2009). Exposure assessment to fungal allergens has been rendered more difficult by recurrent identification of numerous speciesand genus-specific, but minor allergens (Crameri, 2011; SoeriaAtmadja et al., 2010). To avoid this problem, we have focused on major proteins present on the surfaces of spores, spore- and hyphal fragments, present in numerous strains of the target fungi collected over a wide geographic area and screened with a large collection of human sera (e.g. Wilson et al., 2009; Xu et al.,
2007). Using this approach (Liang et al., 2011), an allergen from A. versicolor was identified. The protein was approved by the I.U. I.S. allergen nomenclature sub-committee as Asp v 13. This fungus is characteristically found growing on particular damp building materials worldwide. It produces a number of toxins as well as triple helical glucan that affect lung biology (Liang et al., 2011; Miller et al., 2008; Rand et al., 2010). A number of researchers have reported that proteins from A. versicolor yield IgM monoclonal antibodies (mAb; Liang et al., 2011). Assays for allergens based entirely on IgM antibodies have generally not been successful (Abebe et al., 2006). However, our process includes the development of an enhanced polyclonal antibody (pAb) to the target fungus (e.g. Xu et al., 2008; Liang et al., 2011; Wilson et al., 2009). As an alternative approach to developing a mAb based assay, this paper reports a method based on a novel method using the enhanced pAb developed during the discovery process (Liang et al., 2011). This produced a capture assay reported suitable for measuring the allergen in settled dust. 2. Materials and methods 2.1. Polyclonal antibody preparation
⁎ Corresponding author. Tel.: + 1 613 520 2600x1053; fax: + 1 613 520 2749. E-mail address: [email protected] (J.D. Miller). 0022-1759/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2011.06.034
A goat anti-Asp v 13 polyclonal antibody (pAb) was produced according to the procedure described by Xu et al.
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(2007). Briefly, a goat was injected with 5 mg of A. versicolor spores. After 23 days, the goat was boosted using 1 mg purified Asp v 13 from A. versicolor filtrate, to increase the quantity of the resulting target polyclonal antibody (Liang et al., 2011). Goat boosting was completed at Cedarlane Laboratories, Ltd. (Hornby, Ontario; meets the requirements of the Canadian Council on Animal Care). The final antigen-specific pAb (hereafter PAb-Asp v 13) was purified and decontaminated as follows (Fig. 1, panel A). Step 1:20 ml boosted goat serum was adjusted to pH 7.4, and then loaded to 5 ml Protein G affinity column (GE Healthcare) for affinity purification. After a 100 ml buffer A (50 mM PBS buffer containing 200 mM NaCl) wash, antibody was eluted by buffer B (20 ml 20 mM pH = 2.5 sodium acetate). The eluted solution was immediately neutralized by buffer C (400 mM pH = 8.0 PBS). The antibody solution was then concentrated using a 15 ml 30 kDa molecular weight cutoff filter (Millipore) and the buffer exchanged with buffer A for future usage. Step 2: purified Asp v 13 was prepared as previously reported (Liang et al, 2011). Two milligrams biotinylated Asp v 13 was then applied to a 1 ml streptavidin affinity column (GE Healthcare). The column was washed
with 20 ml Buffer A, loaded with pAb from Step 1 and incubated for 5–10 min at 4 °C, and then washed by 2 × 20 ml buffer A. The antigen-specific pAb was then eluted with buffer B and immediately neutralized by buffer C to pH around 7.0. Step 3: 5 mg antibody from step 1 was biotinylated and applied to 1 ml streptavidin affinity column. After the column was washed with 20 ml Buffer A, pAb from step 2 was loaded to the antigen affinity column and incubated for 5–10 min. Step 4: The flow-through from Step 3 was then loaded to an empty 1 ml streptavidin column to get rid of any leached biotinylated Asp v 13/antibody from the former streptavidin treatment. The resultant antibody from the above 4 step purification process was concentrated, buffer exchanged with buffer A and 50% glycerol added for storage for ELISA and immune blot assays. 2.2. Biotinylation of antibody and antigen Purified 1 ml Asp v 13 (2 mg/ml), pAb purified from protein G sepharose (Step 1) or 0.5 mg pAb-Asp v 13 were reacted with 80 mol excess NHS-LC-LC-Biotin (Pierce) in DMSO according to the manufacture's instruction. The
Fig. 1. pAb-Asp v 13 purification and characterization. Panel A, Cartoon representation of pAb-Asp v 13 purification. Please see the Materials and methods for detailed procedure. Panel B, Western blot characterization of pAb-Asp v 13 using A. versicolor spore extract. Lane M: protein standard with molecular weights marked left; lane 1: CBB staining of spore protein; lane 2: western blot with crude pAb after step 1; lane 3: western blot with pAb-Asp v 13. Panel C and D, Asp v 13 capture ELISA for antigen (C) and A. versicolor spores (D) using pAb-Asp v 13 as coating antibody and biotinylated pAb-Asp v 13 as primary antibody.
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reaction was stopped by adding 0.2 ml 1 M pH = 7.0 Tris buffer and the buffer exchanged using a 5 ml G-25 column (GE Healthcare) to remove unbound biotin molecules. Biotinylated antigen and pAb were then loaded to streptavidin columns to prepare the antigen-biotin and pAb-biotin binding streptavidin columns for antigen-specific pAb purification and antigen-specific pAb decontamination above respectively, while biotinylated pAb-Asp v 13 was used as the primary antibody in the antigen capture ELISA assay. 2.3. SDS-PAGE and Western blot A. versicolor spores were produced as follows. Rice (50 g; Uncle Ben's™) was placed in 500 ml wide-mouth Erlenmeyer flasks, 30 ml of water was added and the mixture was autoclaved for 30 min. Cultures were inoculated with A veresicolor DAOM 235361 and incubated at 25 °C in the dark for 3 weeks, and dried at 30 °C. The provenance of other strains is provided elsewhere (Liang et al., 2011; Xu et al., 2008). Spores were harvested by physical removal from the rice grains and stored in vials at 4 °C. This method is known to produce allergens and toxins reflective of those present on building material (e.g. Wilson et al., 2009). For each test, spores (20 mg) were placed in a vial with 3/8 methacrylate beads (ATS Scientific Inc.), which was milled for 30 min with a Spex-Certiprep mixer mill (Model 5100, Metuchen, NJ). The spore fragments were then dissolved in 0.1 ml of PBS buffer with 0.1% Tween-20 (PBST) and sonicated for 2 h at 4 °C. The resulting solution was centrifuged at 10,000 ×g. SDS-PAGE and Western blot were performed following the methods of Xu et al. (2007). Briefly, 10 μg proteins were separated by 12% SDS-PAGE with SeeBlue® Plus2 Pre-Stained Standard (Invitrogen). The gel was visualized with GelCode Blue Stain Reagent (Pierce) according to manufacturers' instructions or transferred to a Hybond-PVDF membrane (Amersham Biosciences) with a Hoefer miniVE electrotransfer unit (Amersham Biosciences). The transfer was carried out at a constant current of 350 mA for 60 min. 1/20000 or 1/5000 dilutions of pAb-Asp v 13 (1 mg/ml) or 1/2000 dilution of pAb (5 mg/ml) from step 1 was used as primary antibodies followed by corresponding anti-goat-IgG conjugated with alkaline phosphatase (Sigma). Detection was achieved by incubating membranes with BCIP/NBT (Sigma) for 5 to 10 min.
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pH 9.6 carbonate bicarbonate buffer (Sigma) to each well of microplates (NUNC Maxisorp, Nalgene). The plate was incubated overnight at 4 °C, then washed three times with 200 μl PBS buffer with 0.1% Tween-20 (PBST) and blocked with 200 μl PBST solution with 1% BSA for 4 h at room temperature. After washing twice with 200 μl PBST, 100 μl 1:8000 biotinylated pAb-Asp v 13 (0.5 mg/ml) in 1% BSA/ PBST was added and the plate was incubated at room temperature for 2 h. After washing twice with 200 μl PBST, 100 μl streptavidin conjugated with horseradish peroxidase (Sigma) in 1% BSA/PBST (1 mg/ml, 1:16000) was then applied to each well and incubated at room temperature for 30 min. After 4× final wash with 200 μl PBST, 100 μl TMB substrate (Sigma) was added per well and the plate was incubated for 10 min at room temperature for the blue color development. The enzyme reaction was stopped by adding 50 μl 0.5 M H2SO4. The optical density at 450 nm was read using a Molecular Devices Spectra Max 340PC reader (Sunnyvale, CA). Antigen Asp v 13 capture ELISA was performed by coating 20 ng capture antibody pAb-Asp v 13 in 50 mM pH 9.6 carbonate bicarbonate buffer each well in the microplate, which was incubated overnight at 4 °C. After the plate was blocked by 1% BSA, 100 μl different concentrations of Asp v 13, spore extract or dust sample extracts in 1% BSA/PBST were added for antigen capture at room temperature for 2 h. The samples were then analyzed using the indirect ELISA outlined above. 2.6. Analysis of A. versicolor spores in house dust Fine dust was used from samples collected as part of a study where floors were repeatedly vacuumed with a HEPA filter equipped vacuum. The dust was sieved to b300 μm (stainless steel test sieve model 50, Fisher Scientific) and weighed using a Sartorius balance (A120‐S 4; accurate to N0.1 mg (Salares et al, 2009). The dust was spiked with different amounts of A. versicolor spores only or with both A. versicolor and A. ochraceus were milled as above. The spore fragments and spiked dust samples were dissolved in PBST containing 5 μl of fungal protease inhibitor (Sigma) and sonicated for 2 h at 4 °C. The solution was then centrifuged at 10,000 ×g. The supernatants were then loaded to ELISA microplates for indirect and capture ELISA detection.
2.4. Protein concentration assay 3. Results Bradford method was applied to determine the protein concentrations. Briefly, reaction mixtures were prepared in a 96 well microtiter plate (Fisher Scientific) consisting of 150 μl of Quick Start Bradford Dye Reagent (Bio-Rad) mixed with 5 μl of detected samples or 5 μl PBS buffer as the negative control. After the addition of ddH2O to final volume of 300 μl, the plate was incubated at room temperature for 10 min. Absorbance readings were taken at 595 nm using a microplate spectrophotometer (Molecular Devices SpectraMax 340PC). 2.5. Indirect and Asp v 13 capture ELISA Indirect ELISA was carried out by coating 100 μl aliquots of different concentrations of antigen/spore extracts in 50 mM
3.1. Purification, decontamination and characterization of PAb-Asp v 13 Approximately 40 mg pAb was purified from 20 ml Asp v 13 boosted goat sera by protein G sepharose column (Fig. 1A, step 1). Purity of the pAb in this step was N99% by SDS PAGE with CBB staining (data not shown). As would be expected, most of the pAb antibodies were not specific to Asp v 13 antigen. Further purification by allergen affinity chromatography was done using a biotinylated allergen-/streptavidin column (Fig. 1A, step 2). Yield from this step was ~ 5% Asp v 13-specific pAb. After removal of self-interacting pAb using the biotinylated antibody-bound streptavidin column and elimination of any residual biotinylated Asp v 13 or pAb from
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step 3 and step 2, there was a modest loss (henceforth pAbAsp v 13). The final, purified pAb was characterized by Western blot and capture ELISA. The response to the pAb by Western blot using the pAb is shown in Fig. 1. This was purified from protein G sepharose (1:2000 dilutions, Fig. 1B, lane 2) and PAb-Asp v 13 (1:20,000 dilution, Fig. 1B, lane 3). Lane 2 had multiple low density bands without a dominant band, while lane 3 had only a single dominant band around 80 kDa, the dimer of Asp v 13 (Shi and Miller, 2011). The pAb-Asp v 13 was used as the capture antibody and the biotinylated pAb-Asp v 13 as the detection antibody in capture ELISA. Titration curves were established with serial dilutions of the allergen and A. versicolor spores with the optimized capture antibody (20 ng/well, primary at 1:8000 dilution and secondary antibody concentrations at 1:16000 dilution; Fig. 1C and D). The limit of detection (LOD) of the capture ELISA was 24 pg/ml allergen (p = 0.045; Fisher's LSD) with a linear range of 0.05–0.4 ng/ml (R 2 = 0.990). For spores, the LOD was 120 ng/ml (p = 0.0381) with a linear range 0.06–0.48 μg/ml (R 2 = 0.997). The detection limit for the spores in the capture ELISA was ca. 110 spores per well. 3.2. Cross-reactivity Using indirect ELISA, the cross-reactivity of pAb-Asp v 13 to 22 selected fungi spores was examined. Compared to the response from A. versicolor spores (which was set at 100%), the spores of the closely-related A. sydowii gave the highest
response around 71%. A. ochraceus and A. penicillioides and P. chrysogenum and P. decumbens gave intermediate crossreactivity; the remaining species did not cross react (Fig. 2A). When pAb-Asp v 13 was used as the capture antibody for capture ELISA, the intermediate cross-reactive spores such as A. ochraceus, A. penicillioides, P. chrysogenum and P. decumbens had modest cross-reactivity b of the A. versicolor/A. sydowi response. Cross reactivity to the other species was not observed (Fig. 2B). 3.3. Analysis of house dust samples spiked with A. versicolor spores Recovery of A. versicolor spores in dust was reduced to 7.8 μg/g dust and 9.8 μg/g dust when extracted with 50 and 10 mg/ml dust, respectively (Fig. 3A). In similar experiments, the recoveries of allergen were 84.0% ± 5.0 and 85.5% ± 5.5 respectively. The linear ranges for 50 mg/ml and 10 mg/ml dust were between 2 and 15.6 μg/g dust (R 2 = 0.99), and between 9.8 and 78 μg/g dust (R 2 = 0.99), respectively. When A. versicolor and A. ochraceus spores were both present, the response of the capture ELISA was not significantly reduced until a very large proportion of A. ochraceus spores were present. When 250 μg A. ochraceus spores per gram dust sample with half as much A. versicolor spores, the detection limit dropped by 50% (Fig. 4B). As the proportion of A. versicolor spores was increased below 125 μg, (i.e. 50%) there was no effect on the detection of A. versicolor (Fig. 4C).
Fig. 2. The cross-reactivity of pAb-Asp v 13 to 23 selected spores. Panel A, 100 μl selected spore extracts per well was coated directly in plate at 0.156 μg/ml for indirect ELISA; panel B, Asp v 13 capture ELISA response to selected spores and 3.906 ug/ml selected spore extracts were applied. 1:8000 biotinylated pAb-Asp v 13 was used as the primary and 1:16000 Strep-HRP used as the secondary.
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Fig. 3. Analysis of A. versicolor spore-spiked dust by Asp v 13 capture ELISA and demonstration of cross-reactivity. Panel A, A. versicolor spore-spiked dust sample. Diamond: spores only; square: spore-spiked 50 mg/ml dust and triangle: spore-spiked 10 mg/ml dust. Long arrow indicates quantification limit of 0.39 μg/ml with 50 mg/ml dust (p = 0.026 with Fisher's LSD model), while the short arrow indicates quantification limit of 0.098 μg/ml with 10 mg/ml dust (p = 0.000 with Fisher's LSD model). Panel B, the affect of A. versicolor spore detection by A. ochraceus spores. 25 (circle), 5(triangle), 1 (square) or 0 (diamond) μg/ml A. ochraceus spores were applied in the analysis.
4. Discussion Our previous studies using a multistep PCR protocol (Shi and Miller, 2011; Shi et al., 2011) had shown that the dominant allergen from A. versicolor is Asp v 13 (provisionally Avs41; Liang et al., 2011). The allergen is a 403 amino acid protein with multiple antigenic and allergenic epitopes on its surface (Shi and Miller, 2011). A BLAST search of the sequence of Asp v 13 indicated that it is a subtilisin-like alkaline serine protease similar to Asp f1 1 and other alkaline serine proteins from Aspergillus spp. and Penicillium species (Shi and Miller, 2011; Chou et al., 1999). After a goat was immunized with spores of A. versicolor, followed by two sequential boosts with the purified Asp v 13 protein, the response of the polyclonal antibody against Asp v 13 was enhanced. This is due to an increase of particular antibodies resulting from the spore immunization related to the target protein and those not boosted declined (Liang et al., 2011). This response was observed for other fungi (e.g. Wilson et al., 2009; Xu et al., 2008). The polyclonal antibody purified directly from a protein G column was not specific. However using pAb and biotinylated after one purification
Fig. 4. Combinative effect of both cross-reactive spores (A. ochraceus as the representative) and dust to the detection of A. versicolor spores in dust samples. Panel A, A. versicolor spore's assay in dust sample without A. ochraceus spores. Arrow showed the detection limit of 7.8125 μg spores per gram dust (p b 0.05). Panel B, A. versicolor spore assay in dust sample with 250 μg A. ochraceus spores per gram dust. Arrow shows the detection limit of 15.625 μg spores per gram dust (p b 0.05). The variant data and the notched plot box with 95% confidence were calculated by SYSTAT. Panel C, statistical analysis of the effect of 250 μg A. ochraceus spores to different A. versicolor spore's assay. P-values in each concentration with/without A. ochraceus calculated from SYSTAT were shown in the top of the related A. versicolor spore's concentration respectively.
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(step 1, Fig. 1A) as the capture and primary antibody respectively gave high background in the Asp v 13 capture ELISA. This resulted from the strong interaction between capture antibody and primary antibody that obscured the capture ELISA signal (data not shown). Although using pAb after antigen affinity purification (Fig. 1A, step 2) as the capture and primary antibody significantly decreased the noise, the sensitivity of the assay remained inadequate (data not shown) possibly from the leaching of biotinylated antigen from the streptavidin column and trace goat auto-antibodies. The specificity of the pAb to Asp v 13 was significantly improved by the antigen-affinity purification and decontamination steps (Fig. 1A & B). The Asp v 13 limit of quantification was ~ 1 ng Asp v 13 antigen per gram sieved dust (representing ~ 7.8 μg or ~ 7 × 10 4 spores per gram dust). This is more sensitive than our assay for S. chartarum (Xu et al., 2008), to A. fumigatus Asp f1 (Dillon, et al., 2007; Ryan et al., 2001), and is a much more sensitive a published assay for than A. alternata allergen Alt a1 (Salo et al., 2005). The panel that produced “Clearing the air” (NAS, 2000) the desirable method for assessing exposure to fungi must be based on measurement of allergens (antigens). Unlike serious cross-reactivity problem between allied species of Alt a1 polyclonal antibodies (Schmechel et al., 2008; Peters et al., 2008), pAb-Asp v 13 prepared by our method is specific with negligible cross-reactivity to the 21 tested fungi species tested. This included the closely related species such as A. niger, A. fumigatus, A. ochraceus, A. flavus and A. penicillioides. Only A. sydowii spores gave similar signal to A. versicolor. These are closely related taxa (Peterson, 2008) that often co-occur (Miller et al., 2008). As with the closely related S. chartarum and S. chlorohalonata (Xu et al., 2008), the Asp v 13 capture ELISA would detect both A. sydowii and A. versicolor if one or both were present. In summary, we report a new pAb protocol in this case for the allergen Asp v 13. This was achieved by using the enhanced pAb in a capture ELISA detection of A. versicolor spores in dust. The method is suitable for the measurement of the dominant A. versicolor allergen Asp v 13 in house dust. Acknowledgments This work was funded by an NSERC IRC to JDM and a MITACs award to Ms. Natacha Provost and JDM. We are grateful for the contributions of Cedarlane Laboratories, Ltd. who made the antibodies. References Abebe, M., Kumar, V., Rajan, S., Thaker, A., Sevinc, S., Vijay, H.M., 2006. Detection of recombinant Alt a1 in a two-site, IgM based, sandwich ELISA opens up possibilities of developing alternative assays for the allergen. J. Immunol. Methods 30 (312), 111. Chou, H., Lin, W.L., Tam, M.F., Wang, S.R., Han, S.H., Shen, H.D., 1999. Alkaline serine proteinase is a major allergen of Aspergillus flavus, a prevalent airborne Aspergillus species in the Taipei area. Int. Arch. Allergy Immunol. 119, 282.
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