e-mail+to+Kathy+3-19-12


 * **Kurt Whittemore**  || Mon, Mar 19, 2012 at 5:32 PM ||
 * To: Kathryn Sykes  ||
 * || Well when I first saw your question, I thought we had already checked whether the peptide matches the mouse strain for sure. However, it looks like a major mistake was made. The Ova peptide motif does not match the MHC of our mouse strain. The Ova peptide (SIINFEKL) is a MHC Class I (H-2Kb) restricted peptide epitope as seen here:

@http://www.sigmaaldrich.com/catalog/product/sigma/s7951?lang=en&region=US

Our cell lines and mice were Balb/c which has the H-2Kd haplotype as seen here:

http://www.imgt.org/IMGTrepertoireMHC/Polymorphism/haplotypes/mouse/MHC/Mu_haplotypes.html#notes

How could such a major mistake have been made? I think I and the people working with me were focusing on a lot of other details at the time, and we didn't know enough to realize the importance of this issue. I also remember checking into some MHC haplotypes before as well, but that was to make sure that our cell lines matched our mouse strain rather than to also make sure that the mouse strain was appropriate for the peptide.

So now I think we have two major options. We could switch to a new mouse strain such as C57BL/6 which is H-2Kb and new cell lines (perhaps B16F10?) and redo the immunization as before. Alternatively, we could pick a different peptide from ovalbumin which would be compatible with the H-2Kd of Balb/c. I'm thinking it would be easier to change the mouse strain and cells rather than the peptide since changing the peptide might require other changes to the immunization scheme as well (different part of sequence in the plasmid, different part of protein in the protein boost, etc.).

What do you think is the best strategy to take next?

-Kurt

On Mon, Mar 19, 2012 at 10:19 AM, Kathryn Sykes  wrote: [Quoted text hidden] ||  ||


 * **Kathryn Sykes**  || Mon, Mar 19, 2012 at 5:35 PM ||
 * To: Kurt Whittemore  ||
 * || I agree.


 * From: ** Kurt Whittemore [mailto:Kurt.Whittemore@asu.edu]

**Sent:** Monday, March 19, 2012 5:32 PM

 **To:** Kathryn Sykes

 **Subject:** Re: ELISPOT problems

Well when I first saw your question, I thought we had already checked whether the peptide matches the mouse strain for sure. However, it looks like a major mistake was made. The Ova peptide motif does not match the MHC of our mouse strain. The Ova peptide (SIINFEKL) is a MHC Class I (H-2Kb) restricted peptide epitope as seen here: [Quoted text hidden] [Quoted text hidden] ||  ||


 * **Kurt Whittemore**  || Mon, Mar 19, 2012 at 5:37 PM ||
 * To: Kathryn Sykes  ||
 * || Okay so I guess fairly soon we'll need to decide how many mice to order, which strain (probably C57BL/6), and which cell lines we'll want to use.

-Kurt [Quoted text hidden] ||  ||