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> Dear Paul/Steve;
>
> I am looking for references detailing a procedure for the titration of
> monoclonal antibodies used for two and/or three color staining of
> lymphocyte subsets in whole blood.
We do a 2- or 3-fold dilution series, bind these dilutions to cells for 15
min at room temp, rinse, (then FITC-anti-IgG 15 min on ice if the antibody
is not directly conjugated, rinse), optionally fix, then analyze.
Generally for monoclonals the results nicely fit the Michaelis-Menten
1-site binding equation. Using a program which fits the data to the
equation (we use Fig P from Biosoft, 314-524-8029), you get the
concentration which half-saturates (Ch) and the maximum intensity at
theoretical saturation (Imax) in an objective way which uses all data
points. The equation then lets you predict that e.g. at a concentration of
4Ch, you'll get 80% of saturation. The equation is simply I = Imax (C / (C
+ Ch)), where I is the intensity observed at concentration C. Note that
you must use the antibody CONCENTRATION not dilution, e.g. convert 1/1,000
to 0.1%. (I don't have a reference for this approach, which we arrived at
ourselves, but I'd like to know a reference if someone else has seen it in
print.)
/*- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
Eric Martz, Professor of Immunology emartz@microbio.umass.edu
Dept Microbiology Voice: 413-545-2325 FAX: 413-545-1578
Morrill IVN 203, Box 35720, Univ Massachusetts, Amherst MA 01003-5720
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