Objective: To test IgG and IgG4 titration in CSF of anti-NF155 positive CIDP patients.
Samples: 216-09, 16-031, 16-042, 19-221.
MATERIALS:
- ELISA plates NUNC ref: 442404 Thermoscientific
- Coating buffer: sodium carbonate (pH 9.6)
- Recombinant Human NF155 protein (1ug/ml)
- MAH-HRP IgG4 secondary antibodies 1:1000 or RAH IgG secondary antibody.
- 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. To perform dilutions, add 100uL from the first well to next well until a dilution of 1/270.
- 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.
- 216-09: 1/160 for IgG4, 1/40 for total IgG
- 19-221: 1/160 for IgG4, 1/40 for total IgG
- 16-031, 16-042, negative for total IgG –> we perform ICC.
ICC NF155 in CSF:
- Sample 16-031: Negative
- Sample 16-042: Positive
- Sample 19-221 (positive control): Positive
- Negative control: Negative
