Page 560 - Review of Medical Microbiology and Immunology ( PDFDrive )

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nal fluid. mebooksfree.com
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mebooksfree.com mebooksfree.com mebooksfree.com CHAPTER 64 Antigen–Antibody Reactions in the Laboratory 549 mebooksfree.com
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Counter-Immunoelectrophoresis—This method relies
on movement of antigen toward the cathode and of anti-
body toward the anode during the passage of electric cur-
rent through agar. The meeting of the antigen and antibody
is greatly accelerated by this method and is made visible in
30 to 60 minutes. This has been applied to the detection of
A
bacterial and fungal polysaccharide antigens in cerebrospi-

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mebooksfree.com mebooksfree.com mebooksfree.com Radioimmunoassay (RIA) mebooksfree.com mebooksfree.com

This method is used for the quantitation of antigens or
B
haptens that can be radioactively labeled. It is based on the
competition for specific antibody between the labeled
(known) and the unlabeled (unknown) concentration of
material. The complexes that form between the antigen and
antibody can then be separated and the amount of radioac-
tivity measured. The more unlabeled antigen is present, the
C
tion of the unknown (unlabeled) antigen or hapten is deter-
FIGURE 64–5
Immunoelectrophoresis. A: Human serum
mined by comparison with the effect of standards. RIA is a
placed in the central well is electrophoresed, and the proteins
highly sensitive method and is commonly used to assay
migrate to different regions (orange ellipses). Antiserum to human less radioactivity there is in the complex. The concentra-
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hormones or drugs in serum. The radioallergosorbent test
serum is then placed in the elongated trough (gray areas). B: Human
(RAST) is a specialized RIA that is used to measure the
serum proteins and antibodies diffuse into agar. C: Precipitate arcs
amount of serum IgE antibody that reacts with a known
(orange lines) form in the agar. (Reproduced with permission from Stites
DP, Terr A, Parslow T, eds. Basic & Clinical Immunology. 9th ed. Originally published by
Appleton & Lange. Copyright 1997 McGraw-Hill.)
Enzyme-Linked Immunosorbent
Precipitation in Agar with an Electric Field
Assay (ELISA)
Immunoelectrophoresis—A serum sample is placed in a
well in agar on a glass slide (Figure 64–5). A current is
This method can be used for the quantitation of either
passed through the agar, and the proteins move in the elec-
tric field according to their charge and size. Then a trough
covalently linking an enzyme to a known antigen or anti-
body, reacting the enzyme-linked material with the patient’s
is cut into the agar and filled with antibody. As the antigen
and antibody diffuse toward each other, they form a series antigens or antibodies in patient specimens. It is based on
specimen, and then assaying for enzyme activity by adding
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the substrate of the enzyme. The method is nearly as sensi-
of arcs of precipitate. This permits the serum proteins to be
characterized in terms of their presence, absence, or
tive as RIA yet requires no special equipment or radioactive
labels (Figure 64–6).
unusual pattern (e.g., human myeloma protein).
EE E
EE E
Antigen is
the enzyme is
human IgG
patient’s
attached to
serum
added, which
attaches to
the bottom Antibody in Antibody to Substrate for
of the well attaches to patient’s IgG; changes color
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when acted
antigen
the antibody to
human IgG is
upon by the
enzyme
enzyme-linked
FIGURE 64–6
Enzyme-linked immunosorbent assay (ELISA). The term enzyme-linked refers to the covalent binding (linking) of an
enzyme to antibody to human IgG. If the patient has antibodies to the microbial or viral antigen, those antibodies will bind to the microbial or
viral antigens. The antibody to human IgG linked to the enzyme will then bind to the patient’s antibodies. Then when the substrate of the
enzyme is added, the substrate changes color, indicating that the patient’s serum contained antibodies.
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