OBJECTIVE:
- To test IgG positivity towards NF155 in a CMT patient (positive for ICC).
- Titulate the sera of 3 assistential patients: 18-860, 19-061, 19-162.
Samples: For titration: 18-860, 19-061, 19-162. For confirmation: 101, C- 11, C- 7, C+LAC
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 (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 only positive patient by titration is 19/162 and the titers are 1/300. Patients 18-860 and 19-061 are not positive for IgG4 (maybe is positive for another subclas of IgG?). We are going to repeat ICC and maybe ELISA for NF140.
Patient 101 is positive by ICC and ELISA, we are going to titrate it.
