we are getting false negative in virus samples. Please share your ideas.
Tried 0.5% chicken RBC, 0.75% guinea pig RBC and 0.4% chicken+guinea pig RBC. Also tried with young chicken blood. every time getting RBC precipitation in positive control wells.
pH and temperature is also okay.
using 'U' shaped cell culture plate (Nuclon)
protocol followed:
50μL PBS in each well, 50μL virus (influenza) in 1st well then serially diluted
finally 50μL RBC in each well.
If you could provide more data on which viruses are you testing. For example NDV or low pathogenic influenza virus, etc will be more helpful. However, If this is diagnostic sample which you expect contains hemagglutinating virus, I would recommend you to amplify your virus in susceptible host like embryonated chicken eggs in case of aforementioned viruses to get high titre sufficient to agglutinate Red blood cells.
Dear Nagy, I'm working on influenza virus. Sorry that I missed the point.
Okay, just try to do aforementioned trick and once you do the assay incubate at 4 c for 40 minutes. Also, try to use a new aliquots of your positive control.
Dear Nagy, virus titre is high enough I think. Will you please share what is the reason for 4°C incubation? Do I require a more long time at room temp. after 4 C for 40 mins?
Some influenza strains have high neuraminidase activity, so we prefer to incubate at 4 C for 40-60 minutes. However, once you add RBS in final step, you have to mix all wells gently and incubate at room temperature for at least 20-30 minutes will be enough.
I was not aware about this phenomena. So the steps will be, after virus dilution in PBS> RBC in each well> mix all wells > incubate at 4 C for 40-60 minutes > later at room temperature for 20-30 minutes .
Is that so or I misunderstood something? Pardon, if I make this confusing.
Hi Abdullah, simply you could follow these steps:
1. Add 50ul PBS to all wells (A1-A12)
2. Add 50ul of your stock virus in first well (A1), then mix well the virus with PBS for 4-5 times by pippetting then transfer 50 ul of well A1 to well A2, mix well and transfer 50 ul from well A2 to well A3, etc
3. Add 50 ul of 0.5-1% chicken RBC to all wells from A1 to A12 and mix all wells gently
4. seal the plate and incubate at 4 C for 40-60 minutes
5. Read the results by titling the plate with 45 angle
Good luck
Followed the above mentioned process but result still same. Now I am completely puzzled.
We are using 96 well cell culture plate, is that okay? Though when we tried this assay for the first time we got result in this plate but when tried to repeat facing problem since then.
Are you investigating human or avian influenza virus?
In recent years, human influenza virus especially H3, would give false-positive in HA assay. Human red blood cell would gives a better result.
Thank you all. We already identified the problem. It was due to the strain it's self.
We are using H3N2, which poorly bind with RBC.
Using a positive control along with negative control might help you to understand the assay is ok or not.
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