Objective:
- Titration of 4 samples: 18-605, 18-860, 19-061, 101.
- Confirmation NF140: 19-061, C-, C+
MATERIALS:
- ELISA plates NUNC ref: 442404 Thermoscientific
- Coating buffer: sodium carbonate (pH 9.6)
- Recombinant Human NF155 protein
- RAH-HRP IgG secondary antibody, Dako P0214
- MAH-HRP IgG4 secondary antibody REF: A-10654
- TMB solution (BioLegend)
- Non-fat dry milk
- H2SO4
- PBS-Tween 0.1%
PROCEDURE:
- Coat ELISA plate with NF155 protein or NF140 (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 RAH IgG 1/5000 for confirmation and MAH IgG4 for titration 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 figure of the ELISA is here.
Sample 18-605 is clearly positive, the titers are 1/900. Sample 18-860 and 1/100 are positive by ELISA (1/900) but they are negative in pig-teased nerve fibers.
We are going to repeat ICC in non-transfected cells and we are going to perform an LRP4 ICC (with c-myc).
