Detection

IFA Protocol for Detection of Rubella Virus in Clinical Specimens

This is an indirect immunofluorescence assay using commercially available monoclonal antibody(ies) to rubella virus virion proteins, E2 and C. Reagents are very important since rubella virus does not produce large amounts of antigen. Highly cross-adsorbed fluorescent antibody works very well.

1. Grow Vero cells to about 50% confluence in chambered slides, e.g. Lab-Tek Chamber Slide (catalogue number 177445). Typically this is done in DMEM with 10% fetal calf serum (FCS) and gentamycin in 35 C, CO2 incubator.
2. Remove medium and add 100 ul of specimen (typically supernatant from 1 week culture of a clinical specimen on Vero cells) to one chamber and 100 ul of medium to control chamber. Add sufficient medium to both chambers to insure coverage of Vero cells. A known positive specimen (or lab virus) should also be included.
3. Incubate 1 hour in 35C, CO2 incubator, in order to allow virus attachment
4. Fill chambers with DMEM, 10 % FCS, containing penicillin and streptomycin.
   • Comment: Penicillin and streptomycin are used in place of gentamycin to insure against bacterial contamination. If monolayers of Vero cells are more than 50% confluent, reduce FCS to 5% so cells will not become overgrown. IFA will still work if cells are overgrown, but results will not be as pretty.
5. Incubate about 3 days in 35C, CO2 indubator.
   • Comment: If there is lots of virus present, medium in virus containing wells will typically become acidic.
   • Comment: Fixation of cells is by 2% paraformaldehyde at 0C. Cells are perneabilized with -20C methanol. This protocol also works with more fragile cell types.
6. Remove chambered slide and place on ice for 10 minutes; typically by laying slide on metal or foil strip in contact with ice.
7. Remove medium, wash 1X with cold PBS.
8. Add 2% paraformaldehyde in PBS for 30 minutes on ice.
9. Remove paraformaldehyde and wash 1X with cold PBS.
10. Add -20C methanol for 10 minutes to permeabilize cells.
    • Comment: This is easily done on lab bench by placing slide on -20C freezer pack, adding -20C methanol, and placing another -20C freezer pack on top of slide(s).
11. Remove methanol and wash 1X with room temperature PBS.
    • Comment: Fixed cells may be stored at this point, covered with PBS, at about 5C for at least 36 hours.
    • Comment: Blocking, antibody incubations, washes, and propidium iodide staining of nuclei are all done in 1% BSA, 0.5% FCS, 0.1% Tween 20 in PBS at room temperature. Final washes after propidium iodide are with PBS.
12. Block nonspecific antibody reactions by adding 1% BSA, 0.5% FCS, 0.1% Tween 20 in PBS for 1 hour at room temperature.
13. Remove block and add monoclonal antibody(ies) in block buffer. Incubate 1 hour at room temperature.
    • Comment: Typically this is a mix of two antibodies from Viral Antigens Incorporated, 26-24 to the E2 protein and 2-36 to the C protein, both at a 1/100 dilution (e.g. 10 ul 26-24 and 10 ul 2-36 to 1 ml block buffer).
14. Wash 2 (or more) times with block buffer, and then add second fluorescent antibody in block buffer. Incubate 30 minutes at room temperature;cover slide with foil to keep it in the dark.
    • Comment: Typically this is Alexa Fluor 488 goat anti-mouse IgG (H+L) “highly cross-adsorbed” from Molecular Probes, at a 1/100 dilution.
15. Wash 2 (or more) times with block buffer, and then add propidium iodide at 0.5-1 ug/ml in block buffer. Incubate 15+ minutes at room temperature in the dark.
16. Remove chambers and gasket from slide (chamber and gasket can also be removed before propidium iodide since all wells will get nuclei staining.
17. Wash 2 (or more) times with PBS, wick off excess PBS with “Chem Wipe” paper, and mount with mount made for fluorescent work (e.g. Dako Fluorescent Mounting Medium, #S3023) and cover slip(s).
    • Comment: Observe results with fluorescent microscope using blue light (e.g. Zeiss, Axiovert BlueH 485 filter). Antibodies will be green, propidium iodide stained nuclei will be red. There should be no background antibody staining in uninfected cell lawns, although there is sometimes some staining near edges of lawn, presumably due to gasket. If there is background antibody staining, most likely it is too much fluorescent second antibody. Utility of IFA test is dependent on background antibody staining being low, hopefully zero, in uninfected cells.

    Rubella antibody staining patterns should be punctate staining with C antibody throughout cytoplasm and intense staining of golgi with E2 antibody. Depending on how much virus was in innoculum, all cells may not be stained.

Note: Use of trade names and commercial sources in this protocol does not imply endorsement by the Centers for Disease Control and Prevention.

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