OBJECTIVE:
To test Caspr1 reactivity after serum pre-absorption with HEK cells transfected with CNTN1 or Caspr1 or CNTN1-Caspr1 contrasfection
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:
- Coat ELISA plate with 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%
- Block with milk 5% in PBS-Tween for 1 hour, 200uL per well).
- Add patients sera diluted 1/100 (200uL/well)
- Incubate 1h at RT.
- Wash 3xPBS-Tween 0.1%
- Add secondary antibody RAH-HRP IgG 1/2000 in milk 5% in PBS-Tween 0.1% and incubate 1h at RT.
- Wash 3xPBS-Tween
- Add TMB solution 10′
- Stop reaction with H2SO4
- Lecture at 450-620 nm.
RESULTS:
see results here ELISA Caspr1 26.2.20 comprobación ABSORC
