Ch10 (5)

Clinical Immunology & SerologyA 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 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.



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.



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.



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.



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.



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.



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.



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.



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.



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.



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.



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.



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.



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.



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.



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.



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.



Labeled ImmunoassaysFigure 10-1



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.



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.



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.



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.



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.



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).



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.



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.)



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.



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.



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.



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.



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.



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.



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.



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.



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.



Labeled Immunoassays Excess labeled immunoreactant migrates to

the absorbent pad.

Test results are most often qualitative rather

than quantitative.



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.



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.



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.



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.



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.



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.



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.



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.



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.



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.



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).



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.



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.



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.



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.



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.



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.



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.



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.



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.



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.



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.



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.

Ch10 (5)

Science

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