Objective:
- Titrarion of samples included in NF155+ CIDP patients study.
- Evaluate CASPR1 positivity in samples from Luuk dataset (ICOS_biobank).
SAMPLES:
- Titration NF155: 24.1, 24.2, 24.3, 407
- CASPR1: 507, 363, 350, 356, 357, 361, 344, 362, 391, 392, neg control and pos control.
MATERIALS:
- ELISA plates NUNC ref: 442404 Thermoscientific
- ELISA plate ref. 1581
- Coating buffer: sodium carbonate (pH 9.6)
- Recombinant Human NF155 protein
- Recombinant Human CASPR1 protein
- MAH IgG4 for NF155 titration, MAH total IgG for CASPR1.
- TMB solution (BioLegend)
- Non-fat dry milk
- H2SO4
- PBS-Tween 0.1%
PROCEDURE:
- Coat ELISA plate with NF155 protein (1ug/ml) or CASPR1 protein (5ug/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. For titration: Make serial dilutions 1:3 of the samples in a ELISA plate ref. 1581 (8 wells). In the first well, add 200uL of the sample (diluted at 1/100), in the other wells add 140uL of milk 5% in PBS-Tween and 70uL of the previous well (serial dilutions). Add the dilutions in the blocked ELISA plate (ref. 442404) and incubate 1h at RT.
- Wash 3xPBS-Tween 0.1%
- Add secondary antibody MAH IgG4 1/1000 or MAH IgG 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 results of the ELISA were here.
All samples were negative for CASPR1. Sample 407 was negative for NF155 (IgG4 and total IgG).NF155 titration: 1/8100 for 24.1, 24.2 and 24.3.