|
| Status |
Public on Dec 13, 2016 |
| Title |
patient 3.028|Pre-vaccination IgM |
| Sample type |
protein |
| |
|
| Source name |
Serum
|
| Organism |
Homo sapiens |
| Characteristics |
time point: Pre-vaccination
survival months: 59.8
event at time of survival analysis: Deceased
|
| Treatment protocol |
Diluted 1:50
|
| Growth protocol |
N/A
|
| Extracted molecule |
protein |
| Extraction protocol |
Serum was obtained from peripheral blood samples
|
| Label |
DyLight 649
|
| Label protocol |
After serum was incubated on the array, bound anti-glycan antibodies were labeled for 2 hours with a combination of DyLight549 conjugated anti-human IgG (Jackson 109-505-008) and DyLight 649 conjugated anti-human IgM (Jackson Immuno, 109-495-043).
|
| |
|
| Hybridization protocol |
Arrays were blocked with 3% BSA in PBS buffer for overnight, and serum samples (diluted 1:50) was incubated on the array for 4 hours at 37 C and gentle agitation (100 RPM). Slides were then washed with PBS contain 0.05% Tween (PBST). After washing, bound antibodies were detected with fluorescently labeled secondary antibodies. Slides were then washed with PBST, spun dry in a centrifuge, and scanned.
|
| Scan protocol |
Slides were scanned with a Genepix 4000B fluorescence scanner at two gain settings (typically 430 and 520) in order to increase dynamic range. Signal from ch1 (532 nm) was analyzed to determine the level of bound DyLight 549 conjugated secondary antibody specific for human IgG. Signal from ch2 (635nm) was analyzed to determine the level of bound DyLight 649 secondary antibody specific for human IgM. Scanned images were analyzed with Genepix Pro (v6.0) to determine the fluorescence and background signal for each array component.
|
| Description |
3.028_V1
IgM
|
| Data processing |
First, median pixel intensity of each feature was background subtracted. Second, signals for features that were saturated at the high photomultipler tube (PMT) setting were calculated by proportionally scaling the value from the low PMT setting according to a correction factor, which was calculated based on mid-intensity signals measured at the high and low PMT settings. To account for slide-to-slide variations in array processing, microarray data were normalized by median centering based on a reference serum sample analyzed on each slide. Additionally, a minimum signal of 150 was set as the floor. Since array features were printed in duplicate and all samples were analyzed on two slides, pre-processing averaged normalized data from 4 technical replicates. Final data are reported on a log 2 scale.
|
| |
|
| Submission date |
Jun 07, 2016 |
| Last update date |
Dec 13, 2016 |
| Contact name |
Jeff Gildersleeve |
| Organization name |
National Cancer Institute
|
| Street address |
376 Boyles St.
|
| City |
Frederick |
| State/province |
MD |
| ZIP/Postal code |
21702 |
| Country |
USA |
| |
|
| Platform ID |
GPL21991 |
| Series (1) |
| GSE83087 |
Whole-Cell Cancer Vaccines Induce Large Antibody Responses to Carbohydrates and Glycoproteins |
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