Biology Reference
In-Depth Information
EAS FOR INTRACELLULAR MOLECULES
Intracellular molecules are also important to assess by ¯ow cytometric analysis,
but the di½culty with ¯uorescent signals for these antigens is even more pro-
nounced than with surface molecules. Consequently, we have developed EAS
for intracellular molecules. CEM cells were stained for bcl-2 using a speci®c
murine monoclonal antibody. For the standard ampli®cation procedure, we
added an anti-murine Ig antibody conjugated with FITC. For EAS, we used a
kit from Flow-Amp Systems, Ltd., (Cleveland, OH) which included the same
¯uorochrome for detection. Although the standard technique gave only mar-
ginal staining of bcl-2, we obtained excellent ampli®cation of the ¯uorescent
signal with EAS ( Fig. 17.3). The mean ¯uorescence channel in the sample treated
with an isotype control was 2 in the standard ampli®cation method, whereas
the mean ¯uorescence channel in the sample treated with the speci®c anti-bcl-2
monoclonal antibody was 3. In contrast, EAS gave mean ¯uorescence channels
of 2 and 142 for the isotype control and the speci®c monoclonal antibody.
Thus, EAS gave an approximate fold enhancement of approximately 140
compared with the standard ampli®cation procedure.
We have succeeded in amplifying signals for organellar, cytoplasmic, and
nuclear antigens. Similar to the situation with cell-surface molecules, ampli®-
cation of the signal from more than 10 antigens has been achieved without en-
countering a single molecule whose signal could not be ampli®ed.
USES OF EAS IN THE INVESTIGATION OF HIV-1 INFECTION
Flow cytometric analysis has played an important role in the investigation of
infection by human immunode®ciency virus type I ( HIV-1) from the beginning
of the epidemic to the present time. In the initial characterization of the infec-
tion, ¯ow cytometry was important in recognizing the selective loss of CD4
T
lymphocytes from the peripheral blood. Analysis of the cell surface of mono-
cytes revealed low-level CD4 expression and thereby explained the potential for
these cells to be infected by the virus. Investigations of various induced cell
surface molecules on CD8
T cells demonstrated a high level of expression of
activation antigens, indicating the responsiveness of these cells to the virus. More
recently, the availability of GFP-expressing recombinant HIV-1 has provided
an important research tool for the identi®cation of infected cells. Moreover, the
construction of tetramers of MHC class I molecules and HIV-1 peptides has
demonstrated the large proportion of CD8
T cells from the peripheral blood
of infected persons that have speci®city for the virus.
Nevertheless, there are many circumstances in the investigation of HIV-1
disease with ¯ow cytometric analysis that may be clari®ed with the enhanced
resolution of EAS.
