20170116: CNTN-1+CASPR-1 IgG ICC in paraneoplastic neuropathies

CNTN-1+CASPR-1 IgG ICC IN HEK 293 TRANSFECTED CELLS IN PARANEOPLASTIC NEUROPATHIES

Objectives

To test sera IgG positivity towards human CNTN1+CASPR1 complex in HEK 293 transfected cells.

Materials

• Human CNTN-1+ CASPR-1-transfected HEKs 293 grown in coverslips. Fixed with PFA 4% and blocked with RABBIT serum 1/40 in PBS.
• PBS
• Blocking: RABBIT 1/40 in PBS
• Patient’s sera diluted 1/100 with RABBIT 1/40 in PBS
• Primary Ab: Goat CNTN-1 (AF904 ) (1/1000)
• Secondary Abs: RAG 488 (1/1000)+ RAH 594 (1/1000)
• Vectashield mounting medium

Patients

• A1) 95-43
• A2)94-292
• A3) 97-543
• A4) 93-40
• A5) 92-147
• A6) 94-55
• B1) 93-38
• B2) 92-133
• B3) 01-848
• B4) 00-1289
• B5) 98-14
• B6) 03-1167
• C1) 04-2
• C2) 13-1127
• C3) 15-2420
• C4) 15-1131
• C5) 11-1167
• C6) 00-307
• D1) 15-2373
• D2) 08-530
• D3) 00-1248
• D4) 90-92
• D5) 04-726
• D6) 92-38
• E1) 98-164
• E2) 03-838
• E3) 05-409
• E4) 04-1323
• E5) 16-3512
• E6) 01-203
• F1) 93-94
• F2) 97-506
• F3) 02-616
• ct MRVM

Methods

1. Transfected HEKS grown in coverslips were fixed and blocked with 1/40 Rabbit serum and frozen at -80ºC.
2. Thaw these cells with a little 1/40 Rabbit serum in PBS.
3. Perform ICC:
   1. Add patient’s sera 1/100 1h at RT.
   2. Wash 3 times with PBS
   3. Add primary Ab 1/1000 and incubate 1h at RT.
   4. Wash 3 times with PBS
   5. Add secondary Abs 1/1000 each  and incubate 1h at RT.
4. Wash 3 times with PBS
5. Mount with Vectashield mounting medium

Results

The experiment must be discarded since positive control MRVM did not stain CNTN1- CASPR1 complex, quite shockingly the same that happened in the 20170112 experiment with the HVH1 positive control. Since these assays share the secondary antibodies RAG488 (which stains OK in both of them) and RAH594 (the one that does not stain), our belief is that something must be wrong with the latter. In forthcoming experiments we will test the antibody efficacy. Samples will be repeated in future experiments.