Samples: 507, 363, 350, 356, 357, 361m 344, 362, 391, 392, 367, 366, 476, 477. 475. 478, 499, 500, 501, 496, 394, 395, 497, 498, 396, 480, 481, 374, 479, 482, 487, 509, 508, 513, 505, 512, 371, 377, 373, 376, 483, 485, 484, positive control and negative control.
Materials:
- ELISA plates NUNC ref: 442404 Thermoscientific
- 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% (last one only with PBS)
- Block with milk 5% in PBS-Tween for 1 hour, 200uL per well).
- Add patients sera diluted 1/100 (100uL/well)
- Incubate 1h at RT.
- Wash 3xPBS-Tween 0.1%
- Add secondary antibody RAH-HRP IgG 1/5000 in milk 5% in PBS-Tween 0.1% and incubate 1h at RT.
- Wash 3xPBS-Tween (last one only PBS)
- Add TMB solution 10′
- Stop reaction with H2SO4
- Lecture at 450-620 nm.
RESULTS:
The results of the ELISA are here http://s811335031.mialojamiento.es/lab/wp-content/uploads/2020/08/ELISA-04.08.xls
