Samples:
- For NF155: 15-192, 19-200, neg control, pos control.
- For CNTN1: ATR, 19-204; 19-209; 19-212; 19-216; 19-186; neg control, pos control.
MATERIALS:
- ELISA plates NUNC ref: 442404 Thermoscientific
- ELISA plate ref. 1581
- Coating buffer: sodium carbonate (pH 9.6)
- Recombinant Human CNTN1 protein
- RAH-HRP IgG secondary antibody DAKO
- TMB solution (BioLegend)
- Non-fat dry milk
- H2SO4
- PBS-Tween 0.1%
PROCEDURE:
- Coat ELISA plate with CNTN1, NF155 or NF140 protein (1ug/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 5% in PBS-Tween for 1 hour, 200uL per well).
- Add patients sera diluted 1/100 (200uL/well) and incubate 1h at RT. For titration: Make serial dilutions 1:3 of the samples in a ELISA plate ref. 1581 (8 wells). In the first well, add 200uL of the sample (diluted at 1/100), in the other wells add 140uL of milk 5% in PBS-Tween and 70uL of the previous well (serial dilutions). Add the dilutions in the blocked ELISA plate (ref. 442404) and incubate 1h at RT.
- Wash 3xPBS-Tween 0.1%
- Add secondary antibody MAH IgG4 1/2000 in milk 5% and incubate 1h at RT.
- Wash 3xPBS-Tween
- Add TMB solution 10′
- Stop reaction with H2SO4
- Lecture at 450-620 nm.
RESULTS: The results of the ELISA were here.
Sample 15-192 is positive for NF1500 (1/300)
Sample ATR is positive for CNTN1 (1/8100)
Sample 19-216 seems a positive week for CNTN1.
Sample 19-200 is negative for NF155 and NF140 and it seems positive for ICC. We are going to perform teasing.
