OBJECTIVE: To test IgG positivity towards PLEXIN D1 in small fiber neuropathy patients.
SAMPLES: IVIg 1, 2 and 3, control 1 and 2, Ab anti-plexin D1
MATERIALS:
- ELISA plates NUNC ref: 442404 Thermoscientific
- Coating buffer: sodium carbonate (pH 9.6)
- Recombinant Human PLEXIN D1 protein
- Ab anti-plexin D1 (ab28762)
- RAH-HRP IgG secondary antibody, Dako P0214
- DAG-HRP
- TMB solution (BioLegend)
- Non-fat dry milk
- H2SO4
- PBS-Tween 0.1%
PROCEDURE:
- Coat ELISA plate with PLEXIN D1 protein (5ugr/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. OR Ab anti-plexin D1 (ab28762) 1/2000
- Wash 3xPBS-Tween 0.1%
- Add secondary antibody RAH IgG 1/5000 in milk 5% (except for the Ab anti-PLEXIN D1, using DAG 1/5000) and incubate 1h at RT.
- Wash 3xPBS-Tween
- Add TMB solution 10′
- Stop reaction with H2SO4
- Lecture at 450-620 nm.
RESULTS:
| sample 1 | sample 2 | sample 3 | control 1 | control 2 | Ac anti-plexinD1 | ||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| 0.135 | 0.037 | 0.386 | 0.036 | 0.428 | 0.037 | 0.091 | 0.046 | 0.111 | 0.034 | 0.047 | 0.06 |
