Objective: to do an immunoabsorption of CNTN1/Caspr1 in sera from 1 patients with antibodies anti-CNTN1/Caspr1
20.05.2019: 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: 240.000 cells
- Incubate at 37ºC for 24h.
21.05.2019: 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.
22.05.2019 : Fixing and Immunoabsorption
- Fix with PFA 4%. Incubation at RT during 10min.
- Wash with PBS 1x.
- Dilute serum 1/500 in Goat 5%. Samples:
- Sample: 16047
- 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
Note: serum preabsorbed with Caspr1 was only incubated in 3 wells
28.05.2019: Comprobation (teasing):
(teasing protocol):
Sample 16.047 1:500 pre-absorbed with:
1.CNTN1+Caspr1: NEGATIVE
2. CNTN1: NEGATIVE
3. Caspr1: POSITIVE
4. NON-TRANSFECTED HEK: POSITIVE
5.CONTROL: 16047 1:500 non-absorbed: POSITIVE
CONLUSION: I don’t know why sera preabsorbed with CNTN1 did not have paranodal reactivity, altough the nerve did not look very good. Maybe serum was too diluted now, we will repeat the same process with serum diluted 1:250
