An immunoassay is a test that uses
antibody and antigen complexes as a
means of generating a measurable result
* It is the light emission that is produced in
certain chemical oxidation reactions.
[A] + [B] → [◊] → [Products] + light
*About 1000 B.C., a Chinese emperor possessed
a magic paint on which the image of an ox
appeared at sunset. This is the first known
case of a man-made substance capable of
storing daylight for later recovery.
*Robert Boyle first estabilished the significance
of oxygen in bioluminescence
*luminous fungus and luminous bacteria was
first reported by Heller in 1843
*In 1877, Radziszewski reported for the
first time on the CL exhibited by the
synthetic organic compound lophine
*The term “chemiluminescence” was first
coined by Eilhardt Weideman in 1888.
*Albrecht, who described the intense
luminescence associated with the alkaline
oxidation of luminol
*Seitz and Neary reported in the same year
on the advantages of CL and BL for chemical
*Chemiluminescence (CL)defined as the
emission of electromagnetic radiation
produced by a chemical reaction.
*reactions generally yield one of the
reaction products in an electronic
excited state producing light on falling to
the ground state
*the emission intensity is a function of the
concentration of the chemical species
involved in the CL reaction
*In general, a chemiluminescent reaction
can be generated by two basic
*In a direct reaction, two reagents, usually a
substrate and an oxidant in the presence of
some cofactors, react to form a product or
intermediate, sometimes in the presence of a
catalyst. Then some fraction of the product or
intermediate will be formed in an electronically
excited state, which can subsequently relax to
the ground state with emission of a photon. The
catalyst, enzyme or metal ions, reduces the
activation energy and provides an adequate
environment for producing high CL efficiency
out of the process.
*indirect or sensitized CL is based on a process
of transfer of energy of the excited specie to a
fluorophore. This process makes it possible for
those molecules that are unable to be directly
involved in CL reactions to transfer their excess
of energy to a fluorophore that in turn is
excited, releasing to its ground state with
*derivatives of lucigenin
*Transition meta ion
*reaction must be exothermic to
produce sufficient energy to form the
electronically excited state.
*reaction pathway must be favorable
to channel the energy for the
formation of an electronically excited
*chemical reaction with other species
*Intramolecular energy transfer
*intermolecular energy transfer
*chemical structure of the CL precursor
*nature and concentration of other substrates
affecting the CL pathway
*presence of metal ions, especially transition
metals involved in the processing of the oxidant
*pH and ionic strength
*presence of energy transfer acceptors
*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.
* A large number of molecules are capable of
chemiluminescence, but some of the most common substances
used are luminol, acridinium esters, 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 emitted light is measured by using luminometer.
*In a CL reaction, A and B react to form a product C, some
fraction of which is present in an electronically excited state,
C∗, which subsequently relaxes to the ground state emitting a
A + B → C∗ → C + hν
*The fraction of molecules emitting a photon on return to
the ground state is the quantum yield (φcl). It is the
product of three ratios:
φcl = φc × φe × φf
Φc:fraction of reacting molecules giving an excitable
Φe:fraction of such molecules in an electronically excited
Φf:fraction of these excited molecules that return to the
ground state by emitting
In heterogeneous assays, competitive and
sandwich formats are the ones most often
used. Smaller analytes such as therapeutic
drugs and steroid hormones are measured
using competitive assays, while the sandwich
format is used for larger analytes such as
protein and hormones.
*Two different types of nonseparation
chemiluminescent immunoassays have
been described. One is based on the
enhancement of light emission when a
labeled antigen binds to a specific
*The other exploits an energy transfer
between chemiluminescently labeled
and a fluorophore-labeled assay
*Chemiluminescent assays have an excellent sensitivity,
comparable to EIA and RIA
*The reagents are stable and relatively nontoxic.
*The sensitivity of some assays has been reported to be
in the range of attomoles (10-18 mol) to zeptomoles
*Because very little reagent is used, they are also quite
inexpensive to perform.
*The relatively high speed of detection also means a
faster turnaround time.
*Detection systems basically consist of photomultiplier
tubes, which are simple and relatively inexpensive.
*false results may be obtained if there is
lack of precision in injection of the
hydrogen peroxide or if some biological
materials such as urine or plasma cause
quenching of the light emission.
*Determination of bioanalyte in clinical specimens
*Chemiluminescence detection in Liquid
*Chemiluminescence detection in capillary
*Bioanalytical applications of chemiluminescent
*Chemiluminescence in DNA analysis
*Determination of drugs using direct
•Chemiluminescence in analytical chemistry
by Ana M Gracia Campana,
•Immunoassay by Eleftherios P. Diamandis,
Theodore K. Christopoulos
•Tietz Text Book of Clinical Chemistry and