Quantification of C. globosum spores in house dust samples
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Department of Chemistry, Carleton University, Canada
David Miller   

Department of Chemistry, Carleton University, Canada
Ann Agric Environ Med. 2014;21(3):525–530
Chaetomium globosum is one of the most common fungi that grows in damp buildings and occurs in agricultural and forestry workplaces. Using sera from atopic patients, we characterized and purified an extracellular chitosanase (Chg47) from C. globosum that is antigenic to humans. The study reports the production of monoclonal antibodies to the protein. Three capture ELISAs were developed for Chg47 for detection of spores and spore and mycelial fragments in dust samples using different mono- and polyclonal antibody combinations. One method is based on an enhanced biotinylated polyclonal antibody as the secondary antibody and coating anti-IgM to capture one of two clones of IgM monoclonal antibodies as the capture antibody. The other method makes use of an enhanced rabbit polyclonal antibody as both the primary and capture antibody. The detection limit of the double PAb method for the Chg47 antigen was 7.6 pg/ml. When the anti-IgM+10B3 clone was used, the detection limit was 61 pg/ml and for anti-IgM+5F12, 122 pg/ml. The detection limit of double PAb method is comparable to methods for the allergen and spores of Aspergillus versicolor in house dust and is more sensitive than other immunoassays for allergens in house including for Stachybotrys chartarum, Aspergillus fumigatus and Alternaria alternata. All three methods had limited cross-reactivity to fungi common in house dust representing a diverse array of taxa.
Miller JD, Rand TG, McGregor H, Solomon J, Yang C. Mold ecology: recovery of fungi from certain moldy building materials. In: Prezant B, Weekes D, Miller JD (eds.). Recognition, Evaluation and Control of Indoor Mold.. American Industrial Hygiene Association (Fairfax, VA), 2008.p.43–51.
Xu J, Jensen JT, Liang Y, Belisle D, Miller JD. The biology and immunogenicity of a 34 kDa antigen of Stachybotrys chartarum sensu lato. Int Biodeterior Biodegrad. 2007; 60(4): 308–318.
Shi C, Smith ML, Miller JD. Characterization of human antigenic proteins SchS21 and SchS34 from Stachybotrys chartarum. Int Arch Allergy Immunol 2011; 155(1): 74–85.
Luo W, Wilson AM, Miller JD. Characterization of a 52 kDa exoantigen of Penicllium chrysogenum and monoclonal antibodies suitable for its detection. Mycopathologia 2010; 169(1): 15–26.
Liang Y, Zhao W, Xu J, Miller JD. Characterization of two related exoantigens from Aspergillus versicolor isolated using sera from atopic patients. Int Biodeterior Biodegrad. 2011; 65(1):217–226.
Shi C, Miller JD. Characterization of the 41 kDa allergen Asp v 13, a subtilisin-like serine protease from Aspergillus versicolor. Molecular Immunol. 2011; 48(15–16): 1827–1834.
Provost NB, Shi C, She, Y-M, Cyr TD, Miller JD. Characterization of an antigenic chitosanase from the cellulolytic fungus Chaetomium globosum. Medical Mycology 2013; 51(3):290-299.
Xu J, LiangY, Belisle D, Miller JD. Characterization of monoclonal antibodies to an antigenic protein from Stachybotrys chartarum and its measurement in house dust. J Immunol Methods. 2008; 332(1–2): 121–128.
Shi C, Belisle D, Levac S, Miller JD. The development of an assay for the quantification of the P. chrysogenum allergen Pch 52. J Occup Environ Hygiene. 2012; 9(4): 211–216.
Shi C, Belisle D, Miller JD. Quantification of the Aspergillus versicolor allergen in house dust. J Immunol Methods. 2011: 372(1–2): 89–94.
Andersen B, Frisvad JC, Søndergaard I, Rasmussen IS, Larsen LS. Associations between fungal species and water-damaged building materials. Appl Environ Microbiol. 2011; 77(12): 4180–4088.
Flannigan B, Miller JD Microbial growth in indoor environments. In: Flannigan B, Samson RA, Miller JD (eds.). Microorganisms in home and indoor work environments: Diversity, Health Impacts, Investigation and Control, second edition. Taylor & Francis, New York, 2011.pp. 57- 107.
Hoover-Litty H, Hanlin RT. The mycoflora of wood chips to be used as mulch. Mycologia. 1985; 77(5): 721–731.
Patovirta RL, Reiman M, Husman T, Haverinen U, Toivola M, Nevalainen A Mould specific IgG antibodies connected with sinusitis in teachers of mould damaged school: a two-year follow-up study. Int J Occup Med Environ Health. 2003; 16(3): 221–230.
Niedoszytko M, Chełmińska M, Jassem E, Czestochowska E. Association between sensitization to Aureobasidium pullulans (Pullularia sp.) and severity of asthma. Ann Allergy Asthma Immunol. 2007; 98(2): 153–156.
Beezhold DH, Green BJ, Blachere FM, Schmechel D, Weissman DN, Velickoff D, Hogan MB, Wilson NW. Prevalence of allergic sensitization to indoor fungi in West Virginia. Allergy Asthma Proc. 2008; 29(1): 29–34.
Provost NB. Isolation, purification and characterization of proteins from indoor strains of Chaetomium globosum thatare antigenic to humans. MSc Thesis, Carleton University (Ottawa, ON, Canada), 2010.
Wilson AM, Luo W, Miller JD. 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 2009; 168(5): 213–226.
Salares VR, Hinde CA, Miller JD. Analysis of settled dust in homes and fungal glucan in air particulate collected during HEPA vacuuming. Indoor Built Environment. 2009; 18(6): 485–491.
WHO. Guidelines for Indoor Air Quality: Dampness and Mould. World Health Organization, Regional Office for Europe, DK-2100, Copenhagen, Denmark, 2009.
Health Canada. Fungal Contamination in Public Buildings: Health Effects and Investigation Methods (Ottawa, ON, Canada), 2004.
National Academy of Science. Clearing the Air. Asthma and Indoor Air exposures. National Academy Press (Washington, DC), 2000.
Abebe M, Kumar V, Rajan S, Thaker A, Sevinc S, Vijay HM. 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. 2006: 312(1–2): 111–117.
Matthew WD, Reichardt LF. Development and application of an efficient procedure for converting mouse IgM into small, active fragments. J Immunol Methods. 1982; 50(3): 239–253.
Gautam S, Loh KC. Immunoglobulin-M purification – Challenges and perspectives. Biotechnol Adv. 2011; 29(6): 840–849.
Gagnon P. Monoclonal antibody purification with hydroxyapatite. Nature Biotechnol. 2009: 25(5): 287–293.
Mukherjee S, Casadevall A. Sensitivity of sandwich enzyme-linked Immunosorbent assay for Cryptococcus neoformans polysaccharide antigen is dependent on the isotypes of the capture and detection antibodies. J Clin Microbiol. 1995; 33(3): 765–768.
Panichakul T, Chawengkirttikul R, Chaiyaroj SC, Sirisinha S. 2002. Development of a monoclonal antibody-based enzyme-linked immunosorbent assay for the diagnosis of Penicillium marneffei infection. Am J Trop Med Hyg. 2002; 67(4): 443–447.
Rand TG, Miller JD. Immunohistochemical and immunocytochemical detection of SchS34 antigen in Stachybotrys chartarum spores and spore impacted mouse lungs. Mycopathologia 2008; 165(2): 73–80.
Dillon HK, Boling DK, Miller JD. Comparison of detection methods for Aspergillus fumigatus in environmental air samples in an occupational environment. J. Occup Environ Hygiene. 2007; 4(7): 509–513.
Ryan TJ, Whitehead LW, Connor TH, Burau KD. Survey of the Asp f 1 allergen in office environments. Appl Occup Environ Hyg. 2001; 16(6): 679–684.
Salo PM, Yin M, Arbes SJ, Jr, Cohn RD, Sever M, Muilenberg M, Burge HA, London SJ, Zeldin DC. Dustborne Alternaria alternata antigens in US homes: results from the National Survey of Lead and Allergens in Housing. J Allergy Clin Immunol 2005; 116(3): 623–629.
Peters JL, Muilenberg ML, Rogers CA, Burge HA, Spengler JD. Alternaria measures in inner-city, low-income housing by immunoassay and culture-based analysis. Ann Allergy Asthma Immunol. 2008; 100(4): 364–369.
Schmechel D, Green BJ, Blachere FM, Janotka E, Beezhold DH. Analytical bias of cross-reactive polyclonal antibodies for environmental immunoassays of Alternaria alternata. J Allergy Clin Immunol. 2008; 121(3): 763–768.