Objective: To determine the titration of NF155+ CIDP patients.
Samples: 19-035; 19-036; 18-882.
MATERIALS:
- ELISA plates NUNC ref: 442404 Thermoscientific
- ELISA microtest plate 96wells, ref. 1581
- Coating buffer: sodium carbonate (pH 9.6)
- Recombinant Human NF155 protein
- MAH IgG4-HRP
- TMB solution (BioLegend)
- Non-fat dry milk
- H2SO4
- PBS-Tween 0.1%
PROCEDURE:
- Coat ELISA plate with NF155 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 5% in PBS-Tween for 1 hour, 200uL per well).
- 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 using MAH IgG4-HRP 1/2000 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 titer is 1/900 for all patients.
The figure of the ELISA is here.
