Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo

What is titration of viral progeny?


  • Please log in to reply
8 replies to this topic

#1 Curtis

Curtis

    Metaller Scientist

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,112 posts
59
Excellent

Posted 11 July 2012 - 12:28 AM

Does any body know what titration of viral progeny means? A journal reviewer is asking me to include this in my manuscript. I work on viruses and the apoptosis they cause after infection.

#2 leelee

leelee

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 652 posts
53
Excellent

Posted 11 July 2012 - 12:32 AM

I'm confused...in what context? Are you saying that you don't typically titrate your virus before using it?

#3 Curtis

Curtis

    Metaller Scientist

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,112 posts
59
Excellent

Posted 11 July 2012 - 01:18 AM

I do HA assay with red blood cells.

Plaque assay is really difficult to do in our case. Although my virus leaves plaques in some certain cell type, it kills most other cell lines. therefore, the only titration I can do is HA test.

#4 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,666 posts
396
Excellent

Posted 11 July 2012 - 01:41 AM

Plaque assay is really difficult to do in our case. Although my virus leaves plaques in some certain cell type, it kills most other cell lines. therefore, the only titration I can do is HA test.

You can explain this in a rebuttal/covering letter for the re-submission. Potentially you could do some sort of kill curve where you work out the amount of virus that kills 50% of the cells by doing some sort of serial dilution onto (96 well) plated cells.

#5 leelee

leelee

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 652 posts
53
Excellent

Posted 11 July 2012 - 05:07 AM

Ok. So presumable you use the HA titre to determine that you are using the same amount of virus in each experiment yes?

I presume that you have mentioned this in your manuscript and the reviewer has a problem with it because it isn't a measure of infectious virus?

You can do as bob1 says, and do a TCID50 assay.

As for plaque assay, why not just use the cell line that your virus does cause plaques in then?

Which virus are you working with? What do other labs that use this virus typically use to quantify stocks?

#6 Curtis

Curtis

    Metaller Scientist

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,112 posts
59
Excellent

Posted 11 July 2012 - 08:41 AM

Leelee, i work on NDV. Most people do plaque assay, but their strains are not as deadly as mine.

Also, im not sure if it is technically correct to use the plaque assay titer that i obtained from Vero cells on human cancer cells, which in my case is HCT116. do you understand what im saying? some might argue that it's alright, but the nature and size of cells are different and i dont like that.

Bob1, thanks as usual mate, that is actually what im gonna do. im going to add some tcid50 data.

Sorry for lowercase words, im typing from mobile.

#7 leelee

leelee

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 652 posts
53
Excellent

Posted 11 July 2012 - 06:36 PM

I know what you mean- you feel that titrating a virus on a non-human line, then using it for experiments on a human line is bad, because the ability of the virus to infect Vero will differ from the ability to infect your human line.

I disagree that this is an issue. You have to think about what the plaque assay is for. I think you are over thinking it.

What you are measuring is the infectivity of that stock, in Vero in your case.
This then gives you a measure for the infectivity of your stock- so that your input dose for your experiments is the same. It doesn't matter if your virus infects the human lines differently to Vero, because the dose of virus that you put in will be the same- as determined by plaque assay.
I don't think I'm explaining this very well Posted Image

Anyway, the point is that using a measure of infectivity (such as TCID50 or plaque assay) will be preferable to HA assays where non-infectious and defective particles can skew your results.

#8 Curtis

Curtis

    Metaller Scientist

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,112 posts
59
Excellent

Posted 11 July 2012 - 08:12 PM

a friend of mine found that 1 HA = 3x10^5 pfu for Vero cells.

Now, we usually infect cells 30 HA for 10^5 cells. This is standard protocol. Maybe I can use pfu to satisfy him?

#9 leelee

leelee

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 652 posts
53
Excellent

Posted 11 July 2012 - 09:19 PM

Is this friend in your lab? Is this correlation with the same virus? Did he do several dilutions of virus to evaluate the nature of the relationship?

I don't think it is reasonable to simply say 1HA = 3x10^5 pfu , so therefore 30 HA = 9x10^6 pfu, the relationship might not be linear.

You could however, in my opinion, take your 30HA dose and perform a plaque assay on it. Then say that your 30 HA dose has a plaque assay titre of x.




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.