OBJECTIVE
Test patients and controls after setting up the assay
PROCEDURE
We only perform live staining because it seems to be more sensitive according to the setting up experiment.
Live staining:
- Culture Schwann cells following protocol (Poly-D-lysine coated plates w/ coverslips up to 80% confluence in 60mm plates
- Transfer coverslips to 12-well plates (1 coverslip per well)
- Dilute CIDP serum (1/100) in NGS 5% in PBS and add to the wells
- We test, in two different days, all sera from our Unit and the ones from outside
- Incubate 2h at 37ºC in incubator.
- Wash 3xPBS
- Fix with PFA 4% in PBS 5 minutes
- Wash 3xPBS
- Prepare secondary ( GAH IgG Alexa 488 1/500) and add to the coverslips
- Incubate 1h at RT
- Wash 3xPBS
- Mount with Vectashield with DAPI
RESULTS
Almost all samples have an unspecific staining in the cytoplasm of the Schwann cells (patients classified as P in the results database).
There are, at least, three staining patterns: the unspecific pucntate pattern, a GDP-like pattern (membrane asymmetric staining) and TMF-like staining (the most frequent, stains Schwann cell processes)
- Seven patients from our Unit have weak staining, four intermediate staining and one (GDP) strong staining
- Five patients from outside our center have weak staining, two, intermediate staining and two strong staining (MIGP and JRSO)
Interestingly, some of the patients known to react against neurons (TMF, MRVM, ATR, TJMB) are positive against Schwann cells too, although not the stronger ones.
The only patient with NF155 antibodies (LAC) does not bind to Schwann cells.
CONCLUSIONS
We will start with GDP and a control for IP.
NOTES
Experiments performed by Fina in two different days
There are several patients that need to be repeated or done (see database)
