Objective: To test reactivity of serum samples from CNTN1/Caspr1+ patients, against CHO cells transfected with Caspr1 or CNTN1+Caspr1 (and see if there is difference with HEK cells?
Samples: MRVM, 19209, 17595, 19205, 19212, 16047, 20002, 20001, Regine, Vieira, Piffort, 19216, 19225, 15198 negative control
Complete Growth Medium: F-12K + 10% FBS + P/S
Transfection with chamber slides pre-coated with Poli-D + laminina
Same protocol as HEK cells
Day 1 : 70.000 cells/well
Day 2: transfection
For every 2 slides (=16 wells) –> 136ul optimen + 6.4ul lipofectamin and 136ul optimen + 4.32 ug DNA
- Incubate at RT for 5 min.
- Unify the DNA solution with the Lipo solution and incubate at RT for 30 min.
- For every well = 17ul
- Incubate O/N at 37ºC.
Day 3: ICC
- Fix with PFA 4% (aprox 5 ml/plate). Incubation at RT during 10min.
- Wash with PBS 1x
- Blocking solution: 5% goat serum for Caspr1 in PBS for 1h at RT (or Rabbit serum 1/40) for CNTN1/Caspr1
ICC same protocol as HEKs:
- Blocking solution: Goat serum 5 % diluted in PBS 1x (or Rabbit serum 1/40 in we use anti-CNTN1 mAb)
- Patient’s sera diluted 1/100
- Primary antibody (anti-CASPR1) diluted 1/250 (or anti-CNTN1 1/1000 in Rabbit serum 1/40).
- Secondary antibodies: GAR 488 + GAH 594 IgG (1/1000) (or RAG 488 + RAH 594)
- Vectashield mounting medium
Results: all samples showed reactivity agaisnt CHO cells co-transfected with CNTN1 and Caspr1. We observed reactivity against CHO cells transfected with Caspr1 in the same samples as with HEKs transfected with Caspr1
