Week 14 - Virology

This is the 14th week already. For the past 4 week, I was at the Immunofluorscence section. This area is mainly on antigen and antibody detection. For antigen detection, it is to detect the particular antigen that is present on the cell. There are 2 methods: direct and indirect.
For direct method, a fluorescent-labeled primary antibody is added and it will react with the antigen. Thus, fluorescence is observed.
As for indirect method, it will employ the use of a specific primary antibody (unlabelled) and a labeled secondary antibody (labeled with fluorescence dye). Therefore, antigen present will bind to primary antibody. And when labeled secondary antibody (also known as conjugate) is added, it will then bind to primary antibody and thereby staining the positive cells. The positive cells will then fluorescence under the UV microscope.
This method is more specific, thus reduce non-specific binding.

As for the antibody detection, it is usually to detect the presence of antibodies in serum.
Commercial antigen slides will be used, which means that the slides are already fixed with the specific antigen. If the corresponding antibody is present, then it will bind to the antigen. Following that, conjugate labeled to secondary antibody is added and it will bind to the primary antibody. Fluorescence will be observed when viewed under the microscope.

Now, I will know talk about the detection of Cytomegalovirus antigen by using cytospin. This is the most common test that is requested for in IF section. The purpose of the test is to identify the lower matrix protein pp65 of cytomegalovirus in the white blood cells. The principle of this test is mentioned above (antigen detection by indirect method).

Cytospin processing for CMV antigen

Processing the blood

1. Transfer the blood (in EDTA tubes) into sterile plastic centrifuge tubes
2. Add separation solution or 6% dextran, ¼ of the total volume of blood to the blood.
3. Place the tube in 37 ̊ C waterbath to allow the separation to take place.
4. Transfer the upper layer of suspension ( contains serum, plasma and leucocytes) into another sterile plastic centrifuge tubes.
5. Centrifuge the tube at 300g for 10 minutes.
6. Discard the serum and add cold water to lyse the red blood cells.
7. Vortex to mix well before adding phophate buffered saline (PBS).
8. Centrifuge at 300g for 10 minutes again.
9. Discard the supernatant, leaving the cell pellet behind.
10. Add an appropriate amount of PBS to the cell pellet and mix well.
11. Determine the white blood cell concentration using the coulter counter.
    
    Generating the cytospin slides
12. Vortex the tube before inoculating 0.2ml into the cytospin funnel.
13. Balance the funnel in the cytocentrifuge container and spin at 800rpm for 5 minutes.
14. Remove slides and dry it on slide warmer before fixing.
    
    Fixing the slides
15. Fix the slides with in fixative solution for 10 minutes.
16. Rinse the slides 2 times with PBSA.
17. Immerse the slides in permeabilization solution for 5 minutes.
18. Rinse the slides 3 times with PBSA again.
19. Dry the slides on slide warmer.
    
    Staining the slides
20. Add monoclonal antibody using a dropper, ensure that it is well spread (Vortex mab before use).
21. Place the slides in a moist chamber and incubate at 37 ̊C incubator for 35 minutes.
22. Immerse the slides in PBS to wash for 10 minutes, before placing it on a shaker.
23. Dry the slides on the slide warmer.
24. Add conjugate to the slides using a dropper (Vortex before use).
25. Repeat step 21-23.
26. Add mounting fluid and mount it with a coverslip.
27. View the slide under fluorescence microscope and record the results

Separation solution or 6% dextran - Acts to separate out the plasma and red blood cells.
Fixative solution- Fix the cells on the slides
Permeabilization solution - Permeabilise to allow antibody to access to intracellular structures of the cells and maintain the morphological characteristics.
Moist chamber- to maintain the humidity condition for incubation


Possible problems encountered:

• The cytospin funnel and chamber were not assembled well. Hence, the cells were not properly fixed as a circle on the slide. This may interfere with the interpretation of results.
• White blood cell concentration is too low, due to high dilution with PBSA. The resolution is to centrifuge the tube again to obtain cell pellet. Then, thus time a smaller volume is added to dilute the cell pellet.
• Cross contamination with other sample. Hence, to minimize this, always work with 1 cell line. And keep the other chambers away when loading cell suspension for 1 sample (As 1 or few drops may splash into other chamber).

For more information on cytomegalovirus, please refer to this website;
http://www.dhpe.org/infect/cytomegalo.html (same as my previous 1st entry)
http://www.cdc.gov/cmv/

That’s what I have for now. Thanks for reading!!

Shihui
0607135A