ICC on NF155 transfected HEKs on Multiple Sclerosis patients (CSF samples 23-44)

OBJECTIVES

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

SAMPLES

1. (23) 161-17. Maximiliano Yvañez.

2. (24) 167-16. Joaquim Maristany.

3. (25) 168-13. Ana M. Acevedo.

4. (26) 169-14. Montserrat Pardo.

5. (27) 171-10. Venacio J. Rodríguez.

6. (28) 172-02. Mirna Peguero.

7. (29) 174-17. Lorena Palet.

8. (30) 175-02. David Mignorance.

9. (31) 175-16. Isabel Valiente.

10. (32) 176-18. David Ramos.

11. (33) 178-10. Silvia Gil.

12. (34) 179-08. Montserrat Quer.

13. (35) 180-16. Mónica Castro.

14. (36) 181-01. Milagros Oliveras.

15. (37) 181-10. Mª Carmen Vera.

16. (38) 186-04. Amelia Gallant.

17. (39) 190-14. Vanesa Carrasco.

18. (40) 191-07. Elisabeth Font.

19. (41) 192-17. Elena Rebollo.

20. (42) 192-18. Eva Ballesteros.

21. (43) 194-02. Carmen Berga.

22. (44) 194-10. Mª Jesús Torres.

23. Negative control (Edu serum)

24. 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. Control sera were added at 1/100 dilution.

• 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. Faint dot-like pattern.

4. Negative.

5. Negative.

6. Negative.

7. Negative. Strong nuclear pattern.

8. Negative.

9. Negative.

10. Negative.

11. Negative. Faint nuclear pattern.

12. Negative.

13. Negative.

14. Negative.

15. Negative.

16. Negative.

17. Negative.

18. Negative.

19. Negative.

20. Non-visible or non-tinted cells. This sample will have to be repeated.

21. Negative.

22. Positive.