Objective:
- Titration of samples included in NF155+ CIDP patients study (IgG4).
SAMPLES:
- Plate 1: 17, 19, 20, 21, 22, 23
- Plate 2: 26, 28, 29, 30, 31, 32
MATERIALS:
- ELISA plates NUNC ref: 442404 Thermoscientific
- ELISA plate ref. 1581
- Coating buffer: sodium carbonate (pH 9.6)
- Recombinant Human NF155 protein
- MAH IgG4 for NF155 titration
- 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 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.
- 17 –> 1/24300
- 19 –> 1/2700
- 20 –> 1/8100
- 21 –> 1/8100
- 22 –> 1/72300
- 23 –> 1/72300
- 26 –> 1/2700
- 28 –> 1/24300
- 29 –> 1/900
- 30 –> 1/2700
- 31 –> 1/2700
- 32 –> 1/8100
