OBJECTIVE: To test IgG reactivity towards CNTN1 in GBS samples from the spanish cohort of IGOS. We modify the previous experiment, using milk 10% diluted in PBS-Tween, because we think that there were a problem with the blocking solution.
SAMPLES: 3 and 25 (CIDP), 44-60 (GBS), CTRL 1,2,4,5, CTRL +
MATERIALS:
- ELISA plates NUNC ref: 442404 Thermoscientific
- Coating buffer: sodium carbonate (pH 9.6)
- Recombinant Human CNTN1 protein
- RAH-HRP IgG secondary antibody Dako P0214
- TMB solution (BioLegend)
- Non-fat dry milk
- H2SO4
- PBS-Tween 0.1%
PROCEDURE:
- Coat ELISA plate with NF40 protein (1ugr/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 10% 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 using RAH IgG 1/5000 diluted in milk 10% in PBS-Tween and incubate 1h at RT.
- Wash 3xPBS-Tween
- Add TMB solution 10′
- Stop reaction with H2SO4
- Lecture at 450nm.
RESULTS: The figure of the ELISA results is here. All samples were negative.
