SAMPLES: 21-2-099, 21-2-0101, 21-2-0102, 21-2-0103, 21-2-0105, 21-2-0106, 21-2-0107, 21-2-0108, positive control, negative control.
MATERIALS:
- ELISA plates NUNC ref: 442404 Thermoscientific
- ELISA plate ref. 1581
- Coating buffer: sodium carbonate (pH 9.6)
- Recombinant Human Caspr1 His-tag Protein ( 2418-CR R&d Systems)
- RAH-HRP IgG secondary antibody DAKO (P0214)
- TMB solution (BioLegend)
- Non-fat dry milk
- H2SO4
- PBS-Tween 0.1%
PROCEDURE:
- COATING: coat ELISA plate with Caspr1 protein (5ug/ml) in carbonate/bicarbonate buffer (pH 9.6) and incubate O/N at 4ºC rocking.
- BLOQUING: The day after, wash plate 3xPBS-Tween 0.1%
- Block with milk 5% in PBS-Tween for 1 hour (200uL per well).
- SERUM: Add patients sera diluted 1/100 (200uL/well) and incubate 1h at RT.
- Wash 3xPBS-Tween 0.1%
- 2nd ANTIBODY: Add secondary antibody RAH-HRP IgG 1/2000 in milk 5% in PBS-Tween 0.1% and incubate 45min at RT.
- Wash 3xPBS-Tween
- Add TMB solution 10′
- Stop reaction with H2SO4 (50ul) after 5min.
- Lecture at 450-620 nm.
RESULTS: the results were here.
