LA B ELLED IM M U N O A SS AY

Labeled im m unoassays
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 or not specific
binding has taken place.

 Analyte
substance to be measured
are bound by molecules that react

specifically with them(antibody)

Examples:

bacterial antigens, hormones, drugs,

tumor markers, specific
immunoglobulins, and many other
substances.

Im portance ofLabelled
Im m unoassays:
rapid
quantitative
measurement
ability to detect very small
quantities of antigen or
antibody

C H A R A C TER ISTIC S O F
LA B ELED A SSAYS
Current techniques include these

labels:
Fluorescent
Radioactive
Chemiluminescent
Enzyme

There are two major formats for all

labeled assays:
competitive
noncompetitive

C om petitive im m unoassay
Reactants are mixed together
Labeled antigen competes with unlabeled

patient antigen for a limited number of

antibody-binding sites.
Interpretation:
The amount of
bound label is inversely
proportional to the concentration of the labeled
antigen.
This means that the more label detected, the
less there is of patient antigen.

N on-com petitive
Im m unoassay
The antibody, often called 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.
Interpretation:
The amount of label measured is directly

proportional to the amount of patient antigen.

Antibodies
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 ultimate sensitivity of the

immunoassay, in fact, depends

largely on the magnitude of affinity.

Standards or C alibrators
are unlabeled analytes that are

made up in known concentrations of

the substance to be measured.
They are used to establish a

relationship between the labeled

analyte measured and any unlabeled
analyte that might be present in
patient specimens.

Separation M ethods
a partitioning step, or a way of separating reacted from

unreacted analyte
If a separation step is employed in an assay, the efficiency of
the separation is critical to the accuracy of the results.
The bound and unbound fractions are usually separated by

physical means including:

Decanting
Centrifugation
Filtration
Washing
Great care must be taken to perform this correctly, because

incomplete washing leads to incomplete removal of the

labeled analyte and inaccurate results.

 solid-phase vehicles used for separation:

polystyrene test tubes
microtiter plates
glass or polystyrene beads
Magnetic beads
cellulose membranes

Term inology
Heterogeneous or Homogeneous

assays:
Heterogeneous assays called

separation assays
Involve a solid phase
Require multiple steps
Careful washing of surface to remove

unbound reagents and samples.

 Homogeneous assays do NOT require

a separation step.
Consists only of a liquid phase, no

washing required
Mix reagents and patient sample.
Measure the labeled product.
Easier and faster to perform

D etection of the Label

The

last
step
immunoassays

common

to

all

Radioimmunoassays: involves a system for

counting radioactivity
other labels such as enzymes, fluorescence,
or chemiluminescence: change in absorbance
in
a
substrate
is
measured
by
spectrophotometry.

All systems must use stringent quality

controls.

Q uality C ontrol
run a blank tube, usually phosphate-buffered

saline, with every test.

This is not expected to have any detectable label

but serves as a check for nonspecific adsorption

and for inadequate washing between steps.
Any readings indicative of label in the blank are

known as background.
If the background is too high, wash steps need
to be made more efficient.

 Negative control
Positive control(low and high)
This serves as a check on the quality of the

reagents to make sure the label is readily

detectable under current testing conditions.

All controls and the patient sample are

usually run in duplicate.

If any controls are out of range, test
values should not be reported.

Radioim m unoassay(RIA)
radioactive substance--label is used as a label.
Radioactive elements have nuclei that decay

spontaneously, emitting matter and energy.

Several radioactive labels includes:
131I
125I
tritiated hydrogen(3H)

 125I
is the most popular.
has a half-life of 60 days
has a higher counting rate than that of

3H, the total counting time is less.

easily incorporated into protein
molecules, and it emits gamma
radiation, which is detected by a gamma
counter

C om petitive B inding
A ssays
pioneered by Yalow and Berson in the late 1950s.
First used to determine the level of insulinanti-

insulin complexes in diabetic patients.

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.
Interpretation:
The amount of label in the bound phase is

INDIRECTLY PROPORTIONAL to the amount

of patient antigen present.

 Examples of substances that are measured

by RIA include:
TSH(thyroid-stimulating hormone)
total serum IgE

Disadvantages:
health hazard involved in working with

radioactive substances
disposal problems
short shelf life
need for expensive equipment

EN ZYM E IM M U N O A SSAY
Enzymes
are naturally occurring molecules that

catalyze certain biochemical reactions.

react with suitable substrates to produce
breakdown products that may be
chromogenic, fluorogenic, or
luminescent.

Spectroscopyused for

measurement

 Advantages:
cheap and readily available
long shelf life
easily adapted to automation
can be measured using inexpensive

equipment.
without disposal problems or the health
hazards of radiation

 Enzyme labels can either be used:

Qualitatively
to determine the presence of an antigen

or antibody

Quantitatively
to determine the actual concentration of

an analyte in an unknown specimen.

 Typical enzymes that have been used

as labels in colorimetric reactions

include:
horseradish peroxidase
glucose-6-phosphate dehydrogenase
alkaline phosphatase
-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

H ETER O G EN EO U S EN Z Y M E
IM M U N O A SS AY S

C om petitive EIA
Steps:
1. Enzyme-labeled antigen competes with

un-labeled patient antigen for a limited

number of binding sites on antibody
molecules that are attached to a solid
phase.
2. WASHING to remove Solid-phase
antibody labeled and specific binding any
non-specifically bound antigen
3. Measurement of enzyme activity

 Interpretation:
Enzyme activity is inversely proportional to the

concentration of the test substance.

The more patient antigen is bound, the less enzymelabeled antigen can attach.

In this manner, a sensitivity of nanograms (10-9

g)/mL can be achieved.

Used for measuring small antigens that are

relatively pure such as:

Insulin
Estrogen

N on-com petitive EIA

are often referred to as indirect Enzyme-

linked Immunosorbent Assays (ELISA)

The

enzyme-labeled reagent does not

participate in the initial antigenantibody
binding reaction.

one

of
the
most
frequently
used
immunoassays in the clinical laboratory due
to its sensitivity, specificity, simplicity, and
low cost.

 Either antigen or antibody may be

bound to solid phase.

solid-phase supports used includes:
microtiter plates
nitrocellulose membranes
magnetic latex beads

When antigen is bound to solid phase, patient serum

with unknown antibody is added and given time to react.

After a wash step, an enzyme-labeled antiglobulin is

added. This second antibody reacts with any patient
antibody that is bound to solid phase.

If no patient antibody is bound to the solid phase, the

second labeled antibody will not be bound.

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

 This technique remains the preferred

screening method for detecting

antibody to:
HIV
Hepatitis A
Hepatitis C

Also used to identify Epstein-Barr

specific antibodies produced in

infectious mononucleosis.

C apture A ssays
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.
Excess antibody attached to solid phase is allowed to combine

with the test sample to capture any antigen present.

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

Capture/Sandw ich
im m unoassay

 Capture assays are best suited to antigens that have

multiple determinants:
Antibodies
polypeptide hormones
Proteins
tumor markers
Microorganisms especially viruses

When used with microorganisms, the epitope must

be unique to the organism being tested, and it must

be present in all strains of that organism.
Use of monoclonal antibodies has made this a very
sensitive test system.

 Clinical applications:
Detection of Rotavirus in stool and

respiratory syncytial virus in respiratory

tract secretions
useful for identifying fungi such as
Aspergillus, Candida, and Cryptococcus
Measurement of immunoglobulins,
especially those of certain classes

Types of ELISA

R A P ID IM M U N O A SS AY

M em brane-B ased C assette

A ssays
are a relatively new type of enzyme

immunoassay
They are rapid, easy to perform, and
give reproducible results.
for point-of-care or home testing use
can be made semi-quantitative for
use in a clinical laboratory
designed as single-use, disposable
assays in a plastic cartridge

 Nitrocellulose
membrane
able to easily immobilize proteins and nucleic acids.

The rapid flow through the membrane and its

large surface area enhance the speed and

sensitivity of ELISA reactions.
Either antigen or antibody can be coupled to
the membrane
Interpreted by presence of a colored reaction
product

Im m unochrom atography
The analyte is applied at one end of

the strip and migrates toward the

distal end, where there is an
absorbent pad to maintain a
constant capillary flow rate.
The labeling and detection zones are

set between the two ends.

 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 test.
This may be in the form of a plus sign.

Im m unochrom atography
Apply sample to one

end, migrates
forward.
Sample dissolves
labeled antigen or
antibody to which it
binds.
Migrates towards
detection zone
where it will bind to
immobilized antigen
or antibody.
Color change occurs.

 Clinical application:
Identification of Streptococcus pyogenes

and Streptococcus agalactiae

Pregnancy test
Troponin in a heart attack
Hepatitis B surface antigen screening test

Test results are most often qualitative

rather than quantitative.

H om ogeneous Enzym e
Im m unoassay
no separation step is necessary.
are
generally
less
sensitive

than
heterogeneous assays, but they are rapid,
simple to perform, and adapt easily to
automation.
No washing 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.

 are based on the principle of change in

enzyme activity as specific antigenantibody

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 measurable
loss of activity.

 Free

analyte (antigen) competes with

enzyme-labeled analyte for a limited number
of antibody-binding sites, so this is a
competitive assay.

Enzyme activity is directly in proportion to

the concentration of patient antigen

hapten present in the test solution.

or

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 antibodys binding
(4) susceptibility of the assay to interference from
endogenous enzyme activity, cross-reacting
antigens, or enzyme inhibitors.

Sensitivity:micrograms per milliliter

far less than that achievable by heterogeneous

enzyme assays

 Disadvantages of Enzyme

Immunoassays:
some specimens may contain natural

inhibitors
size of the enzyme label may be a
limiting factor in the design of some
assays
Nonspecific protein binding

FLU O R ES C EN C E
IM M U N O A SS AY S

 1941, Albert Coons

Fluorophores or Fluorochromes

can absorb energy from an incident light source

convert that energy into light of a longer wavelength
lower energy as the excited electrons return to
ground state.
are typically organic molecules with a ring structure,
each has a characteristic optimum absorption range.

and
and
the
and

The time interval between absorption of energy

and emission of fluorescence is very short and can

be measured in nanoseconds.

 Fluorescein
Absorbs maximally at 490 to 495 nm and emits a green

color at 517 to 520 nm

It has a high intensity, good photostability, and a high
quantum yield.

Tetramethylrhodamine
absorbs at 550 nm and emits red light at 580 to 585 nm.

Other compounds used are:

Phycoerythrin
europium (-naphthyl trifluoroacetone)
lucifer yellow VS

Im m unof u
l orescent assay
(IFA )
a term restricted to qualitative observations

involving the use of a fluorescence microscope.

In this manner, many types of antigens can be

detected either in fixed tissue sections or in live cell

suspensions with a high degree of sensitivity and
specificity.
The presence of a specific antigen is determined by

the appearance of localized color 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,

D irect Im m unof u
l orescent
A ssays
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.

Antigens are typically visualized as bright

apple green or orange-yellow objects

against a dark background.

 best suited to antigen detection in

tissue or body fluids, while indirect

assays
Examples of antigens detected by this

method include:
Legionella pneumophila
Pneumocystis carinii
Chlamydia trachomatis
respiratory syncytial virus (RSV)

Indirect
Im m unof u
l orescent A ssays
involve two steps:
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.
In this manner, one antibody conjugate can be used

for many different types of reactions, eliminating the

need for numerous purified, labeled reagent
antibodies.

 result in increased staining, because

multiple molecules can bind to each

primary molecule, thus making this a
more sensitive technique.
can be used for both antigen and

antibody identification.

Fluorescent Im m unoassay
Complex must form for fluorescence to occur.

Fluorescent Im m unoassay
Antibodies and bacteria are fixed on a glass-plate.
The surplus i.e. non-bounded antibodies are washed out,

antibody-bacteria-complexes ("sandwiches") remain.

The "sandwich" becomes visible by adding fluorescent anti

bovine immunoglobulin which can be seen as green light in

the fluorescence microscope.

Fluorescent Im m unoassay
Direct immunofluorescence
Tagged antibody added to unknown antigen

fixed to slide
If patient antigen present = fluorescence

Indirect immunofluorescence sandwich

assay
Patient plus known fixed antigen
Allow to react and wash off unbound reactants
Add tagged anti-antibody
Fluorescence

Fluorescent Im m unoassay

Positive Im m unofl
uorescence
Cryptosporidium parvum oocysts
Photo Credit: H.D.A Lindquist, U.S. EPA

 Clinical application:
used to detect treponema, antinuclear,

chlamydial, and toxoplasma antibodies,

as well as antibodies to such viruses
such as herpes simplex, Epstein- Barr,
and cytomegalovirus.

Direct and indirect Immunofluorescence

O TH ER FLO U R ES C EN T
IM M U N O A SS AY S

Q uantitative f u
l orescent
im m unoassays (FIA s)
can

be
classified
as
Heterogeneous or Homogeneous,
corresponding to similar types of
enzyme immunoassays.

In

this case, the label is

fluorescent, and such a label can
be applied to either antigen or
antibody.

 Solid-phase

Heterogeneous
Fluorescent Assays
have

been
developed
for
the
identification of antibodies to nuclear
antigen, toxoplasma antigen, rubella
virus, and numerous other virus
antigens.

 Homogeneous Fluorescent Immunoassay

requires no separation procedure, so it is rapid and

simple to perform.

There is only one incubation step and no wash step, and

usually competitive binding is involved.

There is a direct relationship between the amount of

fluorescence measured and the amount of antigen in the
patient sample.

As binding of patient antigen increases, binding of the

fluorescent analyte decreases, and hence more
fluorescence is observed.

 Fluorescence polarization immunoassay

(FPIA)
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, however, 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 antigen 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

that will be detected.
Hence, the degree of fluorescence polarization is inversely

proportional to concentration of the analyte

 Advantages:
high sensitivity and versatility
Simple methodology
No problem with disposal of hazardous waste

Disadvantages:
separation of the signal on the label from

autofluorescence produced by different organic

substances normally present in serum.
nonspecific binding to substances in serum can
cause quenching or diminishing of the signal and
change the amount of fluorescence generated.

C H EM ILU M IN ESC EN T
IM M U N O A SSAYS
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.
most
common
chemiluminescent
substances used:
Luminol
acridinium esters
ruthenium derivatives
nitrophenyl oxalates

 Catalyst used:
Hydrogen peroxide
Enzymes

These intermediates are produced and spontaneously return

to their original state, giving off energy in the form of light.

Light emissions range from a rapid flash of light to a more

continuous glow that can last for hours.

Acridinium ester--emit a quick burst or flash of light
Luminol and dioxetane--light remains for a longer time

Different types of instrumentation are necessary for each

kind of emission.

Chem ilum inescent

Im m unoassays
The process of chemiluminescence occurs when

energy in the form of light is released from

matter during a chemical reaction.

Chem ilum inescent

Im m unoassays
Large number of molecules capable of

chemiluminescence
Luminol
Acridium esters
Ruthenium derivatives
Nitrophenyl oxalates

Use sodium hydroxide as a catalyst

Light emission ranges from quick burst or

flash to light which remains for a longer time.

Different types of instruments are required
based on emission.

Chem ilum inescent

Im m unoassays
Can be used for heterogeneous or

homogeneous assays.
Can attach label to antigen or
antibody.
Heterogeneous assays use
competitive and sandwich assay.
Competitive assays used to measure
smaller analytes.
Sandwich assays are used to
measure larger analytes.

Chem ilum inescent

Im m unoassay

Many applications.
Can measure antigen or antibody.
Add chemiluminescently tagged analyte.
Measure light which is emitted which is directly related to

concentration although competitive binding assays are

available.

Chem ilum inescent

Im m unoassays

Best known application of chemiluminescense is

luminol
Luminol reacts with the iron in blood hemoglobin.

 Advantages:
excellent sensitivity
Stable and nontoxic reagents
inexpensive to perform
High speed
Inexpensive instrumentation