Ch10 (5)

Clinical Immunology & Serology
A Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis CompanyCopyright © 2010 F.A. Davis Company
Labeled Immunoassays
Chapter Ten

Copyright © 2010 F.A. Davis Company
 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.

 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.

 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.

 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.

 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.

 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.

 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.

 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.

 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.

 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.

 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.

 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.

 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.

 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.

 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.

 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.

 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.

Figure 10-1

 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.

 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.

 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.

 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.

 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.

 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).

 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.

 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.)

Figure 10-2

 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.

 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.

Figure 10-3

 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.

 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.

 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.

 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.

 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.

 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.

 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.

Figure 10-4

 Excess labeled immunoreactant migrates to
the absorbent pad.
 Test results are most often qualitative rather
than quantitative.

 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.

 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.

 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.

 Typically, sensitivity of homogeneous
immunoassays is far less than that
achievable by heterogeneous enzyme assays,
because the amplification properties of
enzymes are not utilized.

 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.

 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.

 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.

 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.

 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.

 This method is used for rapid identification of
microorganisms in cell culture or infected
tissue, tumor-specific antigens on neoplastic
tissue, and transplantation antigens.

 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).

Figure 10-5

Indirect 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.

 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.

Figure 10-6

 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.

 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.

 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.

 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.

Figure 10-7

 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.

 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.

 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.

 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.

 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.

 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.

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