Fluorescent Antibody Techniques JABU

Fluorescent Antibody Techniques
Learning Objectives
 Describe the benefits of immunofluorescent antibody assays in comparison to

nonfluorescent assays
 Compare direct and indirect fluorescent antibody assays
 Explain how a flow cytometre 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 Figures 2.19-2.20.
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 21.28). 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.
Indirect Fluorescent Antibody Techniques
Indirect fluorescent antibody (IFA) tests (Figure 21.29) are used to look for antibodies in patient
serum. For example, an IFA test for the diagnosis of syphilis uses T. pallidum cells isolated from
a lab animal (the bacteria cannot be grown on lab media) and a smear prepared on a glass slide.
Patient serum is spread over the smear and anti-treponemal antibodies, if present, are allowed to
bind. The serum is washed off and a secondary antibody added. The secondary antibody is an
antihuman immunoglobulin conjugated to a fluorogen. On examination, the T. pallidum bacteria
will only be visible if they have been bound by the antibodies from the patient’s serum.
The IFA test for syphilis provides an important complement to the VDRL test discussed in
Detecting Antigen-Antibody Complexes. The VDRL is more likely to generate false-positive
reactions than the IFA test; however, the VDRL is a better test for determining whether an
infection is currently active.
IFA tests are also useful for the diagnosis of autoimmune diseases. For example, systemic lupus
erythematosus (SLE) (see Autoimmune Disorders) is characterized by elevated expression
levels of antinuclear antibodies (ANA). These autoantibodies can be expressed against a variety
of DNA-binding proteins and even against DNA itself. Because autoimmunity is often difficult
to diagnose, especially early in disease progression, testing for ANA can be a valuable clue in
making a diagnosis and starting appropriate treatment.
The IFA for ANA begins by fixing cells grown in culture to a glass slide and making them
permeable to antibody. The slides are then incubated with serial dilutions of serum from the
patient. After incubation, the slide is washed to remove unbound proteins, and the fluorescent
antibody (antihuman IgG conjugated to a fluorogen) added. After an incubation and wash, the
cells can be examined for fluorescence evident around the nucleus (Figure 21.30). The titre of
ANA in the serum is determined by the highest dilution showing fluorescence. Because many
healthy people express ANA, the American College of Rheumatology recommends that the titre
must be at least 1:40 in the presence of symptoms involving two or more organ systems to be
considered indicative of SLE.[1]

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Fluorescent Antibody Techniques

 Describe the benefits of immunofluorescent antibody assays in comparison to

Direct Fluorescent Antibody Techniques

Indirect Fluorescent Antibody Techniques

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