CHEMILUMINESCENCE
Moderator :
Dr S.S.Ronad
Presenter: Dr Smitha .Patil
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Chemi
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Luminescence
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Chemiluminescence Is light produced by a chemical
reaction
Reactions that are biological in origin bioluminescence
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Eg the firefly luciferin-luciferase-ATP reaction
Electrochemiluminescence or electrogenerated
chemiluminescence (ECL) is a kind of luminescence
produced during electrochemical reactions in solution,
where one/all reactants are produced electrochemically
on the electrodes.
GENERAL PRINICPLES
INSTRUMENTATION
CLASSIFICATION OF CL SYSTEM :
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in static sytem discrete portions of the CL
reagent and the sample are mixed rapidly in the
reaction cell, often at a controlled temperature,
usually the final reagent that initiates the CL
reaction is added with a syringe or using a
automatic injector to provide a more reproducible
injection speed and volume and a syncronisation
of data collection in relation to initiation of
reaction
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continuous flow CL systems are used in gas phase
and solution phase reactions the sample and the CL
reagents are continuously pumped and mixed together
using a merging zones and the emmission is observed
in an integral reactor flow cell . the signal is observed
when the cell is totally filled with reaction mixture , at
a fixed period after mixing
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Stopped flow approaches have interesting
features for chemiluminescence monitoring in
very fast CL reactions . Here the sample and
reagent are efficiently mixed in a reaction
chamber and rapidly forced to an observation cell
in which the flow is violently stopped , allowing
monitoring of the full intensity time curve and
allowing kinetics measurements which can be
treated to the analyte concentration with a
higher precision.
LIMITATIONS OF CHEMILUMINESCENCE
INSTRUMENTATIONS :
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Light leaks , light piping and high background
luminescence from assay reagents and reactive
vessels
Stringent controls on the purity of reagents and
solvents
Efficient capture of the light emission from
reactions that produce flash of light requires an
efficient injector .
Pulse pile up in photomultiplier tubes leading to
underestimate of the true light emision intensity
ADVANTAGES OF CL REACTIONS
Relatively simple instrumentation
 Low detection limits and wide dynamic range
detection limits
 Amenability to remote detection by means of a
laser or fiber optic probes
 Applicable to complex solutions
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APPLICATIONS
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Chemiluminescent methods have become
established in both
routine clinical analysis
– clinical research application
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Routine clinical laboratory applications.
The most important routine application of
chemiluminescent in the clinical laboratory is in
immunoassay
– nucleic acid assays.
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IMMUNOASSAY
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Chemiluminescent immunoassay test kits and
automated immunoassay analyzer have been
developed and commercialized by several of the major
invitro diagnostics companies.
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The comprehensive test menus cover analytes
routinely measured in
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Clinical chemistry,
immunology,
toxicology,
virology, and
endocrinology laboratories for the assessment of
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thyroid function, fertility, myocardial
damage, anemia, therapeutic drug levels,
and diagnosing and monitoring drug
abuse, cancer and infectious diseases (e.g.
hepatitis, HIV
The three major chemiluminescent technologies are:
(1) acridinium ester and sulfonamide labels in
chemiluminescent immunoassay
(2) chemiluminescent detection techniques for
horseradish peroxidase labels
luminol based assay
acridian based assay
(3) chemiluminescent detection techniques for alkaline
phosphatase labels in enzyme immunoassay
dioxetanes based
acridan enol phosphates
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EXAMPLES OF AUTOMATED
IMMUNOANALYZERS ARE :
ADVIA centaur and ACS 180 automated CL
systems
 Lumipulse 1200
 Immulite2000
 Lumigen APS-5
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NUCLEIC ACID ASSAYS
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The first major success for chemiluminescence in
nucleic acid testing was the non-separation
chemiluminescent hybridization protection assay for
detecting specific DNA or RNA sequences.
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This assay utilizes an acridinium ester labeled DNA
probe. The probe is readily hydrolyzed to
nonluminescent products but is protected from
hybridization when it is bound to specific target, and
only this form of the probe produces a
chemiluminescent signal.
This type of chemiluminescent test is now widely used
to test for infectious agents (e.g. Chlamydia)
A chemiluminescent end-point is also used in the
branched chain DNA assay for hepatitis C virus RNA
 Other assays developed include hepatitis B virus
DNA, and HIV-1 RNA
 Assays have been developed for Chlamydia
trachomatis and Neisseria gonorrhea DNA, human
cytomegalovirus (CMV) DNA, and human
papillomavirus (HPV) DNA
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CLINICAL RESEARCH
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In clinical research the sensitivity, dynamic
range and diversity of chemiluminescent assays
has led to a vast range of applications, notably in
immunoassays ,protein and nucleic acid blotting,
microarray-based assays, monitoring reactive
oxygen species, and as detection reactions for
substances separated by HPLC, capillary
electrophoresis (CE), and flow-injection analysis.
RESEARCH IMMUNOASSAYS
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A large number of research immunoassays and
emerging clinical tests have been developed in a
chemiluminescent format
cytokines (IL-4, IL-5, IL-6, IL-10)
endothelin-1 (0.5 ng/l) ,
epidermal growth factor (2 ng/ml)
granulocyte colony-stimulating factor (1 ng/ml)
Interferon (0.2 pg/ml)
and vascular endothelial growth factor-C
LOCI
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sensitive non-separation (homogeneous or mix
and measure assay) called the luminescent
oxygen channeling immunoassay (LOCI) .
This assay has been commercialized as the
AlphaScreen and exploits the in situ production
of a chemiluminescent compound due to singlet
oxygen transfer between a donor and an acceptor
antibody-coated microbead (250 nm diameter)
brought into contact as a result of specific
binding with the test antigen
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The assay has been adapted for
competition assays (cAMP assay),
 interaction assays (ligand/receptor, protein/protein,
protein/DNA),
 enzyme assays (e.g. protease, kinase, helicase), and
 immunoassays (thyrotropin, hepatitis B surface
antigen, digoxin)
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NUCLEIC ACID ASSAYS
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The LOCI-type assay has also been adapted for
nucleic acid assays,e.g. single-nucleotide
polymorphism typing
Other chemiluminescent nucleic acid-based
assays include infectious disease testing in
combination with nucleic acid amplification
techniques such as the polymerase chain reaction
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e.g. telomere DNA, herpes simplex , Lassa fever virus
(reverse transcriptase-polymerase chain reaction
method), papillomavirus in cervical scrapes , and
Trichomonas vaginalis (detects <12 organisms)
BLOTTING—WESTERN, NORTHERN AND
SOUTHERN
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Western blotting for proteins
Southern blotting for DNA and
Northern blotting for RNA, are important
techniques in clinical research
chemiluminescent end-points have been
developed for each of these types of assay. These
include
enhanced luminol- and acridan-based reagents for
peroxidase labels, and
– 1,2-dioxetane and acridan reagents for alkaline
phosphatase labels
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REPORTER GENE-BASED ASSAYS
Chemiluminescent dioxetane-type substrates are
available to detect and quantitate expression
products of genes for placental alkaline
phosphatase, galactosidase (GAL), and
glucuronidase (GUS).
 These assays are sensitive and have a linear
range over several orders of magnitude.
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CELLULAR CHEMILUMINESCENCE
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The luminol- or lucigenin-enhanced light
emission from polymorphonuclear neutrophils or
phagocytes, or biological fluids (e.g. whole blood)
is an important tool for cell-based studies
including investigations of the respiratory burst.
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The scope of cellular chemiluminescence studies is
large and includes investigations of
reactive oxygen species produced by human spermatozoa ,
• defects in the production of reactive oxygen intermediates ,
• responses of cells (e.g. PMN-leukocytes, neutrophils) to
drugs and different agents such as H1-antagonists , and
complement receptors , polyunsaturated fatty acids ,lectins
, lysophosphatidic acid , Helicobacter pylori
lipopolysaccharide , and endotoxins
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GENERAL
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Examples of chemiluminescent assays include
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urinary N-acetyl--d-glucosaminidase
cholesterol (1 mg/ml) ,
Vitamin C in seminal plasma ,
and blood spot screening tests for phenyl ketonuria (PKU),
galactosemia, and maple syrup urine disease ,
nasal nitric oxide for the screening of primary ciliary
dyskinesia
and phenol and 4-methylphenol in urine
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Chemiluminescent reactions are also useful as
detection reactions for components of mixtures
separated by techniques, such as HPLC ,
capillary electrophoresis , and flow injection
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Examples include—
detection of polymerase chain amplified hepatitis C
virus DNA separated by capillary electrophoresis;
post-column detection of phosphatidylcholine and
phosphatidylethanolamine hydroperoxides in red
blood cells and the b-lactam antibiotic,
cefaclor,activity in human erythrocytes separated by
liquid chromatography
chemiluminescent assays for choline containing
phospholipids in serum separated by flow injection
and post-column detection of catecholamines and
their metabolites in rat plasma separated by HPLC on
an ODS column
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Nucleic acid and protein microarrays are
important tools in genomic and proteomic
investigations, respectively.
Although fluorescence is the dominant detection
technique, chemiluminescence imaging using a
charged coupled detection device (CCD) camera
can also be used to detect molecules bound to arrays .
for example for the detection of allergen-specific IgE ,
cytokines , and for differential gene expression
profiling .
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Another emerging application of imaging coupled
with chemiluminescence is in microscopy , and
monitoring assays in high-density
microwell plates, e.g. chemiluminescent drug
assays
REFERENCES
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Teitz textbook of clinical chemistry and molecular
diagnostics – 4th edition
Varley’s clinical biochemistry – 6th edition
Lawrence .A.kaplan et al - Clinical biochemistry -4th
edition
W.R.G. Baeyens et al – “Chemiluminescence-based
detection: principles and analytical applications in flowing
streams and in immunoassays”-Journal of Pharmaceutical
and Biomedical Analysis 17 (1998) 941–953
L. J. Kricka ;Clinical Applications of
Chemiluminescence; Analytica Chimica Acta Vol:
500, No: 1-2, December, 19( 2003 )279-286
Thank you