ANTINUCLEAR ANTIBODIES AND TESTING
Section I. IMMUNOFLUORESCENT MICROSCOPY
Immunofluorescence is a method of detecting an antigen or antibody in tissue by
the pattern of fluorescence resulting when the tissue is exposed to the specific antibody
or antigen labeled with a fluorochrome such as fluorescein. The technique of
immunofluorescence was introduced by Coons and his associates in 1941.
Immunofluorescence, the use of fluorochrome-labeled antibodies for the detection of
antigens, can be qualitative or quantitative. In a qualitative procedure, a fluorescent
microscope is necessary to visualize the presence of a labeled antigen or antibody in
the specimen. Fluorescence is the emission of light of one color or wavelength, while a
substance is irradiated with light of a different color or wavelength. The emitted
wavelength is at a lower energy level than the incident or absorbed light. Fluorescein
isothiocyanate is one of the most common fluorochromes with an absorption maximum
of 490-495 nm and a yellow-green emission maximum of 517 nm.
The two most common methods used in the performance of immunofluorescent
microscopy are the direct and indirect techniques.
a. Direct Method. In the direct method, the antibody is labeled with a
fluorescent compound and is used to detect the presence of antigen in tissue fixed to a
slide. The direct technique utilizes biopsy material obtained from a patient. The
fluorescent-labeled antibodies are added to the antigen in an optimal dilution and
allowed to react. The preparation is washed to remove any unreacted labeled
antibodies. The tissue sections are blotted and the preparation mounted with buffered
glycerol for examination with the fluorescent microscope (Figure 6-1a).
b. Indirect Method. The indirect method is used for the detection of serum
antibodies utilizing an antigen-containing substrate and a fluorescein-labeled antibody
specific for human immunoglobulins. The specific antigen-antibody (unlabeled) reaction
may be visualized by the addition of labeled antihuman globulin directed against the
antibody in the primary reaction. The antigen substrate plus patient's serum antibody
plus labeled antihuman immunoglobulin complex results in fluorescence and detection
of the specific patient's antibody in question (Figure 6-1b).