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Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis CompanyCopyright © 2010 F.A. Davis Company
Labeled Immunoassays
Chapter Ten
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays Labeled immunoassays are designed for
antigens and antibodies that may be small in
size or present in very low concentrations.
The presence of such antigens or antibodies is
determined indirectly by using a labeled
reactant to detect whether specific binding has
taken place.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays The substance to be measured is known as
the analyte.
Analytes can be bacteria antigens, hormones,
drugs, tumor markers, specific
immunoglobulins, and many other substances.
One reactant, either the antigen or the
antibody, is labeled with a marker so that the
amount of binding can be monitored.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays Current techniques include the use of
fluorescent, radioactive, chemiluminescent,
and enzyme labels.
The underlying principles of all these
techniques are essentially the same.
There are two major formats for all labeled
assays: competitive and noncompetitive.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays In a competitive immunoassay, all the
reactants are mixed together simultaneously,
and labeled antigen competes with unlabeled
patient antigen for a limited number of
antibody-binding sites.
The amount of bound label is inversely
proportional to the concentration of the
labeled antigen.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays In a typical noncompetitive immunoassay,
antibody, often called a capture antibody, is
first passively absorbed to a solid phase.
Unknown patient antigen is then allowed to
react with and be captured by the antibody.
After washing to remove unbound antigen, a
second antibody with a label is added to the
reaction.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays In noncompetitive immunoassays, the
amount of label measured is directly
proportional to the amount of patient
antigen.
Radioactivity, enzymes, fluorescent
compounds, and chemiluminescent
substances have all been used as labels.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays In any immunoassay, it is essential for the
antibody used to have a high affinity, or
strength of the primary interaction between a
single antibody-combining site and an
antigenic determinant or epitope for the
antigen.
In competitive binding assays, there is random
interaction between individual antigen and
antibody molecules.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays The higher the affinity of antibody for
antigen, the larger the amount of antigen
bound to antibody and the more accurately
specific binding can be measured.
The antibody used should also be very specific
for the antigen involved in the reaction.
Monoclonal antibodies have been very
beneficial in this regard.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays Calibrators, or standards, are used to
establish a relationship between the labeled
analyte measured and any unlabeled analyte
that might be present in patient specimens.
Differing amounts of standards are added to
antibody–antigen mixtures to ascertain their
effect on binding of the labeled reagent.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays Most instruments then extrapolate this
information and do a best-fit curve to
determine the concentration of the unknown
analyte.
In most assays, once the reaction between
antigen and antibody has taken place, there
must be a partitioning step, or a way of
separating reacted from unreacted analyte.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays Currently, most immunoassays use a solid-
phase vehicle for separation, such as
polystyrene test tubes, microtiter plates, glass
or polystyrene beads, magnetic beads, and
cellulose membranes.
If a separation step is employed in an assay,
the efficiency of the separation is critical to the
accuracy of the results.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays If this is the case, the bound and unbound
fractions are usually separated by physical
means, including decanting, centrifugation, or
filtration.
This is followed by a washing step to remove
any remaining unbound analyte.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays The last step common to all immunoassays is
detection of the labeled analyte.
This is accomplished by counting radioactivity
in RIA methods, or by the use of enzymes,
fluorescence, or chemiluminescence, which
typically measure a change in absorbance by
spectrophotometry.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays A negative control, high and low positive
controls, and a blank tube (usually phosphate-
buffered saline) are typically run as quality-
control samples.
The number, type, and frequency of controls
needed vary among instruments and
methodologies.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays The first type of immunoassay developed
was radioimmunoassay (RIA).
Several radioactive labels, including 131I; 125I;
and tritiated hydrogen, or 3H, have been used,
but 125I is the most popular.
It is easily incorporated into protein molecules,
and it emits gamma radiation, which is
detected by a gamma counter.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays RIA was originally based on the principle of
competitive binding.
Thus, the analyte being detected competes
with a radiolabeled analyte for a limited
number of binding sites on a high-affinity
antibody.
The concentration of the radioactive analyte is
in excess, so all binding sites on antibody will
be occupied.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays If patient antigen is present, some of the
binding sites will be filled with unlabeled
analyte, thus decreasing the amount of bound
radioactive label. (See Fig. 10-1.)
The amount of label in the bound phase is
indirectly proportional to the amount of
patient antigen present.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled ImmunoassaysFigure 10-1
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays RIA is an extremely sensitive and precise
technique for determining trace amounts of
analytes that are small in size.
Its disadvantages include the health hazard
involved in working with radioactive
substances, low-level waste-disposal
problems, and short shelf life of some
reagents.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays Enzymes used as labels in immunoassays
react with suitable substrates to produce
breakdown products that may be
chromogenic, fluorogenic, or luminescent.
Typical enzymes include horseradish
peroxidase, glucose-6-phosphate
dehydrogenase, alkaline phosphatase, and β-
D-galactosidase.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays Alkaline phosphatase and horseradish
peroxidase have the highest turnover
(conversion of substrate) rates, high
sensitivity, and are easy to detect, so they are
most often used in such assays.
Enzyme assays are classified as either
heterogeneous or homogeneous on the
basis of whether a separation step is
necessary.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays Heterogeneous enzyme immunoassays
require a step to physically separate free from
bound analyte.
In homogeneous enzyme immunoassays,
no separation step is necessary, because
enzyme activity diminishes when binding of
antibody and antigen occurs.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays The first enzyme immunoassays (EIAs) were
competitive assays based on the principles of
RIA.
Enzyme activity is inversely proportional to the
concentration of the test substance, meaning
that the more patient antigen is bound, the
less enzyme-labeled antigen can attach.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays Although competitive tests have a high
specificity, noncompetitive enzyme
immunoassays are more common currently.
This is because they offer high sensitivity and
specificity, simplicity, and low cost.
Noncompetitive assays are often referred
to as indirect enzyme-linked
immunosorbent assays (ELISA).
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays Either antigen or antibody may be bound to
solid phases such as microtiter plates,
nitrocellulose membranes, and magnetic latex
beads.
When antigen is bound to solid phase, patient
serum with unknown antibody is added and
incubated.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays After a wash step, an enzyme-labeled
antiglobulin (AHG) is added. This second
antibody reacts with any patient antibody that
is bound to the solid phase.
After a second wash step, the enzyme
substrate is added.
The amount of enzyme label detected is
directly proportional to the amount of antibody
in the specimen. (See Fig. 10-2.)
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled ImmunoassaysFigure 10-2
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays ELISA remains the preferred screening
method for detecting antibody to HIV, hepatitis
A, hepatitis C, and Epstein-Barr virus.
If antibody 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.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays After an appropriate incubation period,
enzyme-labeled antibody is added.
This second antibody recognizes a different
epitope than the solid-phase antibody and
completes the “sandwich.”
Enzymatic activity is directly proportional to
the amount of antigen in the test sample.
See Figure 10-3 for more on this assay.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled ImmunoassaysFigure 10-3
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays Capture assays are best suited to antigens
that have multiple determinants, such as
antibodies, polypeptide hormones, proteins,
tumor markers, and microorganisms,
especially viruses.
Use of monoclonal antibodies has made this a
very sensitive test system.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays Heterogeneous enzyme assays, in general,
achieve a sensitivity similar to that of RIA.
Possible problems include nonspecific protein
binding or the presence of antibodies to
various components of the testing system.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays Sandwich assays are also subject to the hook
effect, an unexpected fall in the amount of
measured analyte when an extremely high
concentration is present.
This typically occurs in antigen excess, where
the majority of binding sites are filled. All
patient analyte cannot bind in this case.
If this condition is suspected, serum dilutions
must be made and then retested.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays Membrane-based cassette assays are a
relatively new type of enzyme immunoassay.
Typically these are designed as single-use,
disposable assays in a plastic cartridge.
The membrane is usually nitrocellulose, which
is easily able to immobilize proteins and
nucleic acids.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays Either antigen or antibody can be coupled to
the membrane, and the reaction is read by
looking for the presence of a colored reaction
product.
Some test devices require the separate
addition of patient sample, wash reagent,
labeled antigen or antibody, and the substrate.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays Another type of rapid assay, called
immunochromatography, combines all the
previously mentioned steps into one.
The analyte is applied at one end of the strip
and migrates toward the distal end, which
contains an absorbent pad to maintain a
constant capillary flow rate.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays As the sample is loaded, it reconstitutes the
labeled antigen or antibody, and the two form
a complex that migrates toward the detection
zone.
An antigen or antibody immobilized in the
detection zone captures the immune complex
and forms a colored line for a positive result.
See Figure 10-4 for an illustration of
immunochromatography.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled ImmunoassaysFigure 10-4
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays Excess labeled immunoreactant migrates to
the absorbent pad.
Test results are most often qualitative rather
than quantitative.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays A homogeneous enzyme immunoassay is
any antigen–antibody system in which no
separation step is necessary.
Homogeneous assays are generally less
sensitive than heterogeneous assays, but they
are rapid, simple to perform, and adapt easily
to automation.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays No washing steps are necessary.
Homogeneous assays are based on the
principle of change in enzyme activity as
specific antigen–antibody combination occurs.
Free analyte (antigen) competes with enzyme-
labeled analyte for a limited number of
antibody-binding sites, so this is a competitive
assay.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays When antibody binds to specific determinant
sites on the antigen, the active site on the
enzyme is blocked, resulting in a measurable
loss of activity.
Enzyme activity is indirectly proportional to
the concentration of patient antigen or hapten
present in the test solution.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays Typically, sensitivity of homogeneous
immunoassays is far less than that
achievable by heterogeneous enzyme assays,
because the amplification properties of
enzymes are not utilized.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays Enzyme immunoassays have achieved a
sensitivity similar to that of RIA without
attendant health hazards or waste disposal
problems.
There is no need for expensive
instrumentation, and reagents are inexpensive
and have a long shelf life.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays Disadvantages include the fact that some
specimens may contain natural inhibitors.
The size of the enzyme label may be a limiting
factor in the design of some assays.
Nonspecific protein binding is another potential
difficulty encountered with the use of enzyme
labels.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays In fluorescent immunoassay techniques,
fluorophores or fluorochromes absorb energy
from an incident light source and emit light of a
longer wavelength and lower energy as the
excited electrons return to the ground state.
The two compounds most often used are
fluorescein and rhodamine, usually in the form
of isothiocyanates, because these can be
readily coupled with antigen or antibody.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays Because their absorbance and emission
patterns differ, fluorescein and rhodamine can
be used together.
Other compounds commonly used are
phycoerythrin, europium (β-naphthyl
trifluoroacetone), and lucifer yellow VS.
Fluorescent tags or labels were first used for
histochemical localization of antigen in tissues.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays This technique is called immunofluorescent
assay (IFA).
These techniques are restricted to qualitative
observations involving the use of a
fluorescence microscope.
The amount of fluorescence is graded against
a dark background.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays This method is used for rapid identification of
microorganisms in cell culture or infected
tissue, tumor-specific antigens on neoplastic
tissue, and transplantation antigens.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays In a direct immunofluorescent assay,
antibody that is conjugated with a fluorescent
tag is added directly to unknown antigen that
is fixed to a microscope slide.
After incubation and a wash step, the slide is
read using a fluorescence microscope.
This technique is useful in demonstrating the
presence of pathogens in patient samples (see
Fig . 10-5).
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled ImmunoassaysFigure 10-5
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled ImmunoassaysIndirect immunofluorescent assays involve
two steps.
The first step is incubation of patient serum
with a known antigen attached to a solid
phase.
The slide is washed, and then an antihuman
immunoglobulin containing a fluorescent tag is
added.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays This combines with the first antibody to form a
sandwich, which localizes the fluorescence.
Such assays are especially useful in antibody
identification in patient samples.
Figure 10-6 depicts the difference between
the two techniques.
Immunofluorescent assays in general face the
problem of subjectivity in the reading of slides.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled ImmunoassaysFigure 10-6
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays Fluorescence polarization immunoassay
(FPIA) is based on the change in polarization
of fluorescent light emitted from a labeled
molecule when it is bound by antibody.
Incident light directed at the specimen is
polarized with a lens or prism so the waves
are aligned in one plane.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays If a molecule is small and rotates quickly
enough, the emitted light is unpolarized after it
is excited by polarized light.
If the labeled molecule is bound to antibody,
the molecule is unable to tumble as rapidly,
and it emits an increased amount of polarized
light.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays Thus, the degree of polarized light reflects the
amount of labeled analyte that is bound.
In FPIA, labeled antigens compete with
unlabeled antigens in the patient sample for a
limited number of antibody binding sites.
The more antigen that is present in the patient
sample, the less the fluorescence-labeled
antigen is bound and the less the polarization
will be detected.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays Hence, the degree of fluorescence polarization
is inversely proportional to concentration of the
analyte (see Fig.10-7).
FPIA has been used mainly to determine
concentrations of therapeutic drugs and
hormones.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled ImmunoassaysFigure 10-7
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays The main problem with fluorescent
immunoassays has been separation of the
signal on the label from autofluorescence
produced by different organic substances
normally present in serum.
Another difficulty lies in nonspecific binding to
substances in serum causing quenching or
diminishing of the signal and the amount of
fluorescence generated.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays Chemiluminescence is another technique
employed to follow antigen–antibody
combination.
It is the emission of light caused by a chemical
reaction, typically an oxidation reaction,
producing an excited molecule that decays
back to its original ground state.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays Some of the most common substances used
are luminol, acridiniumesters, ruthenium
derivatives, and nitrophenyl oxalates.
When these substances are oxidized, typically
using hydrogen peroxide and an enzyme for a
catalyst, intermediates are produced that are
of a higher energy state.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays These intermediates spontaneously return to
their original state, giving off energy in the
form of light.
The light emitted may exist as a short-lived
flash or for a longer period of time.
This type of labeling can be used for
heterogeneous and homogeneous assays,
because labels can be attached to either
antigen or antibody.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays In heterogeneous assays, competitive and
sandwich formats are most often used.
Smaller analytes such as therapeutic drugs
and steroid hormones are measured using
competitive assays.
The sandwich format is used for larger
analytes, such as protein hormones.
Clinical Immunology & SerologyA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis Company
Labeled Immunoassays Chemiluminescent assays have an excellent
sensitivity, comparable to EIA and RIA, and
the reagents are stable and relatively nontoxic.
However, false results may be obtained if
there is lack of precision in injection of the
hydrogen peroxide.
Also, some biological materials in urine or
plasma can cause quenching of the light
emission.