Microbiology, 2021

20.5 • Fluorescent Antibody Techniques 841
In addition to false negatives, false positives can also occur, usually due to previous infections with other
viruses that induce cross-reacting antibodies. The false-positive rate depends on the particular brand of
test used, but 0.5% is not unusual. 10 Because of the possibility of a false positive, all positive tests are
followed up with a confirmatory test. This confirmatory test is often an immunoblot (western blot) in which
HIV peptides from the patient’s blood are identified using an HIV-specific mAb-enzyme conjugate. A
positive western blot would confirm an HIV infection and a negative blot would confirm the absence of HIV
despite the positive ELISA.
Unfortunately, western blots for HIV antigens often yield indeterminant results, in which case, they neither
confirm nor invalidate the results of the indirect ELISA. In fact, the rate of indeterminants can be 10–49%
(which is why, combined with their cost, western blots are not used for screening). Similar to the indirect
ELISA, an indeterminant western blot can occur because of cross-reactivity or previous viral infections,
vaccinations, or autoimmune diseases.
• Of the 1300 patients being tested, how many false-positive ELISA tests would be expected?
• Of the false positives, how many indeterminant western blots could be expected?
• How would the hospital address any cases in which a patient’s western blot was indeterminant?
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20.5 Fluorescent Antibody Techniques
Learning Objectives
By the end of this section, you will be able to:
• Describe the benefits of immunofluorescent antibody assays in comparison to nonfluorescent assays
• Compare direct and indirect fluorescent antibody assays
• Explain how a flow cytometer can be used to quantify specific subsets of cells present in a complex mixture
of cell types
• Explain how a fluorescence-activated cell sorter can be used to separate unique types of cells
Rapid visualization of bacteria from a clinical sample such as a throat swab or sputum can be achieved
through fluorescent antibody (FA) techniques that attach a fluorescent marker (fluorogen) to the constant
region of an antibody, resulting in a reporter molecule that is quick to use, easy to see or measure, and able to
bind to target markers with high specificity. We can also label cells, allowing us to precisely quantify particular
subsets of cells or even purify these subsets for further research.
As with the enzyme assays, FA methods may be direct, in which a labeled mAb binds an antigen, or indirect, in
which secondary polyclonal antibodies bind patient antibodies that react to a prepared antigen. Applications
of these two methods were demonstrated in Figure 2.19. FA methods are also used in automated cell counting
and sorting systems to enumerate or segregate labeled subpopulations of cells in a sample.
Direct Fluorescent Antibody Techniques
Direct fluorescent antibody (DFA) tests use a fluorescently labeled mAb to bind and illuminate a target
antigen. DFA tests are particularly useful for the rapid diagnosis of bacterial diseases. For example,
fluorescence-labeled antibodies against Streptococcus pyogenes (group A strep) can be used to obtain a
diagnosis of strep throat from a throat swab. The diagnosis is ready in a matter of minutes, and the patient can
be started on antibiotics before even leaving the clinic. DFA techniques may also be used to diagnose
pneumonia caused by Mycoplasma pneumoniae or Legionella pneumophila from sputum samples (Figure
20.28). The fluorescent antibodies bind to the bacteria on a microscope slide, allowing ready detection of the
bacteria using a fluorescence microscope. Thus, the DFA technique is valuable for visualizing certain bacteria
that are difficult to isolate or culture from patient samples.
10 Thomas, Justin G., Victor Jaffe, Judith Shaffer, and Jose Abreu, “HIV Testing: US Recommendations 2014,” Osteopathic Family
Physician 6, no. 6 (2014).