OBJECTIVE
To test 3 new patients with CIDP suspicion for anti-CNTN1 -NF155 antibodies
MATERIALS
- NF155-HEK cell line, grown in coverslips and frozen (see previous experiment)
- CNTN1-transfected HEK cells, made by Miquel (19/10/2013)
- Sera from JHH, MASP and FRC and from two positive controls ATR and LAC
PROCEDURE
- Take the coverslips out of the freezer. Let sit 5-10mins
- Add blocking buffer to the plates in which the coverslips sit.
- Prepare first primary antibody (sera). Serum concetration 1/50 in blocking buffer. Prepare moister chamber with parafilm and add a drop (40uL) of each serum preparation. Incubate coverslips with sera for 1h RT
- Wash 3xPBS
- Prepare second primary antibody (commercial): Chicken antiNF155 (1/1000) and mouse anti-CNTN1 (1/5000). Add them and let sit 1h at RT.
- Wash 3xPBS
- Prepare secondary antibodies. Two preparations: GAC 488+GAH594 1/1000 and GAM 488+GAH594 1/1000. Add them and incubate 1h RT
- Wash 3xH2O
- Mount with vectashield.
RESULTS
Patient MASP is clearly positive for anti-CNTN1 antibodies.
The other two are clearly negative.
CONCLUSIONS
– New CNTN1 positive patient: MASP, from Dr Bárcena, Cruces, Bilbao.
