Re[2]: saturation binding & fluorescence intensity
James Weaver 301-594-5879 FAX 301-594-3037 (WEAVER@cder.fda.gov)
Wed, 16 Oct 1996 09:15:27 -0400 (EDT)
While the good Dr. Roederer is correct in that fluorescence
intensity is usually proportional to antigen density, there are
significant excepetions to this observation. When antigens are at a
high local concentration, there exists the possiblily for
significant non-linearity of fluorescence due either to
self-quenching as is observed with FITC, or due to energy transfer
possible with molecules with wide excitation & emmision spectra such
as PE. With multi-color labeling, the possibility for inter-label
energy transfer (eg FITC -> PE) could also occur.
I will freely concede that it does take very high antigen
density for this to occur. This is less likely for integral membrane
proteins but is clearly possible for antigens in membrane bound
systems such as secretory vesicles or lysosomes. It is also possible
to load DNA with sufficient amounts of fluorochrome for this to
occur. If memory is correct, there was a paper in Cytometry
concerning a method to quantitate DNA binding of daunorubicin by
measuring the decrease of HO33342 fluorescence due to energy
transfer between HO & DN. Therefore if a fluorochrome with
self-quenching was used, non-linear responses would clearly be
possible. While this is clearly not the case with PI, the
possibility exists with some othe labels or label combinations.
Under most circumstances the assumption of linearity will be
reasonable, however we need to be aware of the occasional
circumstances where the assumption will not be correct.
-Jim Weaver
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* James L. Weaver Ph.D. *
* Division of Applied Pharmacology Research *
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