Objective:
- To test subclases IgG in a NF155 positive CMT patient (101).
- To test NF140 IgG and NF155 IgG in a patient that has a mild positivity towards NF155 and NF40 in ICC (19-061)
- To confirm positivity in 19-172 (positive by ICC).
MATERIALS:
- ELISA plates NUNC ref: 442404 Thermoscientific
- Coating buffer: sodium carbonate (pH 9.6)
- Recombinant Human NF155 protein
- RAH-HRP IgG secondary antibody, Dako P0214
- MAH-HRP IgG4 secondary antibody REF: A-10654
- TMB solution (BioLegend)
- Non-fat dry milk
- H2SO4
- PBS-Tween 0.1%
PROCEDURE:
- Coat ELISA plate with NF155 protein or NF140 protein (1ug/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 IgG 1/5000 for confirmation and MAH IgG4 for titration 1/2000 in milk 5% 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 the ELISA is here.
The ELISA is not valid. We think there was a problem with the blocking solution; we are going to repeat it with milk 10% in PBS-Tween 0.1%.
