Samples: CIDP anti-CNTN1/Caspr1+ patients
- MRVM (78CIDP)
- 19209
- 17595
- 19204
- 19212
- 16047
- 20002=19186
- Regine
- Vieira
- Pifort
- 19216
- 20001=19003
- 15198 (CNTN1+)
- CIDP44 (CNTN1+)
- Negative control
- Caspr1 + control (Piffort). Only Caspr1 protein
- 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 and CNTN1 proteins at equimolar concentratios: Caspr1 protein (5ug/ml)+ CNTN1 protein (3.8ug/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) and 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: All samples were positive (including two CNTN1+ samples), except the negative control
