CNTN-1, CASPR-1, CNTN-1+CASPR-1, NF-140, NF-155, NF-186, CNTN-2, CASPR-2, CNTN-2+CASPR-2 ICC IN HEK 293 TRANSFECTED CELLS IN PARANEOPLASTIC NEUROPATHIES (15-2373)
Objectives
To test patient’s 15-2373 (paraneoplastic neuropathy) sera IgG positivity towards human CNTN-1, CASPR1, CNTN-1+CASPR-1, NF-140, NF-155, NF-186, CNTN-2, CASPR-2 & CNTN-2+CASPR-2 in HEK 293 transfected cells in . Sample’s background staining will be analyzed by testing it in non-tarnsfected HEK cells.
Patient
- Jesús Nicolás Martín Ibáñez; 15-2373 Hospital Clínic
Each ICC was performed with its due positive control: (no positive controls are available for CNTN-2 or CNTN-2+CASPR-2 ICCs)
- CNTN-1 & CNTN-1+CASPR-1: HVH1
- CASPR-1: 16-047 (BOX ICQ NUM.5)–> confirmation pending
- CASPR-2: 15-199
- NF-155 : LAC 15-195CASPR-2: 15-199
- NF-140 & NF-186: 16-069
MATERIALS & METHODS
CNTN-1 & CNTN-1+CASPR-1 ICC
- 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
CASPR-1 ICC
- Human CASPR-1-transfected HEKs 293 grown in coverslips. Fixed with PFA 4%, permeabilized and blocked with 5% goat serum in PBS.
- PBS
- Blocking: 5% goat serum in PBS
- Patient’s sera diluted 1/100 with5% goat serum in PBS
- Primary Ab: Mouse CASPR-1 (105571 ) (1/1000)
- Secondary Abs: GAM 488 (1/500)+ GAH 594 (1/500)
- Vectashield mounting medium
CNTN-2 & CNTN-2+CASPR-2 ICC
- Human CNTN-2 & CNTN-2+ CASPR-2-transfected HEKs 293 grown in coverslips. Fixed with PFA 4%, not permeabilized and blocked with 5% goat serum in PBS.
- PBS
- Blocking: 5% goat serum in PBS
- Patient’s sera diluted 1/100 with5% goat serum in PBS
- Primary Ab: Rabbit CNTN-2 (1/200)
- Secondary Abs: GAR 488 (1/500)+ GAH 594 (1/500)
- Vectashield mounting medium
CASPR-2 ICC
- Human CASPR-2-transfected HEKs 293 grown in coverslips. Fixed with PFA 4%, permeabilized and blocked with 5% goat serum in PBS.
- PBS
- Blocking: 5% goat serum in PBS
- Patient’s sera diluted 1/40 with5% goat serum in PBS
- Primary Ab: Rabbit CASPR-2 (33994 ) (1/500)
- Secondary Abs: GAR 488 (1/1000)+ GAH 594 (1/1000)
- Vectashield mounting medium
NF-140, NF-155 & NF-186 ICC
- 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
Non-transfected HEK ICC
- Human non-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
- Secondary Abs: GAH 594 (1/1000).
- Vectashield mounting medium
Results
- All of patient’s 15-2373 ICCs have proven to be negative in spite of their remarkable background, which presumably may be caused by the patient having a lymphoma.
- All postive controls are positive except for CASPR-1 ct 16-047 (BOX ICQ NUM.5), which turned out not to stain CASPR-1 at all. As with CNTN-2 and CASPR-2+CNTN-2, we do not have a CASPR-1 positive control.
