ICC on NF155 transfected HEKs on Multiple Sclerosis patients (CSF samples 45-54 and repeated serum samples)

OBJECTIVES

To determine the existence of anti-NF155 antibodies in a subset MS patients.

SAMPLES

CSF

1. (45) 192-01. Isabel M. López.

2. (46) 196-05. Concepción Ciurana.

3. (47) 197-01. Mieria Costa.

4. (48) 197-02. Michael Labatzki.

5. (49) 197-05. Víctor Pérez.

6. (50) 197-19. Olga Teixidó.

7. (51) 198-11. David Poca.

8. (52) 199-09. Beatriz Núñez.

9. (53) 201-10. Pascual Meléndez.

10. (54) 201-14. Joan Bernal.

Serum

11. 109

12. 111

13. 114

14. 115

15. 171

16. 214

17. 215

18. 221

19. Negative control (Edu serum)

20. Positive control (15-195)

MATERIALS

• Human NF155-transfected HEKs 293 grown in coverslips. Fixed with PFA 4% and blocked with GOAT serum 5% in PBS.

• PBS.

• Blocking: GOAT 5% in PBS.

• Patient’s CSF diluted 1/1 with GOAT 5% in PBS. Control sera diluted 1/100 with GOAT 5% in PBS.

• Primary Ab: Chicken anti-NF155: AF3235 (1/1000).

• Secondary Abs: GAH 594 (1/1000) + GAC 488 (1/1000).

• Vectashield mounting medium.

PROCEDURE

• NF155-transfected HEKs were grown and fixed on coverslips and frozen at -80ºC. They where thawed before the ICC with Goat Serum 5%.

• Added patient’s CSF at 1/1 dilution in Goat Serum 5% and incubated 1h at RT. Sera were diluted at 1/100.

• Washed 3 times with water.

• Added primary commercial anti-NF155 Ab: AF3235 at 1/1000 dilution and incubated 1h at RT.

• Washed 3 times with water.

• Added secondary Abs: GAH 594 and GAC 488 1/1000 each and incubated 1h at RT.

• Washed 3 times with water.

• Added Vectashield mounting medium and put onto microscope slides.

• The slides were observed with a fluorescense microscope to determine wether or not the patients’ CSF presented anti-NF155 antibodies.

RESULTS

1. Negative.

2. Negative.

3. Negative.

4. Negative.

5. Negative.

6. Negative.

7. Negative.

8. Negative.

9. Negative.

10. Negative.

11. Non-visible or non-tinted cells.

12. Negative.

13. Negative.

14. Negative.

15. Non-visible or non-tinted cells.

16. Negative.

17. Negative.

18. Negative.

19. Negative.

20. Positive.