The light emission in a chemiluminescent reaction is
influenced by the quantity of signal used for generation of
light. The manufacturing capabilities are limited globally
and hence a prohibitive cost in procuring the signal for use
in commercial scale. This limits the volume of signal for
generation and also the sensitivity (lesser quantum of light
produced, compromising the assay sensitivity).
The solution for this impediment can be achieved
by increasing the quantity of signal generated in the
reaction process. This is best done by using enhancers,
which increase the intensity of signal produced. In 1985,
chemiluminescence about 1000 times, while also
prolonging the duration of chemiluminescence.
Since the appearance of enhanced chemilumine -
scence, where enzymes like iodophenol, phenothiazine,
etc. are employed to improve the light output of reactions,
enzyme-sensitive chemiluminescent compounds have
been the basis of several new clinical laboratory tests.
These compounds increase the duration and quantum
of signal produced by the reaction. Both peroxidase
(HRP) - and phosphatase-sensitive chemiluminescent
tags are commercially available. More tests employing
these compounds can be expected to reach the clinical
output has resulted in clinical tests with much-improved
This process of enhancement improves the performance of chemiluminescence immunoassay kits.
Equally important is the fact that the light produced by
the reaction process be measured within a specific time.
The chemiluminescent reactions can be of two types
depending on the duration of light produced.
586 Concise Book of Medical Laboratory Technology: Methods and Interpretations Flash
In this, the addition of signal causes the immediate
emission of light, typically over milliseconds or seconds.
The instrumentations generating this type use a module
for injecting the signal into the reaction system (injector
module). These systems have moderate efficiencies. These
systems have the benefit of a traditional chemiluminescent
systems by increased sensitivity and dynamic range,
but with its inherent inadequacies like homogenization
effect, difficult for photon counting and impossibility
of repeat measurements in a reaction. Particularly the
repeat measurement is important because, it gives more
confidence in reporting. This is not possible by these
systems and one has to repeat the entire test for second
The emission of light builds and reaches a maximum.
The emission is stable for a longer period of time making
remeasurement possible. Glow type systems are excellent
for quantitative systems such as immunoassays and
detection of proteins. In the case of glow reactions,
procedure development is relatively simple and the timing
of reagent addition and reagent/sample mixing are not
critical as in flash reactions.
The instrumentations perform the function of
quantification of emission and read out design. There
are many ways of doing this depending on the level of
sensitivity and sophistication required. The instrument
employs a photomultiplier tube (PMT) for this purpose.
These devices can be used in either a current measuring or
photon-counting mode. Photon-counting systems are the
latest development in chemiluminescence technology and
provide greater sensitivity and long-term stability than the
traditional current measuring chemiluminescent systems.
Different types of PMTs exhibit different sensitivities
to different wavelengths and it is, therefore, important
to select the PMT with maximum spectral response for
maximum sensitivity. There are a very few good manufacturers of PMT present globally.
The instrumentations are available from simple one,
which can count photon emissions from a single tube to
fully automated systems capable of counting microplates
by photon-counting mode. These often carry the software
on board to be able to perform data reduction of standards
and samples. The PMT count every single electron
generated by secondary emission from the system in the
form of a pulse and gives the output.
These pulse chemiluminescent systems are better than
other chemiluminescent systems.
Comparison with Other Technologies
The detection of antigen-antibody binding can be done
by many ways. Methods like RIA, ELISA, and fluorescence
immunoassay have been used widely. Of this, ELISA is
adopted commonly for many parameters.
Drawbacks of Other Technologies
¾ Disposal issues, health hazard pertaining to radioactivity
¾ Limitation of photometric measuring range
¾ Low sensitivity in 2nd generation assays
¾ Smaller dynamic range and linearity.
¾ Sensitivity to temperature, pH
¾ Interference from hemoglobin, bilirubin.
“Interference from light scattering, background fluorescence
and quenching can reduce the potential sensitivity of
fluorescence immunoassay by factors between 100 and
“Fluorescent EIAs are identical to other EIAs. There may
be substances in the system that emit fluorescent light.
These substances increase the background signal which
may interfere with the assay’s sensitivity” (Fig. 22.19).
FIG. 22.19: Relative sensitivity
Advantages of Chemiluminescence Technology
1. Linearity: In chemiluminescence, since the individual
photons are counted, there is very high linearity. Very
high values can be obtained without dilution.
2. Stability: The signal generated in chemiluminescence
is stable for long time making it better than other
3. Sensitivity: The lower detection limit is more in
chemiluminescene than other technology.
4. Convenience: There is no second incubation in
chemiluminescence since there is no substrate
5. Cost: Since less signal quantity is used in “Enhanced
pulse chemiluminescence” systems, the reagent
and instrumentation cost are less than the closed
Overall, enchanced pulse chemiluminescence is
favored for the following reasons:
¾ No excitation source is required
¾ Chemiluminescent substrates have a shelf-life of about
a year, whereas those of fluorescence (which contain a
fluorescein molecule) will last only about a week
¾ The level of detection is also lower with that of
chemiluminescence—femtogram level has been well
¾ Fluorescence due to its limited availability is very
expensive. Chemiluminescence is much more affordable
¾ Extraordinary sensitivity; a wide dynamic range;
inexpensive instrumentation; and the emergence of novel
luminescent assays make this technique very popular
¾ Superior sensitivity and low background distinguish
chemiluminescence from other analytical methods
¾ Chemiluminescence is up to 100,000 times more
sensitive than absorption spectroscopy and is at least
1,000 times more sensitive than fluorometry
¾ The background light component is much lower in
chemiluminescence than in other analytical techniques
such as spectrophotometry and fluorometry
¾ Wide dynamic range and low instrument cost are also
distinct advantages of chemiluminescence. Samples
can be measured across decades of concentration
without dilution or modification of the sample cell.
Enhanced pulse chemiluminescence immunoassays
1. Impulse 9.0: An open semi automated chemiluminescent immunoassay system (Fig. 22.20).
¾ First of its kind in the category of chemiluminescent
¾ No protein quenching problem as in fluorescence
¾ Better sensitivity out of all available immunoassay
¾ Simple operation, performs single tests
¾ Robust instrument design. Ideal for distant locations
¾ Alpha Prime LS: Fully automated walkaway chemiluminescent immunoassay system (Fig. 22.21).
¾ Fully automated multiparametric immunoassay system
¾ Can run up to 384 samples at a time
¾ Can perform 18 different parameters simultaneously
¾ Can operate in CLIA and EIA technology also (for
infectious and autoimmune diseases parameters).
PCR stands for the Polymerase Chain Reaction (Fig. 22.22)
and was developed in 1987 by Kary Mullis and associates.
It is capable of producing enormous amplification (i.e.
identical copies) of a short DNA sequence from a single
FIG. 22.20: Impulse 9.0 enhanced pulse chemilunescence system.
DNA (target) sequence lying between known positions
(flanks) on a double-stranded (ds) DNA molecule.
The amplification process is mediated by oligonucleotide
primers that, typically, are 20–30 nucleotides long.
The primers are single-stranded (ss) DNA that have
sequences complementary to the flanking regions of the
target sequence. Primers anneal to the flanking regions
by complementary-base pairing (G=C and A=T) using
The amplified product is known as an amplicon.
Generally, PCR amplifies smallish DNA targets 100–
1000 base pairs (bp) long. (It is technically difficult to
amplify targets > 5000 bp long.)
PCR has many applications in research, medicine and
One PCR cycle consists of three steps:
Heat (usually >90°C) separates double-stranded DNA into
two single strands, referred to as “denaturation”. Since
the hydrogen bonds linking the bases to one another are
weak, they break at high temperatures, whereas the bonds
between deoxyribose and phosphates, which are stronger
covalent bonds, remain intact.
FIG. 22.22: Polymerase chain reaction
Annealing Primer Binding to Target
Primers are short, synthetic sequences of single-stranded
target region of the organism. Two primers are included
in the PCR, one for each of the complementary single
DNA strands that was produced during denaturation. The
beginning of the DNA target sequence of interest is marked
by the primers that anneal (bind) to the complementary
Annealing temperature: Annealing usually takes place
between 40 and 65°C, depending on the length and base
sequence of the primers. This allows the primers to anneal
to the target sequence with high specificity.
Once the primers anneal to the complementary DNA
sequences, the temperature is raised to approximately
72°C and the enzyme Taq DNA polymerase is used to
replicate the DNA strands. Taq DNA polymerase is
a recombinant thermostable DNA polymerase from
the organism Thermus aquaticus and, unlike normal
polymerase enzymes is active at high temperatures.
Taq DNA polymerase, begins the synthesis process
at the region marked by the primers. It synthesizes new
double-stranded DNA molecules, both identical to the
original double-stranded target DNA region, by facilitating
the binding and joining of the complementary nucleotides
that are free in solution (dNTPs). Extension always begins
at the 3’ end of the primer making a double strand out
of each of the two single strands. Taq DNA polymerase
synthesizes exclusively in the 5’ to 3’ direction. Therefore,
free nucleotides in the solution are only added to the 3’ end
of the primers constructing the complementary strand of
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