A multi-center ring trial of allergen analysis using fluorescent multiplex array technology.

BACKGROUND: Consistent performance of allergen assays is essential to ensure reproducibility of exposure assessments for investigations of asthma and occupational allergic disease. This study evaluated intra- and inter-laboratory reproducibility of a fluorescent multiplex array, which simultaneously measures eight indoor allergens in a single reaction well. METHODS: A multi-center study was performed in nine laboratories in the US and Europe to determine the inter-laboratory variability of an 8-plex array for dust mite, cat, dog, rat, mouse and cockroach allergens. Aliquots of 151 dust extract samples were sent to participating centers and analyzed by each laboratory on three separate occasions. Agreement within and between laboratories was calculated by the concordance correlation coefficient (CCC). RESULTS: Results were obtained for over 32,000 individual allergen measurements. Levels covered a wide range for all allergens from below the lower limit of detection (LLOD = 0.1-9.8 ng/ml) to higher than 6800 ng/ml for all allergens except Mus m 1, which was up to 1700 ng/ml. Results were reproducible within as well as between laboratories. Within laboratories, 94% of CCC were ≥ 0.90, and 80% of intra-laboratory results fell within a 10% coefficient of variance (CV%). Results between laboratories also showed highly significant positive correlations for all allergens (~0.95, p<0.001). Overall means of results were comparable, and inter-laboratory CV% for all allergens except Rat n 1 ranged between 17.6% and 26.6%. CONCLUSION: The data indicate that performance criteria for fluorescent multiplex array technology are reproducible within and between laboratories. Multiplex technology provides standardized and consistent allergen measurements that will streamline environmental exposure assessments in allergic disease.

Production, characterization and utility of a panel of monoclonal antibodies for the detection of toluene diisocyanate haptenated proteins.

Diisocyanates (dNCOs) are highly reactive low molecular weight chemicals used in the manufacture of polyurethane products and are the most commonly reported cause of occupational asthma. Mechanistic disease studies and development of biomonitoring and research tools, such as monoclonal antibodies (mAbs) have been hampered by dNCOs' ability to self-polymerize and to cross-link biomolecules. Toluene diisocyanate (TDI)-specific monoclonal antibodies (mAbs), with potential use in immunoassays for exposure and biomarker assessments, were produced and reactivities characterized against mono- and diisocyanate and dithioisocyanate protein conjugates. In general, TDI reactive mAbs displayed stronger recognition of isocyanate haptenated proteins when the NCO was in the ortho position relative to the tolyl group, and were capable of discriminating between isocyanate and isothiocyanate conjugates and between aromatic and aliphatic dNCOs. Preliminary studies using TDI vapor exposed cells suggest potential utility of these mAbs for both research and biomonitoring.

Monoclonal antibodies to hyphal exoantigens derived from the opportunistic pathogen Aspergillus terreus.

Aspergillus terreus has been difficult to identify in cases of aspergillosis, and clinical identification has been restricted to the broad identification of aspergillosis lesions in affected organs or the detection of fungal carbohydrates. As a result, there is a clinical need to identify species-specific biomarkers that can be used to detect invasive A. terreus disease. Monoclonal antibodies (MAbs) were developed to a partially purified preparation of cytolytic hyphal exoantigens (HEA) derived from A. terreus culture supernatant (CSN). Twenty-three IgG1 isotype murine MAbs were developed and tested for cross-reactivity against hyphal extracts of 54 fungal species. Sixteen MAbs were shown to be specific for A. terreus. HEA were detected in conidia, hyphae, and in CSN of A. terreus. HEA were expressed in high levels in the hyphae during early stages of A. terreus growth at 37°C, whereas at room temperature the expression of HEA peaked by days 4 to 5. Expression kinetics of HEA in CSN showed a lag, with peak levels at later time points at room temperature and 37°C than in hyphal extracts. Serum spiking experiments demonstrated that human serum components do not inhibit detection of the HEA epitopes by MAb enzyme-linked immunosorbent assay (ELISA). Immunoprecipitation and proteomic analysis demonstrated that MAbs 13E11 and 12C4 immunoprecipitated a putative uncharacterized leucine aminopeptidase (Q0CAZ7), while MAb 19B2 recognized a putative dipeptidyl-peptidase V (DPP5). Studies using confocal laser scanning microscopy showed that the uncharacterized leucine aminopeptidase mostly localized to extracellular matrix structures while dipeptidyl-peptidase V was mostly confined to the cytoplasm.

Production and characterization of IgM monoclonal antibodies against hyphal antigens of Stachybotrys species.

Stachybotrys is a hydrophilic fungal genus that is well known for its ability to colonize water-damaged building materials in indoor environments. Personal exposure to Stachybotrys chartarum allergens, mycotoxins, cytolytic peptides, and other immunostimulatory macromolecules has been proposed to exacerbate respiratory morbidity. To date, advances in Stachybotrys detection have focused on the identification of unique biomarkers that can be detected in human serum; however, the availability of immunodiagnostic reagents to Stachybotrys species have been limited. In this study, we report the initial characterization of monoclonal antibodies (MAbs) against a semi-purified cytolytic S. chlorohalonata preparation (cScp) derived from hyphae. BALB/c mice were immunized with cScp and hybridomas were screened against the cScp using an antigen-mediated indirect ELISA. Eight immunoglobulin M MAbs were produced and four were specifically identified in the capture ELISA to react with the cScp. Cross-reactivity of the MAbs was tested against crude hyphal extracts derived from 15 Stachybotrys isolates representing nine Stachybotrys species as well as 39 other environmentally abundant fungi using a capture ELISA. MAb reactivity to spore and hyphal antigens was also tested by a capture ELISA and by fluorescent halogen immunoassay (fHIA). ELISA analysis demonstrated that all MAbs strongly reacted with extracts of S. chartarum but not with extracts of 39 other fungi. However, four MAbs showed cross-reactivity to the phylogenetically related genus Memnoniella. fHIA analysis confirmed that greatest MAb reactivity was ultrastructurally localized in hyphae and phialides. The results of this study further demonstrate the feasibility of specific MAb-based immunoassays for the detection of S. chartarum.

Characterization of recombinant terrelysin, a hemolysin of Aspergillus terreus.

Fungal hemolysins are potential virulence factors. Some fungal hemolysins belong to the aegerolysin protein family that includes cytolysins capable of lysing erythrocytes and other cells. Here, we describe a hemolysin from Aspergillus terreus called terrelysin. We used the genome sequence database to identify the terrelysin sequence based on homology with other known aegerolysins. Aspergillus terreus mRNA was isolated, transcribed to cDNA and the open reading frame for terrelysin amplified by PCR using specific primers. Using the pASK-IBA6 cloning vector, we produced recombinant terrelysin (rTerrelysin) as a fusion product in Escherichia coli. The recombinant protein was purified and using MALDI-TOF MS determined to have a mass of 16,428 Da. Circular dichroism analysis suggests the secondary structure of the protein to be predominantly ß-sheet. Results from thermal denaturation of rTerrelysin show that the protein maintained the ß-sheet confirmation up to 65°C. Polyclonal antibody to rTerrelysin recognized a protein of approximately 16.5 kDa in mycelial extracts from A. terreus.

Monoclonal antibodies against toluene diisocyanate haptenated proteins from vapor-exposed mice.

Toluene diisocyanate (TDI) is an industrially important polymer cross-linker used in the production of polyurethane. Workplace exposure to TDI and other diisocyanates is reported to be a leading cause of low molecular weight-induced occupational asthma (OA). Currently we have a limited understanding of the pathogenesis of OA. Monoclonal antibodies (MAbs) that recognize TDI bound proteins would be valuable tools/reagents, both in exposure monitoring and in TDI-induced asthma research. We sought to develop toluene diisocyanate (TDI)-specific MAbs for potential use in the development of standardized immunoassays for exposure and biomarker assessments. Mice were exposed 4 h/day for 12 consecutive weekdays to 50 ppb, 2,4;2,6 TDI vapor (80/20 mixture). Splenocytes were isolated 24 h after the last exposure for hybridoma production. Hybridomas were screened in a solid-phase indirect enzyme-linked immunosorbent assay (ELISA) against a 2,4 TDI-human serum albumin (2,4 TDI-HSA) protein conjugate. Three hybridomas producing 2,4 TDI-HSA reactive IgM MAbs were obtained. The properties of these MAbs (isotype and reactivity to various protein-isocyanate conjugate epitopes) were characterized using ELISA, dot blot, and Western blot analyses. Western blot analyses demonstrated that some TDI conjugates form inter- and intra-molecular links, resulting in multimers and a change in the electrophoretic mobility of the conjugate. These antibodies may be useful tools for the isolation of endogenous diisocyanate-modified proteins after natural or experimental exposures and for characterization of the toxicity of specific dNCOs.

Murine models of airway fungal exposure and allergic sensitization.

Inhalation of common indoor filamentous fungi has been associated with the induction or exacerbation of allergic respiratory disease. The understanding of fungal inhalation and allergic sensitization has significantly advanced with the use of small animal models, especially mouse models. Numerous studies have employed different animal exposure and sensitization techniques, each with inherent advantages and disadvantages that are addressed in this review. In addition, most studies involve exposure of animals to fungal spores or spore extracts while neglecting the influence of hyphal or subcellular fragment exposures. Recent literature examining the potential for hyphae and fungal fragments to induce or exacerbate allergy is discussed. Innate immune recognition of fungal elements and their contribution to lung allergic inflammation in animal models are also reviewed. Though physical properties of fungi play an important role following exposure, host immune development is also critical in airway inflammation and allergy. We discuss the importance of environmental factors that influence early immune development and subsequent susceptibility to allergy. Murine studies that examine the role of intestinal microflora and prenatal or early life environmental factors that promote allergic sensitization are also evaluated. Future studies will require animal models that accurately reflect natural fungal exposures and identify environmental factors that influence immune development and thus promote respiratory fungal allergy and disease.

Discrimination of Penicillium isolates by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry fingerprinting.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to generate highly reproducible mass spectral 'fingerprints' for twelve Penicillium species. Prior to MALDI-TOF MS analysis, eight replicate cultures of each Penicillium species were subjected to three one-minute bead-beating cycles in an acetonitrile/trifluoroacetic acid solvent. The mass spectra contained abundant peaks in the range of m/z 5000-20 000, and allowed unambiguous discrimination between species. In addition, a biomarker common to all Penicillium mass spectra was observed at m/z 13 900. Discriminant analysis using the MALDI-TOF MS data yielded classification error rates of 0% (i.e. 100% correct identification), indicating that MALDI-TOF MS data may be a useful diagnostic tool for the objective identification of Penicillium species of environmental and clinical importance.

Discrimination of Aspergillus isolates at the species and strain level by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry fingerprinting.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to generate highly reproducible mass spectral fingerprints for 12 species of fungi of the genus Aspergillus and 5 different strains of Aspergillus flavus. Prior to MALDI-TOF MS analysis, the fungi were subjected to three 1-min bead beating cycles in an acetonitrile/trifluoroacetic acid solvent. The mass spectra contain abundant peaks in the range of 5 to 20kDa and may be used to discriminate between species unambiguously. A discriminant analysis using all peaks from the MALDI-TOF MS data yielded error rates for classification of 0 and 18.75% for resubstitution and cross-validation methods, respectively. If a subset of 28 significant peaks is chosen, resubstitution and cross-validation error rates are 0%. Discriminant analysis of the MALDI-TOF MS data for 5 strains of A. flavus using all peaks yielded error rates for classification of 0 and 5% for resubstitution and cross-validation methods, respectively. These data indicate that MALDI-TOF MS data may be used for unambiguous identification of members of the genus Aspergillus at both the species and strain levels.

Prevalence of allergic sensitization to indoor fungi in West Virginia.

Exposure to indoor fungi is of growing concern in residential and occupational environments in the United States. The purpose of this study was to determine the prevalence of sensitization to common indoor fungal species in an atopic population. We evaluated 102 patients (73 female and 29 male patients) for immunoglobulin E (IgE) reactivity to a panel of skin-prick test (SPT) reagents used for routine allergy testing. Patients also were tested for six additional fungi that are common indoor contaminants. All patients had symptoms consistent with allergic rhinitis or asthma. The presence of specific IgE against the fungal species was determined using immunoblotting. Of the 102 eligible patients, 68% had at least one positive skin test. The most prevalent positive SPTs were to dust mites, cats, vernal grass, and short ragweed. Overall, 21/102 (21%) patients with asthma or allergic rhinitis were skin test positive to at least one fungal extract. Of the patients with a positive SPT to fungi, 12/21 (58%) showed sensitivity to one or more of the newly tested species; most notably Trichoderma viride (8%), Chaetomium globosum (7%), Paecilomyces variotii (7%), and Acremonium strictum (6%). Immunoblotting revealed specific IgE against a number of protein bands belonging to these fungal species. The prevalence of fungal sensitization was common, particularly for indoor fungal contaminants that are not routinely included in SPT panels. Cross-reactivity with other fungi may partially explain our results; however, skin testing for these indoor fungi may provide useful diagnostic information.

Analytical bias of cross-reactive polyclonal antibodies for environmental immunoassays of Alternaria alternata.

BACKGROUND: Alternaria alternata is recognized as an important aeroallergen indoors and outdoors, and exposure to the fungus has been identified as a risk factor for asthma. Two recent publications concluded that 95% to 99% of American homes contained detectable amounts of Alternaria antigens when analyzed with a polyclonal antibody (pAb)-based ELISA. OBJECTIVES: We investigated the cross-reactivity of the commercially available pAbs that were used in those studies. METHODS: Reactivity to 24 fungal species commonly found in indoor environments was analyzed by inhibition ELISA by using solid-phase A alternata antigen. The pAbs were also tested by immunoblotting and halogen immunoassay for a subgroup of fungi. RESULTS: Spores of 7 fungi including species of Alternaria, Ulocladium, Stemphylium, Epicoccum, Drechslera, and Exserohilum strongly inhibited the binding of the pAbs when tested by ELISA. Six other fungi reacted in the ELISA at a lower level, and 11 fungal species including several Penicillium, Aspergillus, Fusarium, and Cladosporium species failed to show inhibition. The immunoblots and the halogen immunoassay staining confirmed the cross-reactivity patterns of the ELISA. CONCLUSION: The pAbs against A alternata were found to cross-react broadly with related and nonrelated fungi. The prevalence data previously reported for A alternata should be considered to be fungal-reactive rather than A alternata-specific.

A two-stage cyclone using microcentrifuge tubes for personal bioaerosol sampling.

Personal aerosol samplers are widely used to monitor human exposure to airborne materials. For bioaerosols, interest is growing in analyzing samples using molecular and immunological techniques. This paper presents a personal sampler that uses a two-stage cyclone to collect bioaerosols into disposable 1.5 ml Eppendorf-type microcentrifuge tubes. Samples can be processed in the tubes for polymerase chain reaction (PCR) or immunoassays, and the use of multiple stages fractionates aerosol particles by aerodynamic diameter. The sampler was tested using fluorescent microspheres and aerosolized fungal spores. The sampler had first and second stage cut-off diameters of 2.6 microm and 1.6 microm at 2 l min(-1)(geometric standard deviation, GSD = 1.45 and 1.75), and 1.8 microm and 1 microm at 3.5 l min(-1)(GSD = 1.42 and 1.55). The sampler aspiration efficiency was >or=98% at both flow rates for particles with aerodynamic diameters of 3.1 microm or less. For 6.2 microm particles, the aspiration efficiency was 89% at 2 l min(-1) and 96% at 3.5 l min(-1). At 3.5 l min(-1), the sampler collected 92% of aerosolized Aspergillus versicolor and Penicillium chrysogenum spores inside the two microcentrifuge tubes, with less than 0.4% of the spores collecting on the back-up filter. The design and techniques given here are suitable for personal bioaerosol sampling, and could also be adapted to design larger aerosol samplers for longer-term atmospheric and indoor air quality sampling.

Airborne fungal fragments and allergenicity.

Exposure to fungi, particularly in water damaged indoor environments, has been thought to exacerbate a number of adverse health effects, ranging from subjective symptoms such as fatigue, cognitive difficulties or memory loss to more definable diseases such as allergy, asthma and hypersensitivity pneumonitis. Understanding the role of fungal exposure in these environments has been limited by methodological difficulties in enumerating and identifying various fungal components in environmental samples. Consequently, data on personal exposure and sensitization to fungal allergens are mainly based on the assessment of a few select and easily identifiable species. The contribution of other airborne spores, hyphae and fungal fragments to exposure and allergic sensitization are poorly characterized. There is increased interest in the role of aerosolized fungal fragments following reports that the combination of hyphal fragments and spore counts improved the association with asthma severity. These fragments are particles derived from any intracellular or extracellular fungal structure and are categorized as either submicron particles or larger fungal fragments. In vitro studies have shown that submicron particles of several fungal species are aerosolized in much higher concentrations (300-500 times) than spores, and that respiratory deposition models suggest that such fragments of Stachybotrys chartarum may be deposited in 230-250 fold higher numbers than spores. The practical implications of these models are yet to be clarified for human exposure assessments and clinical disease. We have developed innovative immunodetection techniques to determine the extent to which larger fungal fragments, including hyphae and fractured conidia, function as aeroallergen sources. These techniques were based on the Halogen Immunoassay (HIA), an immunostaining technique that detects antigens associated with individual airborne particles >1 microm, with human serum immunoglobulin E (IgE). Our studies demonstrated that the numbers of total airborne hyphae were often significantly higher in concentration than conidia of individual allergenic genera. Approximately 25% of all hyphal fragments expressed detectable allergen and the resultant localization of IgE immunostaining was heterogeneous among the hyphae. Furthermore, conidia of ten genera that were previously uncharacterized could be identified as sources of allergens. These findings highlight the contribution of larger fungal fragments as aeroallergen sources and present a new paradigm of fungal exposure. Direct evidence of the associations between fungal fragments and building-related disease is lacking and in order to gain a better understanding, it will be necessary to develop diagnostic reagents and detection methods, particularly for submicron particles. Assays using monoclonal antibodies enable the measurement of individual antigens but interpretation can be confounded by cross-reactivity between fungal species. The recent development of species-specific monoclonal antibodies, used in combination with a fluorescent-confocal HIA technique should, for the first time, enable the speciation of morphologically indiscernible fungal fragments. The application of this novel method will help to characterize the contribution of fungal fragments to adverse health effects due to fungi and provide patient-specific exposure and sensitization profiles.

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

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 species-specific 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 (IgG(1)) 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.

Characterization of nigerlysin, hemolysin produced by Aspergillus niger, and effect on mouse neuronal cells in vitro.

Aspergillus niger produced a proteinaceous hemolysin, nigerlysin when incubated on sheep's blood agar (SBA) at both 23 and 37 degrees C. Nigerlysin was purified from tryptic soy broth (TSB) culture filtrate and found to have a molecular weight of approximately 72 kDa, with an isoelectric point of 3.45. Nigerlysin is heat stable up to 65 degrees C but unstable at 75 degrees C when incubated for 10 min. Circular dichroic analysis revealed that nigerlysin has an alpha helical structure. Exposure of mouse primary cortical neuronal cells to 0.1 microg ml(-1) of nigerlysin resulted in the rapid loss of their viability, approximately 50% in 24 h. The IC50 is estimated to be 0.037 microg ml(-1), or between 0.034 and 0.041 microg ml(-1) at the 95% confidence level.

Enumeration and detection of aerosolized Aspergillus fumigatus and Penicillium chrysogenum conidia and hyphae using a novel double immunostaining technique.

The identification of collected airborne unicellular fungal conidia and hyphae using nonviable techniques is subjective and an imprecise process. Similarly, to determine whether an individual is allergic to a particular genus requires a separate immunodiagnostic analysis. This study demonstrates the development of a novel double immunostaining halogen assay, which enables (1) the simultaneous identification of collected airborne fungal conidia and hyphae of Aspergillus fumigatus and Penicillium chrysogenum using monoclonal antibodies and (2) the demonstration of patient-specific allergy to the same particles using human serum IgE. The results demonstrate that when conidia were ungerminated the binding of antibodies was homogeneous and localized in close proximity around the entire conidia for both species. However, when conidia were germinated, the proportion expressing antigen increased (P < 0.0001) for both species and the sites of binding of the two antibodies changed with double immunostaining restricted to the hyphal tips for A. fumigatus, in addition to the sites of germination for P. chrysogenum. The described immunoassay has the potential to identify fungal particles in personal environmental air samples, provided species-specific monoclonal antibodies are available, while simultaneously demonstrating allergic sensitization to the same particles by co-staining the samples with the patient's own serum. Such an immunoassay can use those fungi that the patient is actually exposed to and potentially avoids many problems associated with extract variability based on the performance of current diagnostic techniques for fungal allergy.

Detection of aerosolized Alternaria alternata conidia, hyphae, and fragments by using a novel double-immunostaining technique.

A double-immunostaining halogen immunoassay was developed to identify aerosolized conidia, hyphae, and fragments of Alternaria alternata by using an anti-Alternaria polyclonal antiserum, while, simultaneously, allergy to these components was concurrently determined by using human immunoglobulin E antibodies.

Clinical use of immunoassays in assessing exposure to fungi and potential health effects related to fungal exposure.

OBJECTIVE: To review and summarize current evidence regarding the proper role of immunoassays in clinical assessments of exposure to fungi and health effects related to fungal exposure. DATA SOURCES: We reviewed relevant scientific investigations and previously published reviews concerning this topic. STUDY SELECTION: The authors' clinical, laboratory, and public health experiences were used to evaluate relevant data for scientific merit. RESULTS: Testing to determine the presence of IgE to specific fungi may be a useful component of a complete clinical evaluation in the diagnosis of illnesses that can be caused by immediate hypersensitivity such as allergic rhinitis and asthma. Detection of IgG to specific fungi has been used as a marker of exposure to agents that may cause illnesses such as hypersensitivity pneumonitis. However, the ubiquitous nature of many fungi and the lack of specificity of fungal antigens limit the usefulness of these types of tests in the evaluation of potential building-related illness and fungal exposure. Specific serologic tests (such as tests for cryptococcal antigen, coccidioidal antibody, and Histoplasma antigen) have been shown to be useful in the diagnosis of some fungal infections, but these are the exception not the rule. CONCLUSIONS: There is currently not enough scientific evidence to support the routine clinical use of immunoassays as a primary means of assessing environmental fungal exposure or health effects related to fungal exposure. Health care providers who care for persons expressing concerns about the relationship of symptoms to potential exposure to fungi are advised to use immunoassay results with care and only as an adjunct to a comprehensive approach to patient care.

Fungal fragments as indoor air biocontaminants.

The aerosolization process of fungal propagules of three species (Aspergillus versicolor, Penicillium melinii, and Cladosporium cladosporioides) was studied by using a newly designed and constructed aerosolization chamber. We discovered that fungal fragments are aerosolized simultaneously with spores from contaminated agar and ceiling tile surfaces. Concentration measurements with an optical particle counter showed that the fragments are released in higher numbers (up to 320 times) than the spores. The release of fungal propagules varied depending on the fungal species, the air velocity above the contaminated surface, and the texture and vibration of the contaminated material. In contrast to spores, the release of fragments from smooth surfaces was not affected by air velocity, indicating a different release mechanism. Correlation analysis showed that the number of released fragments cannot be predicted on the basis of the number of spores. Enzyme-linked immunosorbent assays with monoclonal antibodies produced against Aspergillus and Penicillium fungal species showed that fragments and spores share common antigens, which not only confirmed the fungal origin of the fragments but also established their potential biological relevance. The considerable immunological reactivity, the high number, and the small particle size of the fungal fragments may contribute to human health effects that have been detected in buildings with mold problems but had no scientific explanation until now. This study suggests that future fungal spore investigations in buildings with mold problems should include the quantitation of fungal fragments.