20200116-17: ELISA Caspr1 in healthy controls

Samples: healthy controls

1. 1
2. 3-1
3. 5
4. 3-2
5. 16
6. 4
7. 5
8. 7
9. 10
10. 2
11. Caspr1+ control (Piffort)

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)  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: al samples were NEGATIVE except Caspr1+ control

ELISA Caspr1 170120