Objective: To test IgG positivity towards NF155 and NF140 in GBS samples from the spain cohort of IGOS.
SAMPLES: 43, 85-102, CTRL – (1-4) and CTRL + (LAC).
MATERIALS:
- ELISA plates NUNC ref: 442404 Thermoscientific
- Coating buffer: sodium carbonate (pH 9.6)
- Recombinant Human NF155 protein
- Recombinant Human NF140 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 NF155 protein (1ugr/mL) and NF140 protein (1ugr/mL) in carbonate/bicarbonate buffer (pH 9.6) and incubate O/N at 4ºC rocking. Half of the plate will be coated with NF155 and the other half with NF140.
- 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.
- Wash 3xPBS-Tween 0.1%
- Add secondary antibody using RAH IgG 1/5000 diluted in milk 5% in PBS-Tween and incubate 1h at RT.
- Wash 3xPBS-Tween
- Add TMB solution 10′
- Stop reaction with H2SO4
- Lecture at 450-620 nm.
RESULTS: The figure of the ELISA results is here.
All samples were negative except sample 97 for NF140. We are going to perform ICC for NF140 in this sample.
