Objective: To test subclases IgG and titration in anti-NF155 positive CIDP patients.
Samples:
- 44 and 45 (samples from Sevilla S20-23 and S20-28).
MATERIALS:
- ELISA plates NUNC ref: 442404 Thermoscientific
- Coating buffer: sodium carbonate (pH 9.6)
- Recombinant Human NF155 protein (1ug/ml)
- MAH-HRP IgG1, IgG2, IgG3 and IgG4 secondary antibodies 1:1000
- TMB solution (BioLegend)
- Non-fat dry milk
- H2SO4
- PBS-Tween 0.1%
PROCEDURE:
- 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 MAH IgG4/3/2/1 and incubate 1h at RT.
- Wash 3xPBS-Tween
- Add TMB solution 10′
- Stop reaction with H2SO4
- Lecture at 450-620 nm.
RESULTS: The results of the ELISA were here.
- Sample 45 si clearly positive, titters 1/300, IgG4.
- Sample 44 seem negative, we are going to perform IHC in teasing nerve fibers.
