20181128-29: ELISA NF140 IgG in CMT and CIDP patients (sample 18-866)

Objective: To test IgG reactivity towards NF140 in CIDP and CMT patients that have a doubtful staining in ICC.

Samples:  

MATERIALS:

• ELISA plates NUNC ref: 442404 Thermoscientific
• Coating buffer:  sodium carbonate (pH 9.6)
• Recombinant Human NF140 protein 
• RAH-HRP IgM secondary antibody, Dako P0215
• RAH-HRP IgG secondary antibody, Dako P0214
• TMB solution (BioLegend)
• Non-fat dry milk
• H2SO4 0.25%
• PBS-Tween20 0.1%

PROCEDURE:

• Coat ELISA plate with NF140 protein (5ugr/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.
• Wash 3xPBS-Tween 0.1%
• Add secondary antibody RAH IgG 1/5000 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.

All samples were negative.