Guys please help me out. I was given the protocol below for inactivation of FLU A Pandemic 2009 H1N1 for the HA assay but I get no agglutination on the HA assay.
For step 1. I take 4ml of supernatant, split into 8x500ul and add 500ul of the HEPES-PBS to give 8x1ml. This is pooled into 2x4ml for 2 centrifuge tubes. I re-suspend the 2 pellets in 2ml PBS
Can you pros please advise me on the following:
1. Dilute the virus in PBS containing 100mM Hepes buffer pH7.2 (strong buffer is required as the proiolactone decreases the pH on reaction)
2, Add 50ml beta propiolactone to 200ml water (this does not store)
3. Add 8.3ml this mixture per ml virus suspension with rapid mixing. (final propiolactone concentration 0.166%)
4. Incubate at 33 deg C for 45 min.
5. Pellet virus by spinning at 100,000g 20 min (TL100).
6. Resuspend virus in buffer of choice.
7. Check for inactivation either by serial blind passages in eggs (egg grown viruses) or serial plaque assays (cell grown virus).
*****The inactivated virus doesn't grow and the control does.
I think perhaps the centrifugation time is far too short and perhaps the input volume of supernatant might be too low, but I don't have enough to keep playing around
Really appreciate any advice
Cheers
Mark Q
Hi Mark
You don't say whether you can detect HA activity before you inactivate the virus. If you can, my guess would be that the pH is not being controlled well enough.
The other question is "why"? If you have a BSL2 facility to do the inactivation in, why not simply do the HAI assays with live virus?
Sometimes while inactivating with BPL or Formalin,
Nodoubt, the virus gets inactivated but the its HA titre reduces to nil.
Probably, the surface modification gets to such an extent that RBCs are not able to bind the virus.
Try inactivating again
Reduce the concentration of Beta propiolactone to 0.05%. If it doesnt inactivate, they try 0.1%.
After adding BPL to virus dont agitate too much..
Virus inactivation as per my experience is random. Sometimes it works but sometimes we have to repeat. I am also trying to figure out the reasons
Thanks suspect
Just a few more questions. What about the volume of supernatant I used, is it enough
do you perform a spin step around 10000rpm for 30 minutes prior to inactivation to remove cell debris
At what temp and for how long do you incubate with BPL.
Do you resuspend the pellet in PBS and leave inches fridge overnight to remove clumping
Really appreciate your help. I just want a complete reliable protocol if you have one
Mark
I suggest that you perform a spin step at 6,000 g for 30 minutes to remove cells debris from cell cultures or embryonated eggs before inactivation.
But, I think that the major problem is the ultracentrifugation after inactivation. I have always pelleted influenza viruses by spinning at 80,000 g for 60 minutes. I think you will have success in this way.
J Nelson Couceiro
the problem might be in hemagglutination of influenza virus, some influenza viruses do not agglutinate (agglutinate poorly) or agglutinate only specific RBC (turkey/chicken/human/horse, guinea pig), does your virus agglutinate as a alantoic fluid or medium ? We have pdm09 strain which agglutinate poorly
Hi Mark
You don't say whether you can detect HA activity before you inactivate the virus. If you can, my guess would be that the pH is not being controlled well enough.
The other question is "why"? If you have a BSL2 facility to do the inactivation in, why not simply do the HAI assays with live virus?
Mark I have protocol from T. Rove in our CDC flu team if you need
From my Poxvirus work I remember that not all birds agglutinate poorly
Before inactivation, I ensure that BPL and virus (Allantoic fluid) are at same temperature. Then after adding 0.05% BPL, i gently rock (very slowly- around 1 rocking/min) at 37 deg for 2 hrs. Then i keep at 4 deg overnight for BPL hydroylsis.
If virus doesnt inactivate with 0.05, try increasing BPL to 0.1%
I confirm the HA titre before and after putting BPL. In my case, most of the times i will get one log drop in HA titre after inactivation. But sometimes I observed that HA titre becomes nil after inactivation.
Now if you want to purify, you can proceed with ultracentrifugation. I use 30% sucrose for virus pelleting followed by overnight keeping at 4 deg (let the pellet loosen itself) ad resuspend the pellet in TEN buffer (tris EDTA NaCl)
But before virus pelleting you have to clarify it. You can perform spinning to remove the cell debris
Good luck!
Thanks a lot guys, you given me polenty of options
All the best
I`ll keep you posted
Jaroslav
I have pdm09 and using turkey rbcs, which according to the literature should work
Which rbcs do you use
Cheers
Has anyone used the bead purification kit from oz bioscienceS?
It didn't work either for me
Vladimir
Do you ultra-centrifuge after inactivation.I presume you need to if the virus is for haemagglutination assay?
Can I use HEPES buffer instead of DSP and sodium bicarbonate?
Cheers
Mark
Hi Paul
Unfortunately there are two of us having to process 2300 samples
Would HEPES buffer control the pH well, that's what I've been using?
Hi Guys
Before trying to inactivate again changing the various parameters you kindly recommended, I tried using virus before inactivation and the titre was zero. I simply took fresh infected supernatant |(from MDCK cells), spun the cell debris down at 10,000 RPM for 30 minutes and used the supernatant directly. Should this work?
I`m using turkey red blood cells mixed 1:1 with Alsever`s solution and California/07/pdm09. The blood is from a company called seralab. From the literature turkey RBCs should be suitable?
On a more serious note, does anybody work in a lab in or around London and is inactivating virus and/or performing the HA or HAI assay, I`d really really appreciate it and of course pay for all reagents and your time ??????
Cheers
Mark
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