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chapter
8
Enzymes III: Clinical Applications
of both sensitivity and specificity. The sensitivity, which
is due to the amplification factor introduced by way of
the catalytic activity of the labeled enzyme, can equal
(with comparable precision) that obtainable with RIA. The
specificity is attributable to specific molecular recognition
properties of antigens and antibodies. The ELISA may
be more advantageous than RIA because ELISA reagents
have longer shelf lives than do many radioisotopes (e.g.,
1 2 5
I-labeled reagents) and present no health hazards. The
ELISA also has been adapted to identify antigens or an-
tibodies in tissue slices by the use of protein-peroxidase
conjugates.
Homogeneous EIA procedures have been especially
useful for rapid determination of low-molecular-weight
substances (haptens). Thus, these procedures have found
application in therapeutic drug monitoring and toxicol-
ogy. A commercial reagent for the assay of many drugs
is available under the trade name of EMIT (Enzyme-
Multiplied Immunoassay Technique). Figure
8 - 6
diagrams
the steps involved in this procedure. The principle is
based on competition between the enzyme-labeled hap-
ten and free hapten in the test serum for binding with
a limited amount of a specific antibody. This assay re-
quires a hapten covalently linked to an enzyme (e.g.,
glucose-
6
-phosphate dehydrogenase, lysozyme, or malate
dehydrogenase), with retention of enzymatic activity, and
antibodies raised against the hapten (through injection
into an animal of the hapten covalently linked to a carrier
protein) that inhibit enzyme activity when they combine
with the hapten-linked enzyme reagents. The inhibition
by the antibody of catalytic activity of the enzyme-linked
hapten complex may be due to steric hindrance or to
conformational constraints affecting access of substrate
of the active center. This reaction system consists of
the test specimen, the enzyme-labeled hapten, a limited
amount of the antibody specific to the hapten, and the
substrate. Any haptens present in the test serum com-
pete with the hapten linked to the enzyme for binding
with the antibody. The unbound hapten-linked enzyme
acts on the substrate to convert it to an easily measur-
able product. Thus, the enzyme activity is directly cor-
related with the free hapten concentration in the test
specimen. The method is calibrated with known standard
concentrations of the hapten.
The ELISA and EIA receptor systems measure sub-
stances at concentrations as low as a few nanograms
(ICC
9
g). This sensitivity is not sufficient for detecting
many substances and alternate methods have been devised.
One is chemiluminescence immunoassay (CLIA), which
can measure concentrations in femtogram (
1 0 - 1 5
g) quan-
tities. CLIA depends on the detection of emitted light as-
sociated with the dissipation of energy from a substance
FIGURE
8 -6
Diagrammatic representation of a homogeneous enzyme immunoassay
procedure for the determination of low-molecular-weight compounds
(haptens).
excited electronically as a result of a chemical reaction. An
example of a chemiluminescent tracer is acridinium ester
conjugated to a desired protein, polypeptide, or other or-
ganic molecule (Figure 8-7).
CLIA is similar to EIA and ELISA techniques ex-
cept that the final receptor enzyme assay is replaced with
a chemiluminescent tracer followed by measurement of
light released as a result of the chemical reaction. The
principles of a chemiluminescence competitive binding
assay are shown in Figure
8
-
8
.
8.4 Enzymes as Therapeutic Agents
Enzymes have found a few applications as therapeutic
agents. Some examples are transfusion of fresh blood
or its active components in bleeding disorders, oral ad-
ministration of digestive enzymes in digestive diseases
