20180828-29: ELISA NF155 IgM with CIDP patients

OBJECTIVE: To test IgM positivity towards NF155 in CIDP patients.

Samples: 

MATERIALS:

• ELISA plates NUNC ref: 442404 Thermoscientific
• Coating buffer:  sodium carbonate (pH 9.6)
• Recombinant Human NF155 protein 
• RAH-HRP IgM secondary antibody, Dako P0215
• RAH-HRP IgG secondary antibody, Dako P014
• TMB solution (BioLegend)
• Non-fat dry milk
• H2SO4
• PBS-Tween 0.1%

PROCEDURE:

• Coat ELISA plate with NF155 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 RT
• Wash 3xPBS-Tween 0.1%
• Add secondary antibody RAH IgM 1/2000 in milk 5% (except for the positive control, using RAH IgG 1/5000) and incubate 1h at RT
• Wash 3xPBS-Tween
• Add TMB solution 10′
  
• Stop reaction with H2SO4
• Lecture at 450-620 nm.

RESULTS:

The experiment was not completed because we forget to do the final slep wash before adding TMB solution. We are going to repeat it.