section 8.3
Enzymes as Analytical Reagents
Specific antibody
against the antigen
Adsorption of antibody onto a solid
surface, followed by washing procedure.
An inert solid surface
(e.g., a bead)
Incubation with the test specimen
containing antigen, which binds to the
specific antibody, followed by
aspiration and washing procedure.
Incubation with enzyme-linked specific
antibody, which binds wherever the
antigen is affixed, followed by washing
and aspiration procedure.
Incubation with specific
substrate, and quantitation
of products formed. Under
standardized and optimal
conditions, the concentrations
of products are directly
proportional to the
concentration of antigen.
F IG U R E 8 -5
Diagrammatic representation of an ELISA (solid-phase, sandwich technique) for an antigen.
reagent is separated from the unbound enzyme, enabling
the measurement of either bound or free activity. A pro-
cedure based upon this principle is the enzyme-linked
immunosorbent assay (ELISA), which is analogous to an
An example of a heterogeneous immunoassay that uses
a solid-phase system is shown in Figure 8-5. This method
consists of coating or adsorbing antibodies generated in an
animal (e.g., guinea pig) against a human antigen, whose
concentration is to be determined in a test serum sam-
ple, onto a solid surface (e.g., beads or inner surface of a
test tube) and incubating with the serum specimen. Dur-
ing the first incubation step, the antigen combines with its
specific antibody and is fixed to a solid surface. Subse-
quently, the unreacted serum components are removed by
aspiration of the fluid, and the solid surface (containing
antibody-antigen complexes) is washed with suitable
buffers. Incubation with antibodies covalently linked to
enzyme molecules follows. During this step, the enzyme-
labeled antibody (E-Ab) will combine wherever the anti-
gens are affixed; a washing procedure then removes the
unbound E-Ab conjugates. Finally, incubation with the
substrate specific for the enzyme yields a colored product
that is quantitated by suitable spectrophotometric meth-
ods. The time interval for the substrate incubation step and
other reaction conditions must be kept constant for both
test sera and the standards. The enzyme reaction is stopped
by altering the pH of the reaction mixture by adding an
acid solution (e.g., 1 N hydrochloric acid). The change in
absorbance of color is proportional to the concentration
of antigen in the test sera. A standard curve is obtained
by plotting known antigen concentrations against their
Various modifications of this technique are in use. For
example, if the concentration of an antibody is to be deter-
mined, the process can begin with immobilization of the
antigen onto the solid phase.
The enzymes used as labels should have a high turnover
number, act on substrates that are stable and soluble, yield
easily measurable products, be obtainable in a highly pu-
rified form, and be relatively inexpensive. Enzymes that
fit these criteria are horseradish peroxidase, glucose oxi-
E. coli
or calf-intestine alkaline phosphatase, cata-
lase, and /3-D-galactosidasc. The covalent linkage of the
enzyme to the protein (antigen or antibody) through free
amino groups can be accomplished with the bifunctional
reagent glutaraldehyde. The periodate oxidation method
(Chapter 9), involving the carbohydrate side-chains of pro-
teins, also yields reactive groups useful in the preparation
of enzyme conjugates. The ELISA exhibits high levels
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