OBJECTIVE: To test IgM positivity towards NF155 in CIDP and CMT patients.
SAMPLES:
CODE | DISEASE |
---|---|
18-563 | CIDP |
18-564 | CIDP |
18-565 | CIDP |
18-566 | CIDP |
18-567 | CIDP |
18-568 | CIDP |
18-569 | CIDP |
18-570 | CIDP |
18-571 | CIDP |
18-572 | CIDP |
18-573 | CIDP |
18-574 | CIDP |
18-575 | CIDP |
18-576 | CIDP |
18-577 | CIDP |
18-578 | CIDP |
18-579 | CIDP |
18-580 | CIDP |
1 | CMT |
2 | CMT |
3 | CMT |
4 | CMT |
5 | CMT |
6 | CMT |
7 | CMT |
9 | CMT |
10 | CMT |
11 | CMT |
12 | CMT |
13 | CMT |
14 | CMT |
15 | CMT |
16 | CMT |
17 | CMT |
18 | CMT |
19 | CMT |
20 | CMT |
21 | CMT |
22 | CMT |
23 | CMT |
24 | CMT |
25 | CMT |
26 | CMT |
CTRL - ANA | CTRL - |
CTRL - M35A | CTRL - |
CTRL- A10A | CTRL - |
CTRL - Silvia | CTRL - |
CTRL- 1 | CTRL - |
CTRL +(LAC) | CIDP NF155+ (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.
We have to repeat the experiment. We have a problem with the reactions.