Microbiology, 2021

842 20 • Laboratory Analysis of the Immune Response
Figure 20.28
A green fluorescent mAb against L. pneumophila is used here to visualize and identify bacteria from a smear of a sample
from the respiratory tract of a pneumonia patient. (credit: modification of work by American Society for Microbiology)
LINK TO LEARNING
Watch the animation (https://openstax.org/l/22dirfluorant) on this page to review the procedures of the direct
fluorescent antibody test.
CHECK YOUR UNDERSTANDING
• In a direct fluorescent antibody test, what does the fluorescent antibody bind to?
Indirect Fluorescent Antibody Techniques
Indirect fluorescent antibody (IFA) tests (Figure 20.29) 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 20.30). The titer 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 titer must be at least 1:40 in the presence of symptoms involving two or more organ systems to be
considered indicative of SLE. 11
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