20180806-07: ELISA CNTN1 IgG with CIDP patients from Holland

OBJECTIVE: To test IgG positivity towards CNTN1 in 3 patients with CIDP from Holland, that have a doubtful staining in ICC with HEK293 cells.

Samples: 1, 3 and 16 and CTRL +

MATERIALS:

• ELISA plates NUNC ref: 442404 Thermoscientific
• Coating buffer:  sodium carbonate (pH 9.6)
• Recombinant Human CNTN1 protein (Sino Biological, 10383-H08H)
• RAH-HRP secondary antibody, Dako
• TMB solution (BioLegend)
• Non-fat dry milk
• H2SO4
• PBS-Tween 0.1%

PROCEDURE:

• Coat ELISA plate with CNTN1 protein at different dilutions (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 3% 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 1/10000 in milk 3% and incubate 1h at RT
• Wash 3xPBS-Tween
• Add TMB solution 10′
• Stop reaction with H2SO4
• Lecture at 450-620 nm.

RESULTS: 

CONCLUSIONS: All samples were negative (1,3 and 16), except the positive control.