Labeled Immunoassays

Part 4

Fluorescent & Chemiluminescent

Immunoassays

Fluorescence Immunoassay
In 1944 it was demonstrated that antibodies could be
labeled with molecules that fluoresce.
These fluorescent compounds are called fluorophores or
fluorochromes.
They have the ability to absorb energy from an incident
light and convert that energy into light of a longer
wavelength and lower energy as the excited electrons
return to the ground state.
Each Fluorophore has a characteristic optimum
absorption range.
The time interval between absorption of energy and
emission of fluorescence is very short and can be
measured in nanoseconds.
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Characteristics of Fluorescent
molecule
Ideally, a fluorescent molecule should:
Exhibit high intensity, which can be
distinguished easily from background
fluorescence,
It should also be stable,
and have a high molar extinction coefficient
(a measurement of how strongly a chemical species absorbs light at a given wavelength)

Types of Fluorescent molecules

The two compounds most often used are:
Fluorescein
and Rhodamine,
because these can be readily coupled with
antigen or antibody.

Types of Fluorescent molecules

Fluorescein
absorbs maximally at 490 to 495 nm
and emits a green color at 517 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.

Because their absorbance and emission patterns differ,

fluorescein and rhodamine can be used together.

Fluorescein

Heterogeneous Fluorescent
Immunoassays
Require a separation step, include the
following:
indirect,
competitive
and sandwich assays.

Same principle as those of enzyme

immunoassays, but in this case the label
is fluorescent.
Such label can be applied to either antigen
or antibody.
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Heterogeneous Fluorescent
Immunoassays
Use of solid phase is the typical means of
separation in heterogeneous assays.
Microbeads made of polysaccharides and
polyacrylamides have been used
Either antigen or antibody can be attached to the
beads and reacted with analyte and a
fluorescent labeled analyte.
Then reaction mixture is centrifuged, the
supernatant is discarded, and the beads are
analyzed for fluorescence.

Homogenous Assays
There is only one incubation step and no wash step,
Usually competitive binding is involved.
The basis for this technique is the change that occurs in
the fluorescent label on antigen when it binds to specific
antibody.
Such changes can be related to wavelength emission, or
polarity.
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)
With competitive binding, antigen from the specimen and
antigen-fluorescein (AgF) labeled reagent compete for
binding sites on the antibody.
FPIA is utilized to provide accurate and sensitive
measurement of small toxicology analytes such as
therapeutic drugs, and drugs of abuse, toxicology and
some hormones.

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Fluorescence Polarization
Immunoassay (FPIA)
It is based on the change of 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, when it
is excited by polarized light, the emitted light is
unpolarized.
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.
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Fluorescence Polarization Immunoassay (FPIA)

When polarized light is absorbed by the smaller AgF
molecule the AgF has the ability to rotate its position in
solution rapidly before the light is emitted as fluorescence.
The emitted light will be released in a different plane of
space from that in which it was absorbed and is therefore
called unpolarized light.
Emitted light
Polarized light
Anjtibody or Binding partner - BP
Fluorescent analyte
Free analyte
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Fluorescence Polarization Immunoassay (FPIA)

With the larger sized Ab-AgF complex,

the same absorbed polarized light is released as polarized fluorescence
because the much larger Ab-AgF complex does not rotate as rapidly in
solution.

The light is released in the same plane of space as the absorbed

light energy, and the detector can measure it

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Fluorescence Polarization Immunoassay (FPIA)

Measurement of large complexes using fluorescence,
rotation, and polarized light in FPIA
FPIA results in an inverse dose response curve:
lower levels of patient analyte result in a higher signal (in this
case, the signal is polarized light).
High signal at low patient analyte levels results in a highly
sensitive assay.

490
nm

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Advantages and Disadvantages

Advantages
Sensitivity is higher than those of radiolabels and enzyme
reactions.
The methodology is simple and there is no need to deal with and
dispose of hazardous substances.

Disadvantages
The main problem is the separation of the signal on the label from
background fluorescence because of different organic substances
normally present in serum.
Nonspecific binding to substances in serum can cause
diminishing of the signal and change the amount of fluorescence
generated.
Any bilirubin or hemoglobin present can absorb either the
excitation or emission energy.
It requires expensive dedicated instrumentation, which may limit
its use in smaller laboratories.

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Chemiluminescent Immunoassays
Several recently developed immunoassays use
the principle of chemiluminescence to follow
antigen antibody combination.
Chemiluminescence is the emission of light
caused by a chemical reaction producing an
excited molecule that decays back to its original
ground state.
A large number of molecules are capable of
chemiluminescence, but some of the most
common substances used are:
luminol, acridium esters, peroxyoxalates, ruthenium
derivative and dioxetanes.
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Chemiluminescent Immunoassays
When these substances are oxidized,
typically using hydrogen peroxide and an
enzyme

Intermediates are produced that are of a

higher energy state.

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Chemiluminescent Immunoassays
These intermediates 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.
Different types of instrumentation are necessary for each
kind of emission.

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Advantages and Disadvantages

Advantages
Have an excellent sensitivity comparable to EIA and RIA.
Reagents are stable and relatively nontoxic.
The sensitivity of some assays has been reported to be in the
range of (10-18) to zeptomoles ( 10-21).
Because very little reagent is used, they are inexpensive to
perform.
Detection systems basically consist of photomultiplier tubes
which are simple and relatively inexpensive

Disadvantages
False results may be obtained if there is lack of precision in
injection of the hydrogen peroxide
If some biological materials such as urine or plasma cause
diminishing of the light emission.
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