Microbiology, 2021

844 20 • Laboratory Analysis of the Immune Response
Figure 20.30
In this test for antinuclear antibodies (ANA), cells are exposed to serum from a patient suspected of making ANA and then
to a fluorescent mAb specific for human immunoglobulin. As a control, serum from a healthy patient is also used. Visible fluorescence
around the nucleus demonstrates the presence of ANA in the patient’s serum. In the healthy control where lower levels of ANA are
produced, very faint green is detected. (credit left, right: modification of work by Al-Hussaini AA, Alzahrani MD, Alenizi AS, Suliman NM,
Khan MA, Alharbi SA, Chentoufi AA)
CHECK YOUR UNDERSTANDING
• In an indirect fluorescent antibody test, what does the fluorescent antibody bind to?
• What is the ANA test looking for?
Flow Cytometry
Fluorescently labeled antibodies can be used to quantify cells of a specific type in a complex mixture using
flow cytometry (Figure 20.31), an automated, cell-counting system that detects fluorescing cells as they pass
through a narrow tube one cell at a time. For example, in HIV infections, it is important to know the level of
CD4 T cells in the patient’s blood; if the numbers fall below 500 per μL of blood, the patient becomes more
likely to acquire opportunistic infections; below 200 per μL, the patient can no longer mount a useful adaptive
immune response at all. The analysis begins by incubating a mixed-cell population (e.g., white blood cells from
a donor) with a fluorescently labeled mAb specific for a subpopulation of cells (e.g., anti-CD4). Some
experiments look at two cell markers simultaneously by adding a different fluorogen to the appropriate mAb.
The cells are then introduced to the flow cytometer through a narrow capillary that forces the cells to pass in
single file. A laser is used to activate the fluorogen. The fluorescent light radiates out in all directions, so the
fluorescence detector can be positioned at an angle from the incident laser light.
Figure 20.31 shows the obscuration bar in front of the forward-scatter detector that prevents laser light from
hitting the detector. As a cell passes through the laser bar, the forward-scatter detector detects light scattered
around the obscuration bar. The scattered light is transformed into a voltage pulse, and the cytometer counts a
cell. The fluorescence from a labeled cell is detected by the side-scatter detectors. The light passes through
various dichroic mirrors such that the light emitted from the fluorophore is received by the correct detector.
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