20200113-14: ELISA Caspr1 in CIDP patients

Samples: CIDP anti-CNTN1/Caspr1+ patients

1. MRVM (78CIDP)
2. 19209
3. 17595
4. 19204
5. 19212
6. 16047
7. 19225
8. Regine
9. Vieira
10. Pifort
11. 19216
12. 15198 (CNTN1+)
13. Negative control

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 positive except CNTN1+ and negative control

ELISA caspr1 14.01.20