antigen density
Alice L. Givan (Alice.L.Givan@Dartmouth.EDU)
12 Jun 95 11:03:20 EDT
With regard to Edgar Milford's and Mario Roederer's important comments: good
standardization of the instrument, proper compensation, and saturating
antibody reagents, still allow you to come up with only *relative* antigen
density but not absolute antigen density. Often this is good enough, but
should not be confused with any knowledge about the actual number of antibody
binding sites. Absolute antigen density is much harder to determine as the F/P
ratio determined spectrophotometrically for antibodies does not apply when the
antibodies are bound to cells. MESF (molecular equivalents of soluble
fluorochrome) for a given conjugated antibody bound to a cell is NOT the same
as the actual number of bound fluorescein molecules. As I recall, the
fluorescence of a fluorescein molecule may decrease to one third of its value
when it is bound to a cell. Even more difficult is the fact that
MESF-calibrated beads from different suppliers (and even from the same
supplier...) don't give the same channel calibration values for MESF. This
is a can of worms which perhaps this network may want to discuss at some
time.... If you really want to determine the number of binding sites, I think,
sadly, that it is necessary to go back to radioactive binding measurements.
See papers and ISAC abstracts by Poncelet for some comments on the subject (
his calibration system (for sale in France and Canada, but not yet the US) may
help as, I believe, it goes back to the foundation of radioactive
determinations).
Alice Givan
Englert Cell Analysis Laboratory
Dartmouth Medical School
Lebanon, NH 03756, USA
tel: 603-650-7907
fax: 603-650-6130
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