Biology Reference
In-Depth Information
surface molecules is the relative insensitivity of this technique compared with
functional assays. The number of molecules required to be observed by ¯ow
cytometry has been estimated to be 2000±4000 molecules per cell (Loken and
Herzenberg, 1975), but activity assays require only a few hundred molecules to
be functionally active and signi®cant. For a large number of important mole-
cules, ¯ow cytometric analysis has been uninformative.
ENZYMATIC AMPLIFICATION STAINING (EAS) FOR CELL-SURFACE
MOLECULES
Cell-surface molecules can be detected with speci®c monoclonal antibodies
directly conjugated with a ¯uorochrome. This technique, called direct stain-
ing, is easy to perform because it involves only one step: incubation with the
labeled antibody and washing. Direct staining works well for molecules that are
abundantly expressed on the cell surface and for which there are high-a½nity
antibodies available. Nevertheless, many cell-surface molecules cannot be ade-
quately assessed by this technique.
Ampli®cation of the ¯uorescent signal for the detection of cell-surface mole-
cules by ¯ow cytometry has been a long-sought-after goal. The major mechanism
in current use for the ampli®cation of these signals has been the application of
multiple layers of receptor ligands with the ®nal layer labeled with ¯uorochrome.
This technique is generically known as indirect staining and represents an
ampli®cation over the staining obtained with ¯uorochrome-labeled primary
antibodies.
The simplest scheme for ampli®cation by layers is the use of a ¯uorochrome-
labeled anti-immunoglobulin ( Ig) binding to the Ig with speci®city for the
chosen cell-surface molecule. Ampli®cation is achieved by multiple labeled anti-
Ig binding to the bound primary antibodies, thereby increasing the number of
¯uorescent molecules ®xed to the cell surface. A similar technique involves the
use of biotinylated primary antibodies followed by avidin or streptavidin con-
jugated with the ¯uorochrome. Similarly, the ampli®cation results from multi-
ple, conjugated biotin molecules binding avidin or streptavidin conjugated with
multiple molecules of ¯uorochrome.
Indirect staining has been further developed to include additional layers.
Excellent results have been obtained with unconjugated primary antibodies
followed by a layer of biotinylated anti-Ig antibodies and then by avidin or
streptavidin conjugated to ¯uorochrome (Zola et al., 1990).
Another approach to the ampli®cation of the ¯uorescent signal has been the
development of new ¯uorochromes with brighter relative ¯uorescence intensi-
ties. Fluorescein isothiocyanate (FITC) was the ®rst ¯uorochrome used widely
for the analysis of cell surface molecules by ¯ow cytometry. Fluorochromes
with brighter relative ¯uorescence intensities include phycoerythrin ( PE) and
allophycocyanin.
Peridinin
chlorophyll
protein
is
another
commonly
used
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