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¯uorochrome that is less bright than FITC. Conjugate ¯uorochromes, such as
PE-cyanin 5 and PE-Texas Red among others, have been developed to enhance
the ¯uorescent signal that can be obtained. The use of brighter ¯uorochromes
has increased the capacity to distinguish positive and negative cells, although
the e¨ect is only in the range of a ®ve-fold enhancement.
We have used a di¨erent approach to enhance the ¯uorescent signal in ¯ow
cytometry. Harnessing the potential of enzymes to amplify signals, we devel-
oped an enzymatically catalyzed reporter deposition system for ¯ow cytometric
analysis of cell-surface receptors ( Kaplan and Smith, 1999). A similar system
had been developed for immunoassays, immunohistochemistry, and in situ hy-
bridization but had not been established for ¯ow cytometry (Bobrow et al.,
1989, 1991; van Gijlswijk et al., 1997). The system involves the conjugation of
horseradish peroxidase (HRP) to the primary antibody. Alternatively, uncon-
jugated primary antibodies or biotinylated primary antibodies can be used with
HRP-conjugated anti-Ig or HRP-conjugated streptavidin, respectively. After
HRP is localized on the surface of cells expressing the speci®c molecule of
interest, peroxide is added with a labeled molecule, tyramide. In the presence of
the enzyme with its substrate, it has been postulated that the phenolic com-
pound, tyramide, forms a short-lived, highly reactive free radical that is able to
react covalently with nearby electron-rich moieties such as tyrosines or trypto-
phans on the cell surface. Because the free radical does not persist long enough
to di¨use away from the cell surface where it is formed, only the cell that
has bound the enzyme is labeled with the tyramide. It should be noted that the
labeled tyramide does not simply bind to the targeted molecule. Instead, it also
binds to other molecules in the vicinity of this targeted molecule.
We have adapted this system of tyramide deposition for ¯ow cytometry and
have found that it can provide excellent enhancement of the ¯uorescent signal.
CEM cells from a human T cell tumor line, were stained for surface expression
of CD5 either by a standard ampli®cation procedure (indirect staining) or by
EAS. CD5 is a 67-kD surface glycoprotein of the scavenger receptor cysteine-
rich family. It appears on T lymphocytes early in their development and is
abundantly expressed on all mature T cells. It should be noted that the enzy-
matic ampli®cation procedure di¨ered from the standard procedure only by the
inclusion of the enzyme-catalyzed reporter deposition steps. The primary anti-
bodies and the ¯uorochromes were identical in both procedures. The results
demonstrate that EAS gave a marked ampli®cation of the ¯uorescent signal
compared with standard ampli®cation staining (indirect staining) ( Fig. 17.1).
The mean channel numbers for the cells stained with the speci®c anti-CD5
monoclonal antibodies were 7 for the standard ampli®cation method and 751
for EAS. Cells stained with control Ig demonstrated mean channel numbers of
3 for the standard procedure and 4 for EAS. Thus, in terms of mean channel
separation (speci®c antibody vs. isotype control antibody), EAS enhanced the
¯uorescent signal approximately 140 times over the standard technique. Similar
experiments with cells from Molt-4 and HUT-102, other T-cell tumor lines
