20170111: NF140 NF155 & NF186 IgG ICC in paraneoplastic neuropathies

NF-140/NF-155/NF-186 IgG ICC IN HEK 293 TRANSFECTED CELLS IN PARANEOPLASTIC NEUROPATHIES

Objectives

To test sera IgG positivity towards human NF-140, NF-155 & NF-186 in HEK 293 transfected cells.

Materials

• Human NF-140/NF-155/NF-186-transfected HEKs 293 grown in coverslips. Fixed with PFA 4% and blocked with GOAT 5% in PBS.
• PBS
• Blocking: GOAT 5% in PBS
• Patient’s sera diluted 1/100 with GOAT 5% in PBS
• Primary Ab: Chicken NF155 (AF3235) (1/1000)
• Secondary Abs: GAC 488 (1/1000)+ GAH 594 (1/1000).
• Vectashield mounting medium

Patients

All patients were tested for IgG reactivity against NF-186 unless otherwise stated.

• 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
• F4) CT + NF-155 (LAC) (NF-155)
• G1) 93-94 (NF-140)
• G2) 97-506 (NF-140)
• G3) 02-616 (NF-140)
• G4) 93-94 (NF-155)
• G5) 97-506 (NF-155)
• G6)  02-616(NF-155)

Methods

1. Transfected HEKS grown in coverslips were fixed and blocked with GOAT 5% in PBS and frozen at -80ºC.
2. Thaw these cells with a little GOAT 5% 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

Sample 97-506 (NF-140) will be repeated due to sliding of the lamellae.

The rest of the samples are negative.