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Encyclopedia of Physical Science and Technology EN007I-331 July 3, 2001 18:42

Immunology—Autoimmunity 685

viral antigens in tissue culture monolayers infected with a significant advantage over transmission of light through
varicella and herpes zoster. The human antibody bound to the specimen, as significantly less irrelevant wavelength
viral antigen in the infected cells was detected by a flu- radiation needs to be filtered out. The filtering elements
orochrome covalently linked to antibodies raised against of an epifluorescence microscope consist of an excitation
human immunoglobulin. Although widely used for the filter, an emission filter, and a dichroic mirror (Fig. 2). The
detection of antibody against viral (or nonself) antigens, excitation filter allows transmission of only those wave-
this application was soon followed by studies using the lengths that will excite the specific fluorochrome being
method to detect autoantibodies. Fluorescent anti-human used. The emission (or barrier) filter blocks transmission
globulin was used in 1957 by Friou and collegues, and of the excitatory wavelength light but allows any fluores-
Holborow and his co-workers, to demonstrate staining of cence emitted to pass. The dichroic mirror is coated glass
◦
the cell nucleus by serum from patients with systemic lu- that is positioned 45 to the optical path of the micro-
pus erythematosus (SLE). These studies, using mouse and scope. The dichroic mirror functions as a beam splitter,
human tissue sections respectively, also revealed for the reflecting the excitatory wavelength onto the specimen
first time the species nonspecificity of the antinuclear “fac- but allowing the emitted fluorescence to pass through to
tor” (ANF), allowing tissue from a wide variety of sources the eyepiece (Fig. 2). In most instances these three el-
to be used. Subsequent studies showed the ANF to be an- ements are housed together in a filter cube, with many
tibody, particularly immunoglobulin G (IgG), hence the microscopes able to hold two or more filter cubes. Each
current terminology of ANA (antinuclear antibody). Fol- set of filters in a cube is a matched set and is restricted
lowing closely behind these early studies came reports in its use to a small number of fluorochromes, usually
that the pattern of nuclear staining differed between pa- one.
tients with a single autoimmune disease as well as between
patients with different autoimmune diseases. Thus homo-
3. Method
geneous staining of the nucleus was more likely in SLE,
while nucleolar staining was often found in the serum Immunofluorescence is a relatively straightforward tech-
of patients with scleroderma. During this same period it nique consisting of four major steps.
was shown that patients with organ-specific autoimmune
diseases had serum antibodies which reacted with anti- a. Preparation of cell or tissue substrates. Ava-
gens found in the organ targeted by the disease. Thus pa- riety of cell and tissue substrates from numerous species
tients with Hashimoto’s thyroiditis were shown to have have been used in immunofluorescent microscopy to char-
serum antibody directed against antigens in the thyroid. acterize both autoantibodies and autoantigens. The most
The immunofluorescent test, due to the strong relation- useful of these have been transformed mammalian cell
ship between immunofluorescence “pattern” and autoan- lines grown in tissue culture. These cell lines contain the
tibody specificity, continues to be an important clinical greatest variety of autoantigens and are particularly suited
test. to the detection of autoantibodies in multisystem autoim-
mune diseases where tissue specificity of the autoantibody
is not a confounding consideration. A primary concern in
2. Principle
the preparation of cell or tissue substrates for immunoflu-
The principle of fluorescence is based upon the observa- orescence is that the antigenic integrity of the substrate be
tion that certain substances adsorb radiation and become preserved during the fixation procedure necessary to stabi-
“excited” from a ground state of potential energy to the lize the substrate for experimental use. Another important
first excited state of the molecule. If the excited molecule aspect of the fixation process is that it permeablizes the
is sufficiently stable, it will emit radiation rather than dissi- cell, thereby allowing antibody to enter and interact with
pate energy to the surrounding molecules as it returns to its itscognateantigen.Fixationusuallyinvolvesorganiccom-
ground state. The resulting emission is known as fluores- pounds, such as ethanol and acetone. Determination of the
cence and is almost always of a longer wavelength than optimal fixation conditions for the detection of specific
the exciting radiation. This relationship between excita- antigens may require considerable experimentation.
tion and emission wavelengths is known as Stokes’ law.
Epifluorescence is the most common method employed b. Addition of primary antibody. Primary antibody
influorescencemicroscopyofantibodies.Inthistechnique may consist of an unknown specificity, such as that in a
the excitatory radiation is passed through the objective clinical sample being tested for the presence of autoan-
lens onto the specimen, rather than through the specimen. tibodies, or a known specificity which is being used to
This means that only reflected excitatory radiation needs determine the presence of the cognate autoantigen in a
to be filtered out to detect the emitted radiation. This is particular cell or tissue substrate.