20170202: CNTN1 &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 & CNTN1+CASPR1 complex in HEK 293 transfected cells.

Materials

• Human CNTN-1 & 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

All patients will be tested for CNTN1+CASPR1 reactivity. Only patient 94-55 will be tested for CNTN1 alone.

• 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 HVH1 (positive cotrol for both CNTN1 & CNTN1+CASPR1)
• ct MRVM(weak positive cotrol for CNTN1+CASPR1)

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

All samples (except for positive controls) are negative including the 94-55 sample tested against CNTN-1 alone. Nonetheless, sample 15-2373 caught our attention since it featured some positivity that may be mistaken by background noise. To  discard or confirm this event, the experiment will be repeated including new technical conditions in which we will use non-transfected HEK cells as well as double transfected cells tested with anti-Caspr1 commercial antibody.