|
| Status |
Public on Jan 03, 2012 |
| Title |
Serum sample 13 |
| Sample type |
protein |
| |
|
| Source name |
Human sera
|
| Organism |
Homo sapiens |
| Characteristics |
fluid: Human serum
gender: M
clinical status: non-asthmatic
eczema: negative
conjunctivitis: negative
rhinitis: positive
|
| Extracted molecule |
protein |
| Extraction protocol |
At the recruitment sessions, each subject was interviewed, disease status was ascertained by physical examination, permission was asked to access personal health records, and blood samples were collected
|
| Label |
Alexa 555
|
| Label protocol |
TSA™ Kit #42 *with HRP—streptavidin and Alexa Fluor® 555 tyramide* *50-150 slides* (Invitrogen)
|
| |
|
| Hybridization protocol |
Printed slides were blocked with PBS containing 2% BSA for 1 h at room temperature. Slides were then incubated with serum samples (100 µl) for 60 minutes at 37°C. To reveal bound IgE, the slides were incubated with a secondary mouse monoclonal antibody directed against human IgE (0.14 µg/ml - 100µl) for 45 minutes at 37°C, followed by an incubation with anti-mouse IgG HRP conjugated antibody (1.6 µg/mL - 100µl) for 45 minutes at 37°C and finally incubated with tyramide-Alexa 555 (TSA™ Kit #42 *with HRP—streptavidin and Alexa Fluor® 555 tyramide* *50-150 slides*) (Invitrogen) diluted 1:200 (100µl), for 15 minutes at 37°C. Slides were dried at 37°C before measuring the fluorescence signal.
|
| Scan protocol |
The processed slides were scanned using a fluorescence-detecting scanner ScanArray™ Gx and the images were generated with the ScanArray™ software provided by Perkin Elmer Life Sciences Inc. All the slides were scanned under identical settings: 90 % laser power and 60 % photomultiplier gain.
|
| Description |
no additional information
|
| Data processing |
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 on the GSE20020 record), 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).
|
| |
|
| Submission date |
Jan 24, 2010 |
| Last update date |
Jan 03, 2012 |
| Contact name |
tania dottorini |
| Organization name |
University of Perugia
|
| Department |
Dept. Exp.Medicine and Bichem. Sci.
|
| Street address |
via del Giochetto
|
| City |
Perugia |
| ZIP/Postal code |
06126 |
| Country |
Italy |
| |
|
| Platform ID |
GPL9968 |
| Series (1) |
| GSE20020 |
IgE reactivity profiling in an asthma affected cohort |
|
