|
| Status |
Public on Aug 26, 2015 |
| Title |
Validation patient #5 | Control Arm | Post-treatment | Day 56 | Validation Total Ig 1-200 |
| Sample type |
protein |
| |
|
| Source name |
Serum
|
| Organism |
Homo sapiens |
| Characteristics |
halabi predicted survival (months): 25.0349076
post-vaccination survival (months): 21.2
survival status at last follow-up (1 = alive, 0 = dead): 0
treatment arm: Control
pre-vaccination igg titers for vaccinia: 100
post-vaccination igg titers for vaccinia: 800
post-vaccination igg titer for fowlpox: <100
age at enrollment (years): 55
gleason score: 7
on-study psa: 11
treatment: Control
timepoint: Pre-treatment
molecule: Total Ig
|
| Treatment protocol |
Diluted 1:200
|
| Growth protocol |
N/A
|
| Extracted molecule |
protein |
| Extraction protocol |
Serum was obtained from peripheral blood samples
|
| Label |
Cy3
|
| Label protocol |
After serum was incubated on the array, bound anti-glycan antibodies were labeled for 2 hours with a Cy3 conjugated anti-human total immunoglobulin (IgM+IgG+IgA) (Jackson 109-165-064).
|
| |
|
| Hybridization protocol |
Arrays were blocked with BSA overnight, and serum samples (diluted 1:200) was incubated on the array for 3 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 4000A 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 Cy3 conjugated secondary antibody specific for human total immunoglobulin (IgM + IgG + IgA). Signal from ch2 (635nm) was not analyzed since only one secondary antibody was used. Scanned images were analyzed with Genepix Pro (v6.0) to determine the fluorescence and background signal for each array component.
|
| 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 |
Aug 27, 2013 |
| Last update date |
Aug 26, 2015 |
| Contact name |
Christopher Campbell |
| Organization name |
NCI
|
| Street address |
376 Boyles Street
|
| City |
Frederick |
| State/province |
MD |
| ZIP/Postal code |
21702 |
| Country |
USA |
| |
|
| Platform ID |
GPL16242 |
| Series (2) |
| GSE50239 |
Post-vaccination serum anti-glycan total Ig antibodies of 113 subjects in a clinical trial of PROSTVAC-VF, a therapeutic cancer vaccine. (Dilution = 1:200) |
| GSE50410 |
Anti-glycan humoral responses after vaccination with PROSTVAC-VF |
|
