Antibody titering for flow cytometry

Richard C. Harmon cytact at netcom.com
Sun Oct 9 16:00:56 EST 1994


John.........Daudi has been reported to be Fc receptor positive, so this 
seems to be a good starting point.  If your MAb is allo-specific, you 
could use antigen-negative BLCL lines or monocytes from antigen-negative 
donors.  Would hate to show ignorance in public like this, but, yes, I 
believe that aggregated murine IgG will give you problems with human Fc 
receptors.  Finally, I would also not be surprised if monkey EBV 
transformants were also Fc receptor positive.  A quick medline hunt, 
focusing on human BLCL, should help.  If your MAb is against a framework 
determinant, then your class I negative Daudi, if confirmed Fc receptor 
positive, should control for any concern regarding aggregates.  Good 
luck.  Rick

(p.s. you gave us the troublesome profile for your reagent with the 
monkey cells, but you did not discuss the results with Daudi.  Are they 
clean?)

: 	 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
-- 
-- 
--                                             cytact at netcom.com



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