Last data update: May 20, 2024. (Total: 46824 publications since 2009)
Records 1-8 (of 8 Records) |
Query Trace: Schmechel D [original query] |
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A multi-center ring trial of allergen analysis using fluorescent multiplex array technology
King EM , Filep S , Smith B , Platts-Mills T , Hamilton RG , Schmechel D , Sordillo JE , Milton D , van Ree R , Krop EJ , Heederik DJ , Metwali N , Thorne PS , Zeldin DC , Sever ML , Calatroni A , Arbes SJ Jr , Mitchell HE , Chapman MD . J Immunol Methods 2013 387 89-95 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=0.1-9.8ng/ml) to higher than 6800ng/ml for all allergens except Mus m 1, which was up to 1700ng/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
Ruwona TB , Johnson VJ , Hettick JM , Schmechel D , Beezhold D , Wang W , Simoyi RH , Siegel PD . J Immunol Methods 2011 373 127-35 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
Nayak AP , Green BJ , Janotka E , Hettick JM , Friend S , Vesper SJ , Schmechel D , Beezhold DH . Clin Vaccine Immunol 2011 18 (9) 1568-76 A. 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 IgG(1) 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 antigens were detected in conidia, hyphae and in CSN of A. terreus. HEA antigens were expressed in high levels in the hyphae during early stages of A. terreus growth at 37 degrees C, whereas at room temperature, the expression of HEA antigens peaked by day 4-5. Expression kinetics of HEA antigens in CSN showed a lag, with peak levels at later time points at RT and 37 degrees C compared to hyphal extracts. Serum spiking experiments demonstrated that human serum components do not inhibit detection of the HEA antigen epitopes by mAb 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. |
Comparison of quantitative airborne fungi measurements by active and passive sampling methods
Yamamoto N , Schmechel D , Chen BT , Lindsley WG , Peccia J . J Aerosol Sci 2011 42 (8) 499-507 The present study compared the airborne fungi collection performance of a two-stage cyclone sampler (active method) to the performance of the Personal Aeroallergen Sampler (passive method) using quantitative polymerase chain reaction (qPCR) assays. Indoor air concentrations of the common fungal species Alternaria alternata, Cladosporium cladosporioides, Epicoccum nigrum, and Penicillium chrysogenum were considered. Good correlations between the two sampling methods for the fungi A. alternata, C. cladosporioides, and E. nigrum were observed and the mean effective passive sampling rates (+/-std. dev.) for these species were 0.032 (+/-0.006), 0.058 (+/-0.006), and 0.066 (+/-0.044) l min-1, respectively. Gravitational settling was the dominant collection mechanism for A. alternata and E. nigrum. The root mean square precisions for the passive sampler measurements were also comparable to those of the active sampler (49-73% and 50-102%, respectively). The passive sampler did not allow for the collection of P. chrysogenum, likely due to the insufficient gravitational settling velocity of the fungal particle with its aerodynamic diameter of less than 5 micrometers. The passive sampler, in conjunction with growth-independent qPCR detection methodologies, can be utilized in future exposure assessment studies to deepen our understanding of how individuals are affected by inhalation of airborne fungal pathogens and allergens, especially for those with an aerodynamic diameter greater than 5 micrometers. |
Production and characterization of IgM monoclonal antibodies against hyphal antigens of Stachybotrys species
Nayak AP , Green BJ , Janotka E , Blachere FM , Vesper SJ , Beezhold DH , Schmechel D . Hybridoma (Larchmt) 2011 30 (1) 29-36 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.
Nayak AP , Blachere FM , Hettick JM , Lukomski S , Schmechel D , Beezhold DH . Mycopathologia 2010 171 (1) 23-34 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 beta-sheet. Results from thermal denaturation of rTerrelysin show that the protein maintained the beta-sheet confirmation up to 65 degrees 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
Ruwona TB , Johnson VJ , Schmechel D , Simoyi RH , Beezhold D , Siegel PD . Hybridoma (Larchmt) 2010 29 (3) 221-9 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
Templeton SP , Buskirk AD , Green BJ , Beezhold DH , Schmechel D . Med Mycol 2010 48 (2) 217-28 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. |
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