Objective:
- To test IgM reactivity towards NF140 in CMT and CIDP patients that have a doubtful staining in ICC.
- To test IgG reactivity towards NF140 in 2 CMT patients that have a doubtful staining in ICC.
Samples:
| CODE |
|---|
| 18-532 |
| 18-533 |
| 9 |
| 17 |
| 34 |
| 38 |
| 43 |
| 45 |
| 55 |
| 56 |
| 57 |
| CTRL - Ana |
| CTRL - Aioa |
| CTRL - Xavi |
| CTRL- Silvia |
| CTRL - 1 |
| 23 (IgG) |
| 24 (IgG) |
| CTRL - M35a (IgG) |
| CTRL+ NF140 (IgG) |
MATERIALS:
- ELISA plates NUNC ref: 442404 Thermoscientific
- Coating buffer: sodium carbonate (pH 9.6)
- Recombinant Human NF140 protein
- RAH-HRP IgM secondary antibody, Dako P0215
- RAH-HRP IgG secondary antibody, Dako P0214
- TMB solution (BioLegend)
- Non-fat dry milk
- H2SO4 0.25%
- PBS-Tween20 0.1%
PROCEDURE:
- Coat ELISA plate with NF140 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 sample 23, 24, CTRL – M35a and 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; except for sample 24. We are going to perform teasing in ciatic nerve to confirm it (because in transfected cells the staining was doubtful).
