Page 561 - Review of Medical Microbiology and Immunology ( PDFDrive )

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PART VII Immunology
550
For measurement of antibody, known antigens are fixed to
a surface (e.g., the bottom of small wells on a plastic plate),
sequentially under appropriate conditions for the reaction
to progress. Antigen–antibody complexes are among the
incubated with dilutions of the patient’s serum, washed, and
activators, and the complement fixation test can be used to
then reincubated with antibody to human IgG labeled with
identify one of them if the other is known.
an enzyme (e.g., horseradish peroxidase). Enzyme activity is
The reaction consists of the following two steps
measured by adding the substrate for the enzyme and esti-
mating the color reaction in a spectrophotometer. The
amount of antibody bound is proportional to the enzyme
the other unknown; e.g., use a known antigen and deter-
mine whether a patient’s serum contains antibodies to that
activity. The titer of antibody in the patient’s serum is the (Figure 64–8): (1) Antigen and antibody (one known and
antigen) are mixed, and a measured amount of comple-
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highest dilution of serum that gives a positive color reaction.
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ment (usually from guinea pig) is added. If the antigen and
antibody match, they will combine and use up (“fix”) the
Immunofluorescence
(Fluorescent Antibody)
tized” red blood cells (i.e., red blood cells plus anti–red
Fluorescent dyes (e.g., fluorescein and rhodamine) can be
blood cell antibody), is added. If the antibody matched the
covalently attached to antibody molecules and made visible
antigen in the first step, complement was fixed and less (or
by ultraviolet (UV) light in the fluorescence microscope.
none) is available to attach to the sensitized red blood cells.
Such “labeled” antibody can be used to identify antigens
The red blood cells remain unhemolyzed (i.e., the test is
(e.g., on the surface of bacteria such as streptococci and
positive) because the patient’s serum had antibodies to
treponemes, in cells in histologic section, or in other speci-
mens) (Figure 64–7). The immunofluorescence reaction is
the first step, complement is free to attach to the sensitized
direct when known labeled antibody interacts directly with
red blood cells and they are lysed (i.e., the test is
unknown antigen and indirect when a two-stage process is that antigen. If the antibody did not match the antigen in
negative).
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reactive with all human IgG). mebooksfree.com
mebooksfree.com mebooksfree.com inactivate any human complement activity. The antigen mebooksfree.com
used. For example, known antigen is attached to a slide, the
Complement must be carefully standardized, and the
patient’s serum (unlabeled) is added, and the preparation is
patient’s serum must be heated to 56°C for 30 minutes to
washed; if the patient’s serum contains antibody against the
antigen, it will remain fixed to it on the slide and can be
must be quantitated. The result is expressed as the highest
detected on addition of a fluorescent dye–labeled antibody
dilution of serum that gives positive results. Controls to
to human IgG and examination by UV microscopy. The
determine whether antigen or antibody alone fixes comple-
indirect test is often more sensitive than direct immuno-
ment are needed to make the test results valid. If antigen or
fluorescence, because more labeled antibody adheres per
antibody alone fixes complement, it is said to be
antigenic site. Furthermore, the labeled antiglobulin
anticomplementary.
becomes a “universal reagent” (i.e., it is independent of the
nature of the antigen used because the antibody to IgG is
Neutralization Tests
These use the ability of antibodies to block the effect of toxins
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or the infectivity of viruses. They can be used in cell culture
Complement Fixation
(e.g., inhibition of cytopathic effect and plaque-reduction
The complement system consists of 20 or more plasma
assays) or in host animals (e.g., mouse protection tests).
proteins that interact with one another and with cell
Ultraviolet light
Immune complexes in tissue can be stained with fluores-
Ultraviolet light
cent complement. Immune complexes in serum can be
detected by binding to C1q or by attachment to certain
F
F
F
(e.g., Raji lymphoblastoid) cells in culture.
F
F
F
F
F
Hemagglutination Tests
Many viruses clump red blood cells from one species or
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another (active hemagglutination). This can be inhibited by
B. Indirect fluorescent-
A. Direct fluorescent-
antibody test
antibody test
antibody specifically directed against the virus (hemagglu-
tination inhibition) and can be used to measure the titer of
FIGURE 64–7
Fluorescent antibody test. A: In the direct fluo-
rescent antibody test, the fluorescent dye is attached directly to the
gens and, when mixed with matching antibodies, will
antibody that is interacting with the antigen (dark triangles) on the
clump (this is known as passive hemagglutination, because
surface of the cell. B: In the indirect fluorescent antibody test, the flu-
the red cells are passive carriers of the antigen).
orescent dye is attached to antibody made against human IgG.
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