Atigenic property determination of rDer p 21 and HDM extracts on allergy microarray using allergic patient sera
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Laimis Silimavicius
PROVIDER: E-MTAB-13587 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Current diagnostics for allergies, such as skin prick and radioallergosorbent tests, do not allow for inexpensive, high-throughput screening of patients. Additionally, extracts used in these methods are made from washed pollen that lacks pollen surface materials that may contain allergens. We sought to develop a high-throughput assay to rapidly measure allergen-specific IgE levels in sera and to explore the relative allergenicity of different pollen fractions (i.e. surface, cytoplasmic, commercial extracts). To do this, we generated a protein microarray containing surface, cytoplasmic, and commercial extracts from 22 pollen species, commercial extracts from nine non-pollen allergens, and five recombinant allergenic proteins. Arrays were incubated with <25uL of serum from 176 individuals and bound IgE was detected by indirect immunofluorescence, providing a high-throughput measurement of IgE levels.
2010-09-08 | E-MTAB-177 | biostudies-arrayexpress
Project description:Background: Most asthmatic patients have high serum levels of IgE directed against common environmental allergens such as house dust mite, animal danders and moulds. However the presence of specific IgE against individual allergens alone does not account for asthma. Many individuals are atopic but not asthmatic. Precise knowledge of the serum IgE specificity repertoire in asthmatic and non-asthmatic patients would substantially help in understanding the pathogenesis of the disease and at the same time facilitate the treatment and the implementation of preventive measures. Methods: We developed a microarray immunoassay containing 103 common allergens to study the IgE reactivity profiles of 485 asthmatic and 342 non-asthmatic individuals from families whose members had a documented history of asthma and atopy. The results were analyzed using k-means clustering to investigate whether IgE reactivity profiles correlated with asthma, disease severity and age of onset as well as with other atopic conditions such as rhinitis, conjunctivitis and eczema. We trained an artificial neural network (ANN) using the serum reactivity data to identify individuals as asthmatic and non-asthmatic. Results: Individual sera showed clear differences in the number and the combinations of allergens recognized as well as in the level of specific IgE. While the presence of specific IgE against single allergens correlated poorly with the pathological conditions examined k-means clustering analysis unraveled that a particular profile was significantly associated with asthma (p <1E-8). An ANN-based algorithm, calibrated with the profile reactivity data correctly classified as asthmatic or non-asthmatic 78% of the individual examined. Conclusions: Our analysis demonstrates that asthma may be a higher-order phenomenon related to patterns of response and not attributable to single antibody reactions. This information sheds new light on the risk of developing the disease and can be readily utilized in combination with an ANN-based tool to distinguish asthmatic and non-asthmatic individuals on the basis of their serum reactivity profile. The study consisted of a total of 872 sera samples: 485 individuals were diagnosed with asthma, 342 were classified as non asthmatic, a remaining 45 were classified as unconfirmed asthma diagnosis. The serum IgE reactivity profiles were analyzed against 103 allergens representative of 11 distinct allergen classes. Quantification of bound IgE: The fluorescence signal was acquired using ProScanArray Express™ version 3.0 software. PMC reading values of individual spots were corrected against the internal negative control to identify signals above background. Duplicate measurements of individual allergens were utilized. The signal collected from the allergens was interpolated with an external calibration curve to obtain the IU/ml value (see supplementary file GSE20020_IU_ml values.txt), and translated into a Class Score by plotting the data in a standard reactivity scale (see Sample data tables). Class Score values: (CLASS 0 (less than 0.35 IU/ml); CLASS 1 (0.35-0.7 IU/ml); CLASS 2 (0.71-3.5 IU/ml); CLASS 3 (3.51-17.5 IU/ml); CLASS 4 (17.51-50 IU/ml); CLASS 5 (50.01-100 IU/ml).
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Project description:For determination of reactivity with rDer p 21 and Der p 21 in extracts, a serially diluted rDer p 21 from 100 to 0.00305 µg/mL and 6 different HDM extracts diluted 16 times from their stock concentrations were printed. Human serum albumin, rMBP, HRP and PBS were printed as negative controls. Each allergen and control were printed as a single droplet (~450 pL/drop) in three replicates onto 2D-Epoxy glass slides (PolyAn GmbH, Germany) using sciFLEXARRAYER SX microarray printer (SCIENION GmbH, Germany). The slides were blocked with PBS-T containing 2% BSA (blocking buffer) for 30 min at RT. After that, the slides were incubated for 2 h with MAbs 0.2 µg/mL, diluted with blocking buffer, 80 µL/well). Then the slides were incubated for 30 min with the goat anti-mouse IgG Fc Alexa Fluor® 647 (SouthernBiotech, USA) (1 μg/mL, diluted with blocking buffer, 80 μL/well). The slides were scanned using InnoScan 710 AL microarray scanner (Innopsys, France). The images were analyzed with MAPIX software (Innopsys, France). The average mean ± standart deviation value of each allergen and control was calculated using spots’ median fluorescence intensity (MFI) values
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Project description:For determination of specificity of the MAbs, rDer p 21, thermally denatured rDer p 21 and rMBP-Der p 21 (0.3 mg/mL) were printed. the slides were incubated for 2 h 5 different MAbs (0.5 µg/mL for specificity analysis). Then the slides were incubated for 30 min goat anti-mouse IgG Fc Alexa Fluor® 647. Human serum albumin, rMBP, HRP and PBS were printed as negative controls.
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