Objective: to do an immunoabsorption of CNTN1/Caspr1 in sera from 1 patients with antibodies anti-CNTN1/Caspr1
08.05.2019: Coating HEK293
- Prepare 3 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.
09.05.2019: 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.
09.05.2019 : Fixing and Immunoabsorption
- Fix with PFA 4%. Incubation at RT during 10min.
- Wash with PBS 1x.
- Dilute serum 1/100 in Goat 5%. Samples:
- Sample: 16047
- Put 0’5 ml in the first well of every plate
- Incubate 1 hour in every well (at RT)
- Freeze the immunoabsorpted samples at -20ºC
13.05.2019: Comprobation (ICQ):
(same protocol we use to detec antibodies to CNTN1/Caspr1)
HEK 293 trasfected cells with CNTN1/Caspr1: all pre-absorbed sera 1:100 with CNTN1, Caspr1, and CNTN1+Caspr1 are POSITIVE.
16.05.2019: Comprobation (teasing):
(teasing protocol):
Sample 16.047 pre-absorbed with CNTN1+Caspr1: paranodal stainning (POSITIVE)
Also we tried diferent dilutions with non-absorbed seram from 16.047 patient
- 1:500 POSITIVE
- 1:1000 NEGATIVE
CONLUSION: Sera was not immunoabsorbed because was not diluted enough. We will try with 1:500 dilution
