20140530bis ICC NFASC IgG subclasses

Objective

The aim of this experiment is to determine the igG subclasse present in one positive sera for neurofascin.

Materials

• Transfected HEK cells with neurofascin grown in coverslips
• Patients sera
• Antibodies:
  • chicken-anti-neurofascin
  • mouse-anti-IgG1
  • mouse-anti-IgG2
  • mouse-anti-IgG3
  • mouse-anti-IgG4
  • GAC-488
  • GAH-594
  • GAM-594

Procedure

1. Take one coverslip per condition (5)
2. Prepare sera 1:40 in goat serum. Add a drop (40µl) of each preparation to the parafilm. Incubate for 1h
3. Wash 3 times with PBS
4. Incubate primary antibody chicken-anti-neurofascin 1:2000 together with subclasses Ab at 1:500 for 1h
5. Wash 3 times with PBS
6. Prepare secondary antibody: (1) GAH IgG-594 1:500 + GAC-488 1:500 (2-5) GAM-594 + GAC-488 1:500. Add a drop and incubate 1h.
7. Wash 3 times with H2O
8. Mount with Vectashield (with DAPI)

Results & conclusions

Photo 1.

Photo 2.

The autoantibodies against neurofascin of this patient are mainly IgG2.