Capture Assays

If antibody, rather than antigen, is bound to the solid phase,

these assays are often called sandwich immunoassays or
capture assays. Antigens captured in these assays must have
multiple epitopes. Excess antibody attached to solid phase is
allowed to combine with the test sample to capture any antigen
present. After an appropriate incubation period, enzymelabeled
antibody is added. This second antibody recognizes
a different epitope or binding site than the solid-phase antibody
and completes the “sandwich.” Depending upon the
particular enzyme used, either a colored or chemiluminescent
reaction product is detected (see Fig. 11–2). Enzymatic
activity is directly proportional to the amount of antigen in
the test sample.
Capture assays are best suited to antigens that have multiple
determinants, such as antibodies, cytokines, proteins,
tumor markers, and microorganisms, especially viruses.2,5
When used with microorganisms, the epitope must be unique
to the organism being tested and must be present in all strains
of that organism. The use of monoclonal antibodies has made
this a very sensitive test system. A major use of capture assays
is in the measurement of immunoglobulins, especially those
of certain classes. For instance, the presence of IgM can
be specifically determined, thus indicating an acute infection.
When capture assays are used to measure immunoglobulins,
the specific immunoglobulin class being detected is
actually acting as antigen and the antibody is anti-human
immunoglobulin. Such indirect ELISA tests are quite sensitive
because all patient antigen has a chance to participate
in the reaction.
Heterogeneous enzyme assays, in general, achieve a sensitivity
similar to that of RIA.1 In sandwich assays, capture
antibody on solid phase must have both a high affinity and a
high specificity for this test system to be effective. However,
there may be problems with nonspecific protein binding or
the presence of antibodies to various components of the testing
system.1 If IgG is present, rheumatoid factor can cause
false-positive results. Rheumatoid factor is an IgM antibody
that reacts with IgG. See Chapter 14 for a discussion of the
diseases in which rheumatoid factor may be found. If this is
suspected, serum can be pretreated to avoid this problem.
Sandwich assays are also subject to the hook effect, an unexpected
fall in the amount of measured analyte when an extremely
high concentration is present1. This typically occurs
in antigen excess, where the majority of binding sites are
filled and the remainder of patient antigen has no place to
bind. If this condition is suspected, serum dilutions must be
made and then retested.
Homogeneous Enzyme Immunoassays
Homogeneous enzyme immunoassays are generally less sensitive
than heterogeneous assays, but they are rapid, simple to
perform, and adapt easily to automation.1,2 No washing or
separation steps are necessary. Their chief use has been in the
determination of low-molecular-weight analytes such as hormones,
therapeutic drugs, and drugs of abuse in both serum
and urine.1,2,6 An example of a homogeneous immunoassay
is the enzyme-multiplied immunoassay technique (EMIT) developed
by the Syva Corporation.6
Homogeneous assays are based on the principle of change
in enzyme activity as specific antigen–antibody combination
occurs. Reagent antigen is labeled with an enzyme tag. When
antibody binds to specific determinant sites on the antigen,
the active site on the enzyme is blocked, resulting in a mea -
surable loss of activity.2 Free analyte (antigen) competes with
enzyme-labeled analyte for a limited number of antibodybinding
sites, so this is a competitive assay. Enzyme activity
is directly in proportion to the concentration of patient antigen
or hapten present in the test solution (see Fig 11-3).
A physical separation of bound and free analyte is thus not
necessary.
The sensitivity of homogeneous assays is determined by the
following: (1) detectability of enzymatic activity; (2) change in
that activity when antibody binds to antigen; (3) strength of
the antibody’s binding; and (4) susceptibility of the assay to interference
from endogenous enzyme activity, cross-reacting
antigens, or enzyme inhibitors. This technique is usually applied
only to detection of small molecules that could not be
easily measured by other means because it is far less sensitive
than heterogeneous assays.2
Homogeneous assays have a number of limitations. First,
only certain enzymes are inhibited in this manner. Additionally,
enzymatic activity may be altered by steric exclusion of
the substrate or there may also be changes in the conformation
structure of the enzyme, especially in the region of the
active site. Two enzymes that are frequently used in this type
of assay are malate dehydrogenase and glucose-6-phosphate
dehydrogenase.2
Enzyme immunoassays in general have achieved sensitivity
similar to that of RIA without creating a health hazard or causing
disposal problems. Expensive instrumentation is not
needed because most assays can be read by spectrophotometry
or by simply noting the presence or absence of color. Reagents
are inexpensive and have a long shelf life.
Disadvantages include the fact that some specimens may
contain natural inhibitors. Additionally, the size of the enzyme
label may be a limiting factor in the design of some assays.
Nonspecific protein binding is another difficulty encountered
with the use of enzyme labels.2 However, this technique has
been successfully applied to a wide range of assays.