OBJECTIVE: To test IgM positivity towards NF155 in CIDP patients.
Samples:
| CODE |
|---|
| 18-509 |
| 18-510 |
| 18-511 |
| 18-512 |
| 18-513 |
| 18-514 |
| 18-515 |
| 18-516 |
| 18-517 |
| 18-518 |
| 18-519 |
| 18-520 |
| 18-521 |
| 18-523 |
| 18-524 |
| 18-525 |
| 18-526 |
| 18-527 |
| 18-528 |
| 18-529 |
| 18-530 |
| 18-531 |
| CTRL - |
| CTRL + (LAC) |
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 P014
- 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. We are going to repeat samples 18-520, 18-527 and 18-531.
