Objective: To test subclases IgG in anti-Caspr1 positive CIDP patients
MATERIALS:
- ELISA plates NUNC ref: 442404 Thermoscientific
- Coating buffer: sodium carbonate (pH 9.6)
- Recombinant Human Caspr1 prot (5ug/ml) and CNTN1 (3.8 ug/ml)
- MAH-HRP IgG1, IgG2, IgG3 and IgG4 secondary antibodies
- TMB solution (BioLegend)
- Non-fat dry milk
- H2SO4
- PBS-Tween 0.1%
PROCEDURE:
- Coat ELISA plate with Caspr1 (5ug/ml) + CNTN1 (3.8 ug/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.
- Wash 3xPBS-Tween 0.1%
- Add secondary antibody RAH IgG 1/2000 for confirmation and MAH IgG41,2,3,4 1/2000 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:
ELISA 14.2.20.CNTN1.Caspr1Subclases
