|
| Status |
Public on Mar 07, 2016 |
| Title |
Patient#106-IgM |
| Sample type |
protein |
| |
|
| Source name |
serum
|
| Organism |
Homo sapiens |
| Characteristics |
gender: Female
disease group (nhs-normal human serum; sle-systemic lupus erythematosus ;uctd-undifferentiated connective tissue disease; sjs-sjörgen's syndrome;ssc-systemic sclerosis; psa-psoriatic arthritis): SSc
age in years: 63
printing batches (1,2): 1
slide no.: 8
|
| Treatment protocol |
Serum samples were stored at -70C
|
| Extracted molecule |
protein |
| Extraction protocol |
Serum sample was centrifuged for 6min at 15000g for removing particles
|
| Label |
fluorescent antibodies
|
| Label protocol |
Dried arrays were rinsed in PBS for 15 minutes before use, then incubated with diluted serum at 37°C for 1 hour. Two separated measurements were applied on each sample for detection of bound components. Five-fold serum dilution in 2.5mM Ca2+, 0.7mM Mg2+ and 5% BSA complemented PBS buffer was used for detection of deposited C3 and C4 complement fragments. 125-fold serum dilution in 25mM EDTA, 0.05% Tween 20 and 5% BSA complemented PBS buffer was applied for measurement of bound antigen specific IgG and IgM antibodies. Serum treated slides were washed with PBS containing 0.05% Tween 20, then incubated in the mixture of 1:5,000 diluted FITC-conjugated F(ab’)2 fragment of goat anti-human C3 antibody (Cappel) and 1:1000 diluted Alexa 647 conjugated goat anti-human C4 (Cappel, in-house conjugation) or 1:2500 diluted DyLight 488-conjugated F(ab’)2 fragment goat anti-human IgM (mu chain specific) (Jackson ImmunoResearch) and 1:2,500 diluted DyLight 649-conjugated F(ab’)2 fragment goat anti-human IgG (gamma chain specific) (Jackson ImmunoResearch). Labeling with fluorescent antibodies was carried out at room temperature for 30 minutes in PBS containing 5% BSA and 0.05% Tween 20. After washing in PBS containing 0.05% Tween 20, arrays were dried and scanned
|
| |
|
| Hybridization protocol |
Probe: DyLight 488-conjugated F(ab’)2 fragment goat anti-human IgM (mu chain specific) (Jackson ImmunoResearch)
|
| Scan protocol |
Arrays were scanned by FLAIR Fluorescent Array Imaging Reader (Sensovation).
|
| Data processing |
Local background of spot ('Bkg.Median') was substracted from the value of signal ('Spot.Median'). Values were multiplied by 65535 and rounded to be integers. Negative signals values were clamped to arbitrary value 1.
|
| |
|
| Submission date |
May 29, 2015 |
| Last update date |
Mar 07, 2016 |
| Contact name |
Krisztian Papp |
| E-mail(s) |
pkrisz5@gmail.com
|
| Organization name |
MTA-ELTE Immunology Research Group
|
| Street address |
1/C Pazmany P.
|
| City |
Budapest |
| ZIP/Postal code |
1117 |
| Country |
Hungary |
| |
|
| Platform ID |
GPL20261 |
| Series (2) |
| GSE69371 |
Autoantibodies and nucleic acids skew complement consumption in systemic lupus erythematosus [IgM] |
| GSE69372 |
Autoantibodies and nucleic acids skew complement consumption in systemic lupus erythematosus |
|
