Antibody titering for flow cytometry

[Richard C. Harmon]

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

John Ladasky 
(ladasky at leland.Stanford.EDU) wrote: : Greetings, everyone,

: 	Using a flow cytometer and an antibody at various dilutions, I've
: just analyzed the fluorescence intensity of a particular cell-antibody 
: combination.  I now have a nice, asymptotic curve.

: 			    Mean
: 	ug MAb added	Fluorescence
: 	----------------------------
: 	    0.00	       8
: 	    0.63	     364
: 	    1.25	     541
: 	    2.50	     812
: 	    5.00	    1050
: 	
: 	Now what?  At what level is an antibody considered to be "titered"?
: Any advice or references would be greatly appreciated.  Thanks!


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