Antibody titering for flow cytometry

[John Ladasky]

In article <cytactCx9G3t.JxA at netcom.com>,
Richard C. Harmon <cytact at netcom.com> wrote:
>John........need more info.  I take it that the antibody has been 
>purified?  Does your cell type express Fc receptors?  Purification often 
>leads to aggregates which can and do bind to low affinity receptors for 
>IgG.  I have never been convinced that saturation of _any_ Ag requires 
>more than 1 ug/10E6 cells.  So.....what is the cell type that you are 
>testing, and have you titered the same reagent on Ag-negative cells?  If 
>you can find an Ag-negative cell _with_ Fc receptors, so much the 
>better.  Are the cells human?  Mouse?  Is your MAb conjugated, or are 
>you using a labeled secondary?  If your cells are mouse and if you're 
>using a direct conjugate or if your primary is not rat G2b, you can 
>often clean up an experiment by running it in the presence of MAb 2.4G2 
>(anti-CD32/CD16).  Need more info!  Cheers, Rick

	Thanks for a thought-provoking response, Richard.

	The antibody I'm using is an unlabeled, purified mouse monoclonal,
which is known to react to human class I MHC.  I'm using a goat anti-mouse
IgG FITC conjugate for my fluorescent second step antibody.  The cell line
I've used for the titration is an EBV-transformed B cell line from a New
World monkey (these are the cells I'm studying, so don't tell me something
like "you should titer with a human cell line.").  As a negative control, I
have been using Daudi cells (a human lymphoma-derived cell line which does
not express beta-2 microglobulin, and thus no Class I MHC).  

	I like your idea about using anti-Fc receptor antibodies to block
non-specific staining, but I'm not sure whether it's necessary.  Do mouse
antibodies cross-react with human Fc receptors?  I don't expect the monkey
cells to express Fc receptors, but I'm not so sure about Daudi.

-- 
Unique ID : Ladasky, John Joseph Jr.
Title     : BA Biochemistry, U.C. Berkeley, 1989
Location  : Stanford University, Dept. of Structural Biology, Fairchild D-105
Keywords  : immunology, music, running, Green