The development of species-specific immunodiagnostics for Stachybotrys chartarum: The role of cross-reactivity

Journal of Immunological Methods 309 (2006) 150 – 159 www.elsevier.com/locate/jim

Research paper

The development of species-specific immunodiagnostics for Stachybotrys chartarum: The role of cross-reactivity Detlef Schmechel *, Janet P. Simpson, Donald Beezhold, Daniel M. Lewis Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, U.S. Department of Health and Human Services, 1095 Willowdale Road, M/S L-4020, Morgantown, WV 26505, USA Received 2 September 2005; received in revised form 21 November 2005; accepted 5 December 2005 Available online 3 January 2006

Abstract Mold contamination and exposure to fungi in indoor environments has been associated with various adverse health effects but little is known about the significance of individual fungal species in the initiation or exacerbation of such effects. Using Stachybotrys chartarum as a model fungus we sought to demonstrate that monoclonal antibodies (mAbs) can provide speciesspecific diagnostic reagents and also be used to investigate immunological cross-reactivity patterns among fungi. Mice were immunized with S. chartarum spore walls and monoclonal antibodies were screened against 60 fungal species and 24 different isolates of S. chartarum using an indirect ELISA. One species-specific mAb (IgG1) reacted only with spore preparations but not mycelium of S. chartarum or propagules of any other fungus. Five cross-reactive mAbs (IgM) documented extensive cross-reactivity among nine related Stachybotrys species and several non-related genera including several species of Cladosporium, Memnoniella, Myrothecium and Trichoderma. We also found that the ELISA reactivity for cross-reactive antigens and different isolates of S. chartarum differed considerably for normalized total amounts of mycelial antigen. We demonstrate that mAbs and immunoassays have the potential to detect S. chartarum species-specifically. The observed reactivity patterns with cross-reactive mAbs suggest that several fungi may share common antigens and that the majority of antigens are expressed by spores and mycelia. The observed cross-reactivity patterns need to be considered for accurate interpretations of environmental and serological analyses. D 2005 Elsevier B.V. All rights reserved. Keywords: Monoclonal antibodies; Stachybotrys chartarum; Fungi; Immunological cross-reactivity; ELISA

1. Introduction Fungi are ubiquitous in nearly every environment and over 600 species have been identified from indoor Abbreviations: CCB, carbonate coating buffer; ELISA, enzymelinked immunosorbent assay; mAb, monoclonal antibody; PBS, phosphate-buffered saline; PBST, PBS containing 0.05% Tween 20; PBSTM, PBST containing 1% non-fat dry milk powder; RT, room temperature; SIT, substrate incubation time. * Corresponding author. Tel.: +304 285 6024; fax: +304 285 6126. E-mail address: [email protected] (D. Schmechel). 0022-1759/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2005.12.001

environments in laboratory studies (Li and Yang, 2004). Exposure to fungi in domestic and occupational environments has been associated with adverse health effects such as infections, allergies, mycotoxicoses or irritations (Vijay and Kurup, 2004; Kuhn and Ghannoum, 2003; Bardana, 2003). A recent report by Li and Yang (2004) lists 112 fungal genera that have been reported to produce allergens and 46 that may produce mycotoxins. In co-exposures with other common environmental allergens, molds have also been suggested to enhance the risk of sensitization to other

D. Schmechel et al. / Journal of Immunological Methods 309 (2006) 150–159

common allergens leading to an increase in atopy (Savilahti et al., 2001). Although the allergenic potential of fungi has been well documented, the effects of mycotoxins or other fungal products on human health are much more controversial (Kelman et al., 2004; Chapman et al., 2003). Moreover, ambient environmental exposures are often ecologically complex and little is known about differential effects of individual fungi. A recent comprehensive literature review on dDamp Indoor Spaces and HealthT by the National Academy of Sciences’ Institute of Medicine (2004) identified the development of valid and standardized quantitative exposure-assessment methods as a high research priority. The report also emphasized the characterization of fungal populations in indoor environments to make progress in clarifying the relationship between damp and/or moldy environments and the manifestation of adverse health effects. Current exposure-assessment methods are based on sample cultivation or microscopic spore counts. Although these techniques may be very informative on a case-by-case basis, they are time-consuming, require expert taxonomical skills and have proven impossible to standardize. More recently, molecular techniques based on the polymerase chain reaction (Haugland et al., 2004; Vesper et al., 2004) have been successfully investigated as alternative methods for fungal identification and sample analysis. Our approach for the development of objective sample-assessment methods is based on the detection of fungi using monoclonal antibodies (Schmechel et al., 2005a,b; Green et al., 2005b; Schmechel et al., 2003). Immunoassays have become one of the most common techniques in diagnostic laboratories (Tetin and Stroupe, 2004) and their application in ecological and human exposure monitoring for numerous agents including indoor allergens is rapidly increasing (Andreotti et al., 2003; Van Emon, 2001; Lesnik, 2000; Chapman et al., 2000). They are also being developed for the detection of plant pathogenic fungi (Dewey and Meyer, 2004; Schmechel et al., 1997), food-borne fungi (Li et al., 2000) and environmental and food-borne mycotoxins (Schneider et al., 2004; Dietrich et al., 1995) including macrocyclic trichothecenes produced by Stachybotrys chartarum (Chung et al., 2003). MAb-based immunoassays have been shown to provide highly specific and rapid techniques with proven potential for standardization, automation and the development of on-site dpoint of needT testing formats. However, the clinical use of immunoassays for studying human exposures to fungi remains to be sci-

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entifically documented. Many key issues including sources of assay variability and the loss of assay specificity due to immunological cross-reactivity among fungi need to be resolved before immunoassays can be used on a routine basis for building management or exposure and health assessment studies (Trout et al., 2004). In the present study, we address some of the above research needs by demonstrating the successful application of mAb-based immunoassays for the speciesspecific identification of S. chartarum. We selected S. chartarum as a model fungus because of its toxic potential and its past association with several cases of acute idiopathic pulmonary hemorrhage in infants (Etzel, 2003; Dearborn et al., 2002). Furthermore, S. chartarum is considered to be an indicator fungus for moisture-damaged buildings (Rejula, 2004), and better detection methods for this fungus may help to rapidly identify such environments. We also describe isolate-dependent antigenic variability and cross-reactivity patterns of S. chartarum with other fungi commonly found in indoor environments, and we discuss the implications of biological and analytical variability on the development of immunometric environmental and serological monitoring assays for fungi. 2. Materials and methods 2.1. Culture of fungi and harvesting of spores and mycelium for mAb production and cross-reactivity analyses All fungi were either isolated from indoor air samples, received as gifts from colleagues or purchased from certified culture collections and maintained as sporulating slant cultures. The 24 isolates of S. chartarum were from different geographical regions (isolates # 1–2 from Hungary, isolates # 3–4 from US Virgin Islands, isolates # 5–10 from the continental USA, isolate # 11 from Canada, isolate # 12 from Hawaii, isolates # 13–14 were gifts from Dr. R. Haugland and isolates # 15–24 were gifts from Dr. K.S. Nielsen from Denmark). Fungi were grown in standard unsealed Petri plates on 5 ml of malt extract agar (2% dextrose, 0.1% peptone, 2% malt extract, 2% agar; Difco, Becton Dickinson, Sparks, MD). After 7 days of incubation at room temperature (RT), most culture plates were covered with spores and the agar was completely dried out. Spores were collected from sporulating cultures inside a class II biosafety cabinet (Baker Company, Sanford, ME) by applying 1 g of

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glass beads (0.45 – 0.5 mm in diameter, B. Braun Biotech International GmbH, Melsungen, Germany) per Petri plate. The lid was placed back on the plate and the plates were gently shaken back and forth. This allowed the beads to roll across the sporulating culture and the spores to attach to the beads. The beads were transferred into a 50-ml tube containing 20 ml phosphate-buffered saline (PBS, 10 mM phosphate-buffered saline, 138 mM NaCl, 2.7 mM KCl, pH 7.4; Sigma, St. Louis, MO) for immunogen preparation, carbonate coating buffer (CCB, 60 mM sodium carbonate, 140 mM sodium bicarbonate, pH 9.6) for mAb screening and cross-reactivity tests by enzymelinked immunosorbent assay (ELISA) or PBS containing 0.05% Tween 20 (PBST) for western blot analysis. Spores were suspended and separated from the beads by briefly shaking the tube and decanting the spore suspension. For mycelium production, sporulating slant cultures were washed with 2 ml sterile PBS and 100 Al aliquots were inoculated into flasks containing 200 ml of malt extract broth (2% dextrose, 0.1% peptone, 2% malt extract; Difco, Becton Dickinson, Sparks, MD). Flasks were incubated at RT and rotated at 200 rpm. Mycelium was aseptically harvested after 10–11 days using a fine nylon mesh and aliquots of the mycelium were stored at 20 8C. Thawed mycelium was suspended in 50 ml CCB and 15 ml aliquots were sonicated for 2 min (Sonicator 3000, Misonix, Inc., Farmingdale, NY). The sonicate was cleared by centrifugation at 3500g for 10 min and the supernatant was re-centrifuged at 40,000g for 20 min. Aliquots of the supernatant (extract) were stored at 20 8C until use and protein concentrations were determined according to the method of Lowry et al. (1951). All cross-reactivity tests were carried out with 10 Ag/ml mycelium diluted in CCB. 2.2. Preparation of spore walls for the production of mAbs Spores of S. chartarum were collected from isolate # 5 (classified as non-toxic isolate JS5105 by Jarvis et al., 1998) and isolate # 6 (classified as toxic isolate JS5802 by Jarvis et al., 1998) and equal numbers of spores of both isolates were mixed before being washed twice with PBS by centrifugation for 10 min at 500g. The concentration of the spore suspension was determined by hemocytometer count (Hausser Scientific, Horsham, PA). The final pellet was resuspended in 5 ml PBS and 1 ml aliquots were homogenized using a Mini-Bead Beater (Biospec Products, Bartlesville, OK) and 1 g of

glass beads per aliquot. Spores were homogenized three times for 1-min intervals and kept on ice for 2 min between treatments. Microscopic analysis showed that the resulting homogenate was a mixture of spore debris and intact spores as well as debris of conidiophores and phialides. Aliquots were combined and washed by centrifugation at 1500g for 10 min and the pellet representing the particulate fraction of the homogenate including spore wall fragments was suspended in PBS and used as immunogen. Four 10- to 14-week-old female BALB/c mice were immunized at 2- to 3-week intervals and mice were sacrificed by cervical dislocation 3 days after the final boost. Two mice were immunized 6 times and two mice were immunized a seventh time after being rested for 3 months following the sixth immunization. Mice were immunized intraperitoneally with 0.5 ml of the immunogen containing the spore wall preparations from 106 to 107 spores as determined by spore counts prior to spore homogenization. Hybridomas were produced using standard polyethyleneglycol-based cell fusion techniques (Harlow and Lane, 1988) using SP2/0-AG14 myelomas (ATCC# CRL-1581). Cell cultures were maintained in Dulbecco’s Modified Eagle Medium (Life Technologies, Rockville, MD), supplemented with 1 mM pyruvate, 100 units/ml penicillin, 100 Ag/ml streptomycin and 0.292 mg/ml l-glutamine, 100 AM sodium hypoxanthine, 16 AM thymidine and 10% fetal calf serum (HyClone, Logan, UT) and 100 units/ml IL-6 (Boehringer Mannheim, Germany). Culture supernatants from wells with cell growth were screened by ELISA (see below) and hybridomas from positive wells were cloned twice by limiting dilution. Stable hybridomas were bulk grown in culture plates and aliquots were stored in liquid nitrogen. The antibodies are patented and are available through the Technology Transfer Office of the Centers for Disease Control and Prevention (USA Patent Application No. 10/483,921, USA Publication No. US 2004/0185051 A1, International Patent Application No. PCT/US02/25493, International Publication No. WO 03/016352 A1) and hybridoma 9B4 has been deposited at the American Type Culture Collection (Patent Deposit Designation PTA-4582). Mice were housed under controlled environmental conditions in HEPA-filtered ventilated polycarbonate cages on autoclaved hardwood Beta-chip bedding and were provided feed (autoclaved Prolab 3500 rodent chow) and autoclaved tap water ad libitum. Sentinel mice were free of viral pathogens, parasites, mycoplasmas, and Helicobacter. The program of animal use was accredited by the Association for Assessment and Ac-

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creditation of Laboratory Animal Care International (AAALAC). 2.3. Enzyme-linked immunosorbent assay (ELISA) format for hybridoma screening and cross-reactivity tests Hybridoma screening and cross-reactivity tests were carried out using an alkaline phosphatase-mediated indirect ELISA. Spores of S. chartarum were used as antigen during hybridoma screening (1  104 to 5  104 spores per well) and cross-reactivity tests were carried out with mycelium (12 isolates of S. chartarum and 29 other fungal species) or spores (24 isolates of S. chartarum, 8 isolates of other Stachybotrys species and 52 other fungal species) of fungi commonly found in indoor environments. For the ELISA procedure, spores were collected into CCB, pH 9.6 and uniform spore suspensions were obtained by sonication for 10 s at amplitude 6 using a Sonicator 3000 (Misonix, Farmingdale, NY). PolySorp ELISA plate wells (Nalge Nunc International, Naperville, IL) were incubated for crossreactivity tests with 2  104 to 106 spores or 10 Ag/ml of mycelium overnight at RT and the plates were kept in a plastic box containing moist filter paper. This allowed soluble and secreted antigens to bind to the ELISA well surface and thus to be retained in the ELISA well until the completion of the assay. However, microscopic analysis showed that only small spores from species such as Aspergillus or Penicillium were retained in the ELISA wells until the completion of the ELISA. The retention of the spores, however, did not technically interfere with the spectrophotometric analysis at the end of the ELISA. Spores of genera with larger spores such as Alternaria, Epicoccum, Stemphylium or Stachybotrys were washed out during the washing steps and only their secreted or passively released and adsorbed antigens were retained in ELISA wells. Following overnight incubation and all subsequent ELISA steps, wells were washed 3 by incubating 200 Al of PBST (PBS containing 0.05% Tween 20) per well for 5-min intervals. The plates were blocked by incubating for 1 h at RT in 200 Al of PBST containing 1% non-fat dry milk powder (PBSTM). Hybridoma culture supernatants were incubated for 1 h at 37 8C with 100 Al of mAb culture supernatant diluted 1/5 in PBSTM. Bound antibodies were labeled with 100 Al of Biotin-SP-conjugated AffiPure goat anti-mouse IgG + IgM secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) by incubation for 1 h at 37 8C at a dilution of 1/5000 in PBSTM. Bound biotin was detected with 100 Al of alkaline phosphatase-conjugat-

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ed streptavidin (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) by incubation for 1 h at 37 8C at a dilution of 1/5000 in PBSTM. The extent of the reaction was revealed by incubating 100 Al per well of p-nitrophenyl phosphate-containing buffer [5 mg substrate in 10 ml of buffer (97 ml diethanolamine, 100 mg MgCl2 diluted in 1 l distilled water, pH 9.8)] at RT and the optical density (OD) was determined spectrophotometrically at 405 nm after a substrate incubation time (SIT) of 30 min using an UltraMicroplate Reader, Model ELx800 (BIO-TEK Instruments, Inc., Winooski, VT). The ODs of the negative controls which were processed in parallel by substituting plain culture supernatant for mAb culture supernatant ranged from 0 to 0.06 for different fungi and an OD of z 0.1 (negative + 3 standard deviations) was considered to be a positive result. 2.4. Western blot analysis of mAb reactivity Spores of S. chartarum were collected into PBST and vortexed for 5 min. The suspension was centrifuged and the supernatant was boiled for 5 min in Laemmli reducing sample buffer and proteins were separated by SDS–PAGE on a 15% gel and transferred to nitrocellulose. Sample lanes were treated in acetate buffer, pH 4.25 with or without 50 mM sodium periodate for 2 h at RT. The membrane was then washed in PBST for 30 MWM

1

2

3

–

+

–

4

97.4 66.2 45.0

31.0

21.5

14.5

Periodate

1D4

+ 9B4

Fig. 1. Antigen characterization by Western blot and periodate oxidation. Extracts of S. chartarum spores were separated on a 15% SDS–PAGE gel, blotted onto nitrocellulose and treated with (lanes 2 and 4) or without (lanes 1 and 3) 50 mM sodium periodate. The blots were then probed with mAb 9B4 (IgG1) or mAb 1D4 (IgM) and developed with BCIP/NBT substrate.

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Table 1 The reactivities of mAbs in ELISA with spore preparations of 60 fungal species and 24 isolates of Stachybotrys chartarum Fungal species

Spores/wella Optical density (OD) in ELISA at 405 nm after a substrate incubation time of 30 minb No mAbc

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55.

Alternaria alternata Aspergillus candidus Aspergillus chevalieri Aspergillus clavatus Aspergillus flavus Aspergillus fumigatus Aspergillus nidulans Aspergillus niger Aspergillus parasiticus Aspergillus penicillioides Aspergillus repens Aspergillus restrictus Aspergillus sydowii Aspergillus terreus Aspergillus umbrosus Aspergillus ustus Aspergillus versicolor Aureobasidium pullulans Cladosporium cladosporioides Cladosporium herbarum Cladosporium sphaerospermum Epicoccum nigrum Eurotium amstelodami Eurotium rubrum Fusarium moniliforme Fusarium oxysporum Fusarium solani Fusarium tricinctum Geotrichum candidum Memnoniella echinata Mucor ramannianus Myrothecium verrucaria Paecilomyces variotii Penicillium aurantiogriseum Penicillium brevicompactum Penicillium chrysogenum Penicillium citrinum Penicillium expansum Penicillium fellutanum Penicillium islandicum Penicillium jensenii Penicillium melinii Penicillium purpurogenum Penicillium roqueforti Penicillium simplicissimum Penicillium spinulosum Penicillium variabile Rhizopus stolonifer Scopulariopsis brumptii Trichoderma harzianum Ulocladium chartarum Wallemia sebi Stachybotrys chartarum, # 1 Stachybotrys chartarum, # 2 Stachybotrys chartarum, # 3

51,750 115,000 100,000 875,000 267,000 1,000,000 100,000 458,750 100,000 100,000 61,500 38,000 875,000 1,000,000 7750 100,000 715,500 961,250 371,250 807,500 60,500 5750 29,750 48,750 1,300,000 845,000 8750 34,000 220,000 262,500 26,750 40,750 535,000 100,000 1,000,000 633,333 1,045,000 1,000,000 100,000 521,250 100,000 100,000 306,000 466,250 100,000 100,000 1,000,000 481,250 47,500 1,000,000 31,500 205,250 71,750 33,000 62,000

0.01 0.01 0 0.01 0.01 0.03 0 0.03 0 0.01 0 0 0.02 0.01 0.01 0.01 0 0 0.02 0.06 0.01 0 0.01 0 0.01 0.01 0.01 0.02 0.02 0.01 0.01 0.01 0.05 0 0.01 0.01 0.01 0.01 0 0 0 0 0.01 0.01 0 0 0.01 0.05 0 0 0.03 0.01 0.01 0.01 0.02

mAb 1D4

mAb 3B2

0.02 0.02 0 0.01 0.01 0.02 0 0.06 0 0.01 0.02 0 0.03 0.02 0.02 0.01 0.03 0 0.01 0.04 0.01 0.02 0.01 0.01 0.08 0.18 0.02 0.04 0.01 3.25 0 0.2 0.08 0 0.01 0.06 0.01 0.02 0.01 0.02 0 0 0 0.08 0.01 0 0 0.03 0 0.82 0.08 0.01 2.57 2.73 2.7

0 0.02 0.01 0.01 0.01 0.06 0.01 0.03 0 0.01 0.02 0 0.02 0.01 0.01 0 0.01 0.29 1.19 2.1 0.45 0.01 0 0.02 0.02 0.03 0.03 0.01 0.03 1.74 0 1.16 0.05 0 0.03 0.02 0.01 0.01 0 0.01 0 0.03 0.01 0.05 0.01 0 0.01 0.02 0 0.01 0.03 0 1.2 1.3 0.78

mAb 4E12 0 0.04 0 0.02 0 0.02 0 0.07 0 0 0.01 0 0.03 0 0 0 0.03 0.1 0.38 1.26 0.11 0.01 0 0.01 0.01 0 0 0.02 0 0.48 0.01 0.66 0.03 0 0.02 0.02 0 0 0 0.02 0 0 0.02 0.02 0.01 0 0 0.06 0 0.01 0.03 0 0.31 0.46 0.19

mAb 9B4

mAb 9F9

0 0.01 0 0 0 0 0 0.01 0 0.01 0 0.01 0.01 0 0 0.01 0 0 0.01 0 0 0 0.01 0 0 0.01 0.01 0.01 0.02 0 0 0 0 0 0 0.01 0 0 0.01 0 0 0 0.01 0.01 0 0 0 0 0 0.01 0.01 0 1.23 0.79 0.72

0.01 0.09 0.01 0.04 0.01 0.04 0 0.14 0 0.01 0.01 0 0.08 0.01 0.01 0 0.05 0.15 0.47 1.58 0.14 0.01 0.01 0.02 0.03 0.01 0.02 0.05 0.03 0.66 0.01 0.69 0.1 0 0.01 0.03 0.01 0.01 0 0.02 0 0.01 0.05 0.03 0 0 0.01 0.14 0.01 0.02 0.1 0.01 0.42 0.59 0.27

mAb 10A5 0.01 0.03 0.01 0.01 0.01 0.07 0 0.03 0 0.01 0.01 0 0.03 0.01 0.01 0 0 0.24 1.05 1.88 0.36 0 0 0.01 0.01 0.01 0.01 0 0.02 1.71 0 1.23 0.03 0 0 0.03 0.01 0.01 0 0.01 0 0 0.01 0.04 0 0 0.01 0.02 0 0.01 0.01 0.01 1.02 1.22 0.75

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Table 1 (continued) Spores/wella Optical density (OD) in ELISA at 405 nm after a substrate incubation time of 30 minb

Fungal species

No mAbc 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84.

Stachybotrys Stachybotrys Stachybotrys Stachybotrys Stachybotrys Stachybotrys Stachybotrys Stachybotrys Stachybotrys Stachybotrys Stachybotrys Stachybotrys Stachybotrys Stachybotrys Stachybotrys Stachybotrys Stachybotrys Stachybotrys Stachybotrys Stachybotrys Stachybotrys Stachybotrys Stachybotrys Stachybotrys Stachybotrys Stachybotrys Stachybotrys Stachybotrys Stachybotrys

chartarum, # 4 chartarum, # 5d chartarum, # 6 chartarum, # 7 chartarum, # 8d chartarum, # 9 chartarum, # 10 chartarum, # 11d chartarum, # 12 chartarum, # 13 chartarum, # 14 chartarum, # 15e chartarum, # 16f chartarum, # 17e chartarum, # 18f chartarum, # 19e chartarum, # 20f chartarum, # 21e chartarum, # 22f chartarum, # 23f chartarum, # 24e albipes bisbyi cylindrospora echinata microspora nephrospora parvispora subsimplex

89,250 32,500 29,500 78,000 36,250 64,750 66,250 21,500 79,250 100,000 100,000 20,000 20,000 20,000 20,000 20,000 20,000 20,000 20,000 20,000 20,000 6500 6250 35,750 109,000 27,000 22,500 42,250 2000

0.02 0.01 0.06 0.02 0.04 0.03 0.02 0.03 0.01 0 0 0.01 0 0 0 0 0 0.01 0.01 0 0.01 0.02 0.02 0.01 0 0.03 0.01 0 0.02

mAb 1D4

mAb 3B2

3.27 3.32 2.88 2.87 2.72 2.98 2.97 3.05 2.75 2.79 2.57 2.7 1.79 1.76 2.37 2.69 2.44 2.5 1.76 0.91 1.6 3.24 3.23 2.26 2.54 3.42 2.51 1.67 2.63

1.33 2.07 1.03 1.14 0.88 1.89 1.34 1.2 0.91 0.85 0.83 n.t. n.t. n.t. n.t. n.t. n.t. n.t. n.t. n.t. n.t. 1.89 2.23 1.52 1.66 2.56 2.47 n.t. 1.76

mAb 4E12 0.31 0.71 0.35 0.23 0.27 0.63 0.44 0.26 0.2 0.11 0.14 n.t. n.t. n.t. n.t. n.t. n.t. n.t. n.t. n.t. n.t. 0.92 1.02 0.36 0.4 1.99 1.8 n.t. 0.78

mAb 9B4

mAb 9F9

1.58 1.65 0.83 0.91 0.33 1.48 0.65 0.75 0.86 0.515 1.075 1.63 1.0 1.03 1.6 1.61 1.6 1.76 1.21 0.56 0.96 0.02 0.03 0 0 0.04 0 0 0.02

0.48 0.8 0.45 0.34 0.39 0.79 0.54 0.39 0.31 0.17 0.19 n.t. n.t. n.t. n.t. n.t. n.t. n.t. n.t. n.t. n.t. 1.02 1.07 0.5 0.58 2.11 1.9 n.t. 0.72

mAb 10A5 1.4 1.87 1.04 0.99 1.02 2.0 1.29 3.05 0.83 0.82 0.76 n.t. n.t. n.t. n.t. n.t. n.t. n.t. n.t. n.t. n.t. 1.76 2.19 1.36 1.46 2.48 2.34 n.t. 1.52

a

Spore numbers differed according to the degree of in vitro sporulation of individual fungi. The results show the background-corrected average optical densities (ODs) of 4 ELISA well repeats, n.t. indicates dnot testedT. ODs z 0.1 are shown in bold and mAb reactivities specific for spores but not mycelium are also underlined. c dNo mAbT indicates blank-corrected assay background in which plain culture supernatant instead of mAb supernatant was processed in parallel with test samples for each fungus in ELISA. d Indicates non-toxin-producing strain. e Indicates satratoxin-producing strains. f Indicates atranone-producing strains. b

min and blocked with 3% non-fat dry milk and individual lanes were reacted overnight at RT with supernatant of mAb 9B4 (IgG1) or mAb 1D4 (IgM) diluted 1/5 in PBST. The blot was then reacted for 2 h at RT with alkaline phosphatase-conjugated anti-mouse secondary antibodies (Promega, Madison, WI) diluted 1/ 5000 in PBST and developed with BCIP/NBT substrate. Molecular markers (Bio-Rad, Hercules, CA) were run in parallel. 3. Results 3.1. Production and characterization of mAbs The immunization with the particulate fraction of homogenized spore preparations of S. chartarum

resulted in the production of one IgG1 isotype (mAb 9B4) and five IgM isotype antibodies (mAbs 1D4, 3B2, 4E12, 9F9, 10A5). All mAbs produced n isotype antibody light chains. Western blot analysis following SDS–PAGE of S. chartarum extracts (Fig. 1) indicates that mAb 9B4 recognizes a single 19–20 kDa antigen and that periodate oxidation of carbohydrate residues does not influence the binding of this antibody. In contrast, the cross-reactive mAb 1D4 shows multiple periodate-sensitive bands in the 30 to 100 kDa molecular weight range. This suggests that mAb 1D4 reacts with either identical or cross-reactive carbohydrate moieties of multiple glycoproteins with different molecular weights or it reacts with different isoforms or multimeric aggregates of the same glycoprotein.

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3.2. Cross-reactivity patterns of mAbs with 84 spore and 41 mycelial fungal isolates

antigen saturation for as many fungal species as possible and thus to increase the sensitivity of the assay, spores were used at their highest collectable concentration which ranged for most fungi from 2  104 to 106 spores/well. For some fungal species such as Epicoccum nigrum, Fusarium solani and some Stachybotrys species, comparatively lower spore numbers due to poor in vitro sporulation

In our analyses we did not standardize the amount of spores for different fungi because the number of spores to achieve antigen saturation of ELISA wells is unknown for different fungal species and may vary according to spore size. In order to approach

Table 2 The reactivities of mAbs in ELISA with mycelium of 29 fungal species and 12 isolates of S. chartarum Fungal species

Optical density (OD) in ELISA at 405 nm after a substrate incubation time of 30 mina,b No mAbc

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. a

Alternaria alternata Aspergillus candidus Aspergillus clavatus Aspergillus flavus Aspergillus fumigatus Aspergillus niger Aspergillus restrictus Aspergillus sydowii Aspergillus terreus Aspergillus versicolor Aureobasidium pullulans Cladosporium cladosporioides Epicoccum nigrum Eurotium amstelodami Fusarium tricinctum Memnoniella echinata Mucor ramannianus Myrothecium verrucaria Paecilomyces variotii Penicillium brevicompactum Penicillium chrysogenum Penicillium expansum Penicillium purpurogenum Penicillium roqueforti Penicillium variabile Rhizopus stolonifer Trichoderma harzianum Ulocladium chartarum Wallemia sebi Stachybotrys chartarum, # 1 Stachybotrys chartarum, # 2 Stachybotrys chartarum, # 3 Stachybotrys chartarum, # 4 Stachybotrys chartarum, # 5 Stachybotrys chartarum, # 6 Stachybotrys chartarum, # 7 Stachybotrys chartarum, # 8 Stachybotrys chartarum, # 9 Stachybotrys chartarum, # 10 Stachybotrys chartarum, # 11 Stachybotrys chartarum, # 12

0 0.01 0.01 0.01 0.01 0 0 0.01 0 0 0.01 0.01 0 0.01 0 0 0.02 0.01 0.01 0 0 0.01 0.01 0.01 0.01 0 0 0 0.01 0 0.01 0 0.01 0 0.01 0 0 0.01 0.01 0 0.01

mAb 1D4 0.01 0.01 0 0 0.01 0.01 0 0.03 0 0.01 0 0 0.01 0.01 0 1.08 0.01 0 0 0.01 0.02 0.05 0.05 0.01 0 0 1.13 0 0.01 2.67 2.56 2.63 3.53 2.01 2.33 3.2 3.05 2.22 2.54 2.04 2.2

mAb 3B2 0.02 0.02 0.01 0.01 0.03 0.03 0.02 0.05 0.02 0.02 0.01 2.11 0.01 0.03 0.03 0.73 0 0.54 0.01 0.05 0.02 0.03 0.04 0.03 0 0.02 0.01 0 0.02 2.02 2.04 1.79 2.66 1.01 1.05 2.66 2.04 1.82 1.05 1.41 1.51

mAb 4E12 0 0.01 0 0 0.01 0.01 0 0 0 0 0 1.14 0 0.01 0 0.13 0.01 0.01 0 0 0.01 0.03 0.02 0.01 0.01 0.01 0 0 0 0.73 0.71 0.43 1.34 0.14 0.24 0.84 0.67 0.73 0.15 0.51 0.47

mAb 9B4

mAb 9F9

0 0 0.01 0 0.01 0 0 0.01 0 0 0 0 0 0 0 0 0.01 0 0 0.02 0 0.01 0.02 0 0 0 0 0 0 0 0 0 0 0.01 0 0 0.01 0 0 0 0.01

0 0.02 0 0 0.01 0.03 0 0.01 0 0 0 1.13 0 0 0.01 0.19 0.01 0.01 0 0.02 0.02 0.04 0.03 0 0.01 0.01 0 0 0.01 0.9 1.09 0.57 1.52 0.21 0.31 1.09 0.86 1.03 0.21 0.6 0.63

mAb 10A5 0.01 0.02 0 0.01 0.03 0.02 0.02 0.02 0.01 0.01 0.03 1.72 0.01 0.03 0.02 0.62 0 0.3 0.01 0.03 0.02 0.02 0.01 0.02 0 0.01 0 0.01 0.02 1.79 1.93 1.43 2.45 0.88 0.94 2.38 2.3 1.64 1.11 1.23 1.4

Mycelium of S. chartarum isolate #6 was tested at 50 Ag/ml and all other mycelia were tested at 10 Ag/ml. The results show the background-corrected average optical densities (ODs) of 4 ELISA well repeats. ODs z 0.1 are in bold. c dNo mAbT indicates blank-corrected assay background in which plain culture supernatant instead of mAb supernatant was processed in parallel with test samples for each fungus in ELISA. b

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may have resulted in antigen concentrations below the amount of antigen saturation. Table 1 compares the ELISA reactivity of all mAbs against spores of 24 different isolates of S. chartarum and 60 fungal species including 8 other species of the genus Stachybotrys. Table 2 compares the ELISA reactivity of all mAbs against mycelia of 12 isolates of S. chartarum and 29 fungal species commonly found in samples from indoor environments. All mAbs, except mAb 9B4, cross-reacted with spores and mycelia from several fungi including all isolates of S. chartarum and all other species of the genus Stachybotrys. MAb 9B4 reacted with all 24 spore isolates but did not react with any of 12 mycelial isolates of S. chartarum. MAb 1D4 strongly cross-reacted with spores and mycelium of Memnoniella echinata and Trichoderma harzianum; it also weakly cross-reacted with spores (mycelium not tested) of Fusarium oxysporum and spores but not mycelium of Myrothecium verrucaria. MAbs 3B2 and 10A5 strongly cross-reacted with spores of Cladosporium herbarum and Cladosporium sphaerospermum and spores and mycelium from Cladosporium cladosporioides, M. echinata and M. verrucaria. Both mAb 3B2 and mAb 10A5 also weakly cross-reacted with spores but not mycelium of Aureobasidium pullulans. MAbs 4E12 and 9F9 strongly cross-reacted with spores and mycelium of C. cladosporioides and M. echinata and spores of C. herbarum (mycelium not tested). Both mAbs also strongly cross-reacted with spores but not mycelium of M. verrucaria. MAb 9F9 also weakly cross-reacted with spores but not mycelium of Aspergillus niger and Rhizopus stolonifer. Table 1 also shows that none of the mAbs discriminated between toxic or non-toxic isolates and atranone- or satratoxin-producing chemotypes of S. chartarum, respectively. The quantitative reactivity of all mAbs with normalized amounts of total mycelial antigens (Table 2, 10 Ag/ ml) varied for different antibodies and depended on individual fungal species or isolates of S. chartarum. For example the ODs of mAb 1D4 ranged from 1.08 for M. echinata and 1.13 for T. harzianum to 3.53 for S. chartarum isolate # 4. This may indicate that different fungi express different amounts per unit biomass of the antibody-binding site for mAb 1D4 or that the binding sites in different fungi have differential affinities for mAb 1D4. 4. Discussion Our results demonstrate for the first time that species-specific immunoassays for the detection of fungi are feasible. Using S. chartarum as a model

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fungus we demonstrate that a species-specific monoclonal antibody (mAb 9B4) is capable of differentiating between spore preparations of the target fungus and spores and mycelia of 60 other fungal species including some of the most prevalent fungi found in indoor environments. To the best of our knowledge, this is the most comprehensive study of immunological cross-reactivity among fungi based on monoclonal antibodies. Initial antigen characterization suggests that mAb 9B4 reacts with a 19- to 20-kDa protein which is only expressed in spore preparations but not mycelium of S. chartarum. Most mAbs (5 out of 6) were found to widely crossreact with all species of the genus Stachybotrys and with other fungi such as several Cladosporium, Memnoniella and Myrothecium species. The results confirm earlier studies with mAbs raised against Penicillium brevicompactum (Schmechel et al., 2003) or Aspergillus versicolor (Schmechel et al., 2005a,b) that also showed extensive cross-reactivity with related Aspergillus and Penicillium species and several mAbs even cross-reacted with multiple Cladosporium or Stachybotrys species. The observed cross-reactivity patterns in these and other studies (Bisht et al., 2002; Baldo, 1995; Tee et al., 1987) demonstrate the phylogenetic sharing of common antigenic determinants especially with related fungi but also, to a lesser extent, with non-related fungi. Clinicians, researchers and personnel in diagnostic laboratories need to consider cross-reactivity among fungi when interpreting the results of immunoassays of environmental samples or the reactivity of patient’s serum with a panel of commonly available fungal extracts. The simple detection of IgG antibodies to such extracts may not be indicative of any cause-andeffect relationships since the antibodies may be due to exposures to cross-reactive fungi. For example the high percentage of healthy individuals who presented with stachybotrys-reactive IgG antibodies (Barnes et al., 2002) or the lack of IgG serum antibody reactivity to differentiate between moisture-damaged and control buildings (Patovirta et al., 2003; Immonen et al., 2002) should be viewed with great caution because of the impact of unknown immunological cross-reactivities. The results of such assays should also be referred to as dfungus-reactiveT rather than dfungus-specificT in order to avoid unfounded assumptions of any causative relationships. Unless species-specific antigens can be identified and used to analyze serum reactivity, it will always be difficult to definitively establish cause-and-effect relationships in fungal exposures since even inhibition

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assays may not always unambiguously identify the primary source of antibody production or differentiate between poly-sensitization and true cross-reactivity. In order to ensure the accuracy and validity of fungal immunoassays, we would recommend the application of species-specific antibodies to avoid a biased interpretation of results. For example, the application of the cross-reactive mAb 1D4 in environmental immunoassays would not allow one to differentiate S. chartarum from M. echinata, T. harzianum or other Stachybotrys sp. The species-specific mAb 9B4, on the other hand, avoids such analytical ambiguities and can provide a useful tool for environmental immunodiagnostics. The occurrence of Stachybotrys species other than S. chartarum such as Stachybotrys microspora and Stachybotrys nephrospora has recently been reported from indoor environments in the USA by Li and Yang (2004) and further emphasizes the need for speciesspecific assays to maintain the accuracy of monitoring results for mixed species contaminations and possible exposures. Our results also demonstrate antigenic differences between spores and mycelium. We found that several fungi expressed spore-specific antigenic determinants while other fungi expressed the same determinant in both spores and mycelium. For example, the speciesspecific mAb 9B4 only reacted with spore preparations but not mycelium of S. chartarum and mAbs 4E12 and 9F9 only react with spores of M. verrucaria but with both spores and mycelium of C. cladosporioides, M. echinata and all Stachybotrys species. This suggests that the expression of some antigens is not a general spore or mycelium phenomenon but rather occurs in a species-dependent manner. The differential reactivities of spores and mycelium suggest that fungal extracts for clinical and analytical purposes need to be prepared from source material containing both spores and mycelium in order to ensure the efficacy and validity of the tests. Current work is aimed at the production of additional mAbs against the purified antigen of mAb 9B4. This will allow the development of a mAb-based sandwich assay for environmental sample analysis. Future research will focus on investigating the magnitude and impact of inter- and intra-species antigenic variability on immunoassay performance to enhance the reproducibility and predictive value of quantitative assays. For example, if spores and mycelium express different specific numbers of antibody binding sites per unit biomass, it becomes methodologically very important to select calibrator reagents for quantitative assays that were prepared from similar ratios of spores and myce-

lium in order to obtain valid estimates of fungal contaminations in target environments. Since different environments can be expected to contain different isolates and assays performed in different laboratories may be based on standard curves prepared from different isolates with different ratios of spores and mycelium, knowledge on antigenic variability will be fundamental to ensure progress in the development of reproducible immunoassays. In conclusion, the widespread cross-reactivity among fungi and the observed biological variability are considered to be major challenges for the successful implementation of accurate and quantitative immunoassays. We feel that the development of speciesspecific mAbs, perhaps even spore or mycelium-specific mAbs, will be critical and that standardization of immunoassay methodology and sample processing will be essential before immunoassays can provide a reliable and reproducible alternative approach to current sample analysis techniques. The recent development of a novel double immunostaining technique for fungi (Green et al., 2005b,c) based on the Halogen Immunoassay (Green et al., 2005a,c) that co-identifies fungal aerosols with mAbs and fungal sensitization with patient’s serum IgE in the same sample clearly demonstrates the diagnostic potential of mAb-based immunoassays as an analytical tool for exposure assessment and environmental health studies. Acknowledgements The authors would like to thank Dr. Richard A. Haugland from the Environmental Protection Agency, USA, and Dr. Kristian F. Nielsen from the Technical University of Denmark for providing several isolates of S. chartarum; Dr. David M. Geiser from the Pennsylvania State University, USA, for providing several Fusarium isolates and Dr. Elliott Horner from Air Quality Sciences, USA, for providing several Aspergillus and Penicillium isolates. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the National Institute of Occupational Safety and Health. References Andreotti, P.E., Ludwig, G.V., Peruski, A.H., Tuite, J.J., Morse, S.S., Peruski, L.F., 2003. Immunoassays of infectious agents. BioTechniques 35, 850. Baldo, B.A., 1995. Allergenic cross-reactivity of fungi with emphasis on yeast: strategies for further study. Clin. Exp. Allergy 25, 488.

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