Au r ights reserved donor rbcs coated with anti rho

AU r ights reserved.
Donor RBCs
coated with
anti-RhO+
antibodies
Immunology
Chapter 13 -2

Chapter 13 • Uses of Imm u no l ogy in Diagnostic Medici ne
Immunofluorescent Assays
Immunofluorescent assays use microscopy to visualize the location
and distribution of fluorescent dyes tagged to antibodies in blood
and tissue samples.
2.1 Direct Fluorescent Antibody Test
The direct fluorescent
antibody test (DFA) is used
to identify the presence
of antigen in a patient .
It is used in cases where
the pathogen is difficult
to visualize or difficult to
culture . It is performed
by treating a biopsy
specimen with fluorescent-
tagged ant ibodies against
a microbe. If the tissue
specimen fluoresces,
Immunology
then the diagnosis is
established. Examples of
the use of this test would
include the diagnoses of
Chlamydia, respiratory
A Figure 13-2.1
Direct Fluorescent Antibody Test
syncytial virus, herpes
simplex viruses 1 and 2,
and Pneumocystis.
2.2 Indirect Fluorescent Antibody Test
The indirect immunofluorescent antibody test (IFA) is used to detect
the presence of antibody in a patient, so it is widely applied in the
diagnosis of autoimmune disease. To perform this test, serum from
the patient is incubated with a sample of tissue or cells expressing
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labeled wi th fluorescent dye
the antigens against which
the patient is believed to
be autoimmune. If the
antibodies are present in
the serum, they will bind
to the tissue sample, so
that subsequent addition
of antihuman globulin that
has been labeled with a
fluorescent dye will cause
the tissue to fluoresce.
Diagnoses assisted with
this test include Graves
disease, Goodpasture
syndrome, and systemic
If the test Ag is fluorescent following these steps, then the
patient had antibody against this antigen in his serum.
l upus erythematosus.
A Figure 13-2.2
Indirect Fluorescent Antibody Test
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Oe Vry/ Be cker Ed u ca tion al Development Co rp. A ll rig hts re se rve d.
Chapt er 13- 3

Chapter 13 • Uses of Immunology in Diagnostic Med icine
Radioimmunoassay (RIA) and Enzyme
Immunoassay (EIA)
--------
--------
Antibody
The radioimmunoassay (RIA)
and enzyme immunoassay (EIA)
are two of the most commonly
used diagnostic techniques
Enzyme-linked
antihuman
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Immunology
in medicine today. They are
extremely sensitive, detecting
as litt le as 10·
9
g of materia l, but
differ in the means of visualizing
the result . In the case of EIA,
Antigen
coated on
microti ter
p la te
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an enzyme causes color change
as the result, and in the RIA,
changes in radioactivity are
detected. An EIA is used as the
screening test for HIV infection.
In such testing, the viral p24
antigen is bound to the we l ls of
a microtiter plate, and a serial
dilution of patient sera is added
to each of the we l ls . This is
followed by washing to remove
unbound antibodies, treatment
with an enzyme-linked antihuman
globul in, repeat washing,
and finally the addition of the
substrate for the enzyme. Any
color change in the well reflects
that these binding reactions have
all occurred in sequence, and that
the patient has a titer (def ined
as the reciprocal of the ser um
dilution in the l ast positive we l l) of
antibody to that viral antigen.