Objective: to do an immunoabsorption of CNTN1/Caspr1 in sera from 1 patient with antibodies anti-CNTN1/Caspr1 (MRVM)
29.07.2019: Coating HEK293
- Prepare 4 6-well plates
- Treatment with Poly-D-Lysine (diluted 1/40 with boratte buffer)
- Incubate for 1h (37ºC, 5% CO2).
- Wash with PBS
- Trypsin-EDTA a plate of HEK293 –> For every well: 400.000 cells
- Incubate at 37ºC for 24h.
30.07.2019: Transfection
Transfect 3 6-well plate with CNTN1, Caspr1, CNTN1+Caspr1- CNTN1: DNA 4 µg /well; Lipofectamine 6 µL/well ; Optimen 250 µL/well
- Caspr1: DNA 4 µg /well; Lipofectamine 6 µL/well ; Optimen 250 µL/well
- CTNT1 DNA 4 µg /well (Caspr1 1.35 µL/well ; CNTN1 2,65 µL/well ); Lipofectamine 6 µL/well ; Optimen 250 µL/well
- Incubate the DNA and the lipofectamine at RT for 5 min.
- Unify every DNA with the appropriate amount of Lipo and incubate at RT for 30 min.
- For every well: 2 ml of HEK medium and 250 µL of the mix of DNA and Lipo.
- Incubate O/N at 37ºC.
31.07.2019 : Fixing and Immunoabsorption
- Fix with PFA 4%. Incubation at RT during 10min.
- Wash with PBS 1x.
- Dilute serum 1/800 in Goat 5%. Samples:
- Sample: MRVM
- Put 0’5 ml in the first well of every plate :
- 1. CNTN1
- 2. Caspr1
- 3. CNTN1/Caspr1
- 4. Non-transfected cells
- Incubate 1 hour in every well (at RT)
- Freeze the immunoabsorpted samples at -20ºC
- ICC transfected HEK cells: pre-absorbed sera is still positive
- IHQ teased nerve fiber: pre-absorbed sera with CNTN1/Caspr1 is negative, but not when sera is absorbed with CNTN1 or Caspr1 alone or non-transfected cells
