OBJECTIVE: To test IgM positivity towards NF155 in CMT patients.
SAMPLES:
CODE |
---|
1 |
2 |
3 |
4 |
5 |
6 |
7 |
9 |
10 |
11 |
12 |
13 |
14 |
15 |
16 |
17 |
18 |
19 |
20 |
21 |
CTRL - ANA |
CTRL - M35a |
CTRL - A10A |
CTRL - SILVIA |
CTRL - 1 |
CTRL + LAC (IgG) |
MATERIALS:
- ELISA plates NUNC ref: 442404 Thermoscientific
- Coating buffer: sodium carbonate (pH 9.6)
- Recombinant Human NF155 protein
- RAH-HRP IgM secondary antibody, Dako P0215
- RAH-HRP IgG secondary antibody, Dako P0214
- TMB solution (BioLegend)
- Non-fat dry milk
- H2SO4
- PBS-Tween 0.1%
PROCEDURE:
- Coat ELISA plate with NF155 protein (5ugr/mL) in carbonate/bicarbonate buffer (pH 9.6) and incubate O/N at 4ºC rocking.
- The day after, wash plate 3xPBS-Tween 0.1% and block with milk 5% in PBS-Tween for 1 hour, 200uL per well).
- Add patients sera diluted 1/100 (200uL/well) and incubate 1h at RT.
- Wash 3xPBS-Tween 0.1%
- Add secondary antibody RAH IgM 1/2000 in milk 5% (except for the positive control, using RAH IgG 1/5000) and incubate 1h at RT.
- Wash 3xPBS-Tween
- Add TMB solution 10′
- Stop reaction with H2SO4
- Lecture at 450-620 nm.
RESULTS:
The figure of ELISA results is here.
All samples were negative.