From: Alice L. Givan (Alice.L.Givan@Dartmouth.EDU)
Date: Mon Jun 12 1995 - 10:03:20 EST
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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