Method To Detect Antigen Number by Flow Cytometry

Dick Haugland dick at
Sun Jan 19 12:54:51 EST 1997

Antigen number is usually determined by comparison with standard
microspheres such as are available from Flow Cytometry Standards (in
Puerto Rico) and us (Molecular Probes).  Flow Cytometry Standards
defines this in terms of "soluble fluorescein equivalents."  However, we
have intentionally not done that for our microsphere standards because
to compare the number that is quoted for the microsphere with the
intensity (mean channel) in the flow cytometer and say that equal
intensities mean equal number of receptors requires one to assume that
the quantum yield of the dye on the microsphere and on the antibody is
identical.  However, we know that the quantum yield of fluorescein on
antibodies (usually about 0.2 to 0.3 at DOS of 4-5 dyes per antibody) is
much less than that of pure, unconjugated fluorescein (QY = 0.92), which
is apparently used to calibrate the microspheres.  Furthermore, the
degree of substitution of the fluorescein on each antibody is also not
known and is definitely not 1:1 so there is not a direct correlation
between the fluorescence, the antibody number and the number of
antigens.  One MAY be able to do the experiment by determining the
number of unquenched fluorescein equivalents of your specific antibody
by measuring its total fluorescence yield against a fluorescein standard
then comparing this with microspheres that have been measured against
the same fluorescein standard.  However, it is also known that
antibodies with lower DOS tend to bind better than those with higher DOS
so the cell binding may select out the conjugates from within the
mixture used for the staining so that even this correlation is probably
not completely valid.  One has a better possibility of assessing the
RELATIVE number of receptors on different cells using the same antibody
and same microspheres that the absolute numbers.

Related to your affinity question:  the affinity of fluorescent
antibodies (especially with different DOS) may be different than that of
the unlabeled antibody.  The affinity of antibodies has been estimated
on microspheres using a system that used to be sold by Pandex (now
someone else).  I am sure other people know more about this system than
I do.

Dick Haugland
dick at

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