Objective: Titrarion of samples included in NF155+ CIDP patients study.
SAMPLES: 5.1, 5.2, 5.3, 5.4, 5.5, 15
MATERIALS:
- ELISA plates NUNC ref: 442404 Thermoscientific
- ELISA plate ref. 1581
- Coating buffer: sodium carbonate (pH 9.6)
- Recombinant Human NF155 protein
- MAH IgG4
- 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. 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 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.
- 5.1 –> 1/24600
- 5.2 –> 1/8100
- 5.3 –> 1/2700
- 5.4 –> 1/300-1/900
- 5.5 -> 1/100
- 15 –> 1/2700
